International Review of Cytology, Volume 28

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME28

Contributors to Volume 28 E. C. COCKING WILLIAMP.

JACOBS

ROBERTC. KING R. B. MORETON R. L. MOTT MAUREENOWEN CHRISTIAANP. RAVEN R E N ~SIMARD F. C. STEWARD J. E. TREHERNE

INTERNATIONAL

Review of Cytology EDITED BY

J. F. DANIELLI

G. H. BOURNE I’wkes Regional Primate Research Center Emory University Atlanta, Georgia

Center for Theoretical Biology State U n i i w f i f j of N e w Y o r k at Buffalo Buffalo, New YO^

ASSISTANT EDITOR K. W- JEON Center for Theoretical Biology State University of N e w York at BuflaEo Buffalo, New York

VOLUME28

Prepared Under the Auspice! of T h e International Soiiety jor Cell Biolo

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FIG. 8. The relative conduction velocity (6test/6normal) of the fast axons in the cerebrovisceral connective of the lamellibranch A. cygnea plotted as a function of the square root of the relative sodium concentration (Nat,st/Na,,r,al) %, in solutions in which the sodium ions were diluted with dextran and tris chloride, respectively. This graphic form permits a comparison to be made with data for Carcinus (Katz, 1947) and Loligo axons (Hodgkin and Katz, 1949). The data for Loligo axons was calculated from mea~urement~ made on the maximum rate of rise of action potentials in test solutions of varying sodium concentration. These calculations were made on the assumption that in a simplified theoretical system the conduction velocity can be related to the square root of the rate of rise of action potential (Hodgkin and Katz, 1949) (from Carlson and Treherne, 1969).

most complete glial investiture (Treherne et al., 1969b). On the other hand, Anodonta shows a much more marked regulation of the extraneural environment than does Helix,whereas Helix has the more complex glial system. It is conceivable that sodium ions could be sequestered by large, indiffusible anions in the intercellular spaces, so that their thermodynamic activity normally remains very low, and released, possibly as a result of the electrical charges occurring at the onset of an action potential, to take part in the conduction process.

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Such an extracellular sodium store is apparently not the only source of sequestered sodium in the region of the axons surfaces in Anodonta, however, for it was observed that there was a rapid return of axonal function when preparations were removed from tris or choline chloride to isotonic dextran solutions (Carlson and Treherne, 1969). It appears necessary, as indicated above, to postulate an additional intracellular sodium fraction, perhaps associated with the glial processes, to explain the ability of the fast axons of Anodonta to function in sodium-free nonelectrolyte solutions. It is of particular interest in this respect to note there is histochemical evidence for the existence of nonfreely diffusible sodium in the Schwann cells of cephalopod giant axons (Villegas, 1968). The neurons of the arthropod species that have been studied appear to be conventional in the ionic mechanisms responsible for determining the resting and action potentials. Comparison of the measured resting potentials with the calculated potassium equilibrium potentials for a number of crustacean and insect species indicates that this cation is largely responsible for carrying the outward current associated with the resting potential (cf. Treherne, 1966). Departure from the relation predicted by the Nernst equation with varying external potassium concentrations described by various workers (e.g., Dalton, 1958, 1959; Yamasaki and Narahashi, 1959; Julian et al., 1962a,b; Edwards et al., 1963; Treherne and Maddrell, 1967b) indicates, however, that some other ion species are also involved in determining the resting potential. This state of affairs is essentially similar to that in the squid giant axon (Curtis and Cole, 1942) where, allowing for the permeabilities of potassium, sodium, chloride, and the variations in potential, the resting potential can be well accounted for (Baker et al., 1962). As in the conventional squid giant axon preparation, the action potentials of isolated crustacean (Dalton, 1958, 1959) and insect axons (Boistel and Caroboeuf, 1958; Yamasaki and Narahashi, 1959; Treherne and Maddrell, 1967b) show a striking dependence on the external sodium concentration. In crayfish giant axons, the magnitude of the action potential was close to that which would be predicted for an ideal sodium electrode (Dalton, 1959), although in cockroach axons the departure from the predicted Nernst relation makes it necessary to postulate that the conductances of other ions may participate in determining the peak of the action potential (Yamasaki and Narahashi, 1959; Narahashi, 1963). Voltage-clamp experiments also confirm the dominant part played by sodium ions in the inward current of the action potential in lobster (Julian et al., 1962b) and cockroach giant axons (Pichon and Boistel, 1967; Pichon, 1968). The existence of apparently conventional, largely sodium-induced action POtentials in arthropod axons is of some interest in relation to the bizarre ion

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concentrations encountered in the blood of some insect species (Section 111). In particular, the extremely low sodium concentrations, which are frequently exceeded by those of potassium ions, present difficulties in the interpretation of action protential production in terms of the conventional membrane theory. The efficiency of the physiological mechanisms involved in maintaining appropriate extraaxonal ion concentrations in the central nervous system of Curuzisizls is well illustrated in the experiments in which axonal function was maintained for extended periods in preparations bathed in sodium-free solutions, whereas desheathed preparations showed a rapid decline in excitability under these circumstances (Treherne and Maddrell, 1967b). The ability to function for appreciable periods in the absence of external sodium ions does not, however, appear to be confined to the axons of phytophagous insects, for conduction was maintained for several hours in the intact nerve cord of Periplaneta in sodiumdeficient saline (Twarog and Roeder, 1956) and in isotonic glucose (Yamasaki and Narahashi, 1959). The sodium dependency of the axons of Caruusiiis under these conditions is also confirmed by the rapid effect of dilute tetrodotoxin on action potential production in intact preparations maintained under these conditions (Treherne and Maddrell, 1967b). The neural fat-body sheath (Section 11, A ) which surrounds the central nervous system of CurdzoinJ- (Maddrell and Treherne, 1966) does not appear to be involved in regulating the sodium level in the extraaxonal fluid, for the ability of the axons to function in sodiumfree solutions was not impaired by removal of this structure (Treherne and Maddrell, 1967b). It is also apparent from the preceding section that the ability to function in sodium-free solutions does not result from an appreciable peripheral diffusion barrier associated with the nerve sheath in insect species. It also appears unlikely that the perineuriuin is involved in actively pumping sodium ions into the general extracellular system, as was suggested by Shaw and Stobbart (1963), for it has been shown that the magnitude of the rapidly exchanging sodium fraction, which has been postulated to be largely extracellular (Section HI), is related to the concentration of sodium in the bathing medium (Treherne, 1962b). Finally, it seems reasonable to reject the possibility that elevated concentrations of cations might be maintained at the axon surfaces in Cdr~~u.riaiand Peripbneta because of the presence of extracellular indiffusible anion molecules (Section 111). It has been pointed out (Treherne, 1967) that the thermodynamic activity of sodium ions associated with a fixed-charge system, such as the anion groups associated with extracellular acid niucopolysaccharide (Section 111), could only be equivalent to that in the bathing solution. Thus, the activity of the cation would be no higher than that in the bathing medium. The possibility cannot be eliminated, however, that the sodium associated with such fixed-anion groups could form part of a cation reservoir

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which might be mobilized if local circuit current flow altered the configuration of the anion groups as has been tentatively suggested earlier in respect to molluscan nervous systems. The possibility also exists that regulation of the extraaxonal sodium level might be achieved by the activity of the glial cells (Treherne and Maddrell, 1967b; Treherne, 1967). In both Peripbneta and Carausius, the subperineurial glial system consists of a peripheral cell layer with an extensive system of cytoplasmic processes (Section V, C) (Wigglesworth, 1960; Smith and Treherne, 1963; Maddrell and Treherne, 1967; Treherne and Maddrell, 1967b). The axons are surrounded by at least one glial fold, the glial membranes being closely applied to the axon surfaces so as to leave extracellular clefts about 150 A in width (Fig. 6). The mesaxons pursue a tortuous course so as to form an extended pathway between the perineurium and the axon surfaces. In view of the demonstrated movement of molecules between adjacent glial cells by tight junctions (Section 111), it seems possible that there could be a rapid intracellular movement of sodium ions to glial processes which could be associated with an extrusion of the cations into the very restricted extracellular spaces adjacent to the axon surfaces, perhaps by conventional sodium pumping mechanisms. The efficiency of such a glial sodium-regulating system would depend on some degree of restriction on sodium movements away from the axon surfaces. It would not be unreasonable to suppose that the tortuous nature of the intercellular channels in the neuropile might restrict sodium movements away from the axon surfaces relative to intracellular movements of the cation via the glial processes. The above system would be in accord with the observations that even local desheathing results in a loss of function in preparations maintained in sodium-free solutions, for, as already mentioned (Section 111) , this procedure results in an invasion of the glial system by relatively large peroxidase molecules via the damaged perineurium and the tight junctions between adjacent glial membranes (Lane and Treherne, 1969). Accordingly, it would be possible to postulate that the loss of extraaxonal regulation in desheathed preparations results from the disruption of normal glial function. The response of insect neurons to surgical damage of the preparation contrasts strikingly with the situation in molluscan nervous systems. As already emphasized, isolated neurons of Helix, with the associated glial coverings, and the split connectives of Anodonta are able to maintain action potential production for appreciable periods in sodium-free solutions as compared to the extremely rapid loss of axonal function encountered in insect nerves and ganglia that have suffered damage to the perineurium during the desheathing procedure. It seems possible that this difference might be a reflection of the differences in the extraneuronal sodium-regulating systems in the two invertebrate groups.

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Thus, as has been shown in Anodoiztd (Carlson and Treherne, 1969), the sodium utilized in maintaining action potentials in sodium-free nonelectrolyte solutions results from the utilization of a small sequestered sodium source in the region of the axon surfaces, probably in the form of an accessible extracellular fraction together with an intracellular fraction which cannot be readily displaced by organic cations such as tris or choline. In the insect nervous system, on the other hand, the extreme sensitivity of extraaxonal sodium regulation to glial damage suggests a more dynamic system perhaps, as suggested above, involving local recycling of sodium ions in the region of the axon surfaces so as to maintain axonal function in sodium-deficient solutions. An interesting feature of the cockroach central nervous system is the description of a difference in magnitude of the resting potential and overshoot of the action potential between desheathed and intact preparations (Pichon and Boistel, 1967). The amplitude of the action potential was found to be greater in intact preparations (103.0 t 5.4 mV) than in desheathed ones (85.9 2 4.6 mV) with an external bathing solution containing 2 10.2 mmoles/liter sodium. The effect appears to be associated with a positive potential, which in a subsequent paper was found to average as much as 28.2 mV (Pichon and Boistel, 1968), between the extracellular fluid and the Lathing solution or hemolymph, for potentials of this magnitude were recorded when the tip of the microelectrode was withdrawn into an apparently extracellular position following impalement of axons in intact preparations (Pichon and Boistel, 1967). This positive potential, recorded between the indifferent electrode and the recording electrode with the tip in an apparently extracellular position, cannot be wholly attributed to variations in tip potential obtained as a result of differing ion concentrations at the tip of the electrode (cf. Adrian, 1956). These results are somewhat difficult to interpret in view of the possible damage caused to the giant axons in the desheathing procedure (although it should be emphasized that isolated giant axons do maintain steady resting and action potentials for appreciable periods) or to other possible effects such as swelling of the axons in desheathed preparations. If it is assumed, however, that the intracellular concentrations of sodium and potassium remain unaffected by the desheathing procedure, then it seems clear that larger action potentials recorded in the intact preparations could result from higher concentrations of sodium ions in the fluid bathing the axonal surfaces in intact as compared with desheathed preparations. If it is further assumed that the concentration of sodium ions in the extraaxonal fluid in desheathed preparations is similar to that in the bathing medium, then it is possible to calculate the concentration of sodium in the fluid immediately surrounding the axons in intact preparations. In saline containing 210.2 mmoles/ liter sodium, the values for the resting and action potentials, 67.4 and 85.9 mV,

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respectively, give an overshoot of 18.5 mV in desheathed preparations. In the intact preparation, the resting and action potentials were 103.0 and 58.1 mV, respectively, with a positive extracellular potential of 6.7 mV. If the positive potential is added to the measured resting potential of the axon, these figures yield a value of 38.2 mV for the overshoot of the action potential in intact preparations. This would correspond to a difference of 19.7 mV between the measured overshoot in intact and desheathed preparations. From the data of Yamasaki and Narahashi (1959), it can be calculated that the active membrane potential of cockroach giant axons shows a 48.0 mV increase per 10-fold increase in external sodium concentration. A difference of 19.7 mV would thus correspond to a concentration of sodium ions at the axon surfaces which would be greater, by a factor of about 2.3, in intact as compared to desheathed preparations. With a bathing medium of 210.2 mmoles/liter sodium, this would correspond to a concentration of 483.0 mmoles/liter sodium in the fluid bathing the axon surfaces in intact preparations used by Pichon and Boistel (1967). In view of the possible secondary effects produced by the desheathing procedure, it would obviously be unwise to examine the calculated values obtained above too closely. These considerations do show, however, that there is the possibility that the extraaxonal fluid niay well have a socliuiii concentration veiy much higher than that of the hemolymph or bathing medium. It is of some interest to consider the above in relation to the suggested mechanism involving a regulation of the extraaxonal sodium level by the activity of the glial cells (Treherne and Maddrell, 1967b; Treherne, 1967). The polarity of the extracellular potential described by Pichon and Boistel (1967, 1968) is the opposite of that which would be expected if it resulted from the outward diffusion of cations maintained by Donnan forces in the extracellular fluid. A positive extracellular potential would be obtained, however, from the presence of an electrogenic sodium pump extruding the cations into the extracellular system in the vicinity of the axonal surfaces. If such an electrogenic sodium pumping system involved glial cells, and perhaps also the perineurium, then the loss of the extracellular positive potential (which, as we have seen, is also correlated with the loss of ability of the axons to function in sodium-deficient media) could be explained by changes in the glial system produced by the desheathing procedure. The fact that the glial cytoplasm in desheathed preparations becomes accessible to molecules as large as those of peroxidase (Lane and Treherne, 1969) clearly indicates that there must be a rapid leakage of small water-soluble ions and molecules from within the glial cells which would produce a dramatic interference with normal glial function. It is possible, therefore, to relate the electrophysiological findings of Treherne and Maddrell (1967b) and of Pichon and Boistel (1967, 196s) to the ultrastructural evi-

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dence (cf. Smith and Treherne, 1963; Treherne and Maddrell, 1967b) and to recent findings concerning the accessibility of insect central nervous tissues to large extracellular indicator molecules (Lane and Treherne, 1969) .

V. General Conclusions It is apparent from the preceding discussion that there is considerable variation in the degree of regulation of the extraneuronal environment as far as inorganic ions are concerned. In the leech central nervous system, for example, movements of inorganic ions take place rapidly through the narrow intercellular channels between the bathing medium and the extraaxonal fluid, and the glial cells appear to play very little part in regulating the immediate ionic environment of the neurons. An essentially similar state of affairs exists in the cephalopod giant axons. In some molluscan and insect species, however, there is clear evidence of regulation of the content of inorganic ions in the extraneuronal fluid. This generalization does not imply that all neurons in the nervous systems exist in a regulated environment. In the freshwater lamellibranch A . cygzea, for example, the majority of the axons in the cerebrovisceral connective respond to changes in the ionic composition of the bathing medium in much the same way as has been described for the leech neurons, and only relatively few large axons show evidence of functioning in a controlled ionic environment. Despite the extremely specialized nature of the blood and the demonstrated ability of some molluscan and insect neurons to function for appreciable periods in preparations bathed in sodium-free solutions, it appears, nevertheless, that the ionic basis of action potential production is conventional in that the inward current is largely carried by sodium ions. There also appears to be a ready access of inorganic ions from the bathing medium to the neuronal surfaces in at least one such molluscan species. In the case of a gastropod and a lamellibranch species it seems likely that the sodium utilized by these neurons is sequestered in the region of the neuronal surfaces and is released into the extraneuronal fluid, thus sustaining neuronal function in preparations bathed in sodiumdeficient media. The available evidence indicates that this extraneuronal sodium store, which has been shown to be depleted by stimulation of the neurons in sodium-free conditions, may exist in two fractions : an extracellular fraction, one perhaps associated with indiffusible anion molecules, which is accessible to and can be displaced by small organic cations, and an intracellular fraction, most probably situated in glial elements, which cannot be easily displaced by small organic cations but which can be mobilized to maintain an appropriate sodium level in the fluid immediately surrounding the neurons. Extraneuronal sodium regulation in insect species appears to be achieved by

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physiological mechanisms somewhat different from those in the molluscan nervous systems that have been investigated. In particular, the ability of axons to function in sodium-deficient media is dependent upon the integrity of the glial system. Loss of the ability to regulate the sodium concentration in the fluid immediately surrounding the axons, observed in desheathed preparations, does not appear to result, as was originally supposed, from the disruption of a peripheral diffusion barrier associated with the nerve sheath, but from effects on the underlying glial system. Thus, even relatively large molecules can move from the region of the damaged perineurium in desheathed preparations into the glial processes surrounding axons within the neuropile by means of transversely permeable tight junctions between adjacent glial membranes. It is envisaged, therefore, on the basis of the currently available evidence, that there is an active extrusion of sodium ions from the glial cytoplasm into the restricted 150-200 A extracellular channels adjacent to the axon surfaces. It is also suggested that the positive potential shown to exist between the extracellular fluid and the bathing medium could result from the activity of an electrogenic sodium pump situated in the glial membranes. The system of fixed-anion groups, which may be associated with the extracellular acid mucopolysaccharide of insect nerves and ganglia, is unlikely to produce sodium of higher activity in the region of the axon surfaces relative to that in the blood or bathing medium. The possibility cannot be eliminated, however, that the sodium associated with such fixed-anion groups could form part of a cation reservoir which might be utilized if there were a transitory drop in the sodium activity of the extraaxonal fluid or if local circuit current flow altered the configuration of the anion groups.

ACKNOWLEDGMENTS Me are grateful to Drs. B. L. Gupta and N. J. Lane for reading and commenting on some portions of the manuscript for this article and for their help, and that of Dr. R. L. Tapp (Department of Physiology, Cambridge) in preparing some of the electron tnicrographs.

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Ramsay, J. A. (1953). J. Exptl. Biol. 30, 358. Robertson, J. D. (1964). In “Physiology of Mollusca” (K. Wilbur and C. M. Yonge, eds.), Vol. 1, p. 283. Academic Press, New York. Rosenbluth, J. (1963). 2.Zellforsch. Mihmikop. Anat. 60, 213. Sandeman, D. C. (1967). Proc. Roy. Soc. (London) B168, 82. Schlote, F. W . (1957). Z . Zellforsch. Mikroskop. Anat. 45, 543. Schmekel, L., and Wechsler, W. (1968). Z. Zellforsch. Mjkroskop. Anat. 89, 112. Shaw, J., and Stobbart, R. H. (1963). Advan. Insect Physiol. 1, 315. Simpson, L., Bern, H. A., and Nishioka, R. S. (1966). Anz. Zoologjst 6, 123. Smith, D. S., and Treherne, J. E. (1963). Advan. Insect Phy.rio1. 1, 401. Sorokina, 2. A. (1966). Fiziol. Zh. Akad. Nauk Ukr. RSR 12, 776. Sorokina, 2. A., and Zelenskaya, V. S. (1967). Zh. Evolyzltsjonnoi Biokhim. Fiziol. 3, 25. Stephens, P. R., and Young, J. 2. (1969). Phil. Trans. Roy. SOL. Londoiz B255, 1. Treherne, J. E. (1961a). J. Exptl. Biol. 38, 315. Treherne, J. E. (1961b). J. Exptl. Biol. 38, 629. Treherne, J. E. ( 1 9 6 1 ~ ) .J. Exptl. Biol. 38, 729. Treherne, J. E. (1961d). J. Exptl. B i d . 38, 737. Treherne, J. E. (1962a). J. Exptl. B i d . 39, 193. Treherne, J. E. (1962b). J. Exptl. Biol. 39, 631. Treherne, J. E. (1965). 1. Exptl. Biol. 42, 7. Treherne, J. E. (1966). “The Neurochemistry of Arthropods.” Cambridge Unii . Press, London and New York. Treherne, J. E. (1967). In “Insects and Physiology” (J. W. L. Beament and J. E. Treherne, eds.). Oliver & Boyd, Edinburgh and London. Treherne, J. E., and Maddrell, S. H. P. (1967a). J. Exptl. Biol. 46, 413. Treherne, J. E., and Maddrell, S. H. P. (1967b). J. Exptl. Biol. 47, 235. Treherne, J. E., Mellon, D., and Carlson, A. D. (1969a). J . Exptl. Biol. 50, 71 I . Treherne, J. E., Carlson, A. D., and Gupta, B. L. (1969b). Natirre 223, 337. Tristram, G . R. (1953). I n “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. 1, p. 224. Academic Press, New York. Trujillo-Cenbz, 0. (1962). Z . Zellforsch. Mikroskop. Anat. 56, 649. Twarog, B. M., and Roeder, K. D. (1956). B i d . Bull. 111, 278. Van Harreveld, A., and Maihotra, S. K. (1966). J . Cell Sci. 1, 223. Veprintsev, B. H., Gerasimov, V. D., Krasts, I. V., and Magura, I. S. (1966). BiofiziFn 11, 1000. Villegas, G. M., and Villegas, R. (1960). J. Ulrtruct. Res. 3, 362. Villegas, G. M., and Villegas, R. (1968). J. Gen. Phyriol. 51, 44s. Villegas, J. (196s). J . Gen. Phy.riol. 51, 61s. Villegas, J., Villegas, L., and Villegas, R. (1965). J. Gen. Phy.riol. 49, 1 . Villegas, J., Villegas, R., and Gimenez, M. (1968). J . Gen. Physiol. 51, 47. Villegas, R., Caputo, C., and Villegas, L. (1962). J . Gen. I’hysiol. 46, 245. Villegas, R., Villegas, L., Gimenez, M., and Villegas, G. M. (1963). J. Gerz. I-’hyriul. 46, 1047. Wigglesworth, V. B. (1959a). Quart. J . Microscop. Sci. 100, 285. Wigglesworth, V. B. (1959b). Quart. 3. Micro.rroi1. Sci. 100, 299. Wigglesworth, V. B. (1960). J. Exptl. Bid. 37, 500. Wyatt, G. R. (1961). Ann. Rev. Entomoi. 6, 75. Yamasaki, T., and Narahashi, T . (1959). J. Insect Physiol. 3, 146.

Virus Uptake, Cell Wall Regeneration, and Virus Multiplication in Isolated Plant Protoplasts E. C. COCKING Department of Botany, University o/ Nottinghnnz, Notzingham, England

I. Introduction: The Isolated Protoplast System . . . . . . . . . . . 11. Uptake of Viruses by Isolated Protoplasts . . . . . . . . . . . . . . A. Entry of Macromolecules into Plant Cells: Evidence for Pinocytosis in Isolated Protoplasts . . . . . . . . . . . . . B. Virus Uptake and the Initiation of Infection . . . . . . . III. Cell Wall Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Virus Multiplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

89 92 92 100 10s 115 122

I. Introduction : The Isolated Protoplast System One of the distinguishing characteristics of most higher plant cells is that they possess a rigid cell wall. The phenomenon of plasmolysis in which it is possible to observe the protoplast of the cell drawing away from the cell wall is dependent in part on the presence of this rigid wall. Yotsuyanagi (1953) observed that plasmolysis of the cells of an Elodea leaf in a solution of CaC1, or Ca(N03) produced protoplasts within the cells which often survived for more than 50 days. He also observed (Fig. 1) small spherical fragments of cytoplasm which had become isolated spontaneously from the protoplast during this prolonged plasmolysis. Similar small spherical fragments of cytoplasm (balls of protoplasm) were produced in the microdissection studies of Plowe (1931) on the cells of the epidermis of bulb scales of Bermuda onions. Strips of epidermis were plasmolyzed in 18% (0.56 M) sucrose for about 2 0 minutes; during this time the protoplasts were reduced to about half their original volume and were well rounded away from the end walls. A strip was then cut with a sharp razor transversely to the long axis of the leaf, and the blade passed between the end walls and protoplasts of many cells, leaving the protoplasts untouched and uninjured. This mechanical method of isolating protoplasts was originally described by Klercker in 1892. Plowe noted that when the solution in which plasmolyzed, sectioned material was mounted was diluted, the protoplasts moved toward the open end of the outer cells as they swelled until they partially protruded or were even set free into the surrounding medium. Frequently, she was able to pinch these protruding protoplasts in two using a needle, and initially a thin strand of plasmalemma connected the two portions (Fig. 2 ) . She noted that one portion was enucleate, yet streaming in it con89

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FIG. I. Cell of Elodea plasmolyzed for a long time in a solution of CaCI, or Ca(N03)2. Small spherical fragments of cytoplasm (subprotoplasts) are present as well as the large subprotoplast (Yotsuyanagi, 1953).

tinued in exactly the same manner as in protoplasts containing a nucleus even after the connecting thread was broken. The strand or connecting thread is apparently self-sealing. Each of the portions of the original protoplast was surrounded by part of the plasmalemma that covered the protoplast initially, and the plasmalemma was unbroken. Somewhat similar division of the protoplast on plasmolysis, particularly in the presence of Ca++ ions, has been observed by Yoshida (1962), Kamiya (1959), and Stadelmann (1956). These portions of the protopIast have been named subprotoplasts by Cocking (1963). Mechanical methods for the isolation of plant protoplasts, although providing suitable material for investigating, for instance, the iiifluence of environmental factors on the osmotic behavior of isolated protoplasts (Vreugdenhil, 1957), have always been limited by the small number of protoplasts that can be

FIG.2. A protoplast protruding from the cut end of a cell is seen dividing into two. The needle (arrow) is pulling one of these subprotoplasts away from the other, but at this stage the two subprotoplasts are still connected by a thin strand of plasinalemina (Plowe, 1931).

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readily isolated. The introduction of a method by Cocking (1960) for the isolation of protoplasts which involved the enzymic degradation of the cell wall by Myrothecium verrucaria cellulase has permitted far greater numbers of protoplasts to be readily isolated from a wide range of different tissues and their

FIG.3. Isolated tobacco leaf protoplasts together with isolated vacuoles (Power and Cocking, unpublished observations).

properties investigated. The use of cellulases for the isolation of root, cotyledon, and fruit protoplasts has been described (Cocking, 1960, 1961a; Gregory and Cocking, 1963) , and Ruesink and Thimann (1965) have also reported the use of M. verrucaria cellulase for the isolation of protoplasts from Avena coleoptiles and from a wide range of higher plant tissues including tissue culture cells (Ruesink and Thimann, 1966). The large-scale isolation of protoplasts from immature tomato fruit using commercially available pectinase to

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degrade the highly pectinaceous cell walls of tomato locule tissue has also been described (Gregory and Cocking, 1965). Recently, Power and Cocking (1968) have reported a simple method for the isolation of very large numbers of leaf protoplasts involving the use of mixtures of commercially available Trichoderma viride cellulase and pectinase to degrade the cell walls, chiefly those of the mesophyll cells of the leaf. Isolated vacuoles are often produced as well, and a typical preparation of such isolated leaf protoplasts and vacuoles is shown in Fig. 3. The ready availability of these enzymically isolated protoplasts has greatly stimulated work particularly in relation to studies on the entry of macroniolecules into protoplasts (see Section II), on cell wall regeneration (see Section HI), and on the ability of such regenerated isolated protoplasts to support the multiplication of plant viruses (Section IV) . Although it will not be discussed further in this review the ability of isolated protoplasts to fuse (Michel, 1937), which has recently been more fully investigated by Binding (1 9 6 6 ) , should be noted. This work was carried out using mechanically isolated protoplasts, and it could well be that far more extensive fusion is possible with the greater range of isolated protoplast types now available. To some extent, the use of enzymes to liberate protoplasts from cells by digestion of the cell wall can be regarded as one of the simplest forms of cell fractionation and, indeed, pectinase and cellulase have been used in more extensive degradation procedures on leaf tissue to obtain readily large quantities of nuclei fractions uncontaminated with other organelles (D’Alessio and Trim, 1968). 11. Uptake of Viruses by Isolated Protoplasts A. ENTRYOF MACROMOLECULES INTO PLANTCELLS:EVIDENCE FOR PINOCYTOSIS IN ISOLATED PROTOPLASTS Several workers, including Whaley et al. ( 1 9 6 4 ) , have commented on the numerous bays and infoldings in the plasmalemma of plant cells, observable by electron microscopy, and have noted that many of these membrane irregularities are to be seen in cells in which there is no conspicuous secretion of a Golgi product. These workers have concluded that it seems quite likely that a process similar to pinocytosis may occur in plant cells despite the presence of the cellulosic wall. Other workers, particularly Bradfute et al. ( 1 9 6 4 ) , have observed that morphological evidence suggestive of vacuole formation by the cell membrane may be found as a result of phenomena other than pinocytosis. The liquid endosperm of Piszm sativum was employed in these studies so that the presence of a rigid highly impermeable cell wall was not a complicating factor in following the uptake of fluorescent-labeled basic proteins by phase and fluorescent microscopy. The isolated protoplast system is one in which marker mole-

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cules can be presented directly at the surface of the plasmalemma to a relatively uniform population of protoplasts which can be readily handled for electron microscope observations, whether by thin-sectioning or by freeze-etching, or for biochemical assays. Since the cell wall is absent, it is not a complicating factor and it is possible to devise experiments to follow readily any pinocytic uptake of ferritin, viruses, or suitably labeled material. The extent, however, to which pinocytosis detected in isolated protoplasts is comparable to that in the cell itself is more difficult to ascertain (Clowes and Junniper, 1968). Isolated protoplasts of higher plants, because of their special osmotic relationships, have to be kept in a suitable plasmolyticum. Usually this is 20% sucrose together with small amounts of various salts and special nutrients. From work with amoebas and mammalian cells, it is known that pinocytosis is induced by a wide range of different substances and inhibited by others (ChapmanAndresen, 1964), and it could well be that on occasion the extent of pinocytosis in isolated protoplasts is greatly in excess of that in the cell itself. The real difficulty in comparing these two systems is that, as previously mentioned, the cell wall acts as a very efficient ultrafilter largely preventing the penetration through it of ferritin (Barton, 1964) or of certain viruses (Cocking and Pojnar, 1969) and, as a result, it is impossible to use marker molecules with cells as it is with protoplasts. It seems likely that all modes of vesicle formation by the plasmalemma may have a basic course. Threadgold (1967) has provided a very lucid account of the various processes involved in this vesicle formation as far as animal cells are concerned. Phagocytosis i s the ingestion of large particles such as bacteria and results in the formation of relatively large vacuoles (Fig. 4 ) . Pinocytosis proper is also regarded as involving the formation of active fringes by the cell surface (plasma membrane), but these occasionally fall back into the cell (Fig. 4). In micropinocytosis no pseudopodia or veils of cytoplasm are formed; in this instance there is invagination of the plasma membrane. Rhopheocytosis involves attachment of macromolecules to the plasma membrane as an essential sequel before uptake takes place (Fig. 5 ) . Cytopemphis involves the formation of vesicles as in rhopheocytosis but these vesicles are then discharged into a main control vacuole system (Fig. 5 ) . As Threadgold has pointed out, pinocytosis in animal cells has come to be used as a term to describe the formation of any small vacuole formed by invagination of the cell surface regardless of whether or not prior attachment to the plasma membrane takes place. Holter (1959) has repeatedly stressed that there seems little effectual difference between pinocytosis and phagocytosis since the main diff ereiice does not appear to be the mechanism of the process but more the dimensions of the vesicles formed and the nature of the material being taken up. Moreover, in relation to pinocytosis itself, Holter (1963) has pointed out that pinocytosis by invagi-

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nation is fairly variable with regard to the shape of the cavities formed and also extremely variable with regard to dimensions. In animal cells, pinocytosis and phagocytosis has also frequently been directly implicated in intracellular digestive processes. Gordon et al. (1965) were able a

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( a ) Serial drawings illustrating phagocytosis. The opposing evaginated pseudopodia eventually fuse and trap the particle. ( b ) Pinocytosis. (c) Micropinocytosis (Threadgold, 1967).

FIG. 4 .

to show in electron microscope studies the phagocytic uptake of DNA-protein coacervates containing colloidal gold by strain L fibroblasts. From studies of the progressive morphological alterations of these phagocytized gold-inarked coacervates, these workers were able to formulate the structural pathways involved in their degradation. Various forms of lysosomes were found to be in-

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volved, and a schematic scheme of the proposed structural pathways of intracellular digestion in these strain L cells is shown in Fig. 6. The origin and development of a possible lysosomal apparatus in higher plants has been investigated by Matile and Moor (1968) using the freezeetching technique in rootlets of corn. Vacuolation was seen to take place by four main processes (a diagrammatic representation of these processes is shown in Fig. 7 ) : first, formation of provacuoles derived from the endoplasmic reticulum ( 1 ) ; second, fusion of vacuoles ( 2 ) and the expansion of the vacuolar a A

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( a ) The process of rhopheocytosis. (b) Cytopemphis (Threadgold, 1967).

volume; third, invagination of the tonoplast ( 3 ) ; and fourth, encapsulation of the larger Golgi-derived vesicles by invaginations of the tonoplast ( 4 ) . These workers do not comment on possible involvement of the plasmalemma in vesicle or vacuole formation but clearly, as is shown in Fig. 7 , the possibility also exists for vacuole formation by pinocytic vesicle formation. Direct evidence for pinocytosis in isolated protoplasts was provided from studies in which isolated fruit protoplasts were incubated in 5% ("/v) ferritin for several hours prior to fixation and embedding for electron microscope studies. The pH of the incubation mixtures was maintained at 4.5, since in comparable studies in amoebas Nachimias and Marshall (1961) observed that

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the initial bending of ferritin to the cell surface was optimal at a pH near the isoelectric point of ferritin ( 4 . 4 ) . Accumulation of ferritin particles was observed in the cytoplasmic vesicles of these protoplasts. No initial binding of ferritiii to the plasinalemina was, however, observed. It was considered that pinocytosis was taking place in these isolated fruit protoplasts, thereby enabling ferritin molecules to be accumulated in pinocytic vesicles in the cytoplasm. In

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this electron microscope study, particular care was taken to ensure that the finding of ferritin in vesicles in the cytoplasm was not a fixation artifact (Cocking, 1966a). The experimental advantages of this isolated protoplast system for studying pinocytosis were obvious, yet largely because the initial events of this pinocytic uptake remained obscure and also because the relevance of these ob-

FIG. 7. Diagrammatic representation of processes contributing to the formation of vacuoles. For detailed discussion of 1-4 see the text plasmalemma (PL), cell wall (CW), endoplasmic reticulum ( E R ) , dictyosome (D) , dictyosome vesicle (DV) , tonoplast (T) , vescicular body (VB) (Matile and Moor, 1968).

servations on pinocytosis to pinocytosis in plant cells under natural conditions was unclear, botanists were slow to make use of this system. It was stressed (McLaren and Bradfute, 1966) that pinocytosis was a dynamic process and not a static end result and that no one had at that time visually observed the dynamic events with plant systems. Neither ferritin particles nor most viruses, because of their small size, are ideally suited to follow the early stages of pinocytosis in isolated protoplasts. Furthermore, it also seems likely that early stages may differ as between different protoplast systems. The nature of the particles being taken up is also of importance. The ready availability of polystyrene latex particles in high concentrations and in a range of particle sizes suggested their general applicability in these investigations. After incubation, latex particles were readily detected attached to the plasmalemma of protoplasts. Uptake proper was observed to occur in localized regions of the plasmalemma. In these

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regions, several stages of pinocytic uptake were evident and vesicles containing one to several latex particles were seen detached from the plasmalemma within the cytoplasm (Fig. 8) (Mayo and Cocking, 196%). The latex particles employed were usually 0.088 p in diameter, but it was also observed that particles of up to 0.25 p diameter could also be taken into the cytoplasm by pinocytosis. Electron microscope studies were made difficult by the fact that the particles were soluble in the usual embedding media but the use of hydroxypropyl niethacrylate as an embedding medium largely overcame these difficulties. It was observed that the uptake vesicles containing the latex particles were small and in the range 0.1-0.3 p in diameter. The diameter of vesicles of the pinocytic vesicles observed in the cytoplasm of protoplasts after incubation in ferritin or tobacco mosaic virus (TMV) (Cocking, 1966a,b) were, however, much more varied in diameter and often greatly in excess of 0.3 p. Hirsch et al. (1969) have noted that in the case of pinocytic vesicles formed in macrophages from tiny invaginations of the surface membrane these vesicles commonly fuse with one another to form vacuolar structures 0.5-1.0 p in diameter. There thus exists the possibility that during ferritin and virus uptake by fruit protoplasts pinocytic vesicles coalesce, either with themselves or with existing vesicles, shortly after entering the cytoplasm, to form larger vesicles; but the possibility cannot be eliminated that the range of initial size of pinocytic vesicles during ferritin and virus uptake is very much more varied than when latex particles are being taken LIP. The use of phosphotungstic acid (which occasionally stains only regions of the membranes surrounding larger cytoplasmic vesicles) as a selective stain for pinocytic activity (Mayo and Cocking, 1969b) suggests that sometimes coalescence of pinocytic vesicles with existing cytoplasmic vesicles does take place in these protoplasts. Although, as previously mentioned, uptake of ferritin by isolated fruit protoplasts does not appear to involve any binding of ferritin to the plasmalemma, a binding of ferritin to the plasmalemma of isolated leaf protoplasts (Power and Cocking, 1968) has, however, been observed preliminary to pinocytic uptake. Clearly, differences are to be expected in the detailed mechanism of pinocytic uptake depending on the nature of the protoplast surface and the nature of the materials being ingested. What is evident is that the FIG. 8. ( a ) Section of a protoplast following incubation in 2% latex particles for i hours. Material was fixed in glutaraldehyde, postfixed in osmium tetroxide, stained with uranyl acetate during dehydration, and embedded in 95% hydroxypropyl methacrylate. Sections were poststained with 5% phosphotungstic acid for 1 hour. The membranes and latex particles appear in negative contrast. Particles are attached to the plasmalemma and indented into the membrane (Mayo and Cocking, 196%). ( b ) Outer region of a protoplast incubated in 2% latex for 3 hours. Fixation and embedding was as in ( a ) . Sections were poststained with lead citrate. A region of active pinocytosis is shown containing several stages of engulfment of latex particles (arrows) (Mayo and Cocking, 1969a).

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plasmalemma, in relation to pinocytic activity, is not very discriminating with respect to the material being taken in. It is the subsequent fate of pinocytosed material that is of the greatest interest in relation to the overall physiological behavior of the protoplast or cell. Discrimination appears to occur within the pinocytic vesicle. B. VIRUSUPTAKEAND

THE

INITIATION OF INFECTION

Before attempting to survey the available conclusions regarding virus uptake and the initiation of virus infection in the isolated protoplast system, it will be particularly instructive to review briefly the situation in bacteria and in animal cells. Bawden (1964) has called for caution in extrapolating our knowledge of bacteriophages to considerations of plant viruses and plant cell interactions, especially since bacteriophages such as T2 organize their own transmission and spread unaided, whereas plant viruses depend on other organisms to transmit them. Nevertheless, Best (1965) provided strong evidence in favor of the view that in plant virus infections the protein of the virus becomes adsorbed to the cell wall and ejects its RNA which then enters the cell alone and mediates the synthesis of more virus. A diagrammatic representation of Best’s ideas on the sequence of events in the biosynthesis of an RNA virus is shown it1 Fig. 9. Work on isolated plant protoplasts and the detection of pinocytic activity at the plasmalemma in these protoplasts, particularly the pinocytic uptake of virus intdct, has now focused attention on comparisons with the animal cell system in which, in many instances, it has been shown that viruses are taken up intact, become uncoated, and subsequently initiate infection. It appears that in many respects the isolated plant protoplast is similar to the animal tissue culture cell. The forces responsible for holding virus particles on the surface menibrane of animal cells are considered to be electrostatic, and it has been suggested that strongly acidic phosphate groups of the cell surface interact with amino groups in the virus. It is generally held that most animal viruses are taken into cells by pinocytosis after specific interaction of the virus with receptor sites at the cell surface (Dales, 1965). It is interesting to recall that following the demonstration by Hershey and Chase (1952) in which only the infectious component of the T2 bacteriophage was injected into the host, the idea held, in the absence of any direct evidence, that the viral nucleic acid was first released by rupture of particles at the surface followed by direct penetration through the surface membrane into the host cell. The process envisaged was comparable to that represented diagrammatically by Best for the initiation of infection of plant cells (see Fig. 9 ) . As critically discussed by Dales (1965), however, further experiments have indicated that the converse viewpoint, namely that infection occurs after the cell has engulfed the intact virus, is probably the correct one, Vaccinia has been shown to be phagocytized by L cells and fowl pox by chorio-

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allantoic membrane cells. Myxoviruses have also been shown to be taken up into animal cells by phagocytosis, as have adenoviruses, herpesviruses, and reoviruses. The evidence for this comes mainly from electron microscopy, but more recently biochemical techniques applied to the study of viral entry have served to confirm these observations and to indicate that following uptake the virus

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Viral RNA (parent)

Viral RNA (cornplernentary 1 q T r a n s f e r RNA Host chromosomes C 3 Viral RNA replicase N = Nucleus R = Ribosome n = Nucleolus

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FIG. 9. Diagrammatic representation of the sequence of events in the hiosynthesis of an RNA virus (Best, 1965).

particles become decoated. Thus, Silverstein and Dales (1968) were able to show in a combined electron microscope radioautographic and histochemical approach that reovirus type 3 was phagocytized by L cells and rapidly sequestered inside lysosomes. These workers also showed that hydrolases within these organelles were capable of stripping the viral coat proteins but failed to degrade the double-stranded RNA genome, and Silverstein and Dales concluded that “sojourn of reovirus in lysosomes, when the lytic enzymes uncoat its genome, is an obligatory step in the sequence of infection.” Reovirus labeled in its RNA was obtained by infecting cells and allowing the virus to multiply in a medium containing cytidine3H and uridine-3H (Dales and Gomatos, 1965). Since, as we shall see later, plant tissue culture cell studies are not as yet sufficiently

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developed to enable plant viruses to be labeled in a comparable fashion, such studies have not been carried out using these viruses. Nevertheless, Mayo and Cocking (1968) developed a simple and rapid in vitm labeling technique for preparing T M V labeled with 1251. Only the protein of the virus particle is, however, labeled and although very high specific activity virus can be obtained by this in vitro technique the application of such labeled virus in following the uptake and development of plant viruses is necessarily limited. In the future, infection studies using plant protoplasts should clearly allow plant viruses of high specific activity, labeled in their nucleic acid moiety, to be readily obtained (see Section IV). It has recently been suggested, exclusively on electron microscope evidence, that herpes simplex virus and influenza virus can both become uncoated at the cell surface very rapidly, with rupture of the viral core permitting release of nucleoprotein directly into the cytoplasm (Morgan et d.,1968; Morgan and Rose, 1968). As emphasized by Watson (1968), however, “electron microscopy, by itself, disassociated from allied or complementary techniques, can raise new questions, but it cannot provide the answers.” For the present we can still conclude, as did Dales in 1965, that the preponderance of available evidence favors the conclusion that upon attachment to host cells, which may involve specific membrane receptors, animal viruses are transferred intact, with their genomes still protected, into phagocytic vacuoles by an active and unspecific engulfment. In animal cells, this engulfment is as mentioned quite unspecific. Particles of ferritin and colloidal gold, polystyrene latex spheres, high-molecular-weight D N A and DNA-protein coacervates, as well as viruses, are all capable of being engulfed (Dales, 1965). As far as has been investigated, isolated fruit and leaf protoplasts behave comparably (see Section 11, A ) but, as we have seen, the detailed mechanism of this engulfment seenis to vary depending on the nature and size of the material being taken up. Great interest, therefore, centers on the subsequent fate of engulfed, or pinocytosed material in both the animal cell and in the isolated protoplast system. In both instances, lysosomal enzymes may be involved. Intracellular digestion of phagocytized material is known to occur frequently in animal cells, and the observation that heat-denatured vaccinia is degraded within vacuoles is probably another example of intracellular digestion. In the sense that the internal milieu of a pinocytic vesicle differs from that existing on the cell surface, it has frequently been suggested that virus inside vacuoles is no longer extracellular. The release of the infectious genome of the virus particle that occurs in such a vesicle has been termed the “uncoating process.” This uncoating process involves the activities of the host cell as well as the specific nature of the virus coats. Lysosomal enzymes may, in animal cells, participate in the uncoating process of virus nucleic acid after the intact particle has entered the cell (Allison and

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Sandelin, 1963) . Novikoff ( 1963) has demonstrated an accumulation of lysosomes near pinocytic vesicles. The rate at which this uncoating process takes place varies markedly. In the case of Newcastle disease virus and pox virus, the viral nucleic acid is rapidly released into the cytoplasm as a result of prompt lysis of both the outer viral envelope and the pinocytic (phagocytic) vesicle (Dales, 1963; Silverstein and Marcus, 1964). As previously mentioned in the case of reovirus in L cells, Silverstein and Dales (1968) observed that a sojourn of the virus in lysosomes was an obligatory step in the sequence of infection. When viral nucleic acid itself is used to infect cells, clearly no uncoating process is involved. It is, however, far more difficult and usually impossible to follow by the usual electron microscope methods entry of viral nucleic acid into animal cells or isolated plant protoplasts, since in thin-section studies the free nucleic acid of the virus is not readily detected. Cocking and Pojnar (1968a) and Mayo and Cocking (1969a) have suggested that by selective staining of TMV with uranyl acetate or phosphotungstic acid it may be possible to detect stages in the uncoating process before the nucleic acid of the virus is released (see Section I V ) . A large number of electron microscope and cytochemical studies have demonstrated that protein molecules can be taken up by tumor cells in suspension by pinocytosis. One of the difficulties in investigatiiig the effects of viral nudeic acid is that free viral RNA, particularly single-strandcd, and D N A are quite readily degraded by nucleases present in incubation mixtures. It has been demonstrated, for instance, that infectious RNA, obtained from encephalomyocarditis virus is very rapidly degraded on contact with Krebs mouse ascites tumor cells. It has also been demonstrated that nuclease inhibitors such as bentonite, DEAE-dextran and polyamino acids augment the detectable infectivity of various RNA preparations; recently, Stonehill and Huppert (1 968) have detected an endonuclease associated with cell wall preparations obtained from Krebs ascites cells. These workers suggest that mammalian cell walls may be a source of nuclease activity and may contribute to the inactivation of infectious RNA molecules. It has also been observed that short exposure of cell cultures to hypertonic media enhances the biological effects of nucleic acids on animal cells. Ryser (1967) has suggested that maximum enhancement of RNA infection may be the result of a form of severe, but reversible, cell damage. He points out that cell damage caused by hypertonicity and other conditions may modify the usual digestion process of lysosomal enzymes creating new paths of uptake, the cytopathic uptake vacuoles. These large cytopathic uptake vacuoles could account for an increased or an abnormal penetration of nucleic acids into cells exposed for a short time to adverse conditions. Ledoux (1965) has critically reviewed the phenomenon of uptake of DNA by living cells and has concluded that the physicochemical state of the DNA

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preparations used is a variable of paramount importance; more recently, Olenov (1968) has stressed the importance of the physiological state of recipient cells if transformation phenomena are to be observed in somatic cells. Rogers and Pfuderer (1968) added nucleotide sequences (poly A) to TMV RNA and were able to induce polylysine formation in tobacco plants. It was shown that the poly A must be attached to the virus RNA to be effective. This is an instance of a virus (or more precisely its nucleic acid) being modified to transmit added genetic information. The demonstration by Kovics and Bucz (1967) of the isolation of complete virus from yeast and Tetrahymena experimentally infected with picorna viral particles or their infectious RNA serves also to demonstrate the universality of the RNA code. It raises the possibility of the infection of plant cells by animal viruses. As we shall see later, the isolated plant protoplast system may be very well adapted for experimental work along these lines. In this connection, it is of interest that Sander (1964), working with tobacco leaves, claimed that phage could multiply in the leaves provided they were inoculated with the free nucleic acid of the phage. As discussed by Kassanis (1967), plant tissue cultures cannot be infected without wounding the cells, and this factor, together with the rather low virus concentrations usually present in the infected cells, has resulted in the usefulness of tissue cultures to plant virus research being rather limited. An approach to the use of plant cells in culture that could largely overcome difficulties was suggested from the initial studies of Cocking (1966b) using isolated protoplasts that had been incubated with TMV. No cell wall was present around these isolated fruit protoplasts so that one of the principal barriers to the entry of viruses had been removed. The suspension of protoplasts was incubated with a 1% suspension of TMV for varying periods of time up to 7 hours. Protoplasts were fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in butyl methacrylate, and sections were poststained with lead citrate. These protoplasts showed in thin section (Fig. 10) some deep invaginatiom of the plasmalemma where virus particles appeared to be present, some apparently attached to the plasmalemma. After a few hours, virus particles were detected in vesicles in the cytoplasm, some attached to the periphery of the vesicles, others apparently lying free. It was suggested that the virus entered the protoplast by pinocytosis so that vesicles containing virus in the cytoplasm were pinocytic vesicles. As noted earlier (Section 11, A ) , further studies have indicated a general pinocytic activity in these isolated protoplasts which is not restricted to the uptake of virus particles. As correctly pointed out by Esau (1967), the plasmalemma is not clearly visible in this early work in which methacrylates were employed as embedding media; but there is no difficulty in seeing the plasmalemma when better embedding media are used (see Fig. 8). The use of a high concentration of virus facilitates the detection of pinocytic uptake. At lower virus suspension

FIG. 10. ( a ) Outer region of isolated tomato fruit protoplast incubated in a suspension of TMV. A deep invagination of the plasmalemma (arrow) is visible. ( b ) Higher magnification of deep invagination. Thin particles of TMV are clearly visible and pinocytic activity is also evident (arrows) (Cocking, unpublished observations),

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concentrations (0.1 “/o), fewer virus particles are detected (Fig. 11A) in pinocytic vesicles in the cytoplasm, but when detected the characteristic vesicle with virus particles is present (Fig. 11B) . These findings led Cocking (1966b) to suggest that uptake of virus particles intact by pinocytosis might be an initial stage in the infection of these isolated fruit protoplasts by TMV, just as we have seen is the situation in most instances in the infection of animal cells by viruses. It is of interest that a year earlier Mundry (1965) had concluded that although the mechanism of virus uptake by plant cells was still obscure it was not inconipatible with the hypothesis of virus uptake into plant cells by pinocytosis. One of the difficulties in this work was that at that time protoplasts could only be isolated readily in large quantities from the locule tissue of tomato fruit using a commercially available pectinase (Gregory and Cocking, 1965). Little was known about the rate or extent of infection of the locule tissue of tomato fruit during the systemic infection of tomato plants by TMV. Cocking and Pojnar (1968b) showed, however, that the rate of multiplication of TMV in fruit tissues was comparable to that in the leaves and that there were comparable levels of infection in the various tissues of the fruit. More recently, Power and Cocking (1968) were able to obtain from tobacco leaves very large numbers of isolated protoplasts by using a mixture of commercially available cellulase (from Trichodermu viride) and pectinase so that comparable studies are now possible using isolated leaf protoplasts. Since intact particles of TMV were detected within pinocytic vesicles, it seems likely that infecting virus particles were being taken up intact prior to being uncoated, releasing their nucleic acid and initiating infection. However, in the early studies of Cocking (1966a) protoplasts were incubated continuously in very high concentrations of TMV. Moreover, after about 9 hours the highly vacuolated fruit protoplasts began to burst progressively with time. As a result, a detailed study of the fate of pinocytosed virus was impossible in this early work. It was also evident that high levels of ribonuclease were present in the incubation mixtures (Cocking and Pojnar, 1969). As we shall see later (Section III), it was only with the development of incubation conditions that enabled isolated protoplasts to regenerate a new cell wall and the incubation (“inoculation”) of protoplasts with virus for relatively short periods of time under coilditions of low ribonuclease activity prior to wall regeneration, that it became possible to begin to follow the fate of pinocytosed virus and the establishment of infection. Isolated leaf protoplasts are considerably less highly vacuolated than isolated fruit protoplasts and are inherently more stable, and it may prove possible in the future to follow pinocytic uptake of virus, initiation of infection, and virus multiplication while these are still protoplasts.

FIG. 11. ( a ) Region of isolated tomato fruit protoplast incubated in a suspension of TMV (0.1%) for 6 hours. Note virus in pinocytic vesicle (arrow). ( b ) Higher magnification of pinocytic vesicle. The particles of TMV are clearly visible (Cocking and Pojnar, unpublished observations).

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111. Cell W a l l Regeneration Following this work on the uptake of viruses by isolated protoplasts, the idea arose of allowing the virus to enter the protoplast by pinocytosis and then “triggering off” cell wall regeneration so that the isolated protoplast was converted into a cell containing the virus, which could then be cultured under conditions in which multiplication of the virus would result. It seemed likely from the early work of Townsend (1897) that enzymically isolated protoplasts and sub-

FIG. 12. Drawing of plasmolyzed cell of G. Lnceolata. Only the nucleate subprotoplast within the leaf cell regenerates a new cell wall (redrawn from Townsend, 1897).

protoplasts, provided that they were nucleate, would regenerate a new cell wall. Townsend showed that in cells of Elodeu cunadensis and Gaillardiu lanceolata that had been plasmolyzed the plasmolyzed protoplast, or the nucleate subprotoplast within the leaf cell, regenerated a new wall when maintained in 2070 sucrose (Fig. 1 2 ) . H e also showed that “free pieces of protoplasts” (subprotoplasts in the terminology of Cocking, 1,963) isolated by the mechanical method of Klercker (1892) from protonema of mosses, prothecia of ferns, hairs of stems, and leaves of higher plants could be kept alive for days, and even weeks, during which time they built distinct cell walls; pieces without nuclei (enucleate subprotoplasts) never made cell walls (Fig. 13). More recently, Binding (1966) has reported the regeneration of protoplasts isolated by the method of Klercker (1892) froin the musci Fzmaria hygrometrica, Physcomifrizm ezoystom,zlm, and Pipiforme and Brynm erythrocarpum. Here again, wall formation was only carried out by nucleate protoplasts and most of these isolated protoplasts formed a rigid wall after at least 13 days. Some of these protoplasts (plaslocytes) germinated to protonemata. Yoshida (1961), using leaf cells of Elodea densa, investigated the role of the nucleus in some activities of protoplasm by comparing the behavior of nucleated and enucleated halves of protoplasts which he separated by means of plasmolytic treatment of intact cells. H e observed that the membrane that was reformed on the surfaces of divided protoplasmic halves (subprotoplasts) was more solidified and less elastic in the nucleated halves than in the enucleated halves. H e also noted that by treatment with ribonuclease the properties of nucleated protoplasmic halves could be rendered similar to those of the enucleated halves.

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Up to 1967, although the general properties of enzymically isolated protoplasts had been extensively investigated by Cocking (1961b) and by Ruesink and Thimann (1965, 1966), there was little or no evidence of cell wall regeneration. In 1967, Pojnar et ul. reported that isolated fruit protoplasts, when well washed to remove contaminating enzymes and incubated in suitable media, ra-

FIG. 13. Isolated protoplasts of Bvyum caespititium held in contact for 4 days. The protoplasts possessing a nucleus (a and b) formed a cell wall; the protoplast (subprotoplast) without a nucleus ( c ) did not (redrawn from Townsend, 1897).

pidly began to regenerate new cell walls. It was established from electron niicroscope observations on thin sections of suitably fixed and embedded freshly isolated tomato fruit protoplasts that there was no detectable wall material on the surface of the protoplasts; surface replica studies largely confirmed this conclusion. More recently, freeze-etching studies of glutaraldehyde-fixed freshly isolated fruit and leaf protoplasts have further substantiated this conclusion. Evidence was obtained that essentially the entire wall substance was removed from the surfaces of the protoplasts. It should be noted that in a freeze-etch study of the surface structure of yeast protoplasts Streiblova (1968) obtained evidence that, at least in some cases, the entire wall substance was not removed from the surfaces of the protoplasts. She showed that after treatment of the yeast with snail enzymes an innermost thin wall layer, as well as remnants of the fibrillar middle layer, could sometimes be demonstrated. Perhaps the extensive plasmolysis of fruit and leaf cells prior to protoplast release greatly facilitates the removal of the cell wall from the surface of the protoplasts. A general view of a region of a freeze-etched freshly isolated leaf protoplast is shown in Fig. 14. When isolated tomato locule protoplasts that had been in 20% sucrose were transferred to a culture solution consisting of a modified White’s medium (Lam-

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port, 1964) but without 2,4-dichlorophenoxyaceticacid, together with an OSmotic stabilizer, cell wall regeneration was rapidly initiated. After about 3 hours the plasmalemma, but not the tonoplast, was no longer mainly smooth but possessed numerous infolds. Electron-dense material was massed near the plasmalemma and outgrowths from the plasmalemma were being formed (Fig. 1 5 ) .

FIG. 14. Outer region of a freeze-etched isolated tobacco leaf protoplast. Note absence of the cell wall. Plasmalemma (PL), plastids ( P ) , fat body ( F B ) , tonoplast ( T ) , central vacuole ( V ) , and possible pinocytic vesicles (arrows) (Power and Cocking, unpublished observations).

As regeneration proceeded, an initial multilayered wall was formed and, after 3 days, plasmolysis of the protoplast away from this wall was often evident (Fig. 16). Pojnar and Cocking (1968) observed that if these isolated protoplasts were kept in contact with each other during cell wall regeneration, cell aggregates were formed. A typical cell aggregate is illustrated in Fig. 17. Many of the cells within these cell aggregates are no longer spherical and cells of a variety of shapes and sizes are present; it appears that a single common wall is formed at the contact area of their surfaces. It is of interest that NeEas and Svoboda (1967) observed that similar aggregates of yeast cells are formed during the regeneration of isolated protoplasts of Sacchnroniyces cereuiseae growing contiguously in gelatin. These workers showed in an electron microscope study

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that a single common wall was formed at the contact area of the surface of the regenerating yeast protoplasts. These cell aggregates of higher plant cells could be very useful for following movement of virus from cell to cell. Border bodies (lomasomes), which are aggregates of membranes in a matrix between the plasmalemma and the cell wall, are sometimes detected in thin-section studies of these regenerating fruit protoplasts (Fig. 18). In thin-section electron

FIG. 15. Very early stage in the regeneration of a cell wall by isolated tomato fruit protoplasts. Material was stained during dehydration with uranyl acetate and poststained with lead citrate. Note the convoluted plasmaleinina and dense material massed at its surface (Cocking, unpublished observations).

microscope studies, Bowes and Butcher (1967) examined cell wall inclusions in Androgruphis punicalutu callus. These workers detected complex invaginations of the cell wall and plasmalemma into the cytoplasm which they interpreted as border bodies. They suggested that peripheral regions of the cytoplasm could become incorporated into the cell because of the formation of a new plasmalemma. A typical region showing a freeze-etched border body is shown in Fig. 19. It has frequently been suggested that these border bodies may be concerned in wall synthesis as well as playing a role in extracellular enzyme secretion (Calonge et ul., 1969) and micropinocytosis (Bracker, 1967). The marked layering of this newly formed wall is clearly shown by freeze-

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etching in Fig. 20 and after 5 days’ culture a few fibrils (probably microfibrils of cellulose) are frequently visible, being formed at the outer surface of the initially formed wall (Fig. 21). The finding by freeze-etching (of what are in all probability cellulose fibrils on the outer surface) agrees with the surface replica studies of Pojnar et al. (1967) in which fibrillar material (approximately 100 A thick) was detected at the outer surface of regenerating protoplasts.

FIG. 16. Region of regenerating tomato fruit protoplast after 3 days in the cell wall regenerating medium. Plasmolysis of the plasmalemma away from the newly formed wall is evident. Newly formed wall ( W ) , plasmalemma (PL), and toiioplast (T) (Cocking, unpublished observations).

The process of cell wall regeneration in this protoplast system appears to parallel in certain respects the pattern of cell wall formation in the soil amoeba Acanthamoeba during encystation. This amoeba has cyst walls containing cellulose (Tomlinson and Johnes, 1962). Bauer (1967) showed that at an early stage of wall formation during encystation a lamellae pile (Lumellenstupel) was first formed, and later cellulose fibrils. A somewhat similar sequence of events also appears to occur during cell wall formation in Chlorellu (Staehlin, 1966) ; this has been presented in diagrammatic form by Muhlethaler (1967) and is shown in Fig. 22. There is little evidence in the case of Chlorellu for the initial formation of a lamellae pile prior to the formation of the primary wall. In the regenerating protoplast, reverse micropinocytosis (Pickett-Heaps, 1967) has

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been implicated in the deposition of amorphous material on the outside of the plasmalemma (Pojnar et dl., 1967). It is difficult to draw any conclusions concerning the involvement of plasinalemma particles in protoplast wall regeneration, since prior to freeze-etching protoplast material was fixed in glutaraldehyde and these particles are known to be very labile during fixation (Miihlethaler, 1967). Preston and Goodman (1967) have stressed the need for an

FIG. 17. Characteristic cell aggregate which is formed when isolated protoplasts are kept in contact with each other during cell wall regeneration (Pojnar and Cocking, 1 9 6 s ) .

examination of naked protoplasts during the initiation of wall formation. They observed, by freeze-etching of unfixed material, ordered rows of granules with attached fibrils at the surface of Chlamydomonas protoplasts. The detailed investigation of cell wall regeneration by these fruit protoplasts is, as yet, at too early a stage of development to discuss profitably the implications of any finding of a hydroxyproline-rich protein in these cell walls; but it will be particularly instructive to determine whether the bulk of the hydroxyproline present occurs in the cell wall, as is the case in the callus cells of sycamore and bean (Northcote, 1969), or whether it is present mainly as a structural entity in their groundplasm as suggested by Israel et al. (1968) for cultured carrot explant cells.

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C. COCKING

Cocking and Pojnar (1969) have shown that the onset of cell wall regeneration in protoplasts leads to a diminishing pinocytic uptake of virus. This is probably not only associated with the fact that the newly formed wall is highly impermeable to virus, but that Golgi vesicle activity is at this time contributing to cell wall synthesis by reverse pinocytosis. Leaf protoplasts isolated by the

FIG. 18. Outer region of an isolated tomato fruit protoplast after 3-day incubation in cell wall regeneration medium. A border body (BB) (or lomosome) is clearly visible between the plasmalemma and the newly formed wall (W) (Cocking, unpublished observations).

method of Power and Cocking (1968), which involves the use of a concentrated mixture of cellulase and pectinase, do not readily regenerate a new cell wall, yet as previously mentioned, they are stable in suitable culture media for several days. This could permit their use in studies of virus multiplication without their first having to be converted into cells. Svoboda and NeEas (1968) have reported that snail enzyme (which contains cellulase) prevents regeneration of 3. cerevisiue protoplasts, and this observation may explain the difficulty encountered in getting isolated leaf protoplasts to regenerate a wall. Only nucleate fruit protoplasts regenerate a cell wall under the present experimental conditions. As a result, the isolated single cell cultures formed from isolated

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protoplasts that have taken up virus by pinocytosis provide a uniform population of suitable nucleate material for studying virus multiplication.

IV. Virus Multiplication In no instance does a virus divide or increase in size and, therefore, the term “virus replication” would perhaps be better than virus multiplication. Viral nu-

FIG. 19. Freeze-etched outer region of an isolated tomato fruit protoplnst after ?-day incubation in the cell wall regeneration medium. A border body (BB) (or lomosonie) i s clearly visible. Note the suggestion of a multilayered wall which is beginning t o hrenk down (arrows). Plasmalemma (PL) (Willison and Cocking, unpublished observations)

cleic acid and protein are probably synthesized separately and the virus assembled later. It is clear that whatever may be the mechanism of infection of plant cells by viruses, the nucleic acid of the virus must first be freed of its protein coat after entering a susceptible cell. In animal cells, the infectivity drops sharply shortly after infection, and it is difficult to detect the presence of infection units. This is known as the eclipse phase. When dealing with the inoculation of leaves with virus, Bawden (1964) has indicated that after an interval, the latent period, newly produced virus becomes detectable and the content of successive extracts then rapidly increases. Bawden (1964) has indicated that this latent period has close similarities to the eclipse phase evident in animal cell virus infection.

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M e must now consider the facts known about the fate of virus pinocytosed by isolated protoplasts; but before considering this further, it is useful to discuss what is known about the fate of virus pinocytosed by animal cells. As we have seen, uncoating of the virus involves the intracellular release of viral nucleic acid from its protective coat. Joklik (1965) has discussed the two main experimental methods for following the release of viral nucleic acid. First, in

FIG. 20. Freeze-etched outer region of a regenerating protoplast similar to that in Fig. 19. Note the layering of the newly formed wall (W) (lamellae pile). The plasmalemma (PL) is highly granular with some marked depressions (arrows) which are probably pinocytic. Ice crystals (IC) (Willison and Cocking, unpublished observations).

certain systems it is possible to follow this release of viral nucleic acid by measuring the appearance of naked infectious nucleic acid; and second, and more directly, it is possible to follow the fate of labeled virus adsorbed to cells. Studies with 32P-labeled poliovirus added to HeLa cells growing in suspension cells showed that a high proportion of adsorbed virus particles eluted from the cells. Most of the virions that do not elute are uncoated within the cell, The work of Joklik and Darnell (1961) showed that the kinetics of uncoating indicated that over 50% of the virus particles were uncoated within 20 minutes after adsorption and that the liberated viral RNA was largely hydrolyzed to acidsoluble material. Very little of the RNA released from input virus is recoverable

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in a macromolecular RNase-sensitive form; and Joklik and Darnell (1961) have concluded that “this is most probably the uncoated viral genome which escapes enzyme attack and goes on to initiate infection.” This analysis of the fate of polio virus thus provided for the first time an explanation for the very low efficiency with which some viruses initiate productive infection. In plant cell

FIG. 21. Freeze-etched outer region of a fruit protoplast after 5 days of regcncr.itiiig a new wall. Note similarity of the multilayered wall to that in protophsts after i days of cell wall regeneration (Fig. 2 0 ) . A few fibrils, probably of cellulose are, ho\vever, present to the outside of the lamellae pile (LP). Cellulose fibrils (arrows). Nntc graw ular plasmalemma (PL) with pinocytic depressions (Willison a i d Cocking, unpublished observations).

infections, it is possible that a single infectious virus particle is sufficient to initiate an infective center, and a discussion of the data available on the relationship between the number of lesions and inoculum concentration led Siege1 and Zaitlin (1964) to conclude that the available evidence was in favor of thc hppothesis that a single virus particle was capable of initiating infection in most plant host-virus systenis but that the interaction of virus particles with infcctible sites is not a well-understood phenomenon. Evidence that a pinocytosis-like process is involved in infection of leaves has been discussed by Mundry (1963). In experiments on the dilution of infective virus by ultraviolet-inactivated virus, WU et al. (1962) showed that when the same concentration of infective virus

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was inoculated in the presence or absence of up to ~ O O - ~ O O times O the concentration of inactivated virus, no difference in the numbers of infections found was observed. One interpretation of these results is that infection involves the occlusion of a certain voltone of inoculum, as would be the case for a pinocytic vesicle, rather than the adsorption of virus particles which tends to be concentration dependent. It should be noted, however, that Kleczkowski (1950) has

a

b

C

FIG. 22. Scheme of the subsequent steps in cell wall formation of the green alga Chloyella sp. ( a ) A cortical region of the cell with the plasmalemma, covered with particles and a subjacent Golgi complex. ( b ) Accumulation of matrix material, carried to the cell surface by Golgi vacuoles. The plasmalemma particles become detached and move to the outer periphery of the matrix. ( c ) Primary wall formation in the region where the particles are concentrated (Miihlethaler, 1967).

suggested that infectible sites are of variable sensitivity and that Dijkstra (1964) has suggested that viral nucleic acid and intact TMV have different infectible sites on Nicotiunu glzltinosu which seems to argue against a simple theory of nonspecific uptake. Moreover, Jedlinski (1964) has reported that infectible sites for T M V and tobacco necrosis virus are distinct on leaves of Nicotiana sylvestris. The experiments of Shaw (1967), who used T M V reconstituted from 1.Klabeled T M V protein and unlabeled nucleic acid to determine the interval between inoculation and breakdown of the inoculated virus, are particularly pertinent to the question of the rate of uncoating of plant viruses during infection. He demonstrated that within 7-8 minutes 25% of an inoculuin of the 14Cprotein labeled TMV had been uncoated and that this value rose to 50% after several hours. This suggests that uncoating occurs regardless of the infectivity of the virus since, as we have seen, it seems likely that merely one virus particle, under suitable conditions, is capable of initiating infection. Shaw himself concluded that the critical phase of establishment of infection occurs later in the

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infection process. From the work of Cocking and Pojnar (1969), it seems likely at least in the case of isolated fruit protoplasts that uncoating also occurs regardless of the infectivity of the virus. Protoplasts were suspended in a high concentration of TMV (0.1%) for 6 hours to allow pinocytic uptake to occur. They were then washed free of excess T M V and incubated in the cell wall regeneration medium of Pojnar et al. (1967). Virus was detected in pinocytic vesicles in the cytoplasm of protoplasts incubated in virus for 6 hours. Serial sectioning of cells was employed in this fine-structural study and it was shown that at this time there was an average of 239 virus particles per protoplast. By 30 hours, with the onset of cell wall regeneration, all virus taken up by pinocytosis in the first 6 hours had disappeared from the pinocytic vesicles. Even at the 6-hour stage there was some slight suggestion from the appearance of the virus that the virus particles were being degraded in the pinocytic vesicles in a fashion somewhat reminiscent of the degradation of viruses in animal cells after pinocytic uptake. In this electron microscope study, because of the technical difficulties of serial thin-sectioning, only one concentration of virus was used (0.1Cjo) and saniples were taken at rather large time intervals. After an average of 120 hours’ culture, 262,000 T M V particles were present in the cytoplasm of the “inoculated” regenerated fruit protoplasts. It was clearly evident that protoplasts were capable of becoming infected by TMV. These electron microscope studies also strongly suggest that infection was being initiated by the formation of pinocytic vesicles. Moreover, it appeared that the level of infection seemed to be of the same order as that of systemically infected tomato fruit locule tissue (cf. Cocking and Pojnar, 1968b). This work needs to be extended using much lower levels of input virus, and the rate of multiplication followed in detail by local lesion assay. Under these conditions, the effects of adsorption of virus to the newly formed wall should not be a major complicating factor (Kassanis, personal communication) and fewer virus particles will be present in the pinocytic vesicles. The ability of this isolated protoplast system to support virus multiplication means that we can now begin to extend the experimental approach that has been so productive in work on animal virus multiplication in animal cell cultures to plant tissue cuItures. Considerable improvements in tissue and single-cell cultures of higher plants have been made in recent years (Hildebrandt, 1962) ; but although meristem culture has aided greatly the elimination of virus infections, and tissue cultures have helped in other respects as well (Raychaudhuri, 1966), as Kassanis (1967) has emphasized, the present usefulness of tissue cultures to plant virus research is limited. These virus-infected isolated single cells formed from regenerated protoplasts have distinct experimental advantages over infected callus cultures in which the concentration of virus in the cells is very frequently much less than that found in cells of systemically infected plants and

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in which often only 50% of the cells are infected (Hansen and Hildebrandt, 1966). Although work on this infected protoplast system is, as yet, only at an early stage of development, the studies of Takebe et ul. (1968) indicated that virus multiplication can readily be obtained in isolated single cells in culture. These workers developed a procedure using a fungal pectinase which

D ooo

Cutin

5%

Hemisubstances Pectin Wax

0 .mi pjqa

FIG. 23. Simplified scheme of the outer wall of an epidermal cell. D = Ectodesmata as nonplasmatic structures (modified from Franke, 1967).

rapidly released mesophyll cells from tobacco leaves. These isolated cells from TMV-inoculated leaves supported multiplication of the virus during subsequent incubation. It was shown by local lesion assay methods that the virus titer of the extract of cells isolated from TMV-inoculated leaves increased 7-to 1.3-fold during incubation of the cells for 2 hours. Moreover, as would be expected for TMV multiplication, 2-thiouracil markedly reduced the increase in TMV titer, whereas actinomycin D had practically no effect. Intact plant tissues, whether they be the organized tissues of the leaf or callus tissues, are far too complex

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for detailed investigations of the rate of virus multiplication or detailed studies of the early stages of virus infection. A brief glimpse at the complexities of the outer wall of a leaf epidermal cell (Fig. 23) (Franke, 1967) will help to indicate the advantages, at least in the first instance, of dealing with isolated protoplasts to obtain clues as to the early stages of virus infection. Changes in the titer of viral antigen in extracts of leaves (Fig. 2 4 ) (Shalla and Amici,

Days after inoculation Titer of viral antigen in extracts of tomato leaves measured at various intervals after inoculation with TMV (Shalla and Amici, 1968).

FIG. 24.

1967) provide only an average value for cells at various stages of infection and tell us little about the rate of multiplication of virus in the cells of the various tissues of the leaf. As Shigematsu et ul. (1966) have emphasized, cells infected with TMV are only a small part of the total cells of a leaf during the early stages following inoculation with TMV and, moreover, there are major technical difficulties in the leaf system in following the incorporation of a labeled precursor into metabolites of a leaf cell. By isolating protoplasts from plant tissues, a less complex system is obtained in which to study both the initiation of virus infection and subsequent virus multiplication in a uniform population of cells. As our knowledge of such protoplast systems improves, cell regeneration from a whole range of different protoplast types infected with various viruses, both plant and animal, may be possible, as well as the formation of hybrid cell aggregates. The recently observed cytopathic effect of viruses such as TMV in regenerated protoplasts may also come to be the basis for a plaque assay for plant viruses (Cocking and Pojnar, 1969). Ultimately, these studies on virus uptake, cell wall regeneration, and virus multiplication in regenerated protoplasts

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could lead to a more complete understanding of the infection of plants by viruses. REFERENCES Allison, A. C., and Sandelin, K. (1963). 3. Exptl. Med. 117, 879. Barton, R. (1964). Exptl. Cell Res. 36, 432. Bauer, H. (1967). Vierteljahresschr. Nuturforsch. Ges. Zuerich, Juhrgaizg 112( 3), 173. Bawden, F. C. (1964). “Plant Viruses and Virus Diseases.” Ronald Press, New York. Best, R. J. (1965). Enzymologiu 29, 377. Binding, H . (1966). Z . PfEanzenphysiol. 55, 305. Bowes, B. G., and Butcher, D . N. (1967). 2. PJEanzenphyriol. 58, 86. Bracker, C. E. (1967). A m . Rev. Phytopathul. 5, 343. Bradfute, 0. E., Chapman-Andresen, C., and Jensen, W. A. (1964). Exptl. Cell Res. 36, 207. Calonge, F. D., Fielding, A. H., and Byrde, R. J. W. (1969). /. Geiz. Micwbiol. 55, 177. Chapman-Andresen, C. (1964). Meth0d.r Cell Phyriul. 1, 277. Clowes, F. A. L., and Juniper, B. E. (1968). “Plant Cells,” Botan. Monographs No. 8 . Blackwell, Oxford. Cocking, E. C. (1960). Nature 187, 927. Cocking, E. C. (1961a). Biochem. J . 82, 12P. Cocking, E. C. (1961b). Nature 191, 780. Cocking, E. C. (1963). Biochem. J , 88, 31P. Cocking, E. C. (1965). Vieu’points Biol. 4, 170. Cocking, E. C. (1966a). 2.Naturfor.rch. 21b, 5 8 1 . Cocking, E. C. (1966b). Planta 68, 206. Cocking, E. C., and Pojnar, E. (1968a). J . G‘en. Virol. 2, 317. Cocking, E. C., and Pojnar, E. (1968b). Phgopathol. 2 . 63, 364. Cocking, E. C., and Pojnar, E. (1969). 1. Gen. Virol. 4, 305. Dales, S. (1963). J . Cell B i d . 18, 53. Dales, S. (1965). Progr. Med. Virol. 7, 1. Dales, S., and Gomatos, P. J. (1965). Virology 25, 193. DAlessio, G., and Trim, A. R. (1968). 1. Exptl. Botany 19, 831. Dijkstra, J. (1964). Mededel. Lundbouwhogeschool W/ugeningen 64, 2. Esau, K. (1967). A n n . Rev. Phytopathol. 5, 45. Franke, W. (1967). Ann. Rev. Plant Physiol. 18, 251. Gordon, G. B., Miller, L. R., and Bensch, K. G. (1965). J. Cell Biol. 25, 41. Gregory, D. M., and Cocking, E. C. (1963). Biorhem. J . 88, 40P. Gregory, D . M., and Cocking, E. C. (1965). J . Cell Biol. 24, 143. Hansen, A. J., and Hildebrandt, A. C. (1966). Virology 28, 15. Hershey, A. D., and Chase, M. (1952). J . Geiz. Physiol. 36, 39. Hildebrandt, A. C. (1962). Mod. Methods Plarzt Analy. 5, 353. Hirsch, J. G., Fedorko, M. E., and Cohn, 2. A. (1969). J . Cell B i d . 40, 629. Holter, H . (1959). Intern. Rev. Cytol. 8, 481. Holter, H. (1963). Proc. 5th Intern. Congr. Biochem., Moscow, 1961. Israel, H . W., Salpeter, M. M., and Steward, F. C. (1968). J . Cell B i d . 39, 698. Jedlinski, H. (1964). Virology 22, 331. Joklik, W. K. (1965). Progr. Med. Virol. 7, 44.

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The Meiotic Behavior of the Drosophiln Oocyte’ ROBERTC. KING Depniftment of Biological Sciences, Northwestern University, Evanjton, I l h o i s

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Morphology of the Drosophjla Ovary . . . . . . . . . . . . . . 111. The Cytology of the Drosophila Oocyte Nucleus . . . . . . . . A. The Light Microscopy of the Prophase Stages of Meiosis B. The Ultrastructure of Meiotic Prophase . . . . . . . . . . . . C. The c ( 3 ) G Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The sbd105 Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Mitotic Crossing-over without Synaptonemal Complexes . . V. The Formulation of a Hypothesis concerning the Origin and 1:unctioning of Synaptonemal Complexes . . . . . . . . . . . . . . . . A. The Synaptomere-Zygosome Hypothesis of Synapto-

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of Recombinases . . . D . Nonspecificity of Synaptomere-Zygosome Interactions . . E. Intersynaptomerjc Distances and Travel Times F. The Biochemistry of Meiotic Prophase . . . . . . . . . . . . . G. Factors Influencing Crossing-over . . . . . . . . . . . . . . . . . Nondisjunction in Meiotic Mutants Affecting Crossing-over Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction The chromosome number of a given diploid, sexually reproducing species would double with each generation had nature not arrived at a mechanism for halving the zygotic chromosome number at some other point during the life cycle. The production of sex cells containing the haploid chromosome number is brought about by a single chromosomal replication followed by two nuclear divisions. The entire process, meiosis, occurs in animals during gametogenesis and in higher plants during sporogenesis. The rules of gene transmission described in Mendel’s famous hereditary laws (see Stern and Sherwood, 1966) follow from the behavior of chromosomes during meiosis and fertilization. Mendel’s law of segregation refers (in modern terms) to the segregation into different gametes and then into different offspring of the members of a given pair of alleles residing on the homologous chromosomes of the diploid parental organism. A restatement of the law of independen/ 1 This essay is fondly dedicated to Professor Theodosius Dobzhansky on the occasion of his seventieth birthday, January 25, 1970.

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FIG: I. ( A ) Dorsal view of the internal reproductive system of an adult female D. melanogater. Two ovarioles have been pulled loose from the left ovary. Sperm are stored in the ventral seminal receptacle (which is drawn uncoiled) and in paired spermathecae. The uterus is drawn expanded as it would be when it contains a mature egg. ( B ) A diagram of a single ovariole and its investing membranes. The nurse celloocyte complexes are representative of the first six stages (Sl-6) of oogenesis. Within the vitellarium, sectioned egg chambers are drawn so as to show the morphology of the nuclei of the oocyte and 6 of the 1 5 nurse cells. The distribution of nucleolar material

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assortment is that the members of different pairs of alleles are assorted independently into gametes during gametogenesis (provided they reside on different chromosomes), and that the subsequent joining of male and female gametes occurs at random. In addition to providing a diploid organism with haploid gametes, meiosis also affords the mechanism whereby different pairs of alleles located upon the same chromosome can recombine by crossing-over. It is the purpose of this article to review the encyclopedic body of genetic information available concerning meiotic crossing-over and disjunction in the female of Drosophila melanagaster, as well as the pertinent data from cytological studies on oogenesis, and to erect a hypothesis as to the mechanism of crossing-over which is in harmony with most of the facts. Information froin other organisms will be used when available. 11. The Morphology of the Drosophila Ovary An ovary of an adult female consists of a parallel cluster of ovarioles, each of which is differentiated into an anterior germarium and a posterior vitellarium (see Fig. I A ) . The vitellarium is composed of a series of interconnected egg chambers which lie in single file. Each chamber is in a more advanced developmental stage than the one anterior to it, and each contains an oocyte and 15 nurse cells surrounded by a monolayer of follicle cells (Fig. 1B). The egg and its 15 nurse cells are fourth generation descendants of a single germarial cell called an ovarian cystoblast. The cells formed by the mitotic activity of a cystoblast have been named cystocytes, and it is within region 1 of the germariuni that such mitoses occur (Fig. IB). The cystocytes generated from a single germaria1 cystoblast form a branching chain of cells. The 16 cells are joined by 1 5 canals each surrounded by a ring which is attached in turn to the plasma is drawn in the starred nurse cell nucleus, whereas the other five nurse nuclei show the distribution of Feulgen-positive material. The distributions of both D N A and nucleolar RNA are shown for the oocyte and follicle cells. Fragments detach from the oocyte nucleolus during stages 4 through 6 . ( C ) A pro-oocyte nucleus (left) and a nucleus in an adjacent pronurse cell (right) from germarial region 3. Synaptonemal complexes are seen in nuclei of the two prooocytes. The nuclei of the 14 pronurse cells lack synaptonemal complexes and contain more nucleolar material (N). Clouds of particulate matter adhere to the surface of these nuclei. ( D ) The oocyte nucleus (left) and a nucleus from an adjacent nurse cell (right) from a stage-3 egg chamber in the vitellarium. The magnification is the same as in C. Nucleolar material is far more abundant in each of the 1 5 nurse nuclei than in the oocyte nucleus. The synaptonemal complexes have congregated into a central area which under the light microscope ( B ) appears as a Feulgen-positive mass adjacent to the nucleolus. From Koch et al. (1967).

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membranes of the interconnected cells (Koch st ul., 1967). The cystocytes can be characterized by the number of ring canals each contains. Two cystocytes (designated l e and 2e) are interconnected, and each possesses four ring canals; two cells (3e and 4e) contain three canals, four cells ( 5 through 8e) contain two ring canals, and eight cells ( 9 through 16e) have but one canal each. Cells l e and 2e undergo a different type of nuclear differentiation from cells 3 through 16e, possibly because cells l e and 2e possess a unique pattern of cortical structures (Koch and King, 1969). Since only these 2 cells enter meiotic prophase, they have been named pro-oocytes, while the remaining 14 are called pronzlrse cells. The production of fourth generation cystocytes and their differentiation into pro-oocytes and pronurse cells begins during the pupal stage and continues throughout adult life (King et al., 1768). In each 16-cell cluster, however, the anterior pro-oocyte eventually switches to the nurse cell developmental pathway (Brown and King, 1964; Koch et ul., 1967). It is within germarial regions 2 and 3 (see Fig. 1B) that the cluster becomes enveloped by follicle cells. Koch and King (1769) have suggested that competitive interactions between the plasmalemmas of a posterior cluster of follicle cells and cystocytes l e and 2e determine which becomes the oocyte and which a nurse cell. At the boundary between regions 2 and 3, an interleafing of follicle cells will result in the production of a stalk that will form the connection between the germarium and the vitellarium. This interleafing of follicle cells anterior to an egg chamber transfers it to the vitellarium (see Fig. 1B). The major growth of the oocyte and its accompanying cells occurs in the vitellarium. The development of the egg chamber has been subdivided into a series of consecutive stages ending with stage 14, the mature primary oocyte (King et al., 1956; King, 1964; Cummings and King, 1969). During the first six stages (shown in Fig. IB), all 16 cells grow at roughly identical rates. During stage 7 , the chamber elongates and the growth rate decreases. During stages 8 through 11 vitellogenesis occurs and the oocyte grows approximately 10 times faster than at its previous maximum rate. The endopolyploid nurse cells contribute most of their cytoplasm to the oocyte and then degenerate. Concurrently, the follicle cells secrete about the oocyte first the vitelline membrane and then the chorion (King and Koch, 1963). During stages 1 2 through 14, while no further increase takes place in the volume of the oocyte, a variety of chemical changes occurs both in the yolk organelles and in the background cytoplasm (King, 1960; King et al., 1966; Cutnmings and King, 1967). During oogenesis, the amount of cytoplasm per oocyte increases by 90,000 times. Under optimal conditions, the developmental time intervals from cystoblast to S1 and from S1 to SI4 are estimated as 5 and 3 days, respectively (King, 1957; Koch and King, 1966). At the electron microscope level, pro-oocytes may be rea&ly differentiated

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f rorn pronurse cells by their nuclear morphology, since synaptonemal complexes are seen only in pro-oocyte nuclei (Koch et al., 1967; King et al., 1968). In the nucleus of the pro-oocyte destined to become a nurse cell, all synaptonemal complexes degenerate during stage 1. 111. The Cytology of the Drosophiln Oocyte Nucleus

A. THELIGHTMICROSCOPY OF THE PROPHASE STAGES OF MEIOSIS In D. melanogaster, the nuclei of pro-oocytes and young oocytes are unsuitable for detailed cytological study at the light microscope level because of their small size. In the wild-type oocyte, the chromosomal filaments begin to condense during stage 3 and are incorporated into a compact karyosome by stage 4 (Fig. 2 ) . The karyosome persists and becomes still more compact during stages 11 through 13. Late in stage 13, the nuclear envelope breaks down and the karyosome is liberated into the ooplasm. This DNA-containing structure which lacks a nuclear envelope is called a karyosphere (King et ul., 1956).

B. THEULTRASTRUCTURE OF MEIOTICPROPHASE At the electron microscope level, pro-oocytes and oocytes can be readily differentiated from pronurse cells and nurse cells by their nuclear morphology (Koch et ul., 1967). Ribbonlike synaptonemalz complexes appear in the nuclei of pro-oocytes shortly after their formation by the division of third generation cystocytes Id and 2d. Fortunately, there are species with larger chromosomes in which a correlation is possible between light and electron microscope observations of cells in meiotic prophase. It is known, for example, that in the primary spermatocytes of Plethodon cinereus (Moses, 1958), in the primary oocytes of Aedes aegypti (Roth, 1966), and in L i h m longiflorum microsporocytes (Roth and Ito, 1967; Moens, 1968) synaptonemal complexes are first seen during zygonema, reach their maximum lengths during pachynema, and degenerate during diplonema. Wettstein and Sotelo (1967) have shown from reconstructions of serial sections that in the pachytene spermatocytes of Gryllus argentinus an uninterrupted synaptonemal complex extends the length of each pair of homologs. Furthermore, the morphology of the synaptonemal complex is the same in each of the different bivalents. In D . melunoguster, leptonema must immediately follow the postmitotic DNA 2 Three spellings of this adjective are found in the literature (synnptinemal, synaptene n d , and synaptonenial). The first spelling was used by Moses, who coined the term synaptinemal complex (Moses, 1958). W e suggest, however, that the last spelling is the nlma, thread). Throughout etymologically correct one (G. .rynuptos, fastened together this article the terms leptonemu, zygonema, pachynema, and diplonema are used as nouns; whereas leptoterze, zygotene, pachytene, and diplotene are used as adjectives.

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replication in cystocytes l e and 2e, since synaptonemal complexes appear in the nuclei of pro-oocytes shortly after their formation by the division of third generation cystocytes I d and 2d. Synaptonemal coniplexes increase first in number and then in average length. The period during which the synaptonemal complexes appear and grow must be zygonema. This stage thus occurs during

+ and

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Sin

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FIG.2. Drawings of the light microscope cytology of the nuclei from germarial prooocytes (PO) and oocytes from egg chambers in consecutive stages (S,-s,) from wildtype and c ( 3 ) G females. The nucleolus, which is drawn as a solid sphere, bredks into fragments which disappear by stage 9 . From Smith and King (1 9 6 8 ) .

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the time the pro-oocytes move posteriorly through the germarium and during stages 1 and 2. Pachynema is completed in oocytes residing in the anterior portion of the vitellarium, since the combined length of all synaptonemal complexes reaches a maximum between stages 3 and 4. The complexes then degenerate and by stage 7 none are visible. It follows that the oocyte nucleus in stages 7 through 13 is in a modified diplotene stage of meiotic prophase.

C. THEc ( 3 ) G MUTATION The spontaneous mutation referred to as the recessive crossovey si/ppre.rsov in chromosome 3 of G o w e n and symbolized c ( 3 ) G was discovered in 1917 by Marie and John Gowen. Subsequently, J. W. Gowen (1933) reported that the mutant gene was located on the third chromosome at about locus 5 5 . Subsequent data (Lewis, 1948) placed the genetic locus at 58 and the cytological locus between 89A1 and S9B3. Gowen showed that in females homozygous for c(3) G, crossing-over in the entire chromosomal complement is reduced to a small fraction of normal. Externally, the mutation produces no visible effect in either the homozygous or the heterozygous condition. In 1964, G. F. Meyer reported (without giving any details) that he could find no synaptonemal complexes in oocyte nuclei from homozygous c( 3) G females. A thorough study of the structure and functioning of the ovaries of c(3)G females was published subsequently by Smith and King (1968), who found that females homozygous for c(3) G oviposited during the third through the eighth day of adult life an average of 1.5 eggs per ovariole daily. This figure is well within the range observed for various wild-type stocks. The distributions of oocytes within the various developmental stages was determined for c ( 3 ) G / c ( 3 ) G and c(3)G/+ females of four ages (0.5, 3.5, 7, and 10 days). Since no significant differences were observed, it was concluded that oocytes of either genotype spent about the same length of time passing from stages I through 14. O n the other hand, a significant difference did exist between the number of 16-cell cysts in the germaria of c ( 3 ) G and in wild-type germaria. Wild-type germaria contained twice as many clusters of fourth generation cystocytes on the average as did mutant germaria. It was concluded that the only difference in developmental dynamics between female germ cells in c ( 3 ) G and in wild type is that relatively less time is spent by c(3)G oocytes in a stage equivalent to zygonema. That is, 16-cell clusters containing pro-oocytes take a shorter time to pass through the germarium in the mutant than in the wildtype female. The results of a study of the light microscope cytology of the oocyte nuclei of c ( 3 ) G homozygotes is summarized in Fig. 2. In the wild-type oocyte nucleus, the chromosomal filaments begin to condense during stage 3 and are incorporated into the compact karyosome by stage 4. In c(3)G, this process is

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speeded up, since a well-defined karyosome is first seen in stage 3. Thus, the stage in c ( 3 ) G equivalent to pachynema appears to be shortened. In both and c ( 3 ) G the karyosome persists from stages 4 through 13. Subsequently,

+

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I00

f IJ, c

3 50

0

FIG. 3. The combined length of all synaptonemal complexes as a function of nuclear ,, and SGS8 represent data from serially sectioned nuvolume. The points shown as S-S clei from oocytes in egg chambers belonging to stages 1-4 and 6-8 from wild-type and c ( 3 ) G females. Each point represents one nucleus. The numbers above the points give the number of sections analyzed per nucleus, and the numbers in parentheses indicate the number of sections passing through the karyosome. Each point labeled P represents the average value calculated from data derived from sectioned pro-oocytes and the number above the P gives the number of nuclei studied. From Smith and King (1968).

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electron micrographs were taken of serial sections of nuclei of pro-oocytes and oocytes residing in c(3) G ovarioles. The data derived from these photographs are presented in Fig. 3. It is obvious that synaptonemal complexes do not occur in c ( 3 ) G oocytes belonging to the same developmental stages where in wild type the formation of the ribbon begins, reaches a maximum, and ceases.

D. THEsbdlo5 MUTATION E. B. Lewis reported the discovery of stubbloidfo5 in 1948. This deficiency resulted from the X-ray-induced loss of a small segment of chromosome 3. The deficiency is lethal when homozygous, but it is viable in the heterozygous condition. The deficiency includes the locus of c ( 3 ) G . Hinton (1966) has shown that crossing-over is abolished in females of genotype c ( 3 ) G / D f ( 3 ) ~ b das~ ~ ~ , well as in c ( 3 ) G / c ( 3 )G. Crossing-over is reduced to one-half to two-thirds of the control value in females of genotype Df(3)sbd10:/+ (Hinton, 1966, 1967). Smith and King (1968) demonstrated that in these females, while synaptonemal complexes are present in germarial pro-oocytes, they are not present in oocyte nuclei in chambers in the vitellarium which correspond to stages 2 and 3 . Thus, the development of synaptonemal complexes is precociously terminated in Df (3 )sbd205/+ females. Smith found subsequently that coniplexes are also missing from all but the most posterior clusters of fourth gencration cystocytes. The above findings demonstrate that the c ( 3 )G gene behaves more like an amorphic than a hypomorphic allele. In the homozygote, not even the earliest stages in the formation of synaptonenial complexes can be detected. Furthcrmore, whether alone or in double dose, the phenotype is nearly the same i n terms of crossing-over. O n the other hand, c ( 3 ) G + appears to produce different phenotypes in single and in double dose. Another way to describe the crossover results is that c ( 3 ) G+ can function normally when c ( 3 )G is present in the same nucleus but not when its homolog is deficient for the segment missing in D f ( 3 )shd1O5. Two alternative explanations suggest themselves. Tlic first is that c(3)G+ cannot function normally unless it can pair with another c ( 3 ) G gene, but that either c ( 3 ) G or c ( 3 ) G C is acceptable. The second esplanation is that the formation of synaptonemal complexes requires the combined action of two closely linked genes, S and t. Only one dose of S, but two doses of t, are required for proper function. In this model, wild type = St, c ( 3 ) G = sat, and Df(3)sbdIo5= soto (is deficient for both). Females of genotype + / c ( 3 ) G ( S t / s a t ) behave normally, since one S and two t genes occur per diploid nucleus. Females of genotype +/Df (3) sbdl05 (St/s"to) show decreased crossing-over because the requirement of two t genes is not met. Since the development of synaptonenial complexes is shortened in D f (3) sbdl05/+

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females, the hypothetical t gene might determine the interval during which the subunits of the synaptonemal complexes are synthesized, and the length of the interval could depend upon the dosage of t . Since the growth of synaptonemal complexes is precociously terminated in sbd1,df05/+ females at stage 1 and crossing-over occurs at a reduced rate, it is probable (1) that some crossing-over occurs prior to stage 2 in the nuclei of sbd105/+ oocytes (and presumably in wild type as well), and (2) that some crossing-over also takes place after stage 2 in the wild-type oocyte nucleus. The premeiotic replication of D N A occurs within the nuclei of oocytes before they enter the vitellarium (Grell and Chandley, 1965; Chandley, 1966). Thus, meiotic crossing-over is completed long after the massive DNA replication that gives the oocyte its 4C DNA content. Such an argument renders untenable the hypothesis advanced by Pritchard (1960) which attributes recombination in eucaryotes to copying errors at the time of DNA replication.

IV. Mitotic Crossing-over without Synaptonemal Complexes Meiotic crossing-over does not take place in the male of D. nzelanogdster (Morgan, 1912), and synaptonemal complexes do not occur in the prophase nuclei of primary spermatocytes (Meyer, 1961). According to LeClerc (1946), pairing of homologous chromosomes is seen in oogonial metaphases and larval salivary gland nuclei in females homozygous for c(3)G, and somatic crossing over occurs. Somatic pairing and crossing-over take place between the homologous autosomes of male D. melanogaster (Stern, 1936). Salivary gland chromosomes have been investigated at the electron microscope level and, although they are somatically paired, there are no reports of synaptonemal complexes associated with them. Newton and Darlington (1930) have shown that when three or more homologous chromosomes are present in a nucleus at pachyneina only two are synapsed at any particular point. Thus, there is a “saturation of pairing forces” once homologs join by twos. On the other hand, the forces in a nucleus such as that of a larval DroJophiJa salivary gland cell, which bring about the association of chromosomes into a polytene bundle, obviously are not saturated once homologs pair. From the above data, it can be concluded that somatic and meiotic chromosome pairing are basically different phenomena and that somatic pairing and somatic crossing-over can occur in Drosophila in the absence of synaptonemal complexes. Crossing-over between homologs in somatic cells may be widespread in its occurrence in plants and animals, but it takes place at a frequency hundreds or thousands of times lower than is the case for germinal crossing-over. Perhaps somatic crossing-over is common in Drosophita because it is facilitated by the occurrence of pairing of homologs in somatic cells in these and related insects.

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In most eucaryotes, however, such somatic pairing does not occur between homologous chromosomes, and germinal crossing-over generally occurs in both sexes (although often at different rates). Electron microscopists studying a wide range of eucaryotic species (including protozoans, slime molds, fungi, flowering plants, annelids, molluscs, crustaceans, arachnids, insects, fish, amphibians, birds, lower mammals, and man) have observed synaptonemal coniplexes inside the nuclei of germ cells undergoing synaptic stages of meitoic prophase (Moses, 1968). In those species in which gametogenesis in both sexes has been examined with the electron microscope, synaptonemal complexes have been observed in both spermatocytes and oocytes in cases in which crossing-over occurs in both sexes. These facts taken together with the data from c(3)G and .tbdl05 make it seem likely that the formation of synaptonemal complexes is essential for meitoic crossing-over in most eucaryotes. It cannot be argued that the enzymes required for exchanges to occur between the DNA of adjacent chromatids are absent or inhibited in somatic cells, because Taylor (1 9 5 8 ) has demonstrated, using radioautographic techniques, that exchanges do occur between the D N A molecules of sister chromatids in the dividing cells of root meristems. It follows that the synaptonemal complex may orient the nonsister chromatids of homologs in a manner that facilitates enzymatically induced exchanges between their DNA molecules.

V. The Formulation of a Hypothesis concerning the Origin and Functioning of Synaptonemal Complexes The combined length of the interphase, polytene, salivary gland chromosomes of D. melanogaster is about 1200 p, and the combined length of all somatically paired chromosomes seen in oogonial metaphases is roughly 8 p (Bridges, 1935). The combined length of all synapsing meiotic prophase chromosomes should be equal to the maximal combined length of all synaptonemal complexes (110 y, see Fig. 3 ) . Therefore, it is obvious that between interphase and meiotic prophase the chromosomes must undergo some sort of folding which reduces their length by at least a 10-fold factor. The synaptonenial complex forms, and crossing-over takes place while the chromosomes are in this folded state. Subsequently, the chromosomes undergo further coiling and folding (see DuPraw, 1968, Fig. 18-6C) to reach the dimensions characteristic of metaphase. The condensation of chromosomes to metaphase dimensions is known to involve a lysine-rich histone which cross-links DNA-containing fibrils (Mirsky et ul., 1968). Synaptonemal complexes must not play a role in the formation of the karyosome or karyosphere or in the condensation of the oocyte chromosomes to metaphase dimensions, since all these processes occur in femules homozygous for c ( 3 ) G .

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A. THE S Y N A ~ ~ T ~ M E R ~ - Z YHVPOTHESIS G O S O ~ ~ . BO F SYNAPTONEMAL COMPLEXFORMATION Each synaptonemal complex is in the form of a tripartite ribbon consisting of parallel dense lateral elements surrounding a medial complex (Fig. 4 ) . Cytochemical studies at the ultrastructural level (reviewed by Moses, 1968) have

FIG. 4. ( A ) Drawing of a segment of a bivalent as seen under the electron micro. scope. c, Chromatin; cs, central space; le, lateral element; sc, synaptonemal complex; tr, transverse rods of the medial complex. (B) Model illustrating the postulated composition of the synaptonemal complex. The aligned transverse rods of the medial complex are areas where paired zygosomes are attached to synaptomeres. The highly folded chromatids form the masses of chromatin fibers surrounding the synaptonemal complex.

shown that the lateral elements are rich in DNA and proteins (among them histones). The medial complex contains protein, but DNA is scarce or absent. The presence of RNA in the synaptonenial complex is questionable. Moses (1958) was the first to show by combined light and electron microscope studies of adjacent thick and thin sections that the lateral elements lie in the central axes of the paired homologous chromosomes of a meiotic bivalent. The niedial complex contains a system of transverse rods oriented perpendicularly to the lateral elements and separated from them by a clear area, the “central space” (Roth, 1966; Smith and King, 1968). It is reasonable to assume that the transverse rods form a strutwork which holds the lateral elements in their parallel

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configuration. If this is the case, then the clear areas to either side must contain fibrils that bridge the gap, and such fibrils can occasionally be resolved in electron micrographs. Much of what is known concerning the synaptonemal complex can be fitted into a logical framework on the assumption that each chromosome contains polynucleotide segments (hereafter referred to as synaptomeres) scattered along its length. Synaptomeres are involved in synapsis and play no role in transcription. Under the appropriate conditions, a series of coils (similar to those seen in a telephone cord) may be generated in each chromosome. Once in the coiled state consecutive synaptomeres lie in close proximity, and they pair in the manner shown in Fig. 5A. The resultant shortened and thickened chromosomes are seen in earIy zygonema, and presumably correspond to the unpaired “axiaI elements” of the electron microscopist. The hypothesis continues with the proposal that the pairing of homologs is brought about by an association, in the manner shown in Fig. 5C, of rod-shaped subunits hereafter called zygosomes. Zygosomes are assembled in the nucleoplasm, and each is visualized as a protein molecule having a folded “head” end by which it can attach to the central portion of a synaptomere. The “tail” end contains charged sites (represented by four dots in Fig. 5C). The charge distribution allows the zygosome to bind laterally with other zygosonies, but only if they are pointing in opposite directions. The bipolar properties of zygosome bridges offer a reasonable explanation of the previously mentioned “saturation of pairing forces” once homologs join by twos. It is important for the working of our model that a newly synthesized zygosome does not immediately attach to another one. Therefore, it is postulated that the zygosome is synthesized in a coiled state, and that the binding sites on the tail end are exposed by uncoiling only after attachment to the synaptomere takes place (Fig. 5B). Thus, our hypothesis assumes that a synaptomerczygosome system has properties similar to the repressor-operator system of bacteria. Jacob and Monod proposed in 1961 that there exists a class of genes called operators which control the functioning of adjacent cistrons. Such operators can be switched on or off depending upon whether they are free or have repressor molecules bound to them. The repressor molecule is now known to be a protein which binds directly to a region of specific nucleotide sequence in a DNA molecule (Bretscher, 1968). A protein zygosome is postulated to function in a similar manner with respect to a specific polynucleotide segment, a synaptomere. A zygosome, upon undergoing this initial chemical reaction, is postulated to make a configurational readjustment which changes its ability to undergo a future reaction with a third molecule (another zygosome). Repressors are known to behave in a similar fashion, since once they react with molecules of another class (effectors) their reactivity toward operators is modified

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(Jacob and Monod, 1963). A repressor is synthesized under the control of a gene called a regulator. A zygosome is presumably also coded by a specific gene and, according to this argument, the cistron involved in D . melanogasfer is the normal allele of c(3)G. Of course, the DNA code of the cistron must be transcribed to an RNA message, and RNA transcription does indeed occur in meiotic nuclei during synaptic stages (Hotta and Stern, 1963; Henderson, 1964). The synaptomere-zygosome hypothesis of a synaptonemal complex formation is in harmony with the previously cited data provided from electron microscope studies on the morphogenesis of the complexes in Drosophila and other insects. These data indicate that the axial complexes form first, that they are then brought into a parallel arrangement by the synthesis of the medial complex, and that once the synthesis is complete a synaptonemal complex extends the length of each bivalent. The suggestion that synapsis begins at the telomeres (Fig. 5B) is in harmony with Henderson's (1961) observations on spermatogenesis in various orthopterans. For the separation of homologs, zygosome bridges must detach from synaptomeres. Roth and Ito (1967) have demonstrated that this separation of homologs can be prevented in the lily by poisoning microsporocytes at zygonema with deoxyadenosine. Earlier, Roth (1966) observed (in diplotene mosquito oocyte nuclei) organelles which he called polycomplexes (see Fig. 6A). According to his interpretation, these structures were formed by the fusion of synaptonemal complexes that had detached from diplotene chromosomes. These findings suggest that during diplonema the folded end of each tygosome is .. . .

_.

..

~

~

~~~~~~

FIG. 5. Postulated steps in the synapsis of homologs to form a meiotic bivalent. ( A ) The left and right telomeres of a pair of homologous chromosomes (TL and T,:) attach in the manner shown to specific areas of the inner membrane of the nuclear envelope (ne) . The chromosomes shorten and thicken because of the folding that results from the pairing of synaptomeres that are distributed along the chromosomes. In A and B, synaptomeres ( s ) are represented by segments which are slightly wider than the intervening chromosomal regions. ( B ) By the beginning of zygonema the folding is complete. Zygosornes are synthesized in the nucleoplasm, and they attach to synaptomeres and simultaneously uncoil. As a consequence, when each synaptomere possesses a zygosome, a peg extends from the base of each chromosomal fold. Interdigitation of the pegs initially produces short stretches of synaptonemal complex. Eventually, the chromosomes pair throughout their length, and an uninterrupted synaptonemal complex extends from left to right telomeres (see Fig. 4 ) . ( C ) A small segment of the model synapsed bivalent drawn at higher magnification to elucidate the structure and functioning of synaptomeres and zygosomes. Each synaptomere is composed of three segments A, B, and C. The lateral elements A and C will pair with the A and C elements, respectively, of any other synaptomere. The B element is the site where the base of a zygosome can attach. Zygosomes projecting from each homolog interdigitate and bind in an overlapping tail-to-tail fashion. Each chromosome is shown to be divided into two chromatids.

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modified so it can no longer bind to a synaptomere. After this modification has occurred, however, the folded end can bind to the folded end of a zygosome pointing in the opposite direction. As a result, once the ribbons of zygosomes are freed from a bivalent, they can stack up as shown in Fig. 6B.

FIG, 6. ( A ) Drawing of a portion of a polycomplex formed by the fu\ion of synalitonema1 complexes which have detached from diplotene chromosomes. ( B ) Drawing of a magnified section of the hypothetical polycomplex illustrating a way in which the zygosomes might be stacked.

It is the sister chromatids that remain paired during the opening up of il tetrad that occurs during diplonema (Belling, 1929; Callan and Lloyd, 1956). Since the breakdown of the synaptonemal complexes is accompanied by the separation of nonsister chromatids, it seems clear that the complexes hold homologs together. €3.

CHROMOSOMAL ATTACHMENT SITESON

THE

NUCLEAR MEMBRANE

Woollam et al. (1966) have shown from a study of the spermatocytes of various rodents that the pachytene bivalents are attached at both ends to the

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niiclear envelope and that no other parts of the chromosomes are so anchored. Wettstein and Sotelo (1967) reached similar conclusions from a study of the syiiaptoiiemal coinplexes of cricket spernlatocytes. There was no tendency for all of the chromosomal termini to adhere to a restricted area of the nuclear cnvelope, which indicates that in these species the points of attachment are distributed throughout the inner surface of the nuclear envelope. These observations lead to the suggestion that, prior to the formation of synaptonemal coinplexes, specific chromosomal telomeres become attached to specific sites on the nuclear envelope. For example, the left telomeres of two homologous chromosomes may have adjacent points of attachment that differ from the attachment points of the right telomeres of the same chroniosonies and from those of the right and left telomeres of all other chromosomes. Therefore, the left end of a given chromosome will be anchored near the left end of its homolog, and the same applies to its right end. Such an arrangement would facilitate the zipping together of the homofogs by zygosome bridges (see Fig. 5B), and eventually this strutwork would be built from one end of the chromosome to the other (Fig. 4 A ) . If there is any truth in this hypothesis, it should be possible to demonstrate in D. melanagaster that starting at zygonema a three-dimensional pattern of telomeric connections to the nudear envelope is formed. Such a study would require reconstructions drawn from stacked positive transparencies of electron micrographs of serial sections from oocyte nuclei of various karyotypes. l o r example, a normal nucleus should have eight telomeric attachment sites, whereas the nucleus carrying a compound ring X chromosome should possess only six. If allelic telomeres are found always to adhere to neighboring sites on the nuclear membrane, this observation would suggest that the membrane is a 1110saic containing different areas, each with a surface specific for a given telomere. The development of this idea has been influenced by the findings of the i n crobial geneticists, who have demonstrated that genetically different bacterial viruses attach to different specific sites on the surfaces of their hosts. Thus, a male-specific bacteriophage such as R-17 attaches to a different part of an Escherichia coli than does T, (Brinton et al., 1964). W e know from the study by Berendes and Meyer (1968) on the polytenc chromosomes of the larval salivary gland cells of Drosophila hydei that telomeres contain tightly packed DNA fibrils coated with a protein matrix. Perhaps it is this protein that gives the telomere its adhesive properties. The teloinere may also play a role in the synthesis of portions of the nuclear membrane, since it is known that acentric chromosome fragments often become invested in a nuclear envelope (Das, 1962). From studies on autotetraploids, Sved (1966) has come to similar coiiclti-

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sions concerning the binding of chromosome ends to the nuclear envelope. If synapsis begins with the association of allelic telomeres into twos, and the chromosomes then zip together ending at the centromeres, one would predict that in a 4N nucleus two-thirds of the chromosomes would form quadrivalents and one-third bivalents during meiotic prophase. This expectation is realized. Interlocked bivalents and quadrivalents are observed very rarely, however. Sved postulates that this interlocking does not occur because the telomeres are attached to the inner surface of the nuclear envelope. Comings (1968) has sug gested that in mammalian cells there is only one specific attachment site on the nuclear envelope for the X chromosome. In female cells, there is competition for this site between the two X chromosomes, and whichever one randomly becomes bound to this specific site first will be the one that remains synthetically active. The other X chromosome eventually attaches to the nuclear envelope at a nonspecific site and becomes a Barr body. C. CROSSING-OVER THROUGH

THE

ACTJONOF RECOMBINASES

In attempting to explain the sequence of events occurring during crossingover, one has to construct a theory in harmony with the genetic as well as the cytological data. W e know from the results of a series of classic genetic studies dealing with D. melanoguster (T. H. Morgan, 1910; Sturtevant, 1913; Anderson, 1925; Bridges and Anderson, 1925; L. V. Morgan, 1933; Beadle and Emerson, 1935) that meiotic recombination generally involves only nonsister chromatids, that it occurs during a stage at which four chromatids are present, that each crossover event involves only two of the four chromatids and produces precisely reciprocal products, and that during double or multiple crossing-over all four, only three, or only two of the four chromatids may participate in exchange. More recent studies (Chovnick, 1966) demonstrate that fine structure mapping with a resolution approaching microbial systems is possible in Drosophilu and, therefore, one nust conclude that recombination can take place between almost any adjacent nucleotides of certain cistrons. The data of Smith and King (1968) derived from studies of c ( 3 ) G and Df(3)sbd1Q5indicate that crossing-over is occurring throughout the time synaptonemal complexes are being constructed in the oocyte nucleus and that crossing-over cannot occur in the absence of such complexes. Taken together, the data provided by Creighton and McClintock (1931) on Zed, by Stern (1931) and by Smith and King (1968) on Drosophilu, by Taylor (1965) on Romalea, by Rossen and Westergaard (1966) on Neotiella, by Hotta et dl. (1966) on Lilium, and by Chiang and Sueoka (1967) on Chlamydomonas demonstrate that crossing-over occurs between already synthesized chromosomes. It follows from this conclusion that chromatids are en-

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zymically broken and, once they have exchanged segments, enzymically rejoined. Crossing-over is known to produce precisely reciprocal products, and it follows from this fact that the nonsister Chromatids must be in such perfect alignment that enzyme-induced breakage occurs at precisely the same points in both. This requirement suggests that the chromatids should be uncoiled and intimately paired. Yet the chromatids of a synapsing tetrad are obviously in some sort of condensed form, since they are short and thick enough to be observed under the light microscope. Furthermore, the homologs are separated by the medial complex and central space of the synaptonemal complex. The following hypothesis offers a way out of this dilemma. Let us assume that the enzyme system involved in meiotic exchange is incorporated into a single macromolecular structure, a recornbindse, and consider some of its required properties. A recombinase must accept only two chromatids at a time, and the chromatids chosen must be nonsisters. Next, by the time that enzyme-induced breakage occurs the chromatids must be in intimate register, so that each break occurs in the same place in each chromatid. The broken ends must be interchanged and enzymically rejoined soon after. Nothing is known about the molecular structure of recombinases, but it is assumed that they bear many resemblances to DNA polymerases (see Erhan, 1968). One function of the synaptonemal complex could be to hold certain regions of the homologous chromosomes in parallel, but at a specific distance apart. Perhaps only under such conditions can the recombinase attach unerringly only to nonhomologous chromatids. One could, for example, visualize a recombinase as a molecule the length of a zygosome bridge with active sites consisting of grooves, one at each end, which accept chromatids. Assume that such a molecule is positioned (as drawn in Fig. 7) alongside and parallel to a zygosome bridge, with each end projecting toward the region of a chromatid adjacent to the synaptomere. Under these conditions, the recombinase could accept only nonsister chromatids. The recombinase would then slide along the chromatids, and as it did so it would contract, pulling the two chromatids toward one another (see Fig. 7). If the synaptomeres were in proper register, the chromatid loops would contain homologous sequences of nucleotides and would pair. The recombinase would proceed along the loops formed by the paired nonsister chromatids. The loops would extend at right angles from the plane of the synaptoneinal complex. The recombinase would open upon reaching the next zygosome bridge and either detach or continue its journey along the bivalent. In the event that the synaptomeres were not in proper register, the chromatid loops would be nonhomologous. Under these circumstances, it is postulated that pairing of the chromatids would be disturbed, and the recombinase would detach prematurely. The average tetrad undergoes a limited number of exchanges.

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For example, Weinstein (1936) has shown for the Drosophib X chromosome that over 90% of the chromatids recovered originate from single and double crossovers and, consequently, most X chromosome tetrads have only one or two chromatid exchanges. Therefore, one must postulate that a given recombinase

FIG. 7. A drawing illustrating consecutive steps ( 2 through 5 ) i n the niovement of a hypothetical recombinase along nonsister chromatids in the regions between adjacent zygosome bridges. The recombinase is shown in 1 before its attachment. The two daggerlike structures on the recombinase represent the sites on the molecule that are responsible for breakage and recombination between the nonsister strands. The movement of the recombinase occurs above the plane of the synaptoneinal complex (see Fig. 5 f o r orientation). To aid in visualizing this, the zygosome bridges have been inoved apart and the loops which project upward are drawn larger (because they would be nearer the eye of the observer). Only the portion of each synaptomere to which a zygosome attaches is shown.

molecule has a low probability of attaching to homologous loops on a bivalent (because synaptomeres may often be out of register) and of subsequently catalyzing an exchange during its journey along the bivalent. Double exchanges involving three or four chromatids are quite common, and these doubles could be used to support the idea that reconibinases attached to

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different portions of the same tetrad may be moving simuItaneously along different pairs of nonsister chromatids. Double exchanges involving the same two strands could be the work of the same recombinase, however. W e know that the minimium distance between two such exchanges is 10 crossover units (Muller, 1 9 1 6 ) . If one divides the combined length of the genetic maps for the four chromosomes ( 2 5 4 crossover units) into the estimated haploid D N A value (see Section V, E ) , one finds that 1 map unit is equivalent to 7 x l o 5 nucleotide pairs. The minimum distance between exchanges ( 7 x 106 nucleotides) could be a function of the minimum distance a recombinase travels before it is ready to catalyze a second recombinational event and the probability that the enzyme will remain attached to the chromatids in question during the time required for the journey. The fact that interference does not extend across the centromere suggests that recombinases do not travel across centromeres. (For further discussion, see Section V, G).

D. NONSPECIFICITY OF SYNAPTOMERE-ZYGOSOME INTERACTIONS There is considerable evidence from light microscope studies that pairing can occur between nonhomologous chromosomal segments during meiotic prophase. For example, as early as 1931 McClintock reported detecting nonhomologous pairing during the pachytene stage in Zea mays microspores. Such nonspecific pairing occurs over small nonhomologous regions when synapsis is interrupted by structural heterozygosity (see also Section V, G, 5 ) . Nonhomologous pairing also takes place in some monoploid individuals when homologous partner chromosomes are absent. At the electron microscope level, it has been reported that synaptonemal complexes form at sites of such nonhomologous associations. For example, a short complex can form in the region where a univalent folds back and synapses with itself (Moens, 1968). Typical synaptonemal complexes are also seen in the microsporocyte nuclei from hybrids between Lycopessicon ercrllentnm and Solanum lycopersicozdes (Menzel and Price, 1 9 6 6 ) . In such hybrids, synapsis between nonhomologous chromosomes is a common event. Thus, one must assume in the hypothesis that zygosomes attach to any synaptoniere and that zygosome bridges can form a strutwork binding together homologous or nonhomologous chromosomal segments.

E. INTERSYNAPTOMERIC DISTANCES AND TRAVEL TIMES The salivary gland chromosomes of D. melanogaster contain a total of about 5150 bands (C. B. Bridges, 1935; Bridges and Bridges, 1939; P. N. Bridges, 1941a,b, 1942; Slizynski, 1 9 4 4 ) . According to Rudkin (1965b), the average band contains 3 x lo4 nucleotide pairs per haploid chromosome strand. Therefore, the haploid D N A content is approximately 1.54 x 108 nucleotide pairs. This value must be an underestimate, since the D N A of heterochromatic and

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interband regions is omitted from consideration. It is assumed that the euchromatic bands contain about 75% of the total DNA and, therefore, that the C value is 2 108 nucleotide pairs. According to Roth (1966), each transverse rod in the medial complex is about 65 A thick and is separated from its neighbor by a space about 75 A wide. If each rod represents a zygosome aligned as shown in Fig. 4B, the maximum dimensions of the portion of a synaptomere that binds to a zygosome would be about 200 A. This length corresponds to roughly 60 nucleotide pairs, if one assumes that the distance between consecutive pairs is 3.4 A. The value of 100 nucleotide pairs will be used as the length of the entire synaptomere in future calculations, since the synaptomere contains a segment to either side of the one that binds to a zygosome according to the model drawn as Fig. 5C. The maximum length of all synaptonemal complexes in the Drosophilu oocyte is about 110 y (Fig. 3 ) . It is, therefore, estimated that a total of about 8 x 103 zygosomes [1.1 106 A/(75 A 65 A ) ] attach to all the bivalents in the pachytene nucleus. Therefore, there would be an identical number of synaptomeres, making up a total of 8 x 105 nucleotides or about 0.2% of the diploid complement of DNA. The average internemd (the DNA segment between two synaptomeres) would contain 5 x 1 0 4 nucleotides. This value, computed by dividing C by the haploid number of s y m p tomeres ( 4 I@), will be an overestimate if the synaptonemal complex is folded laterally in a plane perpendicular to its long axis (see Roth, 1966, Diagram 2 ) . For example, if zygosome bridges were stacked four abreast, the number of zygosomes would be quadrupled and the average internema would be 12,500 nucleotides long. Let us ignore this further complexity and assume that the average synaptomere is 100 nucleotides long and that the average internema is about 50,000 nucleotides long. Since the internema is about 500 times longer than the synaptomere, synaptomeric pairing such as diagrammed in Fig. 5A and B will reduce the length of a chromosome by a 500-fold factor. Keeping these dimensions in mind, it becomes obvious that the DNA loops projecting from the lateral elements will be greatly convoluted and intertwined rather than neatly laid out as they are drawn in Fig. 4B. If the combined length of all pachytene chromosomes is 110 y, then each of the five arms of the major chromosomes measures 22 y. Therefore, the X chromosome would be about four times as long as the diameter of the nucleus in which it resided (Fig. 2), and chromosomes 2 and 3 would each be eight times as long as the diameter of the nucleus. The minimum distance between exchanges in a Drosophilu chromatid was calculated as 7 1 0 6 nucleotides in Section V, C. This value would amount to a distance of 2400 y on an extended DNA molecule, but to only 5 p if the folding described above took place. Thus, if it were possible to see chiasmata one would predict that no two would

x

+

x

x

x

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be closer together than 5 p. In Schistocerca gregaria, this “interference distance” has been determined by Henderson (1963) to be 7.3 p in diplonema. In a female producing two eggs per ovariole per day, an oocyte spends about 3 days in the germarium and 1 day in the vitellarium in stages during which synaptonemal complexes are growing. Crossing-over presumably takes place during this 4-day interval. Let us assume that it takes the entire 4 days for a recombinase to travel from telomere to centromere and consider only the major chromosomes. Dividing the haploid DNA content by 5 , we obtain 4 x lo7 nucleotides per chromosome arm. To make the trip in 4 days, it is calculated that a recombinase would have to travel at the rate of 100 nucleotides per second, or about 2 p per minute. A trip along one internema would take about 8 minutes. A rate of travel of 100 nucleotides per second is not unreasonable, since Levinthal et al. (1963) have calculated that under optimal conditions an E. coli RNA polymerase can insert 1000 nucleotides per second into an mRNA corresponding to a protein weighing 30,000 daltons. Presumably, the enzyme must travel along 1000 nucleotides of DNA per second in order to transcribe at that rate. F. THERICKHEMISTRYOF MEIOTIC PROPHASE

Herbert Stern and his colleagues (Hotta et al., 1966; Ito et al., 1967; Stern and Hotta, 1967) have conducted studies of the DNA metabolism of in vitrocultured microsporocytes of L. longiflorum passing synchronously through the various meiotic stages. Stern’s group demonstrated that approximately 99.7% of the DNA in these meiotic cells was replicated before they entered prophase. Mitra (1958) had previously shown that a lily chromatid becomes breakable by X-rays and capable of rejoining independently of its sister chromatid at about this time. Late in leptonema, however, according to Stern, there is synthesis of DNA that makes up only about 0.3% of the total. If synthesis of this DNA is inhibited by poisoning the microsporocytes with adenosine deoxyriboside, the cells are arrested in zygonema and synaptonemal complexes fail to form. The newly synthesized DNA was isolated and characterized by denaturation and hybridization experiments. It was shown to be double-stranded, and DNA of equivalent nucleotide sequence was found to be present in somatic cells. These molecules were shown not to be nucleolar DNA. Stern suggested that this DNA is distributed throughout the genome in the form of Iinkers which are not replicated during the S period prior to meiotic prophase, whereas the rest of the chromosomal DNA is. The chromosome thus comes to contain two chromatids joined together by the unreplicated linkers. It is the replication of these linkers that converts the zygotene bivalent into a pachytene tetrad. The synaptomere-zygosomehypothesis can be expanded to include this infor-

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mation by assuming that each unreplicated linker is a synaptomere, and that DNA synthesis occurring late in leptonema replicates the synaptoineres to which the zygosomes will later attach. If this synthesis is prevented, the zygosomes are unable to attach to the axial cores and synaptonemal complexes never form. In Fig. 5C, the chromosomes are shown as containing independent chromatids in the intersynaptomeric regions, corresponding to Mitra’s data. The separation of chromatids to a degree visible under the light microscope does not occur until pachynema. Thus, the D N A segments between linkers must have a mechanism of replication independent of the linkers. If these segments have an average length of 50,000 nucleotides, each could contain several genes. Among them would be included a gene involved in the replication of the segment (see Taylor, 1967, his replicon model), structural genes devoted to the synthesis of different messenger RNA molecules, and a controlling gene similar to the operator referred to previously. Indeed, we know from radioautographic studies, which demonstrate asynchronous D N A synthesis along Drosophila polytene chromosomes, that each chromosome is subdivided into hundreds of independently replicating units whose synthetic activities are coordinated in time (Plaut et ul., 1966; Howard and Plaut, 1968; Mulder et ul., 1968). The most recent paper to come from Stern’s laboratory (Hotta ef d., 1968) demonstrates that certain proteins synthesized at the synaptic stages of meiosis are physically associated with the DNA distinctively synthesized at the same time. If this protein synthesis is inhibited with cycloheximide, a failure of chiasma formation results. Perhaps some of the proteins synthesized specifically during zygonema and pachynenia are components of zygosomes. Bogdanov and his colleagues (1968) have recently reported the results of a quantitative cytophotometric study of D N A and histone synthesis during cricket spermatogenesis. They found that the D N A synthesis that converts the 2C to the 4C amount occurs prior to leptonema. Histone synthesis is delayed, however, so that during zygonema only three times the haploid amount of nucleohistone is detected. Between pachynema and diplonema, the histone concentration rises to 4C and remains at this value throughout the remainder of meiotic prophase. These results may be interpreted as an indication that during the early synaptic stages certain portions of the chromosomes are stripped of a histone that is present at all other times. Perhaps pairing of synaptomeres (Fig. 5A) can occur only in the absence of this histone.

G. FACTORS INFLUENCING CROSSING-OVER Any hyl2othesis bearing on the mechanism of crossing-over must take into account a voluminous literature which documents variations in crossing-over governed by numerous extrinsic and intrinsic factors. Many of these factors have been manipulated experimentally by Drosophilu geneticists.

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Although the various genes are found to lie in the same order on both the salivary chromosome map and the genetic map of D . nielaizogaJter, great discrepancies often exist in the relative distances between the two genes on the two maps. In particular, crossing-over seems to be lower per unit of chromosome length in the vicinity of the centromeres. This is particularly striking in the case of chromosomes 2 and 3. It is difficult to interpret these results, since we have no cytological maps of the chromosomes actually undergoing crossing-over (i.e. those of the primary oocyte nucleus). Therefore, one could argue that crossing-over is constant per unit length of D N A in the oocyte chromosomes, but that in the chromosomes of the salivary gland nuclei the D N A near the centromeres is relatively less compact than elsewhere in the chromosomes. If, on the other hand, one can obtain an accurate estimate of the relative physical distance between genes in the oocyte chromosomes from the relative distance between the same genes in the salivary chromosomes, then one is forced to assume that exchanges occur less often per unit length of D N A near the centromeres of chromosomes 1, 2, and 3. According to the synai~tomere-zygosoiiiehypothesis, this reduced exchange could be explained by assuming that the average internema is shorter in regions adjacent to the centromeres or that thc probability of a recombinase detaching as it travels along two lionsister chromatids increases as it approaches the centromere. 2. Heterochromatin

Since heterochromatin lies to either side of the centromeres, the reduced crossing-over observed near centromeres may really be a function of paracentric heterochromatin. Roberts (1965a) has pointed out that the recombinational map length (0.04%) for the paracentric heterochroniatin of the X chromosome of D . melanogaster is far less than would be anticipated from its mitotic bulk, which is almost half of the chromosonie at metaphase. The genetic map length is also considerably less than would be estimated from its length in the interphase, polytene chromosomes of the cells of the larval salivary glands (17 bands out of 1024). Crossing-over is also low in the paracentric chromatin of Drosophila v i r i h . If, as the result of a translocation, this heterochromatin is moued to a position distant from the ceiitromere, the heterochromatin remains refractory to crossing-over (Baker, 1958). The coiling and replication cycles of the euchromatic and heterochromatic portions of the chromosomes are often out of phase. For example, Barigozzi et al. (1966) have shown that during the D N A replication of somatic cells of D . rrzelanogater cultured in vitro the heterochromatic regions replicate after the euchromatic regions. Rudkin (1965a) reports that the ratio of the amount of

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heterochromatin to euchromatin for the X chromosome is about 3:7 as measured for metaphase chromosomes in ganglion cells of female larvae. The ratio for the major autosomes is about 2:s. In the polytene salivary chromosomes, the ratio is about 1:30 for the X chromosome, however. Rudkin concluded that the heterochromatic D N A undergoes fewer cycles of replication than the euchromatic D N A in these chromosomes. In larval salivary gland cells, all the paracentric heterochromatin adheres to form a chromocenter. Some mechanism must be present that insures the release of each recombinase molecule that has completed its journey along a given chromosome arm. The recombinase would thus be available for attachment elsewhere. A hypothetical method for accomplishing this would be to have the paracentric heterochromatin code for an RNA which causes the release of recombinases from neighboring DNA. The production of recombinase-release RNA (rcrRNA) by paracentric heterochromatin would explain why recombination is reduced in the euchromatin close to the centromeres and why recombination rarely occurs in the paracentric heterochromatin itself. Beadle (1932) and Graubard (1932, 1934) have studied the effects of translocations and inversions that move blocks of genes from positions normally distant from paracentric heterochromatin to positions close to it. Under such circumstances, crossing-over between the genes within the transposed blocks is reduced below the normal values. Since the reduction in crossing-over extends quite far from the breakage point, one can postulate that rcrRNA can diffuse away from its site of synthesis in the heterochromatin. Suzuki (1965) has found that the injection of actinomycin D into Drosophih females increases crossing-over in the euchromatic regions near the paracentric heterochromatin. These findings can be explained by assuming that actinomycin D abolishes the transcription of rcrRNA and, therefore, recombinases are allowed to continue their journeys toward the centromere.

3. Temperature In the mouse, in which elevated temperatures are known to reduce the frequency of chiasmata in spermatocytes, Nebel and Hackett (1961) found that transferring males from 22' to 35OC for 76 hours caused a reduction in the number of synaptonemal complexes in zygotene and pachytene nuclei. In treated nuclei, single axial elements persisted into pachynema (where they are never seen normally). Henderson (1966) has shown that in the desert locust Schistocerca gregariu a constant temperature of 40°C reduces the chiasma frequency and can lead to complete failure of bivalent formation. He determined the time of premeiotic DNA synthesis through radioautographic studies and concluded that the heat-sensitive events that led to a reduction of the chiasma frequency in treated material took place at least 2-3 days (at 40OC) after premeiotic DNA

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synthesis had terminated. In Neurosporo crassa, however, according to McNellyIngle et al. (1966), and in Z. mays, according to Maguire (1968), heat treatments administered at late premeiotic interphase as well as at meiotic prophase influence crossing-over. Plough (1917) was the first to show that crossing-over in D. melanogaster females was influenced by temperature. He found that departures from a certain normal range of temperatures (18-29Oc) in either direction increased recombination in chromosome 2. The euchromatic chromosomal regions proximal to the centromere seemed to respond the most to the treatments. Temperature shocks that increase crossing-over, decrease interference (Hayman and Parsons, 1960). Grell (1966) has made a study of this phenomenon in the X chromosome. The shocks she used generally involved a transfer of the insect from 2 5 O to 35°C for a period of 8-12 hours. The increase in crossing-over observed in regions close to the centromere was demonstrated to be the result of meiotic, not gonial, crossing-over. The cells responding to the treatment were oocytes undergoing their early development in the germarium, and some of them may have been in premeiotic interphase (Grell and Chandley, 1965). It is not certain from any of the above studies that temperature shocks act directly upon the recombinational events. A compound reacting during recombination could be synthesized prior to the time it is to function. A heat shock could prevent its synthesis and, thus, produce an effect at a subsequent period. The findings reported for the mouse and locust are in harmony with the hypothesis that meiotic synapsis and crossing-over depend upon the synthesis of synaptonemal complexes, and that the D N A replication of each chromosome of the bivalent precedes such synapsis. Temperature shock could inhibit the formation of zygosomes or cause their premature breakdown. Since axial complexes are observed to persist in heat-treated mouse spermatocytes, the postulated pairing of synaptomeres is not affected. In Drosophilu, one has to explain the differential response of paracentric euchromatin to both heat and cold shocks. Perhaps the enzyme transcribing rcrRNA (Section V,G,2) is stable only in the 18O-29OC temperature range and functions late in premeiotic interphase and early in meiotic propbase. At temperatures outside the specified range, the enzyme is inactivated and, as a consequence, recombinases later encounter lower concentrations of rcrRNA. Therefore, recombination is elevated in the paracentric euchromatin.

4. Age Bridges (1915) was the first to show that the amount of crossing-over bctween certain genes in D. melanoguster declines during the first week of female life. In the cases of the three major chromosomes, the euchromatic segments that showed most clearly this age effect lie close to the centromeres (Bridges,

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1927; Plough, 1917, Stern, 1926). In R. F. Grell’s more recent study (1966), daily measurements were made of the amounts of crossing-over that had occurred in the proximal halves of the X chromosomes in eggs laid over a 2-week interval. The crossing-over frequency was about 20% in the first sample. The daily values then fell until they reached lo%, a frequency which was maintained for the sixth and subsequent samples. The eggs from the first sample were laid about 2 days after eclosion, and they must have undergone crossingover during the pupal period. The eggs from samples 6 through 14 were laid 8-1 6 days after eclosion, and they underwent crossing-over during the female’s adult life. The above data demonstrate that during metamorphosis crossing-over in the chromosomal regions proximal to the centromeres occurs at a rate double that in the X chromosomes of the oocytes of adults. This conclusion raises the question as to whether or not there are differences in the milieu of pupal and adult oocytes that parallel the dissimilarities observed in crossing-over. One difference of possible significance would be in tissue ecdysone levels. The prothoracic gland degenerates late in the pupal period, but before it does, it secretes large quantities of ecdysone (see the discussion in Aggarwal and King, 1969). Therefore, the ecdysone titer should be much higher in the pupal than in the adult milieu. It is well established that this hormone when injected into the larvae of various dipteran species results in localized puffing of the polytene chromosomes in the cells of the salivary glands and other organs (see reviews by Kroeger and Lezzi, 1966; Clever, 1968). RNA transcription is a necessary requirement for such puff formation, and radioautographic studies demonstrate that this synthesis occurs in the puffs themselves. It follows from these data that ecdysone does influence RNA transcription by Drosophila chromosomes, that this hormone is present at the developmental stage when crossing-over in regions proximal to the centromeres is high, and that it is absent or present in a far lower concentration at a stage when crossing-over in the regions specified is low. Obviously, studies should be carried out on the effects of injected ecdysone upon crossing-over in adult females. Data provided by Redfield (1966) complicate the relation between maternal age and crossing-over still further. She studied crossing-over in the region spanning the centromere of chromosome 3 and found that the crossover values fell from 18% in the first eggs laid to 11% in those laid between the fifth and sixth days. Thereafter, the values rose again and by day 1 2 were back at 18%. The significance of this rise and the reason for its occurrence in chromosome 2 but not in the X chromosome are not understood. 5 . Chromosome Aberrations

The first studies of chromosomal aberrations that influence crossing-over were those by Sturtevant and Beadle (1936) on inversions. Fewer progeny represent-

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ing crossover Chromatids are produced by inversion heterozygotes, both because crossing-over is itself inhibited and because certain types of crossover chromatids are eliminated. It can be shown in the case of paracentric inversion heterozygotes, for example, that each single exchange within the inversion loop generates an acentric and a dicentric chromatid. Such aberrant chromatids were first observed by McClintock (1931, 1933) in corn. In female Drosophilu and other dipterans, such chromatids are eliminated because of their selective distribution to the polar nuclei during oogenesis (Sturtevant and Beadle, 1936; Carlson, 1946). Similar exchanges within pericentric inversion heterozygotes produce monocentric, duplication-deficiency chromatids which are not so eliminated. Since eggs bearing chromosomes with duplicated and deficient regions would be expected to be incapable of completing embryonic development after they are fertilized, females heterozygous for pericentric inversions should be semisterile. Roberts (1967) has documented this expectation by finding pericentric inversions in D . melanogaster which drastically reduce both crossing-over and egg hatch in heterozygous females. Pericentric inversions are known (Alexander, 1952) that greatly reduce the production of crossover progeny zuithozrt drastically lowering the frequency of eggs hatching, however. Here it appears that crossing-over itself has been inhibited, and such an inhibition need not be the result of a disturbance in the synaptic pairing between the homologs of a bivalent. In the very first cytological study on chromosome pairing in an inversion heterozygote, McClintock (193 1) showed that reverse loop pairing did not characterize all inversions. She found that in maize plants heterozygous for a long paracentric inversion most pollen mother cells did show reverse loops, but in the case of shorter inversions there was a nonhomologous association which led to straight pairing. In heterozygotes for pericentric inversions, reverse loops were seen in only 1% of the sporocytes examined. More recently, Nur (1968) reported the results of a study of pachytene spermatocytes of the grasshopper Cumnula pellucidu. Males were chosen that were heterozygous for a paracentric inversion occupying about 10% of one of the longest chromosomes. In 90% of the cells observed, straight pairing occurred. Asynapsis was seen 8% of the time and in the remainder of the cells inversion loops or a similar chromosomal configuration occurred. Obviously, nonhomologous pairing takes place the majority of the time between the inverted segments. According to the synaptomere-zygosome hypothesis of meiotic synapsis, the homologs of each bivalent are zipped together by zygosome bridges which first form near each telomore and proceed proximally (Fig. SB). Since the honiologous telomeres are anchored to nearby regions of the nuclear envelope, the zygosome bridges form in a sequential fashion which brings those synaptomeres into register that link allelic internemal segments. Consider, however, the problems brought about by a short inverted segment lying proximal to the telomere of one homolog. The homologs will zip together starting at each end. When

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the inverted segment is reached, nonhomologous pairing will generally occur. This too presumably results from zygosome bridge formation. In this case, however, the internemal loops brought into register will not contain allelic genes and will not pair intimately. Under these circumstances, recombinases are postulated to detach (see Section V, C) and, thus, exchanges between the inverted segments of the heterozygote would not be catalyzed. Crossing-over is often known to be markedly decreased in adjacent uninverted regions to the left and/or to the right of the limits of an inversion (Sturtevant and Beadle, 1936). This finding can be explained by assuming that, for example, if a traveling recombinase reaches the left limit of an inversion and detaches, the probability of an exchange occurring in the uninverted chromosomal region to the right of the inversion is lower than it would be if no inversion occurred nearby. This is because once the recombinase detaches from the chromosome it may subsequently attach to some distant area on the same bivalent or to another bivalent which happens to be nearby, rather than to the segment in question. Crossing-over within the inverted sections is decreased in inverse proportion to the length of the inversion. Thus, more crossover chromatids are recovered from longer inversions. This finding suggests that if the inverted regions are long enough the chromosome segments can sometimes form reverse loops (as demonstrated by McClintock, 1931). Crossing-over should be abolished at the breakage points because a recombinase traveling toward an inversion loop would be forced to halt its journey upon reaching the base of the loop. Similarly, a recombinase that attaches at some site within the loop would be forced to stop upon reaching the end of the loop. Outside or inside the inversion loop, however, zygosome bridges bring internal loops into register that do contain allelic genes, and crossing-over can then take place. In such cases, the more common single crossover chromatids would be acentric or dicentric and would be eliminated. Double crossover chromatids are recoverable from the two- and threestrand double exchange tetrads. While inversions change the order of genes in a chromosome and their distances from the centromere, they do not alter the length of the chromosome. Aberrations such as deficiencies, duplications, and insertional translocations do cause such alterations, however. In D. melanogaster, aberrations of this sort sometimes reduce crossing-over markedly when heterozygous. For example, Roberts (1966) has shown that a specific tandem duplication when carried in the heterozygous condition reduced crossing-over in the left arm of chromosome 2 from 44.2% to 7.3%. Rhoades (1968) has more recently reported the results of a cytogenetic study of the effects of an insertional translocation upon pachytene pairing and genetic crossing-over in maize. The transposition involved the removal of an interstitial piece from the long arm of chromosome 3 and its in-

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sertion into the short arm of chromosome 9. For purposes of discussion, the normal chromosomes will be symbolized by N9 and N 3 and the aberrations by Df3 and Tp9. Female parents of karyotype TpgN9;N3N3 bearing appropriate marker genes were testcrossed. A pronounced reduction in crossing-over within chromosome 9 was observed throughout all the regions tested (these normally had a combined map distance of about 50 crossover units). Cytological observations were made on pachytene bivalents of Tp9N9 constitution. The portion of the Tp9 chromosome that had no homologous counterpart in the N 9 chromosome was expected to protrude as a buckle from one side of the bivalent. Such buckles were observed, but they occurred at various positions along the short arm of chromosome 9, and the lengths of the buckles were not constant (Fig. SA). Every position of the buckle other than those where homologous regions were synapsed on either side obviously involved pairing of nonhomologous segments. Rhoades’ findings fit rather neatly into the synaptomere-zygosome theory of synapsis. Assume that a synaptonemal complex grew from the telonieres of the short arm of the TpN9 bivalent toward the centromere. The complex zipped together homologous chromosomal regions until it reached the translocated piece of chromosome 3. Subsequently, nonallelic chromosomal segments became attached. Once the transposition was passed, the synaptonemal complex continued to zip nonhomologous regions together; as a consequence, all allelic internemal loops would have been out of register until the synaptonemal complex was met by the synaptonemal complex growing toward it from the opposite telomere. When this meeting occurred a buckle was forced to protrude from the longer chromosome. Here, the zygosome bridges could form within the buckle itself. The position of the buckle would be expected to vary if the relative speed with which the synaptonemal complexes grew toward each other also varied in this chromosome arm. Since such a large amount of illegitimate pairing occurred, one would expect crossing-over to be greatly reduced. The same hypothesis can be used to explain Roberts’ observations on the effects of a tandem duplication upon crossing-over. In the case of Df3N3 bivalents, Rhoades found that the observed buckles were far more uniform in size and position (Fig. SB), and crossover studies showed that recombination was reduced only in regions that contained the deleted segment. These observations suggest that the linking together of nonhomologous segments by zygosome bridges rarely occurred in the Df3N3 bivalent. Since the buckle usually protruded from the middle of the long arm of chromosome 3, one must assume that synaptonemal complexes grew in opposite directions along the long arm of chromosome 3 at such rates that they usually met in the middle. Dobzhansky (1931, 1934) studied the effects of a number of translocations

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when heterozygous upon recombination. He concluded that the greatest reduction in crossing-over is in the neighborhood of the breaks, and that this reduction is least pronounced in those chromosomal regions most remote from the points of breakage. Autosomal translocations having one or both breaks in the

FIG. 8 . ( A ) Camera lucida drawings of Tp9/N9 bivalents at pachynema showing the frequently observed asynapsis and nonhomologous pairing. ( B ) Camera lucida drawings of pachytene configurations of Df3/N3 bivalents showing the fairly uniform position of the buckle. From Rhoades (1968).

chromocentral regions exhibit normal or even increased recombination in these regions (Dobthansky and Sturtevant, 1931). Roberts (1965b, 1968) has found that the most commonly recovered crossover suppressors from X-irradiated Drosophild sperm are not inversions, but reciprocal translocations. When heterozygous, some of these reduce crossing-over more than any known inversion.

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Roberts (1965a) has made a study of translocations in which one break was always in the tiny fourth chromosome. He found that when the other chromosome involved in the translocation had a break close to the centromere, recornhination within the arm was little affected. Distally placed breaks reduced crossing-over drastically, however.

FIG. 9. Diagrammatic representation of the postulated pairing behavior of the second and fourth chromosomes of D. melanogaster under normal conditions ( A ) , in a 2L:4 translocation in which the break in 2L is distal to the centromere (B), and in a 2L:4 translocation in which the break in 2L is proximal to the centromere ( C ) . Centronieres are represented by open circles, telomeres by smaller solid circles. Legitimate pairing is represented by I ] 1 I, illegitimate pairing by X X X . In reality, the length of the second chromosome would be eight times the diameter of the nucleus.

These findings support the idea that the telomeres are attached to the nuclear envelope during the synaptic stages of meiosis. Translocations that have their breakage points near these chromosomal attachment points would lead to difficulties in the synapsis in heterotygotes. For example, if the left telomeres of the two second chromosomes were attached to the nuclear envelope at a point distant from the telomores of the fourth chromosome, synapsis would proceed by the formation of zygosome bridges to the point where the translocation occurred. Hereafter, for most of 2L, the pairing that does occur will be illegitimate (see Fig. 9B). If, on the other hand, the break occurs far enough away from the telomere of 2L, the synaptonemal complexes that form will hold the

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synaptomeres in proper register and hence recombination will show little reduction (see Fig. 9C). 6. Interchromosomal Effects

Sturtevant (1919) first observed that an inversion in the third chromosome of D. melanogaster when heterozygous increased crossing-over on chromosome 2. In subsequent years, many investigators noted that heterozygous inversions in any pair of major chromosomes in Drosophila increased crossing-over in the other pairs (see review by Lucchesi and Suzuki, 1968). According to Rendel (1957), increased crossing-over caused by heterologous inversion heterozygosity is accompanied by reduced interference. White and Morley (1955) reported that in the grasshopper Trimerotropis suffusa pericentric inversions suppressed chiasmata formation within the regions heterozygous for the aberrations. The lowered chiasmata frequency in these chromosomes was compensated for by an increased frequency in other bivalents. Thus, the phenomenon appears to be widespread. An explanation of the interchromosomal effects of inversions can be found in the postulated behavior of recombinases. According to the speculations advanced in Section V, C, recombinases remain attached only to those nonsister chromatids that pair properly, and intimate pairing of the type required only occurs if the nucleotide sequences are nearly identical (i.e., if the genes brought into register are alleles). “Liberated recombinase” molecules, which have detached from sister chromatids that fail to meet these specifications because of an inverted segment, are available to attach to other bivalents. Thus, the probabilities of crossing-over occurring in those normal bivalents of the genome would be greater than those normally observed. Roberts (196513) has demonstrated that an autosomal inversion which when heterozygous almost triples the amount of crossing-over in euchromatin proximal to the centromere of the X chromosome has no effect on proximal heterochromatin. Since it is postulated that this chromatin synthesizes rcrRNA, crossing-over would not be expected to increase here. Translocations that act as crossover suppressors should also promote crossingover in other chromosomal regions for the same reasons that inversions do. In Drosophila, numerous instances of translocations enhancing crossing-over in heterologous chromosomes have been recorded (Hinton, 1965), and Hewitt (1967) has reported that a translocation exists in the grasshopper Cibolucris parviceps that increases the frequency of chiasmata in all chromosomes, including the interchange itself. Chromosome rearrangements that attach homologs or arms of homologs to the same centromere are known in Drosophila. An example of such an aberration is the reversed acrocentric, compound X chromosome studied by Hart and

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Sandler (1961). They found that recombination in chromosome 3 was increased in the presence of this compound X chromosome. One would expect that the intensity of the enhancing effect of any aberration upon crossing-over within the chromosomal elements not involved in the aberration would be proportional to the degree to which crossing-over is suppressed within the chromosomal elements involved in the aberration. This would be because the larger the suppression of crossing-over, the larger the number of liberated recombinases available to attach elsewhere in the genome and catalyze crossing-over. Liberated recombinases would be more concentrated in one region of the nucleus than another, however, and each bivalent may be in a characteristic area. Hence, some may be accessible to recombinases liberated from a given aberration and others inaccessible, and a simple relation between crossover suppression in one bivalent and crossover enhancement in another may not always be found. The euchromatic regions of chromosomes that seem to be particularly sensitive to the enhancing effects of heterologous aberrations upon crossing-over lie near the telomeres and centromeres (Lucchesi and Suzuki, 1968). This enhancement pattern can be accounted for on the basis of the hypothesis presented earlier. Synaptonemal complexes would form first in the telomeric regions of chromosomes and, therefore, the total period of time during which these regions would be available for the attachment of liberated recombinases would be the longest. Recombinases reaching the euchromatin proximal to centric heterochromatin would encounter rcrRNA molecules which would bind to them and cause them to detach from the Chromosome. With the concentration of recombinase molecules raised by the addition of liberated recombinases, the centric heterochromatin may be unable to produce enough rcrRNA to bind to all incoming recombinases. As a consequence, the journey of the average recombinase in the proximal euchromatin would be lengthened, and crossing-over enhanced in this region. 7. Recently Discovered M i i t d o n s Influencing Recombination

Lindsley et al. (1968) and Sandler et al. (1968) have recently described three mutations in D . melanogaster, each of which influences crossing-over in a unique way. Since the wild-type alleles of these mutations presumably control meiotic processes, they have all been given the symbol mei. In the case of meiS51, recombination is reduced uniformly to about half the control value. The mei-S282 mutant, on the other hand, reduces crossing-over in a polarized fashion, the reduction being most pronounced distally. The mei-S332b mutant increases recombination in comparison with controls. Cytological investigations of the salivary gland chromosomes demonstrate that none of the mutations are associated with chromosomal aberrations. As yet, no studies have been made of the chromosomes in mutant oocytes utilizing the electron microscope.

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tb, Philadelphia 2, 556. Higgins, G . M., and Anderson, R. M. (1931). A.M.A. Arch. Pathol. 12, 186. Hurwith, J., Furth, J., Malamy, M., and Alexander, M. (1762). Proc. Nail. Acad. Sci. U S . 48, 1222. Iyer, U. N., and Szybalski, W. (1958). Proc. Natl. Acad. Sci. U S . 44, 446. Iyer, U. N., and Szybalski, W. (1963). Proc. Natl. Acad. Sci. U S . 50, 3 5 5 . Jackson, B., and Dessau, F. I. (1961). Lab. Invert. 10, 909. Jacob, J. (1767). Exptl. Cell Res. 48, 276. Jacob, J., and Sirlin, J. L. (1963). J . Cell Biol. 17, 153. Jacob, J., and Sirlin, J. L. (1964). J. Ultrast~uct.Res. 11, 315. Jezequel, A. M., and Bernhard, W. (1764). J. Microscopie 3, 279. Jkzequel, A. M., Shreeve, M. M., and Steiner, J. W. (1967). Lab. Invest. 16, 287. Jones, J. W. (1965). J. Ultrastruct. Res. 13, 257. Jones, J. W. (1967). J. Ultrastrtlct. Res. 18, 71. Jones, J. W., and Elsdale, T. R. (1964). J. Cell B i d . 21, 245. Journey, L. J., and Goldstein, M. N. (1961). Cancer Res. 21, 929. J L ~J.,, and Kemp, T. (1933). Strahlentherapie 48, 457. Karasaki, S. (1964). J. Ultrastruct. Res. 11, 272. Karasaki, S. (1965). J. Cell Biol. 26, 937. Karasaki, S. (1968). Exptl. Cell Res. 52, 13. Kleinfeld, R. G. (1957). Cancer Res. 17, 954. Kleinfeld, R. G. (1966). Natl. Cancer Inst. Monograph 23, 369. Kleinfeld, R. G., and Von Haam, E. (1959). Cancer Res. 19, 769. Koulish, S., and Kleinfeld, R. G. (1964). J. Cell Bid. 24, 39. Krishan, A., Uzman, B. G., and Hedly-Whyte, E. T. (1967). J. Ultrastwct. Rer. 19, 563. Kuboda, Y., and Furuyama, J. (1963). Cancer Res. 23, 682. Kume, F., Maruyama, S., D’Agostino, A. N., and Chiga, M. (1767). Exptl. Mol. Pathol. 6, 254. Lafarge, C., Frayssinet, C., and de Recondo, A. M. (1965). Bull. SOC. Chim. B i d . 47, 1724. Lafarge, C., Frayssinet, C., and Simard, R. (1766). Compt. Rend. 23, 1011. Laird, A. K. (1953). A ~ c h .Biochern. Biophys. 46, 119. Lane, N . J. (1967). J. Cell Biol. 35, 421.

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The Origin of Bone Cells MAUREEN OWEN Medical Reseurch Council External Scientific Staff, Bone Re.remch Luhma1oty, T h e Churchill Hospital, Oxfovd, Englund

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Osteoprogenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Uptake of Tritiated Thymidine . . . . . . . . . . . . . . . . . . . B. Proliferative Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Kinetics of Differentiation . . . . . . . . . . . . . . . . . . . . . . . D. Histocheinistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Electron Microscope Studies . . . . . . . . . . . . . . . . . . . . . F. The Effect of Parathyroid Hormone . . . . . . . . . . . . . . . G. Cell Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. The Composition of Osteoprogenitor Cells . . . . . . . . . 111. Other Cells with Potential for Hone Formation . . . . . . . . . A. Experiments with Millipore Filters (Closed System) . B. Experiments with Direct Transplants (Open System) . C. Bone Fracture Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . 1V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

213 216 217 21 8 21 S 224 226 226 227 22s 229 230 211

235 231 236

I. Introduction The origin of all cells in the body is the fertilized ovum. Studies of the mechanisms that result in the development of different cell types, tissues, and organs from this single cell represent one of the most active fields of current research. For any particular tissue, whether in the embryo or in the postfetal organism, the problem in its siniplest form can be stated as follows. What is the nature of the cells and of the particular set of inductive stimuli that enable the two to react to form the tissue under consideration? In embryonic systems, this is known as the process of induction, although the term is also borrowed for similar situations in the postfetal organism. The process is not well understood and it is likely that there are many kinds of inductions taking place ,zt all stages of differentiation throughout the life of an organism. Cellular microenvironment and cell-to-cell and cell-to-substrate relationships are all probably iinportant in various inductive processes and in differentiation in general. This article is an attempt to review, for the case of bone, the progress that has been made on one small aspect of this problem in recent years, namely, which cells in the postfetal organism are capable of osteogenesis. This immediately raises the question of cell terminology. Cells were originally named accordiiig to their morphological appearance and in some cases this amounts to 110 inore than a difference in size, shape, or location. Cell terminology will, 213

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therefore, be kept as simple as possible and the emphasis will be on what the cells do, rather than their histological appearance. An attempt will be made, when relevant, to relate the results obtained by newer methods to the previous nomenclature. In classic histology we learn that connective tissue develops from the mesenchyme of the embryo. From the studies of Maximow many years ago, the con-

connective tissue stem cells

Increasing differentiation

I

t tissue stem cells

rl progenitors

(d)

Fully differentiated cells

FIG. 1. Diagram of differentiation of connective tissue.

cept arose that there is present in the body a small pool of undifferentiated mesenchymal cells which have the capacity to differentiate along any one of several lines. Although this concept has never been seriously disputed, there are still many unanswered questions, some of which are outlined in the following discussion with reference to the diagram in Fig. 1. Differentiation of any connective tissue in the body can be represented as occurring in four stages, Pig. 1. This is an obvious simplification since differentiation is more likely to consist of a gradation of stages. Using Fig. 1 as a model, we can ask the following questions concerning the nature of the pool of undifferentiated mesenchymal cells presumed to exist throughout connective tissues (Ham and Leeson, 1961). First, are the cells that compose it multipotential? In other words, are they connective tissue “stem cells”l (a in Fig. 1) and, as such, are they common 1 The definition of “stem cell” is after Caffrey-Tyler and Everett (1966). A stem cell is defined as a cell having the capacity for extensive proliferation resulting in renewal of its own kind as well as giving rise to fully differentiated cells. Strictly speaking this definition might also apply to some of the proliferating progenitors but in general the term is reserved for less differentiated cells.

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proliferative precursors of all connective tissues, i.e., hemopoietic tissue, bone, cartilage, fibrous tissue, and so on? Second, is the pool, on the other hand, made up of a mixture of distinct groups of stem cells for each of the different tissues (b in Fig. I ) , eg., hemopoietic stem cells, osteogenic stem cells, fibroblastic stem cells, and so on, each of which is capable of giving rise to all the different cell types in the tissue? The implication here is that each of the different stern cell types in b is already destined to differentiate in the direction of the tissue concerned. A third possibility is that the pool contains cells from both categories a and b. What is the distribution of these undifferentiated cells, whether they are equivalent to a, or b, or a mixture of both? Are they present in the circulating blood and, if so, what is the tissue of origin? The term “proliferating progenitors” is reserved for a further category of cells (c in Fig. I), which are the more immediate precursors of the fully differentiated cells (d) of a tissue. These cells have reached a more differentiated stage than those represented in a and b and are known to be already determined in the direction of differentiation of the tissue concerned. They still have proliferative capacity and, in addition, may exhibit some recognizable characteristics of the final differentiated tissue cell. A final question is, what is the relation between the ubiquitous pool of relatively undifferentiated mesenchymal cells in the body, represenled by a and b in Fig. 1, and the inore differentiated population of proliferating progenitor cells of bone (c in Fig. 1) ? One difficulty in studying the questions outlined above is the fact that the less differentiated cells in the scheme (a and b in Fig. 1 ) are not yet distinguishable with certainty under the microscope. The morphological appearance of the undifferentiated mesenchymal cell is unknown. Nevertheless, there are inany descriptions of it. In the literature on bone, it is often referred to as a cell with a pale, vesicular, oval, or fusiform nucleus and inconspicuous cytoplasm, but it may well take different forms depending on its surroundings. Neither can the different types of stem cells (b in Fig. I ) , if in fact they do exist as separate entities, be recognized under the microscope. In spite of their elusive morphology, there is nevertheless good evidence from other tests for the widespread existence of undifferentiated cells which can be induced to differentiate in the direction, for example, of either hemopoietic tissue or bone as the case may be. This can be inferred from various experiments which test the functional properties of these cells. In the case of hemopoietic tissue, for example, the existence of stem cells has mainly been demonstrated by their capacity to reseed hemopoietic centers in animals that have lost their own hemopoietic tissue by exposure to radiation (Micklem and Loutit, 1966; Loutit, 1967). In the case of bone, the main source of information on the capacity of undifferentiated cells for bone formation comes from experiments on heterotopic bone induction (i.e., induction of bone in sites outside the slteleton; Bridges, 1959; McLean and Urist, 1968). There is a very

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large literature on the subject of bone induction and no attempt will be made to cover it in detail. In particular, the nature of the environmental conditions that induce osteogenesis in sites outside the skeleton will not be considered. Only information relevant to the problem of which cell types have the capacity for osteogenesis will be reviewed. At the more differentiated end of the scheme (Fig. 1) are the immediate precursors of bone cells. They are the population of dividing cells situated near bone surfaces or sometimes in contact with them. Young (1962b) showed that they were the precursors of both the osteoblasts and osteoclasts, the two main differentiated cells of bone, and he named them osteoprogenitor cells. They belong to the category of proliferating progenitors of bone cells (c in Fig. I ) , but the two terms are not synonymous; proliferating progenitors is a wider term, although the composition of cells it covers is not yet fully defined. It includes osteoprogenitor cells and probably less differentiated precursors of these cells, which may exist particularly in marrow tissues. Osteoprogenitor cells will be used herein, as originally suggested (Young, 1962b), for those precursors of bone cells found near bone surfaces. These cells have been very actively studied in recent years, and an account of their characteristics will be given in Section 11. In Section 111, cells other than osteoprogenitor cells which are capable of bone formation, will be considered. Here, as already mentioned, the evidence is mainly from experiments on heterotopic bone induction. From this work, there is evidence to show that in addition to other cell types, relatively undifferentiated cells, which are mobile and have a widespread distribution in the body, can be induced to form bone. This immediately raises the question as to whether or not these ubiquitous undifferentiated mesenchymal cells are precursors of the proliferating progenitors of bone cells. One might envisage feed-in of cells from this compartment into the progenitor population as required. Whether this happens at all under normal physiological conditions in the postfetal organism is questioned. There is a possibility that the proliferating progenitors of bone are a self-perpetuating population, and that transitions from undifferentiated mesenchymal cells to progenitors of bone cells do not take place in the postfetal organism under normal conditions. It may be that in the mature organism the capacity of the undifferentiated cell for bone formation is reserved only for situations in which reparation or regeneration is needed. The evidence for these ideas will be presented and discussed.

11. Osteoprogenitor Cells These cells were first defined as the population of cells near bone surfaces that are labeled shortly after injection with thymidine-". The thickness of the

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layer of osteoprogenitor cells varies with the age of the animal and the part of the bone studied. The location of these cells, associated as they are with bone surfaces, is the main morphological criterion defining them. Their appearance is indistinguishable from cells previously described under the teriiis inesenchymal cells, spindle cells, fibroblasts, reticulum cells, endothelial cells lining blood vessels, and possibly some other components of bone marrow (Kembcr, 1960; Young, 1962a,b). Consequently, on morphological grounds it was thought these osteoprogenitor cells may be part of a pool of undifferentiated rnesenchymal cells with wider potential. Evidence from the kinetic studies of their differentiation (Young, 1962b), however, has shown that they are the immediate precursors of the differentiated cells of bone, and they have been called osteoprogenitor cells to give them a more meaningful name. Furthermore, recent work has indicated that they are largely made ~ i p of the progenitors of the two main differentiated cell lines in bone, osteoblasts and osteoclasts (Scott, 1967; Bingham et ul., 1969), each line of progenitors exhibiting some of the characteristics of its final differentiated form. Studies of the proliferative activity and the kinetics of differentiation of osteoprogenitor cells have been made using tritiated thymidine and radioautographic techniques. The results are described in the following discussion along with an account of the ultrastructure, histochecnistry, and some aspects of the behavior of these cells under the effect of parathyroid hormone.

A. UPTAKEOF TRITIATED THYMIDINE For a proliferating population, the cell cycle is divided into four phases (Howard and Pelc, 1953). G, is the resting period between the previous mitosis and the commencement of D N A synthesis. S is the period during which DNA is synthesized and the D N A content of the nucleus doubled in preparation for the next division. G2 is a short period before cell division, and &l is the period of mitosis. The total cell cycle time (GI S G2 M ) is represented by T,. Numerous studies (Quastler and Sherman, 1959; Cronkite et ul., 1959; Fry et ul., 1963) have confirmed the above scheme and have shown that in mammalian tissues S, G2, and M vary relatively little (Cattaneo ef dl., 1961; Owen, 1965) ; variations in T , are, therefore, mainly attributable to differences in GI. In proliferating cell populations, tritiated thymidine is taken up by cells during the S period. The fraction I; of the cell population that is labeled a short time after injection of thyniidine-:lH (i.e., before the labeled cells have had time to go through mitosis; about 1 hour is common) is an index of the proliferative activity of the tissue. Assuming a random distribution of the cells throughout the different phases of the cell cycle, it follows that f; is related to S and T , by the equation F = S/T,.

+ + +

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13. PROLIFERATIVE ACTIV~TY The fraction of osteoprogenitor cells that is labeled t hour after injection of thymidine-3H varies with the region of the bone being studied and the species and age of the animal (see Table I). Young (1962a) found that the proliferative activity F was different in different regions of the same bone, Results for the metaphysis of the rat (Keniber, 1960; Young, 1962a,b) showed that F fell dramatically with age, and this was also borne out by the results of Tonna (1961) for mice. Tonna also showed that the thickness of the osteogenic cell layer on the bone surface decreased in a similar way with age. Many of the studies, therefore, have been made in very young animals. As will be shown later, the proliferative activity of the osteoprogenitor cells is roughly correlated with the rate at which they differentiate into more mature forins. Values of S have been measured by several of the authors listed in Table I. Young found a value of about 8 hours for the cells in all three regions of the bones of 6-day-old rats, and a value of 6.2 hours was found by Owen and MacPherson (1963) for 2-week-old rabbits. These values are in very good agreement with previous measurements of S for other mammalian cells (Qnastler and Sherman, 1959; Cronkite et al., 1959; Lesher et al., 1961). Some values for the cell cycle time T , are also shown in Table I. It must be emphasized that only average values for T , can be determined. Individual members of the osteoprogenitor population are likely to be at slightly different stages of differentiation. Consequently, some cells may divide more frequently than others, but there is no information on how widespread the rnnge of values for T , might be for any particular osteoprogenitor population.

C. KINETICS OF DIFFERENTIATION

A study of the kinetics of differentiation of osteoprogenitor cells in 6-day-old rats was made by Young (1962a,b, 1963). His method was to determine the initial labeling ( 1 hour after thymidine-3H injection) of the osteoprogenitor cells and then to follow the rate of appearance of labeled nuclei in both osteoblasts and osteoclasts at later times after injection. Some of his studies in the metaphysis are described later. The metaphysis of young rats is a region of very active bone remodeling in which bone deposition and resorption take place side by side on the surfaces of a network of thin trabeculae. In the spaces between the trabeculae can be found the typical osteoprogenitor cells. A diagram taken from Young (1962b) of the cellular arrangement with regard to one nietaphyseal trabecula is shown in Fig. 2. Young’s results for the metaphysis of the tibia taken from Tables IV and VII of his paper (196213) are plotted in Fig. 3 . About 22% of the osteoprogenitor cells in the metaphysis were labeled after 1 hour, labeling of osteo-

VALUES OF F, S

F Species

Age

(%)a

Rat Rat Rat Rat Mouse Mouse Mouse Mouse Mouse Mouse Rabbit

6-8 weeks 6 days 6 days 6 days 1 week 5 weeks 8 weeks 26 weeks 52 weeks 4 weeks 2 weeks

7

a

b c

d

22 14 7

8.5

2.7 0.7 0.2 0.6 5

10

AND

TABLE I FOR OSTEOPROGENITOR CELLS

T,

S (hours) I

8 8 8

-

-

6.2

F = fraction labeled 1 hour after thymidine-3H administration. S = time required for DNA synthesis. T , = total cell cycle time. These results are for the “osteogenic layer” (osteoprogenitor cells

T, (hours) c

Region of bone

36 57 114 -

Metaphysis Metaphysis Endosteum Periosteum Periosteum

Kember (1960) Young (1962a,b) Young (1962a,b) Young (1962a,b) Tonna (1961)d

Periosteum Periosteum Periosteum Periosteum Metaphysis Periosteum

Tonna (1961) Tonna ( 1961) Tonna (1961) Tonna (1961) Simmons (1963) Owen (1963) ; Owen and MacPherson (1963)

-

-

62

+ osteoblasts)

Reference CI

Ei 8

5 2

B

m

n m P

K

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clast nuclei at this time was zero and that of osteoblasts very low. With increasing time after injection, the percentage of labeled osteoblasts and osteoclasts rose and leveled off at a value approximately equal to the initial labeling index of the osteoprogenitor cells. Since the rise in the percentage of labeled osteo-

RESORPTION O F TRABECULA

TRANSITION ZONE

APPOSITION OF BONE ON CALCIFIED CARTILAGE

RESORPTION OF CALCIFIED CARTILAGE

~

FIG. 2. Diagram of a metaphyseal trabecula. O.P., Osteoprogenitor cells (one of which is undergoing mitosis) ; O.B., osteoblasts; O.C., osteoclasts. Bone is depicted by cross-hatching, calcified cartilage is solid. The dashed arrows indicate the origin of osteoblasts and osteoclasts from osteoprogenitor cells (see Section 11, G ) . (From Young (1962b). Reproduced by permission.)

blasts and osteoclasts occurs at about the same time (Fig. 3 ) , Young concluded that these differentiated cells are both mainly derived from osteoprogenitor cells. In osteoclasts, it was found that one or more nuclei may be labeled, and this suggested that osteoclasts arise from the fusion of precursor cells, a conclusion that had also been reached by Keniber (1960). This disposed of, once and for all, the possibility that osteoclasts might arise either through cell division or through the fusion of osteoblasts, at any rate under normal circumstances. The results (Young, 1962b) showed in fact that there was a continual incorporatioil and shedding of nuclei by the osteoclasts, and it was concluded that the concept of the average lifetime of an osteoclast per se is meaningless-what is meaningful is the average lifetime of the nucleated components. As can be seeii from the width at half-height of the top curve in Fig. 3, the average lifetime of a nucleus in an osteoclast in the metaphysis in these young animals was about 150 hours. In the endosteum and periosteuni of the same animals, the turnover of osteoclast nuclei was slower. As is clear from Fig. 2, bone formation and resorption occur side by side in the metaphysis, and it is not possible to separate the osteoprogenitor cells associated with each proccss. In another system (Owen, 1963; Binghani et d., 1969), the midshaft of the femur of a 2-week-old rabbit, bone formation occurs only on the periosteal surface and bone resorption only on the endosteal surface, so that the two processes can thus be studied separately. This system is shown diagrammatically in Fig. 4. The fully differentiated cells of bone line

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the bone surfaces and behind them are situated the osteoprogenitor cells. Particularly on the periosteal surface, the cells are arranged in well-defined layers with the osteoblasts lining the bone surface; the osteoprogenitor cells (called preosteoblasts after Pritchard, 1952, Section 11, D) are in a layer behind them, Percent labeled osteoprogenitor

5

4 I

2 346810

20 140 60 I100 200 400 30 80 Hours

I

2

3 4 6 8 10

I

20 30 60 1100 200 400 40 80 Hours

FIG. 3. Percent of labeled osteoclast and osteoblast nuclei, ( 0 ) at different times after a single injection of thymidine-3H in the metaphysis of the tibia of a 6-day-old rat. Percent of labeled osteoprogenitor nuclei (U) at 1 hour after injection. (From Youllg (1962b). Reproduced by permission.)

and the whole is enclosed by the fibrous layer of the periosteuin. On the endosteal surface, the cells are not arranged in quite such well-defined layers. Osteoclasts cover about 40% of this surface (Owen and Shetlar, 1968). The osteoprogenitor cells are a layer of uninucleated cells within about 30 p from the endosteal surface. They have the undifferentiated appearance of typical mesencliymal cells. Although kinetic studies of the differentiation of these endosteal mesenchymal cells into osteoclasts have not been made, it is assumed

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that in this situation they are precursors of the osteoclasts. Under the light microscope, they are not distinguishable in appearance from the preosteoblasts on the opposite surface. As will be shown later, however, they are functionally distinguishable, and we have suggested the term preosteoclasts for the osteoprogenitor cells on this particular part of the endosteal surface.

-

Periosteum

FIG. 4. Diagrammatic representation of part of the cross-section of the bone wall from the midshaft of the femur of a 2-week-old rabbit, illustrating ( a ) the cells on the periosteal surface associated with bone growth and (b) the cells on the endosteal surface associated with bone resorption; endosteal mesenchymal cells are taken as equivalent to preosteoclasts.

In a previous paper (Owen, 1963), we have studied the kinetics of cell differentiation on the periosteal surface in the process of bone growth in young rabbits. Our method involved simultaneous injections of glycine-SH to label the position of the bone surface at the time of injection and thymidine-3H to label a proportion of the proliferating population, in this case the preosteoblasts. Information was obtained in two ways. First, glycine is rapidly incorporated into the collagen of bone matrix (Carneiro and Leblond, 1959; Young, 1962~;Owen, 1963) and is left behind as a narrow band of grains in the matrix as the bone grows (Fig. 5 ) . By counting the number of cells between this band and the first layer of fibroblasts of the periosteum at different times after injection, it was possible to determine the increase in the total number of cells and in the different categories of cells. Second, by an analysis of the

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distribution of thymidine-labeled cells with time after injection, it was possible to study the movement and differentiation of cells with respect to the bone surface. The system is advantageous in that bone growth is unidirectional during the period of the experiment (a few days), and it was possible to study

FIG. 5 . Radioautograph of part of the periosteal surface from a cross-section of the midshaft of the femur of a 2-week-old rabbit 2 days after a single injection of glycine-3H. The labeled material is a band of grains in the bone matrix on the right outlining the position of the periosteal surface at the time of injection, about 140 p from the periosteal surface on the left.

the fate and distribution of all cells that were originally on the bone surface at the time of injection. The results from this study were as follows. The main region of cell proliferation was the preosteoblast population on the periosteal surface (Fig. 4 ) . In this system, these cells are continually differentiating in an orderly direction

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to become osteoblasts on the periosteal surface. Subsequently, they become either osteocytes or cells on the surfaces of Haversian canals. The latter have a more flattened appearance than the more active osteoblasts on the periosteal surface. It was shown that cell division in the preosteoblasts on the bone surface could account both for the increase in size of this population attributable to growth and for the loss of cells from it in the process of differentiation. After 3 days, labeled osteocytes began to appear. The percentage of osteocytes incorporated that were labeled, approximately equaled the percentage of preosteoblasts and osteoblasts labeled at earlier times. Within the limits of the experimental results, it was concluded that all preosteoblasts differentiate; there was no evidence for cell death. The fibroblastic layer of the periosteum had a very low proliferative activity (F was about 1% compared with 10% in the case of the preosteoblasts). The increase in size of the fibroblast population by cell division was approximately balanced by the increase resulting from bone growth. It was concluded, therefore, that the fibroblasts contribute little to the preosteoblast population on the periosteal surface during normal bone growth. A similar conclusion was reached by Tonna (1961) and Tonna and Cronkite (1964, 1968) in their studies on mice. Osteocytes are never labeled at short times after thymidine injection. Labeled examples of this cell type are seen at later times, because of the cell having acquired its label when in the progenitor state. The time after thymidine injection at which labeled osteocytes are first seen within bone matrix has been recorded by several of the authors listed in Table I. In the work just described in 2-week-old rabbits (Owen, 1963), labeled osteocytes were first seen 3 days after injection. Young (1962b) reports 2 days in the metaphysis of 6-day-old rats, and Kember (1960) 5 days in the metaphysis of 6- to 8-week-old rats These figures suggest that there is a correlation between the proliferative activity I; of the progenitors of the osteoblasts (Table I ) and the rate at which the osteoblasts are incorporated as osteocytes, i.e., the rate of formation of bone matrix. This concurs with the rapid fall-off in I; with increasing age that has been observed in mice (Tonna, 1961), and the concomitant decrease in the thickness of the cell layers on the bone surface.

D. HISTOCHEMISTRY There are several excellent reviews of histochemical investigations of the pattern of enzyme activity and cell organelles in the different osteogenic cells (Pritchard, 1956; Cabrini, 1961). The results are disappointing from the point of view of tracing metabolic patterns related to differentiation or cell origin (de Voogd van der Straaten, 1966). The main differences are quantitative

THE ORIClN OF BONE CELLS

225

rather than qualitative. Depending on techniques and materials, practically all enzymes studied can be found in all cells. There are, however, several distinctive patterns which have been shown consistently. Alkaline phosphatase predominates in the cells of the bone-forming system, osteoblasts and preosteoblasts. Acid phosphatase and succinic dehydrogenase predominate in the cells of the bone-resorbing system, osteoclasts. Recent studies with the electron microscope (Doty et ul., 196s) have shown interesting variations in location of different phosphatases in the various bone cells, as well as quantitative differences. The association of alkaline phosphatase with the process of osteogenesis has been recognized for a long time (Robison, 1923; Bevelander and Johnson, 1950; Pritchard, 1956). In the embryo, the appearance of alkaline phosphatasepositive condensations of mesenchymal cells is taken as synonymous with the advent of preosteoblasts and subsequent bone formation. In the periosteum of growing bones, a histochemical study of the layer of cells behind the osteoblasts showed that these cells had many features similar to the osteoblasts including a positive alkaline phosphatase reaction. This led Pritchard (1952, 1956) to name these ceIIs preosteoblasts, since it was clear that they were in fact precirrsors of the osteoblasts. This has been confirmed in studies (Owen, 1963) of the kinetics of these cells on the periosteal surface as mentioned above. Apart from the studies of these progenitors of the osteoblasts, there has been little investigation of the histochemistry of osteoprogenitor cells until the recent work by BaIogh and Hajek (1965). In their paper, distribution of the oxidative enzymes of intermediary metabolism in healing fractures and associated bone was described. Several interesting new features emerged from this work. For the first time, certain variations in the enzyme content of different osteoprogenitor cells were observed. The osteoprogenitor cells of the periosteum (presumably preosteoblasts) differ in their content of glucose-6-phosphate dehydrogenase from the osteoprogenitor cells of fracture callus. O n the other hand, young developing chondrocytes of the fracture callus and preosteoblasts both contain a similar amount of this enzyme. It is tempting to conclude that in these cells the appearance of this enzyme may be connected with differentiation. Another striking result was the presence of mononuclear cells exhibiting a reaction for succinic dehydrogenase, thus distinguishing these cells from other osteoprogenitor cells. Succinic dehydrogenase is specific for osteoclasts among bone cells, and it was suggested that these cells may, therefore, be precursors of the osteoclasts. Walker (1961) has also described mononuclear and binuclear cells with strong succinic dehydrogenase activity in bones treated with parathyroid hormone. There is thus histochemical evidence for a “preosteoclast” stage among osteoprogenitor cells (Owen, 1968). This supports the more recent findings described in the next section,

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MAUREEN OWEN

E. ELECTRONMICROSCOPESTUDIES In a recent paper (Scott, 1967), a study has been made of the ultrastructure of the osteoprogenitor cells. The region studied was the proximal epiphysis of 18- to 21-day-old fetal rats, the mothers having been injected with tritiated thymidine 1 hour before sacrifice. Electron microscope radioautographs were made, and the observations were limited to labeled cells on the surfaces of the developing bone trabeculae and along the capillaries in the primary spongiosum. The results demonstrated that this population of proliferating progenitors is composed of two cell types distinguishable on the basis of their appearance under the electron microscope. Type A has extensive endoplasmic reticulum and other features generally associated with cells synthesizing proteins for export. Type B is characterized by an abundance of free ribosomes, mitochondria, and other features similar to those found in neutrophilic leukocytes or phagocytictype cells. It has been suggested that the term preosteoblast is applicable to cells of type A, and preosteoclast has been tentatively proposed for type-B cells. Both types of cell are present in a series of transitional forms, from the most primitive found in pericapillary sites to the most highly organized found nearer bone surfaces. It was also claimed that recognizable transitional forms between A and B subtypes were not observed. Fischman and Hay (1962), from their light microscope studies, also coilcluded that the osteoclast is derived from a cell with the characteristics of a “mononuclear leukocyte,” which is in agreement with the above results.

F. THEEFFECT OF PARATHYROID HORMONE In recent work (Owen and Bingham, 1968; Bingham et al., 1969), a study has been made of the effect of parathyroid extract (PTE) on the different cells of bone. The system used was the same as that illustrated in Fig. 4, i.e., the midshaft of the femur of young rabbits. It was possible to determine the effect of the hormone on RNA synthesis in the four different cell types, osteoblasts and preosteoblasts on the periosteal surface and osteoclasts and endosteal mesendiyma1 cells on the endosteal surface. The results showed that PTE has a similar effect on each differentiated bone cell and its corresponding progenitor cells, but opposing effects on cells of the bone-resorbing and bone-forming systems. There is a stimulation of RNA synthesis in the osteoclasts and endosteal mesenchymal cells and a depression of RNA synthesis in the osteoblasts and preosteoblasts. It was also interesting to note that the effect on the osteoclasts preceded the effect on their precursors, thus suggesting that the osteoclastic activity of the bone surface influences the activity of the immediate precursors. The effect of PTE on the uptake of thyniidine-3H has also been measured (Owen and Williamson, unpublished results) , Again, the results indicate a

THE ORIGIN OF BONE CELLS

227

stimulation of thymidine uptake in the mesenchymal cells on the endosteal surface and a depression in the preosteoblasts. These results show that the proliferating progenitors of the osteoblasts and osteoclasts respond differently to PTE and support the concept that there are two classes of progenitors of bone cells in functionally different states.

G. CELL TRANSFORMATION Young (1962b, 1964) has proposed the hypothesis that the cells of bone are different functional states of the same cell and that they can revert from one state to the other depending on the cellular microenvironment. The origin of osteoblasts and osteoclasts from osteoprogenitor cells, for example, is illustrated in his diagram in Fig. 2. There is one proviso, i.e., that transformations between osteoblasts and osteoclasts go through the osteoprogenitor stage. The arguments in support of the above hypothesis are very strong (Young, 1962b, 1963, 1964, 196S), although the evidence, particularly in vivu, is mainly circumstantial. For example, in the metaphysis of very young animals exceedingly dynamic remodeling of bone occurs. The situation has been diagrammatically represented in Fig. 2, and it has been reported that complete removal of a trabecula in a 6-day-old rat metaphysis, such as is represented here, occurs in 2 days (Young, 1962b). This must involve considerable shifts in cell populations. Since no evidence of cell death has been found in this type of material and since, as can be seen from Fig. 3, there is a rapid turnover of osteoblasts and of nuclei in osteoclasts, it has been concluded that transformations from one cell type to another can readily take place. In earlier papers (Heller et dl., 1950; Kroon, 1958), it was also concluded from morphological evidence that such transformations must occur in PTE-treated bone. The results from tritiated thymidine studies in the metaphysis of young rats treated with PTE (Young, 1964, 1968) have also been cited as evidence in support of the above hypothesis. During the first 24 hours following PTE treatment in young rats, there is a great increase in osteoclastic activity and a depression in osteoblastic activity. In the second 24-hour period, pronounced recovery of osteoblastic activity and a damping down of osteoclastic activity occurs. In Young’s first experiment (Young, 1964), thymidine-3H was given just before administration of PTE; 24 hours later, labeled nuclei were found predominantIy in osteoclasts. In a second experiment, thymidine was given 12-24 hours after PTE, i.e., toward the onset of the recovery period; 36-48 hours later, labeled nuclei were found predominantly in osteoblasts. From these experiments, Young concluded that osteoprogenitor cells are capable of specializing as either osteoblasts or osteoclasts depending on the changing microenvironment of the ceIIs. Although this is a possible explanation, the results are not unequivocal. In the

228

MAUREEN O W E N

previous section, it was shown that two types of osteoprogenitor cells exist and that PTE has an opposite effect on each, and any interpretation of the effects of PTE must take this into account. Further studies are necessary to elucidate these points. Studies of the healing of bone fractures (Tonna and Cronltite, 1961) have shown that osteoblasts can be stimulated to take up tritiated thymidine and presumably to divide. Consequently, in this situation osteoblasts are shown to be capable of reverting to the osteoprogenitor state. Other studies support the concept that the cellular niicroenvironment influences cell differentiation. For example, the availability of oxygen has been shown to play an important role in tissue development z n vitro (Shaw and Bassett, 1967). At low and medium oxygen tensions, chondrogenesis and osteogenesis occur, respectively, whereas at high oxygen tension, chondroclasia and osteoclasia predominate. There is also evidence (Holtrop, 1966; Crelin and Koch, 1967) that hypertrophic chondrocytes may transform into other bone cell types after dissolution of their matrix has taken place.

H. THECOMPOSITION OF OSTEOPROGENITOR CELLS The population of proliferating cells near bone surfaces contains two main cell types which have, to a varying degree, some of the ultrastructural and functional characteristics of their final differentiated forms, osteoblasts and osteoclasts. The extent to which this population of osteoprogenitor cells contains earlier undifferentiated forms, such as a common precursor of preosteoblasts and preosteoclasts or undifferentiated mesenchymal cells which are not yet determined in an osteogenic direction, is not known. If they are present, however, it is likely that these earlier undifferentiated coniponents represent only a small proportion of the osteoprogenitor population. The data available suggest that differentiation of osteoprogenitor cells to osteoblasts and osteoclasts at any one time involves the major proportion of the osteoprogenitor population (Young, 1962b). For example, the results in Fig. 3 show a rapid entry of labeled osteoprogenitor cells into the differentiated cell compartments and a rapid attainment by the differentiated cells of the initial percentage of labeling of the osteoprogenitor population. These two facts favor the conclusion that the proliferating cells near the bone surface behave, broadly speaking, as a single population from the point of view of differentiation, and also that they are comparatively well advanced along the pathway of differentiation. There is no information on the mechanisms that promote preosteoblasts as opposed to preosteoclasts; the key to this presumably must reside in the nature of the environment near forming and resorbing bone surfaces. The question now arises as to the origin of the osteoprogenitor cell in the body. Some authors attribute this role to the endothelial cells lining the small

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capillaries and blood vessels near bone surfaces (Trueta, 1963; Mankin, 1964). Even if this is the case, it only begs the question since the nature or potential of an endothelial cell in this situation is not known. Is it, for example, equivalent to an undifferentiated mesenchymal cell with several potential pathways of development, or i s it already a cell determined in an osteogenic direction? Another possibility is that precursors of osteoprogenitor cells are present in marrow tissues. They may, for example, be members of the “determined osteogenic precursor cells” that have been found in this tissue (Section 111, B) . At the present time, however, no experiments have been performed under normal physiological conditions which demonstrate that osteoprogenitor cells are derived from undifferentiated mesenchymal cells. On the other hand, under abnormal circumstances (e.g., the formation of bone in sites outside the skeleton), the fact that undifferentiated cells can be induced to form bone has been demonstrated many times. An intriguing question concerns the significance of this for normal physiological conditions. Some of the relevant results will be considered in the next section. 111. Other Cells with Potential for Bone Formation Osteogenesis in ectopic sites, i.e., extraneous to skeletal tissue, is an example of tissue induction in an adult organism and might be expected to provide information on the nature of the cells capable of bone formation. Induced osteogenesis always occurs in a connective tissue system. This system, usually a mixture of tissues with at least a proportion of fairly mobile cells can, in addition, be invaded and replenished by cells migrating from the blood. Osteogenic induction can be made to occur under a large variety of circumsta~ices-iinplantation of dead bone tissue, live bone grafts, trauma, and injections of alcohol being a few of the methods that have been successful in producing ectopic osteogenesis (Bridges, 1959; Urist, 1965). The question that is of relevance here, however, is which types of cells can be induced to differentiate in the direction of osteogenesis. In the past, some controversy has centered around whether the cells capable of osteogenesis are cells present locally in the host tissues near the site of induction, whether they are cells brought in by the blood stream, or, in the case of live grafts, whether they may be cells contributed by the donor tissue. Morphological techniques were mainly used, and from the evidence produced it was clear that, depending on the situation, cells of either host or donor tissue or both may take part in bone formation at sites of ectopic osteogenesis (Urist and McLean, 1952; Ray and Sabet, 1963; Burwell, 1964, 1966). Recently, several new techniques have been devised which have provided some more specific information pertinent to these problems. In particular, in

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MAUREEN OWEN

vivo implanted millipore filter chambers have provided a simpler system for the study of bone induction and they have been used to investigate the source of the cells taking part. Furthermore, experiments on direct transplants between animals whose cells can be distinguished by their chromosome karyotype (chromosome marker technique) are now beginning to be performed in connection with bone. Some of the results that have been obtained with these techniques will be described in this section.

A. EXPERIMENTS WITH MILLIPORE FILTERS(CLOSEDSYSTEM) Millipore filters, HA type, with a pore diameter of 0.45 p are impermeable to cells. They were first applied successfully by Goldhaber (1961) to the problem of bone induction. He cultured bone inside small chambers bounded by filters which were implanted in vivo and demonstrated the induction of bone on the outside of the chambers. This showed two things. First, that bone tissue itself was capable of bone induction and, second, that the substance responsible was transmissible across a millipore filter even one as thick as 150 1.1. This result has since been confirmed many times (Friedenstein, 1962, 1963-1964; Post et al., 1966; Buring and Urist, 1967; Heiple et ul., 1968; Friedman et al., 1968). A particularly interesting application of this technique has been the work of Friedenstein and his colleagues (Friedenstein et ul., 1967; Friedenstein, 1968) using the transitional epithelium lining the urinary tract, which is a well-known bone inducer (Huggins, 1930, 1968). Bone is induced in a connective tissue site under the influence of a direct transplant of transitional epithelium. Bone is also induced on the outside of a millipore filter chamber containing a living culture of transitional epithelium within. Bone is induced in certain cells within a millipore filter chamber in the presence of a living culture of transitional epithelium. Friedenstein’s experiments were designed to determine which types of cells were capable of induced osteogenesis. The system he used was as follows. Cells from different tissues in the body were cultured together with transitional epithelium in a millipore chamber implanted intraperitoneally. The tissues within such a chamber are able to derive nutrient from the host tissue but at the same time are isolated from the cells of the host. This constitutes what is called a closed system. It means that the processes of differentiation that develop in the population of cells present in the chamber at the time of its implantation into the recipient can be studied without being complicated by contributions from cells of the host. The different types of cells that have so far been investigated in this system are leukocytes from peripheral blood, peritoneal fluid cells, spleen cells, and cells of subcutaneous connective tissue (Friedenstein et al., 1967). All of these

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were introduced into the chamber as cell suspensions, consequently, cellular organization of the particular tissue concerned is not an important factor. A control experiment with only transitional epithelium in the chamber was also performed. In the case of leukocytes, peritoneal fluid cells, and spleen cells, bone was regularly formed within the chamber under the influence of transitional epithelium. From these results, it was concluded that tissues of hemopoietic origin contain cells that are responsible for bone induction. In the other two cases, subcutaneous connective tissue cells in the presence of transitional epithelium and transitional epithelium alone, no bone formation within the chamber was observed in this particular system (Friedenstein et ul., 1967). This does not necessarily imply that inducible cells are absent from these tissues; it may be that they are not present in large enough numbers. Other results from a different approach to this problem (Urist et d., 1969) demonstrate that the number of inducible cells in different tissues is an important factor in bone induction. In coniparison with cells from the hemopoietic tissues mentioned above, the behavior of marrow cells in this type of system shows several differences. Most important is the fact that the presence of an inducer, such as transitional epithelium, is not necessary. When a piece of marrow or a suspension of marrow cells was placed in a millipore chamber and implanted intraperitoneally, bone was formed inside the chamber even though there was no inducing agent present. On the other hand, the density of marrow cells in the chamber was an important factor; bone was not formed below a certain critical concentration of cells (Friedenstein et ul., 1966). In contrast to these results with marrow cell suspensions, osteogenesis could not be induced in peritoneal fluid cells solely by changing the concentration of cells in the chamber; the presence of an inducer, such as transitional epithelium, was always necessary (Friedenstein et al., 1967).

B.

EXPERIMENTS WITH DIRECT TRANSPLANTS (OPENSYSTEM)

When pieces of bone marrow are transplanted directly under the renal capsule in a host, bone formation with subsequent development of hemopoietic tissue at the grafting site occurs. This situation constitutes an open system since cells of the host are able to contribute. The experiments to be described were performed with a variety of inbred mouse strains and their F, hybrids (Friedenstein et al., 1968), and the questions asked concerned whether cells from the host or donor tissue served as precursors of both the hemopoietic and osteogenic tissue arising at the site of the bone marrow transplants. One of the interesting results is that the bone formed was found to be of donor type, i.e., derived from cells of the donor marrow, whereas the centers of hemopoiesis formed in association with this bone are composed of host-type cells. The experiment that demonstrates these phenomena is described below

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( Friedenstein et al., 1968) , although an earlier experiment by Mawdsley and Harrison ( 1 9 6 3 ) gave essentially the same result. A piece of donor marrow obtained from an inbred mouse strain, CBA-T6T6, was transplanted to a site under the renal capsule of an F, hybrid recipient (the I:, being a cross between the strains A and CBA-T6T6). It is known that, whereas tissues of an inbred animal can be successfully grafted to an F, hybrid, transplantation of I;, to either of the parent types, A or CBA-T6T6, induces an immunological response since the graft possesses antigens not possessed by the host. Following transplantation of the CBA-T6T6 type marrow, healthy bone and marrow were formed in the I;, recipient at the site of the graft. When these tissues were retransplanted back into the donor type CBA-T6T6 animal the hemopoietic tissue was immunologically rejected, whereas the bone tissue was not, thus implying that cells of the bone were of donor type and that cells of the associated hemopoietic tissues were formed from the repopulating elements of the recipient. Supporting evidence came from an examination of the chromosomes of the hemopoietic tissue formed in the F, recipient which showed that these cells were indeed of F, karyotype. Another interesting feature of bone formed from such marrow transplants is that it appears to have an almost unlimited capacity for self-maintenance. Healthy bone sites were still to be found 12-14 months after transplantation of marrow under the renal capsule. This is in contrast to the fate of bone formed under the influence of an inducing agent, such as transitional epithelium, once the inducing agent has been removed. For example, an allogenic transplant of transitional epithelium results in immunological resorption of the latter after a few weeks. This is followed by rapid disappearance of the bone induced under its influence even though there is no immunological action against this bone tissue which has been formed from the host’s own cells (Friedenstein, 1965, 1968). The main conclusion that Friedenstein has drawn from his rcsults, some of which are reported above, is that there are two types of cell in the body capable of bone formation. The evidence for them is briefly summarized below under the names that have been proposed for them (Friedenstein, 1968).

Inducible Osteogenic Precursos Cells (ZOPC) . These cells have been found to be present in some hemopoietic tissues. Osteogenesis occurs only in the presence of an inducer and for it to be maintained, repeated acts of the inducing agent are required. In the experiments so far performed, a self-perpetuating line of cells capable of protracted osteogenesis after the induciiih7 drent b has been removed has not been derived from IOPC cells. Wlicther under prolonged conditions of induction or any other conditions this is possiblc is ttot yet known.

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Determined Osteogenic Preczlrsor C e l b (DOPC). Marrow tissues contain cells that form bone without an inducing agent being present. The fact that cell suspensions are as effective as intact marrow is one factor that rules out the possibility of any bone fragments being present to act as inducing agents. The bone formed is relatively long-lived, and in an open system it was still of donor tissue even at 14 months. This indicates the presence in the original marrow graft of cells that are self-perpetuating and capable of prolonged osteogenesis. In terms of the scheme proposed in Fig. 1, IOPC might be equivalent to the undifferentiated cells represented by a or b or both, and DOPC to the proliferating progenitors of bone cells represented by c. The question to be asked is whether or not transitions between IOPC and DOPC occur under normal conditions in the postfetal organism in vivo? In this context, one may consider the situation that occurs in the case of bone repair, which might be expected to reflect to an exaggerated extent the processes normally occurring iiz oivo. This situation is considered in the next paragraph in the light of the above-mentioned results. C. BONE FRACTURF RFPAIR

The healing of bone fractures has been regarded as an example of bone induction (McLean and Urist, 196S), although this interpretation is not universally accepted. In the first stage, there is hemorrhage and increased vascularization at the fracture site with extensive infiltration of cells. Next, formation of fracture callus, usually of a fibrocartilaginous or bony construction, which bridges the gap between the broken ends of the bone, takes place. This is followed by increased activity, mainly of the osteoprogenitor cells of the periosteutn, with formation of new bone on the surfaces of the fracture callus. The final result is replacement of the callus by bone, ending with a bony union of the fracture pieces. The exact origin of the cells that contribute to the different stages of fracture repair is not known for certain. It is thought that formation of the callus occurs by induction of some of the cells brought in by the increasing blood flow to the fracture site. This might be effected either by trauma or by the broken ends of the bone (both being capable of induced osteogenesis in other circumstances). Formation of new bone and completion of union of the fracture is attributed mainly to the osteoprogenitor cells from adjacent bone surfaces. Subsequently, the callus is resorbed and replaced by this new bone. The temporary nature of the callus would be consistent with its formation from IOPC, i.e., cells capable of osteogenesis in the presence of an inductive stimulus. Formation of callus ceases and the tissue is resorbed when the inductive

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environment is no longer present. On the other hand, the more permanent nature of the bone that constitutes the fracture union would be consistent with its formation by cells which have the characteristics of DOPC, i.e., cells already determined in an osteogenic direction and capable of maintained osteogenesis. This interpretation of bone fracture repair in terms of these two cellular stages is highly speculative. Some aspects of it, however, are open to testing by experiment. Recently, by using the technique of chromosome markers, the origin of the fibroblasts that take part in the inflammation process occurring during skin healing (Barnes and Khrushchov, 1968) has been shown to be blood-borne components from marrow tissues. A similar experiment could be performed to determine the origin of the cells involved in callus formation during bone fracture healing. At the present time, however, there is no evidence to suggest that transitions between the undifferentiated cells and the more differentiated osteoprogenitor cells are part of the fracture-healing process.

IV. Concluding Remarks Certain hemopoietic tissues (peritoneal fluid cells and spleen, and blood leukocytes) contain cells capable of forming induced bone when exposed ro a suitable environment. The same tissues have also been shown to contain hemopoietic stem cells (Barnes and Loutit, 1967; Loutit, 1967). The question immediately arises: Are these hemopoietic stem cells the same cells as the inducible osteogenic precursor cells? In terms of the scheme shown in Fig. 1 are they both members of a pool of multipotential connective tissue stem cells (a)? Alternatively, are they two separate groups of cells each of which has already taken a step in the direction of differentiation of the respective tissxes concerned and in this case could be represented by b in Fig. 1 ? Whether a and b both exist as separate entities has not been resolved. What is certain is the widespread presence, in the circulating blood, hemopoietic tissues, and presumably throughout the vascular channels of connective tissue systems, of undifferentiated mesenchymal cells that can be induced to form either bone or blood. The very ubiquity of their distribution might favor the possibility that they are a pool of true multipotential cells. The bone formed from these undifferentiated mesenchynial cells is of a ternporary nature; repeated acts of inductive activity are required for osteogeiiesis to be maintained, and if the inducing agent is removed the boiie disaiipears. There is no evidence from any source that a self-maintained bone tissue system can be obtained from these cells. Marrow tissues also contain cells capable of bone induction. In this case, tlie bone formed is of a more permanent nature. A self-perpetuating population of

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bone cells capable of relatively unlimited osteogenesis can be derived from these cells, and the presence of an inducing agent is not necessary. It has been suggested, therefore, that these cells are already predetermined along the pathway toward osteogenesis (DOPC). No morphological or ultrastructural studies have been performed on them. The only information available about them is that they will form bone unassisted under favorable conditions and that since cell density is a crucial factor interactions between the cells may therefore be important. The extent to which they are determined in an osteogenic direction is not known. It is possible that they encompass a wide range of the stages of differentiation including both types of osteoprogenitor cell and less diff erentiated precursors of osteoprogenitor cells, and they have, therefore, been equated with the wider term proliferating progenitors c in Fig. 1. In summary, there are two types of cell in the body capable of induced osteogenesis. Our knowledge of them is very meager and can be summed up as follows. There are undifferentiated inesenchymal cells with widespread distribution and there are cells found in marrow tissues which are already predetermined in an osteogenic direction. The evidence for this has been illustrated from examples of the recent work of Friedenstein (Friedenstein et al., 1967; Friedenstein, 1968), although the results from earlier work give pointers in the same direction. Urist and McLean (1952), for example, distinguished between cells that may be “induced” to form bone and cells with inherent “osteogenetic” activity. The superiority of marrow tissues (which contain DOPC) for the purpose of bone grafting has also been realized for a long time (Burwell, 1964). What is still an open question, however, is the relation between these two cell types from the point of view of osteogenesis under normal physiological conditions iiz vivo. One can speculate on the kinetics of osteogenic cells in the body. The only certain feature of this is that replacement of the osteoblasts and osteoclasts, which takes place throughout life, occurs almost entirely by multiplication of the osteoprogenitor cells which are already quite well differentiated in the direction of osteogenesis. Supplementation of this population could take place from the less differentiated cells of the proliferating progenitors and these in turn might be replaced by cells from the ubiquitous compartment of undifferentiated mesenchymal cells. Another possibility is that the compartment of proliferating progenitor cells may be full-sized shortly after birth and the need for replenishment from the undifferentiated mesenchymal cells is zero. The purpose of these latter cells might then be to fill a temporary need for regeneration of osteogenic tissue when necessary, e.g., in the case of bone fracture healing. In the present state of knowledge, it is not possible to decide which of these two possibilities is the more likely. A difficulty is that our techniques for studying the function of undifferentiated incsenchymal cells in vivo are very limited.

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There is no evidence at present, however, for the transformation of iindifferentiated inesenchymal cells into precursors of bone cells and eventually into the mature cells of bone in the postfetal organism in v h o in normal circumstances. In the embryo, one must assume that these transformations do take place. If this no longer occurs in the postfetal organism, it represents an important new concept for osteogenesis in postnatal life.

ACKNOWLEDGMENT I am indebted to Dr. R. W. Young who has allowed me to use his data (Young, 1962b) in preparing the graphs in Fig. 3.

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