proliferation, differentiation and ... -...

P R O L I F E R A T I O S , D I F F E R E N T I B T I O X AND DE- GENERATION I N THE SPl"4L G A N G L I S

O F THE CHICK EMBRYO UNDER XORNAL ASD E X P E R I N E X -

T A L C O S D I T I O S S

VIKTOR HAMBURGER AKD RITA T,EVI-&fO?;TALCIKI TTaslitngtor~ O?ix t i s i ty

NINE FIGL-RES

I. 1NTROI)UCTIOS

The present iiivestigatioii makes ail attempt to integrate observations on the iiormal development of the spinal ganglia with experimental results. The normal developmental process is the ultimate frame of reference for experimental analysis, and the latter should be considered primarily as an effort to elucidate those aspects of iiormal development which a re iiot readily observable. From the combination of direct observation, on all levels, and of causal analysis will emerge a more adequate description of embryological phenoniena than from experimental studies alone.

At present, some aspects of nerve center development a re strongly emphasized and others are neglected. Special atten- tion has been paid to the niodificatioiis which are brought about in the developnieiit of primary nerve centers by changes occurring in the peripheral areas which they innervate. A reduction of the peripheral fields usually results in a size reduction, and a n enlargenieiit results in an overgrowth of the nerve centers, The extensive literature on this topic has been reviewed recently by Piatt ('48).

Supported by n grant f rom tlie Rockefeller Foundation.

4.57

458 VIKTOR HAMBURGER 9 9 D RITA LEVI-MOXTALCINI

The effects on the nerve centers are usually referred to as “hypo-” and “hyperplasia,” respectively. I t has become apparent in recent years that these terms, far from being well defined, cover a number of heterogeneous phenomena.

Our effort in the present study was directed towards a clarification of these terms. Misconceptions derive from an altogether too static approach to these phenomena. Cell counts or volume determinations made at the end of an arbi- trarily chosen observation period give no clues as to the mechanisms which operate to produce them. Yet, it is of great interest to investigate to what extent the observed differences are due to modifications of proliferation, of neuroblast differentiation, of cell growth, and of secondary disappearance of cells. I n other words, hypo- and hyperplasia should not be defined in terms of static end effects, but in termd of modifications of developmental processes. Conse- quently, an analysis of these phenomena should fulfill two major requirements : One, each component in the development of a nerve center should be analyzed separately, and two, the observations should cover as many stages, or phases, of the developmental process as possible, and not merely one terminal stage.

It seemed desirable to re-investigate one of the well-established instances of hypo- and hyperplasia, along these lines. There are only a few nerve centers which lend themselves to an exhaustive analysis. The spinal ganglia of the chick were chosen as particularly favorable objects : they are well cir- cumscribed units which contain within themselves all pro- liferating and differentiating cell elements. Futhermore, regional differences in size exist between different ganglia (cervical, brachial, ctc.) . Finally, considerable preliminary ~7ork has been done on their normal and experimentally modified development. A large material of normal embryos and of limb extirpation and transplantation cases, covering the entire embryonic period was at our disposal. The material was partly silver-impregnated and partly stained in Hema-

DIFFEREXTIATION OF SPIXAL GASGLIA 459

toxylin. Both techniques have to be used if all aspects of nerve tissue development are to be considered.

Our new observatioiis do not give an exhaustive picture. However, they demonstrate the importance of two components which had not been analyzed before, naniely proliferation and degeneration, and they bring into focus the complex inter- play of different components.

We wish to express our appreciation of the very able and competent assistance of Miss Thelma Dunnebacke.

11. MATERIAL AND METHODS

For the study of normal ganglion development, a rather complete series of embryos ranging from two and one-half days to hatching was used.

The analysis of hypoplasia is based on a large number of extirpations of the right wing or leg bud, respecively. All operations were done on embryos of two and one-half to three days of incubation. For the details of the technique see Ham- burger ( '42). The embryos were fixed in a series, ranging from three to 20 days of incubation.

For the study of hyperplasia, approximately 25 cases of wing or leg transplantation were used. The transplantations were performed on two and one-half- to three-day embryos, following the technique described previously (Hamburger, '42). Right wing or leg primordia were transplanted to the right flank of host embryos. They were placed between the wing and leg buds of the host and usually as near to the host wing bud and as closely to the somites as possible to insure an adequate innervation. One series was fixed between the 5th and 8th day of incubation, f o r the purpose of mitotic counts, and another series between 9 and 17 days, f o r the purpose of cell counts.

All embryos to be used for mitotic and cell counts were fixed in Bouin and stained in Heidenhain's Hematoxylin. The embryos to be used for studies of neuroblast differentiation and fiber outgrowth were impregnated with silver, following

460 VIKTOB HAXBURGER AND RITA LEVI-~LIONTALCINI

DeCastro's niodificatioii of Cajal's niethod (for details sec Levi-Montalcini, '49). The younger embryos were sectioned at 8 p, and the older ones at 10 p-14 p.

Following wing extirpation, the brachial ganglia 14 to 16 became hypoplastic; they supplied the major part of the material of this study. In addition, we used hypoplastic lumbosacral ganglia obtained by leg extirpation. For most of the observations on the latter material, ganglion 25, which had been used in a previous study, was chosen (Levi-Mon- talcini and Levi, '43, '44).

The experimental production of hyperplastic ganglia is difficult, fo r several reasons. In tlie case of limb transplantations, it is not possible to control the innervation pattern of the grafted limb. The innervation of transplants is variable even if they are placed in the same position. The different innervation patterns obtained in flank grafts are described and illustrated in Hamburger ( '39a), and additional data are given in Hamburger ('39b). Furthermore, there seein to be intrinsic limitations to a hyperplasia eve11 if a transplant is well innervated. In general, it was found that thoracic ganglia become less markedly hyperplastic, following peripheral overloading than do brachial ganglia. Hence, the transplants were placed as closelv to the host wing as possible, in order to obtain innervation from ganglia 15 and 16. Graphic reconstructions were made of the spinal cord region, ganglia and plexuses involved in the innervation of the transplants, following a method described before (Hamburger, '34). Those ganglia which appeared to be distinctly hyperplastic in the reconstructions were chosen for a special study of hyperplasia. In most instances we used ganglia 16, 17, 18 in varying com- binations. I n a few instances, ganglia 15 01' 19 participated in the plexus of tlie ti*aiisplant and were included in the study.

111. NORMAL DEVELOPMEST

The earliest phases of the development of spinal ganglia, up to two and one-half days, will not be considered in the present investigation. They include : the formation of the

D I F F E R E N T I A T I O N O F S P I N A L GANGLIA 461

neural crest, the migration of the prospective ganglion cells to their final positions, and the segregation of the neural crest derivatives into clusters of cells which will form tlie ganglia.

A. Difere9itintioiz.

A detailed account of the differentiation of spinal ganglia from two and one-half days to hatching was given by Levi- Montalcini and Levi ( '43). W e shall give a brief summary of their results to the extent that they are important for the present investigation. All their observations were made 011

the 25th ganglion which is one of the lumbosacral plexus ganglia.

These authors subdivide the process of spinal ganglion developnient into three periods.

The first period extends approximately to the 8th day. Three important events fall within this period : the process of proliferation, selective degeneration in certain ganglia and the differentiation of one particular group of cells, tlie large ventro-lateral neurons. The first two phenomena will be taken u p in special chapters. The differentiation of the large neuron9 was described as follows: The first neuroblasts begin l o differentiate a t two and one-half days of incubation. At three and one-half days, their number is still small. They i i r ~ bipolar and their proximal nenrites have not yet penetrated into the spinal cord. During the following day, the number of the bipolar cells increases. At 4 and 5 days, the ontprowth of both proximal and distal neiirites is fully nnder way. The dorsal root is established, and a t 5 days, the t ins of the fibers have reached the dermis. (See also Visintini and Levi-Montalcini, '39).

From the beginnin?, the early differentiating cells are concentrated in the ventro-lateral part of the ganglia. At 3 days, a number of these bipolar cells may be found scattered in other parts of the ganglia and mingled with the smaller nndifferentiated cells. How- r ~ e r , it seems that during Ihe following days these scattered bipolar cells migrate to more ventral and lateral positions, because a t 7 and 8 days, the differentiated cells are almo5t completely segregated from the nnclifferentiated, small cells. At the same time, all bipolar neuroblasts increase in size. At the end of the first period (8 days of incubation) the picture is as follows : Two .;..parate. homogeneoiis groups are distinguishablr ; the large differentiated neuroblasts are assembled in the ventro-lateral region of tlie ganglion where they form a cup-shaped strnctnre which appcars siclile-shaped a t the

462 VIKTOR HAMBCRGER AND RITA LEVI-MONTALCINI

cross section. The inner part of the cup, and the mediodorsal region, are occupied by much smaller cells which have not begun to differentiate (fig. 5) .

The second period begins at 8 days and ends approximately at 12 days ; however, the demarcation between this and the third period is not sharp. This phase represents a relatively static condition, in distinction to the preceding and the following periods. The large differentiated cells grow in size and assume gradually their definite pseudo-unipolar form. The small cells grow likewise in size and ac- quire certain properties which characterize them as neuroblasts. Nissl substance is demonstrable in a diffuse distribution, and the nuclei are relatively large and vesicular and contain two nucleoli. On the basis of these features the small cells are no longer considered as indifferent cells but as neuroblasts although they do not show an affinity to silver as yet. One may object against the designation of cells lacking neurofibrils as neuroblasts, or one may be inclined to consider the silver technique as deceptive in this case. However, the subsequent history of these cells lends support to this interpretation. Beginning at 9 to 10 days, a change in the coloration of the silver impregnated cytoplasm from yellow to dark brown can be observed in a number of cells, and this color change is always accompanied by the outgrowth of neurofibrils. From 12 days on, an increasing number of small cells show this differentiation process, and a t the same time a considerable enlargement of the dorsal root has been observed. For further details see Levi-Montalcini and Levi ( '43).

The third period, approximately from 12 days on, is characterized by the progressive cytodifferentiation and neurofibril formation of the smaller cells accompanied by a considerable increase in the size of their caryoplasm. Measurements of cell diameters which were made at 8, 9, 12, 15 and 19 days (op. cit., fig. 5, p. 201) have shown that both the originally smaller cells and the larger cells grow con- spicuously up t o 15 days. The distribution curves retain two distinct peaks which shift gradually towards the larger size clames. From the 15th day t o hatching, it is no longer possible to recognize two distinctly separate populations. There is a great variation in sizes, and some of the originally smaller cells reach the size of the cells of the ventro-lateral group. However, an accumulation of large cells a t the ventro-lateral border is still recognizable after hatching.

The observations of Levi-Rfontalcini and Levi ( '43) which were made on the 25th ganglion, can be readily confirmed on other ganglia. I n particular, the brachial ganglia which were the main object of the present study show a similar developmental pattern.

.

DIFFEREXTIATIOX O F SPIXAL GhNGLIA 463

B. Data o n physiological act iv i ty It has been found in previous iiivestigations that at the end

of 6 days chick embryos respond to tactile stimulations o f the skin (mechanical or electrical) by diffuse contractions. The first proprioceptive responses (reflectory stretching of limbs after mechanical bonding) were detected at 11 days (Visintini and Levi-Montalcini, '39). These observations can be correlated with the time pattern of differentiation of large and small cells. At 6 days, the large, rapidly differentiating cells are the only ones which are connected with the periphery. Their axons have reached the skin at that stage. At this period, the small cells are undifferentiated. There can be no doubt but that the exteroceptive tactile response is mediated by the early differentiating large neurons of the ventrolateral part of the ganglion. However, we do not contend that all large cells serve exclusively this function. We have observed connections between spinal ganglia and sympathetic ganglia (rami communicantes) as early as 5 days, particularly in the thoracic level. Tliese fibers must derive from large cells, since no others are differentiated at that time. This would imply that a fraction of the large, early differentiating cells are visceral sensory neurons.

The time of the first proprioceptive responses coincides with the beginning of neurofibrillar differentiation in the small cells and with the beginning of the differentiation o f muscle spindles in the limb muscles (Tello, ,22). This suggests that at least part of the late differentiating cells are proprioceptive neurons. However, we do not contend that this group is functionally homogeneous. I n fact, after 15 days of incubation this group of originally uniformly small cells gives origin to cells of very different sizes. At hatching, some of them have reached the size of the early differentiating ventrolateral neurons, whereas others have remained small, and all inter- mediate sizes are represented. By analogy with the situation in Slammals, one might expect that those cells which grow extensively and are eventually among the largest ganglionic neurons are the proprioceptive cells, and those which remain

464 VIICTOR HAMBURGER AXD RITA LEVI-MONTALCINI

relatively sniall would mediate exteroceptive sensations of heat, cold arid pain. Oiily the tactile exteroceptive neurons would be derived from the early differentiating large cells, if this interpretation is correct.

C. Prolif e m t i o n

The data in the literature concerning proliferatioii are very scanty. No details are reported, except for the statement that proliferation ends on the 7th day (Olivo, Porta and Barberis, ’32). It was, therefore, necessary to study the niitotic pattern in iiormal ganglia by counts in different stages. Embryos fixed between three to 8 days and stained with Heidenhain’s Heinatoxylin were used for the mitotic counts. The counts were made on each section of a given ganglion. Only meta- phases aiid anapliases were couritecl using a pole counter. It is not feasible to calculate mitotic iiidices because during the peak of mitotic activity a considerable number of cells begin to differentiate and lose their proliferative capacity. There- fore, the ratio of mitoses to all ganglion cells would not be a valid iiidex; and tlie state of differeiitiatioii of a cell could be deterniined oiily froni silver impregnations which, in turn, do not perniit mitotic counts. Therefore, we present in table 1 the absolute figures. They represent “initotic activity” which was defined as “the number of mitotic figures a t a given stage and region,” (Hamburger, ’48, p. 224). The absolute figures perniit a comparison of cliff erent ganglia, ancl they reflect general trends aiid patterns. All data for iiormal ganglia a re presented in columns 5, 11, 17 aiid 18 of table 1. The “coiitrol” ganglia (coluniiis 17 ancl 18) are partly ganglia of iiornial embryos (those with designation “ii ”) and partly ganglia of wing extirpation (“ S”) and wing transplantation (“ t r ”) cahes, which are located in normal, unaffected regions of the operated embryos. I n addition, the partners of hypoplastic and hpperplastic ganglia on tlie left, aiioperated, side of limb extirpation and traiisplaiitation cases were used a s iiornial ganglia (columns 3 and 11) since an effect of a unila- teral opemtion 011 tlie ganglia of the other side is unlikely.

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465

466 VIKTOIX HAMBURGER AKD RITA LEVI-MOSTALCIXI

Time pattern. Mitoses are already present in the neural crest. Our data for the third and 4th days are incomplete. But a comparison of the mitotic activity during the third and 4th days with that of the 5th and 6th days in the same ganglion leaves little doubt but that the mitotic activiy is rising during this period. There is a definite decline on the 7th day. The two ganglia of an 8-day embryo still showed considerable numbers of mitoses, but none were found in several ganglia of a 9-day embryo. I t is evident that the peak of mitotic activity is during the 5th and 6th days and that proliferation is practically completed at 9 days of incubation. Our data do not cover the lumbosacral region, and it is possible that the peak as well as the termination of mitotic activity occur at slightly later stages in the caudal parts of the embryo. The time pattern corresponds rather closely to that in the dorsal (sensory) part of the spinal cord (Hamburger, '48, fig. 5, p.

R e g i o m l pattern. It is possible that during the initial phase of spinal ganglion differentiation a cephalo-caudal gradient of mitotic activity exists. We have no data on this point, but from 5 days on, no such gradient is detectable. From this stage on, the limb innervating brachial (14-16) and lumbosacral ganglia (23-30) are already larger than the cervical, thoracic and posterior sacral ganglia. It is not known whether these regional differences originate when the neural crest is being segregated segmentally into clusters of cells, or whether all ganglia are initially of approximately the same size and the differences become apparent later., as the result of differential mitotic activity. The posterior sacral ganglia are probably from the beginning smaller than the others.

I n table 2, all pertinent data of table 1, for 5- and 6-day embryos, are rearranged according to the cephalo-caudal sequence of the ganglia. Although the material is limited it shows that the figures f o r the brachial level arc on the average, higher than those for cervieal and thoracic levels. The averages f o r cervical ganglia 11 to 13 are 84, for both the 5th and the 6th days. The averages f o r the brachial ganglia 14 to 16

250).

D I F F E R E X T I A T I O N O F S P I N A L GAXGLIA 467

for the same two days are 144 and 147, respectively. These data show that the mitotic activity remains constant during the two-day peak period and that the considerable regional size differences which we find at the end of development are due, in part, to differences in mitotic activity in different ganglia. This does not imply that the mitotic index (that is the ratio of dividing cells to cells which are potentially capable of dividing) is different in different ganglia. Once differences

TABLE 2

X i t o t i c actacrly of riomnul punglia

-~ 5 DAYS 6 DAYS 0 INGT,.

NO. Number of mitoses Are Sutnher of mitoses Are

11 100,99; 71, 68 8.5 85 85 12 66,66; 101,104 84 13 86,82 84

15 183,175; 164; 129 163 136,113, 141 137 16 150,134; 147,188; 114,122 144 196,201,157, 104,139,139; l i 8 1.59 17 172 172 109,104 107 18 118,114 ; 1 3 i ; 103 118

20 75,715; 112 ,120 96 130,139 135 2 1 148,145 143 22 120 120 2.3 172 172

14 114,114; 156; 123 127 123;117 120

19 112 112 120,117; 7 i 105

Averages : 11-1 3 14-16 17-21

84 144 119

84 147 115

in cell numbers are established, the numbers of mitotic figures will be larger in larger ganglia, even if the mitotic index should remain unchanged. As was mentioned above, this point cannot be clarified with the present material.

D. Cellular degeizeratioii

In the course of our study of Heidenhain preparations, we found in the cervical and thoracic ganglia certain cell types which stain deeply in Heidenhain’s Hematosylin but have no

468 VIIiTOB HBMBCRGER A N D RITA L E V I - M O X T A L C I N I

affinity to silver and are neither neurom nor iiidiff erent cells. Their morphological characteristics vary considerably. I n most instances, they are spherical in sliape and consist of a large, deeply stained spherical central par t which is sur- rounded by a thin hyaline surface layer. I n other instances, one observes sniall deeply stained particles in groups of three or more, wltliout a distinct cellular boundary. The latter structures have the appearance of cells in the process of breakdown (figs. 4,7).

Similar structures have been observed before, in embryonic nerve tissue and in other embryonic organs of Vertebrates (Collin, ’06 ; Eriist, ’26 ; Glucksmann, ’30 ; Chang, ’40 ; a.0.). They are usually referred to a s “degenerating cells,” arid they have been given very different interpretations. Exactly the same type of cells a s was found in spinal ganglia was detected in large numbers in tlie lateral motor horn of the cervical region of tlie spinal cord of 4-day embryos, arid also in the white iiiatter lateral to the motor horiis a t the same stages.

The last-mentioned observation that these structures are not limited to nerve cell groups, and the spherical sliape of many of them suggested that not all of them might be nerve cells in the process of breakdown, but that at least the spherical cells niiglit be macrophages. In order to test this assumption, the teclinique of vital staining with trypaii blue was applied u;41icli is considered to be fairly specific for macrophages. Embryos of 4 and 3 clays of incubation were exposed by sawing a window in the shell above the embryo, whose position was determined by candling. A few drops of trvpaii blue solution of 1: 20,000 were injected into the amniotic cavity, using a syringe with a fine needle. The amniotic fluid took immediately a deep blue stain. The window was the11 sealed and tlie embryo incubated for one to two hours. After this period, the amniotic fluid appeared in a much lighter color, and the entire embryo had absorbed some of the stain. The injection w a s repeated 5 or 6 times, at two-hour intervals. After about 1 2 hour?, most of the embryos showed sigiis of impaired

DIFFEl<ESTIATION O F SPIKAL GAKGLIA 463

vitality. They were stained deeply a t that time and were fixed according to Williams ’ modification of the technique of Lav- dowEky (Williams and Frantz, ’48). They were dehydrated in dioxane and sectioned a t 5 to 8 p. No countersta’in was applied in these series. The skin arid mesenchyme showed a weak blue stain wliich was barely perceptible in the sections. On the other hand, the mesonephric tubules, the floor plate of the ependyme of the spinal cord and numerous cells in the ganglia and the spinal cord stood out as distinctly blue structures. The latter cells were found in exactly the same locations as the previously described so-called “degenerative” cells. It seems, then, that a t least some of the latter are actually macrophdd c oes.

It is known that not only macrophages but also secretory cells (for instance the above-mentioned mesonephric tubules) and impaired cells accept the trypan blue. Williams and his co-workers have developed special techniques of counter staining which permit a distinction between these different types of cells, (Williams and Frantz, ’48). llTe were particularly interested in distinguishing between macrophages and impaired cells and have applied the appropriate couiiterstains to a number of trypan blue stained embryos. These preparations indicate that a number of the vital stained cells in the nervous system are macrophages and others are degenerating cells. These results were confirmed by supra-vital staining with neutral red. Ganglia were dissected from living 5- and 6- day embryos, placed immediately into a very dilute solution of neutral red (two drops of 1% solution in 25 em3 of 0.9 NaCl), incubated for 5 minutes and inspected under tlie microscope. Living macrophages with undulating membranes as well as cellular debris were easily recognized by their pink color. They were found only iii normal cervical arid thoracic ganglia and in lumhosacral ganglia of limb extirpation cases, but not in normal brachial and lumbosacral ganglia.2 It is of no importance for tlie present discussion to decide how many of

* W e wish t o express our appreciation of the expert advice of Drs. M. and S. Chbvremont, Unirersity of LiBge, Belgium, who assisted us in this experiment.

470 VIKTOR HAMBURGER AND RITA LEVI-MOKTALCINI

the “degenerating” cells are actually macrophages. The presence of macrophages In itself is evidence fo r the widespread occurrence of cellular breakdown in the nervous system. For the sake of simplicity we shall refer to the cells under consideration as “degenerating cells.”

The distribution of the “degenerating cells” is not at random, but it follows definite patterns. The degenerating cells are limited to certain developmental stages, and there exist striking regional differences in their frequency.

( a ) The t ime pattern. We have found, in agreement with previous investigators (Ernst, ’26 ; a.o.), that the cellular breakdown in the spinal ganglia is limited to certain periods. At 49 days, few “degenerating cells” are present. The peak of cytolysis occurs at 5 and 6 days. This period is followed by a rapid decrease in their numbers, and at the end of the 7th day, they have practically disappeared. This holds at least for the regions up to the lumbosacral level. We have only a few observations on the posterior sacral levels in which degeneration may perhaps continue at later stages.

( b ) Regional pattern. It is difficult to obtain precise quantitative data on the number of “degenerating cells” in different ganglia. Moreover, such data would be of little value, as long as the identity of the individual cells is in doubt, and there is no indication of a constant ratio between actually degenerating nerve cells and macrophages. We have, therefore, contented ourselves with estimates which permit a comparison of the frequencies in different ganglia. The data were obtained in the following way: Outlines were made of each cross section of a given ganglion, and the approximate positions of “degenerating” cells were marked without actually counting them. The overall estimates for different ganglia were grouped in 4 classes, the highest frequency being indicated by 4-+++. The cervical, brachial and thoracic ganglia were examined in over 20 embryos, but the data on the lumbosacral and sacral ganglia were based on a smaller sample. The data for the same ganglia of different embryos were consistent in a high degree. Table 3 which summarizes our data confirms the

D I F F E R E N T I A T I O N O F S P I N A L GANGLIA 471

statements made above concerning the time pattern. It shows, in addition, that the degenerative process seems to proceed in a cephalo-caudal progression ; in the posterior levels, “degenerating” cells were not found in appreciable numbers previous to the 6th day.

TABLE 3 - Put fern of cell degeneratiolz in spinal ganglza of chick embryos

GAXQLIA 5n 1 NO. 41 DAYS

5n 10 5 DAY 4

1 2 3 4

6 7 8 9

10 11 12

7

1 5 16 17 18 19 20 2 2 22

25 26

29 30 31 32 33 34 35

~

br. = brachial ganglia. 1.5. = lumbosacral ganglia.

++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ -

- ++ +++ +++ +++ +++ +++ +++ +++ + - __ - -- __ -

__ -

- _- -

5n 11 6 DAYS

++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ + ++ +++ +++ +++ +++ +++ +++ +++ + + + ++ ++ ++ ++ ++ ++ + + +

-

6n 12 7 WAYS

+ + + + + ++ ++ ++ -t + + + + + + + + + + + + + + + + + + + + + + + + +

-

472 VIKTOR HAMBURGER AND IUTA LEVI-MOXTALCINI

The data give evidence of a definite regional pattern. The large brachial gaiiglia 14 to 16 are either free of degeneration, or they show limited numbers of “degenerating” cells. The same holds for the main ganglia of the lumbosacral plexus (24 to 29). In coiitrast, the cervical arid thoracic ganglia show a very high degree of cellular breakdown. These fiiidings do not confirm the data of Eriist (’26) w7l10 contended that the degenerative process is limited to the brachial and lumbosacral ganglia.

( c ) Topographic distributioqa of “degenerat ing cells.” Tlie “degenerating cells ” are riot scattered a t random within a ganglion, but they are localized in the ventrolateral parts which are occupied by the large and early differentiating bipolar neuroblasts (see fig. 4) . It will be remembered that these cells are the first ones to differentiate, beginning a t two and one-half days, and that the outgrowth of their ncurites to the periphery is fully under way at 4 aiid 5 days of incubation. Furthermore, it was stated tliat the segregation of these cells from the snialler cells which differentiate later takes place during the 4th and 5th days, and that a migration of at least some large cells from a more median to ventrolateral positions is probably instrumeiital in a complete separation of the two cell groups. Accordingly, the “degenerating cells ” are more distinctly localized ventrolatcrally in 6- and 7-day embryos than in earlier embryos. Another observation supports our contention that differentiated neurons are affected by degeii- eration. I f one compares the cell density in a brachial with that of a cervical (or thoracic) ganglion, it is found that the large, ventrolatcral neuroblasts are densely packed in the former but loosely arranged in the latter, with “degenerating cells” occupyiiig the spaces between the neuroblasts. On the other hand, the packing of the small, niedio-dorsal cells is the same in all ganglia.

Our observatioiis on the lumbosacral and sacral ganglia are less extensive than those for the anterior regions. How- ever, the observations 011 6-day embryos are indicative tliat the situation in the leg-innervating ganglia is similar to that

DIFFERENTIATION O F SPINAL GANGLIA 473

in the brachial ganglia. The low frequency of “degenerating cells” in the posterior sacral and tail ganglia may be due to the fact that these ganglia are probably very small from their inception.

The differential cell degeneration process operates in the same direction as the differential mitotic activity and is an additional factor in establishing the size differences as they are found in the adult.

IV. EXPERIMENTAL PART

A. Proliferation Our findings concerning the mitotic pattern in normal gan-

glia (p. 464) have shown that the peak of mitotic activity is reached a t 5 and 6 days of incubation. This period was, therefore, chosen for the study of mitotic activity in hypo- and hyperplastic ganglia. A few additional data were obtained f o r three-, 4-, 7- and 8-day embryos. Altogether 33 ganglia, be- longing to 18 embryos were used f o r mitotic counts, The method of counting was described on page 464. All data are presented on table 1, page 465.

The figures f o r 4 ganglia of 4-day emhryos with wing extirpation (that is one day after operation) indicate the beginning of a reduction of mitotic activity on the operated side; although the data are too few and not statistically significant, they are suggestive in conjunction with the data fo r the older embryos.

The 5-day stage is represented by 6 ganglia from extirpation cases and by 7 overloaded ganglia from transplantation cases. All ganglia of the former group show a reduction of mitotic activity, ranging from 11.8% to 3776, and all ganglia of the latter group show an increase in mitotic activity ranging from 2% to 26%. The differences in the former group are statistically significant f o r all cases but one; those in the latter group are statistically significant in only one case, using the x2 test as a criterion.

The &day embryos give a very similar picture : All ganglia supplying a reduced peripheral field sliow a reduction and the

474 VIKTOR HAMBURGER AKD RITA LEVI-MOKTALCINI

two overloaded ganglia show an increase in mitotic activity. The latter is not statistically significant, but in the former group, 5 of 8 cases show a significant reduction.

Of the 7-day stage, three overloaded ganglia were counted, and of the 8-day stage, two ganglia from extirpations were used. All 5 show statistically significant differences which are consistent with the preceding stages.

If the “pooled x 2 values” are calculated for all ganglia taken together, it is found that the differences in both groups are highly significant; the x2 value f o r extirpation cases is 72.4 with an average reduction of mitotic figures of 22.8%; the xz value for overloaded ganglia is 14.92 with an average increase of mitotic figures amounting to 14.5%.

We conclude from these data that the peripheral field controls the mitotic activity of the spinal ganglia ; its reduction decreases the number of mitotic figures in ganglia which par- ticipate in its innervation, and its enlargement increases it.

Additional though indirect evidence for the peripheral control of mitotic activity was obtained from cell counts of overloaded ganglia in older embryos. The counts were made on 9 ganglia of 5 embryos ranging from 9 to 17 days of incubation, that is, after the termination of mitotic activity. The two younger embryos (tr379 and tr510) were stained in Heidenhain’s Heniatoxylin, and the three older ones were impregnated with silver. All embryos were sectioned at 10 p, except for 48tr119 which was sectioned 14 p. All nuclei showing a nucleolus were counted on each section. The exclusion of nuclei without nucleoli practically eliminates the chance of counting the same cell twice. The counts were made with the help of a grid engraved on a circular coverglass, which was inserted in the ocular; the nuclei were recorded with a pole counter. All data are given on table 4. (The differential counts of large and small cells presented in the same table will be discussed in the next chapter.) The last two columns are pertinent for the present discussion. It is obvious that all overloaded ganglia, with one exception, have higher cell numbers than their controls on the unoperated side; the per-

DIFFENEXTIATIOE O F SPINAL GASGLIA 475

( s 0 0 r i c 1 3 ( 3 o r i Q , 0.1 01 a 0 1 u3 00 c CD L- o a 0.1 d cn 1. L2 m. 0.1 0 0. N bi G 13 U bi 0.1 0.1 ri 0.1 01 hl l-l ri ri ri

00. m m. t- v: w ri 7 + ' +

k I? o m o o C c L - M b + + + +

476 VIKTOR HAMBURGER AKD RITA LEVI-MONTA4LCINI

centage increases range from 8.7% to 20.8%. As was mentioned above, the innervation of transplanted limbs is difficult t o control by the experiniental procedure; each nerve has a clifferent peripheral distribution in o r near the transplant; hence, a considerable variation in the degree of hyperplasia is to be expected. The liypoplasia of ganglion 16 of 48tr79 is not inconsistent with the other results, as will be shown on page 4'79.

There are only two ways by which a numerical hyperplasia of one side, as compared to the other side, can be accounted for: by an increase in the proliferation on the hyperplastic side, or by a differential cell degeneration on the control side. The latter alternative is excluded by our observations reported above, according to m4iich little or no cell degeneration occurs in normal brachial ganglia. Hence, an increase in mitotic activity under tlie influence of peripheral factors must he postulated.

B. haitial differemtiation The process of differentiation in normal ganglia was de-

scribed in the first part. It was shown that one group of cells differentiates early and rapidly between two and a half and 6 days ; the cells of this group establish connections with the dermis early, grow rapidly in size and are aggregated at the lateral and ventrolateral part of the ganglion. Another group of cells begins to differentiate later, from 10 days on, and proceeds to differentiate and to grow at a much slower rate. These smaller cells occupy the median and medio-dorsal regions of the ganglia.

The question arises whether the processes of cellular differentiation are controlled by peripheral factors. It is advis- able to distinguish between the iiaitinl diff ereiztintioiz, that is the first steps in the visible transformation of indifferent cells into neuroblasts, and the following phases, including the growth of the pericaryon and neurite. The second part of the process will be discussed in the following chapter. At this point we inquire: Is tlie differentiation of a certain number

DIFFEREXTIATIOX O F SPINAL GAKGLIA 477

of undifferentiated cells blocked by a reduction of the peripli- era1 area, and is differentiation of indifferent cells stimulated by an increase in the peripheral area?

The cases of hypoplasia cannot give conclusive data on this point, for the following reason. Only the rapidly differentiating ventrolateral cells would lend themselves for observations concerning this problem. Evidence for an inhibitory effect of limb extirpation on the differentiation process of these cells could be obtained only if one were able to show that at a given stage, for instance at 8 or 10 days, the number of large, differentiated cells were deficient on the operated side but that this deficiency was compensated for by an increased number of small, undifferentiated cells, the sum total of cells being equal on both sides. Only in this way could one clemonstrate that a given number of undifferentiated cells has been blocked in its initial differentiation. There is, indeed, a deficit of large, differentiated cells at 8 or 10 days of incubation ; however, the other premise is not fulfilled : the sum total of all cells is smaller 011 the operated side than on the control side. This is due to two factors: the mitotic activity was reduced from 4 days on, and differential degeneration of large cells has operated from 5 days on. These two processes a re synchronous with the niajor phase of the differentiation of the large neurons, and they mask any effect which the periphery might conceivably exert on the initial differentiation process. I n other words, the possibility of the latter effect is not excluded, but no conclusive evidence for or against it can be derived from hypoplastic ganglia.

The situation is unequivocal in the case of peripheral overloading. It can be demonstrated by differential cell counts of large and small cells that more neurons have undergone diff ereiitiation under the influence of the transplant than is normally the case.

The counts were made on 4 ganglia of silver impregnated older embryos, ranging from 13 to 17 days. During this pc- I-iod, the small and large cells are topographically separate w-liich facilitates the clistinctioii between them. Furthermore,

478 VIKTOlX HAMRIWGEB AKD RITA LEVI-MOKTATACIKI

the silver impregiiatioii makes an identification of the large neurons definite. The data are presented on table 4 (last 4 horizontal rows). In all cases, there is an increase in the absolute number of large, differentiated cells, ranging from 36% to 80%. It was pointed out repeatedly that a considerable range of variation in peripheral effects is to be expected iii all transplantation experiments, since tlie pattern of nerves which supply a transplant varies from case to case. The important point is that the numerical liyperplasia concerns the fully differentiated large neurons ; this implies that a t least

Fig. 1 Thirteen-daj- embryo (48ti 79) . Traiisplaiitatioii of iriiig (partly dupli- cated) behind host ning.

part of the supernumerary cells produced by an excessive proliferation have undergone differentiation. Obviously, that is the result of a sequence of events which was initiated bv the experiniental enlargement of the peripliery.

The reaction of the small cells is inconsistent. They show a slight hyperplasia in two ganglia and a liypoplasia of 5.3% and 29%, respectively, in two other ganglia. The independent variation of the large and small cells is a very interesting phenomenon. I t is taken as further evidence f o r our contention that we are dealing ~ i t h different types of neurons

DIFFERENTIATION O F SPINAL GASGLIA 479

which have different fuiictions, the large cells being primarily concerned with tactile, exteroceptive sensitivity and the belatedly differentiating cells, with proprioceptive muscle sensitivity, and possibly with other functions (see p. 463). Particularly suggestive in this respect is case 48tr79 (fig.

Fig. 2 Reconstruction of iiervous system of embryo, shoxvn in figure 1. A = nerves ending at base of transplant.

1) of which two ganglia, 16 and 17, were counted. The sinall neurons in ganglion 16 show a high degree of numerical hypoplasia, those in ganglion 17 are hyperplastic. When the peripheral distribution of the nerves 16 and 17 was traced (fig. 2) , it mas found that nerve 16 did not enter the trans-

480 VIICTOR HAMBURGER SSD RITA LEVI-MOSTALCINI

plant but elided in the skin dorsally to the base of the transplant, whereas nerve 17 had a major share in the innervation of the transplant including its muscles.

The observation that the numerical liyperplasia of the small cells in tlie two instances in which it occurred was considerably lower in degree than that of tlie large cells is uiider- standable if one considers the time patterns of proliferation and differentiation. Proliferation is in full swing when the large cells establish peripheral contacts. A surplus of cells is available for supernumerary clifferentiation, since the en- larged periphery stiiiiulates mitotic activity beyond the normal range. On the other hand, proliferation has terminated at tlie time when the siiiall cells begin their differentiation, a t 9 and 10 days. The only source for supernumerary small neurons that would be available at that period would be (hy- pothetical) indifferent reserve cells.

C. D eg e nc ratio ?z

Few investigators have considered the possibility that a l1~7poplasia following H reduction of the periphery might be attributed, wholly or iii part, to regressive clianges, that is to the atrophy ancl breakdown of iieuroiis. I t s occurrence has now been substantiated by the observation of degenerating cells. Embryos with wing and leg extirpations, stained in Heidenhain’s Hematoxylin, were used for this study. It will be reniembered that in normal embryos only the cervical and thoracic ganglia sliow “ clegeiierating ” cells in conspicuous numbers, whereas these cells are rare or missing in normal brachial arid lumbosacral ganglia. Following wing or leg bud extirpation, they were found in large iiuinbers in the brachial aiid lumbosacral ganglia of the operated side (figs. 3, 6, 7 ) . The experinieiitally produced cell-degeneration affects tlie limb ganglia a t the same stages in which the normal cervical aiid thoracic ganglia undergo degeneration ; in both instances, the peak of degeneration is a t 3 and 6 days (table 5 ) . Like- wise, the topographic location of the degenerating cells is the same in experimentally inciuced and in iiowmally occur-

DIFFERENTIATION O F SPINAL GASGLIA 48 1

ring degeneration ; in both instances, degeneration is almost exclusively limited to the large, diff ereiitiated iieuroiis which are concentrated at the ventrolateral border of tlie ganglia. Occasionally, mitotic figures were observed wliich had an abnormal appearance.

0

A B Fig. 3 Six-day embryo, right wing bud removed. Brachial ganglia (no. 16).

A = right ganglion, showing degenerating cells (D) ; B = left ganglion; M = mitoses.

TABLE 5

Cell degearsation in brachial ganglia follotuing wing eztirpatroii

5 DAYS 6 DAYS 7 D A Y S GANGLIA 4$ DAYS

K . L R I,

11 + + ++ 12 + + ++ 13 + + ++ 14 -- + 15 - ++ -

16 - - + 17 - - ++

-

K

++ ++ +++ +++ +++ ++ ++

1,

++ ++ +i- + + ++

R L R

++ + + ++ + + ++ + + +++ + ++ ++++ + ++ ++ + + ++ + + In order to check further the identity of normally occur-

ring and of experimentally produced degeneration, tlie same vital staining experiments with toluidin blue which were described above for normal embryos, were made on a large

452 VIKTOR HAMBURGER AND RITA LEVI-NOKTALCINI

iiuniber of embryos in wliich wing or leg buds had been extirpated. The results were the same; niany of the “degenerating” cells were identified as macrophages arid found to be identical with the corresponding cells in the cervical and thoracic ganglia with respect to appearance and uptake of dye. The same results u’ci’e obtained in supra-vital staining experiments mitli neutral red (see p. 469). It is implied that under the experimental conditions a “degenerative” process extends to ganglia which are normally spared such degeneration.

At this point, the comparison between normal cervical and thoracic ganglia and limb ganglia of extirpation cases ends. S o further regression is apparent in the former, once the breakdown products are removed. The experimental cases, however, show signs of a continued, though slowly progres- sing atrophy. In other words, limb extirpation results in two types of regressive changes, only one of which has its parallel in normal developnient.

Whereas the first cataclysmic breakdown affects only the large, differentiated cells, signs of further atrophy after 8 days of incubation were found both in the surviving large neurons and in the slowly differentiating small cells which are located in the median and dorsal parts of the ganglion (see figs. 5, 8, 9).

TJTe coiisider first the relatively small group of residual large cells which have survived the breakdown at 5 and 6 days and wliich are located in the ventrolateral part of the ganglion. Cell counts of these cells in the 25th ganglion, following leg bud extirpation, had been reported in a previous paper (Levi-Montalcini and Levi, ’44, p. 534). They indicate that their number does not decline further between 7 and 19 days. MTe assunie that these cells which we consider as exteroceptive (tactile) sensory neurons, supply the dorsal part of the skin that was riot affected by the operation; in fact, fibers from these cells could be traced to the dorsal skin. Xevertheless, signs of atrophy ere found occasionally in this cell group. In silrer impregnated material, the rieurites

D I F F E R E K T I A T I O N O F S P I N A L GAXGLIA 483

are occasionally kinky, and the cell bodies atrophic. Prcpa- rations stained with toluidin blue show in many instances a disappearance of the Nissl substance at 18 days of incubation (Levi-Montalcini and Levi, '44, figs. 5 and 14). However, it should be pointed out that these observations were limited to the 25th ganglion, that the signs of atrophy during this second phase were far less regularly observed than the degeneration in the first phase, and that the most severe atrophy was observed in cases of s7ery radical operations, in which the ganglia were directly exposed to the amniotic fluid and the dorsal skin had failed to cover the wound. It is, therefore, possible that this atrophy is due, in part, t o a direct impairment of the ganglia through adverse environmental conditions.

The srnull cells are in an undifferentiated condition at the time when most of the large cells suffer a cellular breakdown (5-7 days). They seem to be unaffected by the latter, both quantitatively and qualitatively, as cell counts and observations on the 25th ganglion show (Levi-Montalcini and Levi, '44, p. 534). However., it seems that between 8 days and hatching, that is during their differentiation and growth, their development is impaireci. They arc smaller than those on the control side (fig. 9) and the number of fibers is reduced. A further detailed study of this regressive process is necessary. However, it can be stated with certainty that the small cell group never suffers a catastrophic breakdown comparable to the degeneration observed in the large cells.

Both the rapid bi*eakdoww of early differentiating large cells, at 5 and 6 days, a i d the s l o ~ l y proceeding atrophy of the surviving large cells and of all small cells, during the later phases of incubation, are regressive changes which affect neurons that are in the process of differentiation. If we combine these findings with those presented in the preceding chapter, we can state that both the i d i n l cliffwentin- tiow, and the coinyletioii of diffprciitintioii are under the control of peripheral factors.

484 VIKTOR HAMBURGER A S D RITA LEVI-RIONTALCINI

1‘. DIE;CT;SSIOL\

A. Two cell t ypes in clecelopiiig sp i~ ia l ganglia The study of the normal developmeiit of spinal ganglia

has revealed the existence of t x o cell types wliicli are clearly distinguishable from early stages of illcubation on: First , a group of early differentiating neurons, which grow and differentiate very rapidly and are located at the veiitrolateral border of the ganglia, forming a cup-shaped structure there. They will be referred to as “V-L cells.” Second, a group of small neurons wliicli begin to differentiate niuch later and grow and differentiate at a slower rate. They occupy a niedio- dorsal position aiid will be referred to as “ M - D cells” (see

The existence of these two classes of cells was recognized by Levi-Montalcini and Levi (’43) who studied their development in detail. Measurements of cell sizes which were made on the 25th ganglion, showed iridicatioiis of two size-classes at 8 and 9 days, arid clearly bimodal curves at 12, 15 and 19 days. The smaller cells show no signs of neurofibril differentiation up to 9 days of incubation. They are probably iii- different cells up to this stage. This is concluded from the fact that they are capable of undergoing mitotic division and of differentiating either into small (11-D) or large (V-L) neurons, depending on the conditions a t the periphery (see p. 476). Those small cells mhicli have remained undifferentiated until 9 days of incubation begin to differentiate into small neurons. It is important to emphasize that these small neurons are not embryonic or retarded stages of large V-L cells but a special category of neurons. I n other words, we are dealing with two groups of neurons which belong to two structurally and functionally separate categories and which are clearly separate topographically. Not until hatching time do some of the originally smaller iieuroiis approach or sur- pass the size of the larger neurons; from then on, a distinction between the two classes is no longer possible.

Since the contention that we are dealing with two groups supplying separate peripheral fields is of considerable im-

fig. 5).

DIFFERENTIATION O F S P I N A L GANGLIA 485

portance for the interpretation of our results, we have listed below all points which were brought out by Levi-Montalcini and Levi ('43) and in the present study to support this view. We clo not contend that the two classes are homo& oeneous from the functional point of view.

The two cell types differ in their developmeiatal pa t - t erns . The V-L cells differentiate between three and 8 days. They grow rapidly during this plase. The 11-D cells begin to differentiate at 9 days and continue growth and differentiation at a slow rate. They pass through a stage of apolar neuroblasts which never occurs in V-L cells (for all details see p. 462).

2. A correlation was found between the time at which the neurites of the V-L cells in limb ganglia grow out (5-6 days) and the beginning of ez terocept ive reflex; activity of the limb at 7 days (Visintini and Levi-Montalcini, '39). Fur- thermore, neurites of the large cells could be traced to the skin. I t is suggested that the V-L cells are t a c t i k exterocep- t i ve neurons. A similar correlation exists between the beginning of outgrowth of the neurites of M-D cells (10-12 days) ancl the beginning of proprioceptive responses in the limb at 11 days. It is suggested that the group of 11-D cells contains the propriocept ive neurow. However, both groups are likely to contain cells of other functional assignments as well.

The V-L cells and the M-D cells in limb-innervating ganglia give dif ferent ial responses to a reduction of the peripheral fields (limb extirpation) as well as to peripheral overloading (limb transplantation). Following limb extirpation, the majority of differentiated V-L cells undergo degefz- P r a t i m at 5 and 6 days of incubation. KO such breakdown occurs in the 3f-D cells at any time. The latter show merely an atrophic condition toward the later part of incubation. In cases of peripheral overloading, the V-L cells show consist- ently a high degree of nmzerical hyperplasia, whereas the 31-D cells show either a slight degree of hyperplasia, o r even hypoplasia (table 4). The most plausible explanation of this independent variation is the assumption that both cell types

1.

3.

486 VIKTOR HAMBURGER AKD RITA LEVI-MONTALCINI

innervate different peripheral structures arid that their differentiation and their niaintainaiice are controlled by their respective teriniiial areas. Specific iiistaiices supporting this interpretation are given 011 page 478.

A similar difference in the behavior of V-L and If-D cells is found in the developmental process of cervical mid thorac ic ganglia of iiornzal embryos. A considerable number of the V-L cells undergo a rapid degeneration at 5 and 6 days which seems to be identical with the degeneration of V-L cells in limb ganglia following limb extirpation. No such degeneration occurs in the 31-D cells of normal cervical o r thoracic ganglia.

Our contention that tlie V-1, and ;1I-D cells are fuiictionally different types and that tlie latter are, in part, proprioceptive neurons received support from tlie observations of Bue- ker ('48) on the i i i i iei~ation of tumoi. implants. He finds that mouse sarcoma 180 when transplanted in tlie place of the hind limb bud, or between the latter and the somites of three- day embryos, are exclusively innervated by sensory fibers, whereas the lateral niotor columns are hypoplastic. A comparison of cell counts and volunies (weights of cardboard models) of the hyperplastic spinal ganglia showed a very conspicuous discrepancy between numerical and volumetric hyperplasia. Ten of 13 ganglia showed no increase in total cell numbers or only an insignificant iiumerical liyperplasia with an overall average of 6.5%. The volumetric hyperplasia was present in all ganglia: it aniouiited to a n average of 3370, with individual increases up to almost 100%. The author was not aware of tlie cliffereiiccs hetween V-L and 11-D cells and interprets his results in terms of a general cellular hypertrophy. It appears from liis figure 4 that the lipperplastic ganglia contain an cscessive nuruiber of large V-L cells. Differential cell cornits w-oald probably reveal a numerical hypoplasia of 31-D cells and a numerical hyperplasia of V-L cells in all ganglia in wliicli the total cell number was not increased. It is suggestive to attribute the effects on both M-D cells mid lateral motor cells to the complete

4.

DIFFEREXTIATION OF SPIS.SL GASGLIA 487

loss of leg musculature in tlie region of tlie transplanted tumor. (See Bueker, '48, fig. 5.)

Taylor ('43) describes and pictures two types of cells in tlie spinal ganglia of larvae of R a m pipiens: they resemble in size difference, time of differentiation and topographic relations the cell types described above. However, the functional interpretation of these cells is different. The author contends that only fibers from small cells innervate the limb, and that "heavy fibers can be traced to only those encl-organs which are functionally innervated during the embryonic period, namely, somites and skin. . ." (p. 398).

Barron ('44) in his study of the embryonic developnient of the spinal ganglia of the sheep, finds that in this form the first neuroblasts to differentiate are likewise located in the ventral, ventrolateral and ventromedial regions of the ganglia and that these regions contgin for a considerable period all advanced neuroblasts. The precocious differentiation of the ventrolateral marginal cells, corresponding to the V-L cells in the chick embryo, would seem to be a universal feature in vertebrate ganglia.

B. Multiple effects of the periphery o n the deuelopment of s p i m l ganglia

A careful examination of all aspects of the development of spinal ganglia following a reduction or an increase of their peripheral fields of sensory innervation has shown that the following compoiieirts are affected by peripheral changes :

The mitotic activity is reduced by limb extirpation, and increased by peripheral overloading, the changes amounting to approximately 20% in either direction (table 1). This effect of the periphery 011 primary sensory centers is established here for the' first time by direct mitotic counts. I t had been postulated correctly by Detwiler ('20, '23, '36) in his analysis of spinal ganglion developmeiit of Aniblystoma, and by others, on the basis of cell comzts in hyperplastic ganglia. Of particular interest is the work of Carpenter, ('32, '33) who showed that iiunierical hyperplasia of thoracic ganglia

1.

488 VIKTOR HAMBURGER A S D RITS LEVI-MOXTBLCINI

can be produced iii larvae and even after metamorphosis, by limb transplantation. This shows that undifferentiated cells capable of mitosis are present in Grodela a t those late stages, and that the meclianisni of peripheral control operates at least throughout larval life.

2. The i ~ i t i a 2 d i f e r e h a t i o i z of indifferent cells into neuroblasts is controlled by the conditions a t the periphery. Evidence for this effect was obtained from cell counts in hyperplastic ganglia (table 4). It was found that the number of V-L neurons was up to 807% higher than normal. I n other words, the additional cells produced by increased mitotic activity had differentiated almost exclusively into V-L neurons. The enlargement of the periphery must be responsible, in a direct or indirect way, for the differentiation of excessive nuiiibers of indifferent cells into V-L neuroblasts. Det- wiler ('20, '23, '36) has come t: the same conclusion in his analysis of the hyperplasia of ganglia in Amblystoma. It is possible that the reverse effect exists in hypoplastic ganglia. However, this cannot be proved by cell counts, because the effect on differentiation is masked by the simultaneous occurrence of a decrease in mitotic activity and of cell degeneration (see p. 477). The contention of a number of investigators that a cellular hypoplasia is evidence of an interference with the process of cliffereiitiotion is not warranted. These authors do not take into consideration the possibility of a secondary loss of iieuroiis by degeneratiovz which is definitely established in our case. This objection is particularly pertinent with respect to extirpation experiments done a t stages in which the nerve centers were advanced in their differentiation or in those instances in which the cell counts were made several weeks after the operation.

Cowtintied differeiitintioia of m u r o b l a s t s , t h e i r growth and nzaisatainance depend on adequate conditions in the regions in which the lieurites of these cells branch out and establish provisional terminations. Two types of pathological changes were observed in neuroblasts following limb extirpation : a rapid degencrntioiz of V-L cells, and a slow a t r o p h y

3.

DIFFEREXTIATION O F SPINAL GANGLIA 489

of M-D cells. The former break down shortly after their neurites have reached the base of the extirpated limb, at 5-6 days of incubation, without completing their growth and full differentiation. The M-D cells differentiate slowly arid much later and show a less drastic response, iianiely an atrophic condition in late stages of incubation. I t was not possible to determine whether this atrophy is due to a reduction of the growth process o r to a shrinkage of originally large-sized iieuroiis, or to a combination of both.

One might expect a cellular hypertrophy in the case of peripheral overloading of neuroblasts. This point was not yet investigated. Such an effect has been observed by Terni ('20) in the case of tail regeneration in the lizard. The re- generated tail contains no central nervous system, and its innervation is provided by the terminal segments of the spinal cord and spinal ganglia of the stump. The cells of the spinal ganglia supplying the regenerate were found to be approximately three times larger than normal.

Whereas cellular atrophy has been observed or surmised occasionally, the role of izeuron degegzercrtioiz has been gen- erally negle~ted .~ Yet, this effect seems to be of wide occurrence. It was found in sensory ganglia in the chick embryo (Levi-Montalcini and Levi, '44), in tlie ciliary ganglion, following extirpation of the peripheral field ( L4mprino, '43), in secondary sensory centers (Levi-Montalcini, '49) and in primary motor centers (cervical spinal cord, Levi-Mon- talcini, unpublished ; trochlear nucleus, Dnnnebacke, nnpub- lished).

Hall and Schneiderlian ( '45) claim that the liypoplasia which was obserxed in tlic spinal ganglia of the nevly-born rat following limb amputation in late fetal stages is not due t o degeneration but to an inhibition of the process of in- duction of indifferent cells by adjacent neuroblasts. Their assumption is largely based on their failure to detect degenerating cells iu their material. Undoubtedly, nerve fibers were transected a t this operation, and a response of the affected neurons must have occurred. Cellular breakdonn is easilp overlooked, particularly if i t takes place as rapidly as it does in the chick embryo. Furthermoro, it is difficult to believe that, in the mammal, the transformation of indifferent cells into neuroblasts is still actively in progress in late fetal stages.

490 VIIZTOR HAMBURGER ASD RITA LEVI-MOSTALCISI

It should be enipliasized tliat tlie role of peripheral factors in spinal ganglion developnient is limited to the quautitatire regukation of proliferation, growth and neuron differentiation. All these processes are iizitiated by other factors (probably intrinisic factors, residing within the ganglion) and well under way when the peripheral control begins to operate. Furthermore, our observations give no indication that the patterns of proliferation arid differentiation were modified by the extrinsic agents. Proliferation begins probably in the neural crest stage; it reaches its peak at 5 to 6 days and is terminated at 8 to 9 days. This pattern is not altered by changes at the periphery. Under tlie conditions of our experiments merely the quantitative aspect of mitotic activity was changed to the extent of 20% in either direction. Ap- parently, the peripheral factors modify the conditions within the ganglion by which indifferent cells are stimulated to di- vide.

The situation is similar in the case of the initial differentiation of indifferent cells into neuroblasts. Even in cases of a radical limb extirpation, considerable nunibers of both V-L cells and 11-D cells begin to differentiate and to send out neurites. It seenls that the reduction of the periphery results in a blocking of this process, and the increase of the periphery in an extension of this process beyond its normal limits. In no instance did we find a modification of the pattern of differentiation as it was described on page 461.

C. Naunerical and t-oliimetric hypo- and hyperplasia The survey in the preceding paragraph has made it clear

that the terms “hypo- and hyperplasia” cover a combination of heterogeneous factors w+icli come into play at different stages of development. The situation is probably equally complex in the responses of other nerve centers. The use- fulness of the terms “hypo- and l iyperpla~ia,~’ like that of many others, diminishes with advancing knowledge, until they become an impedinient rather tlian an aid to analysis. The ternis under discussion are liable to coiiceal the dynamic

D I F F E R E X T I A T I O N O F S P I K A L GAXGLIA 491

nature of the processes involved ; nevertheless, they are probably not yet dispensable. In using them, one should at least make a distiction between a “numerica l hyper - and hypoplasia,” as determined by cell counts, and a “columetr ic hyper - and ltypoplasiu,”4 as determined by area measure- nients or by weighing of paper models. Those authors who have applied both methods to the same instances have noticed that there is not necessarily a correlation between these two sets of data, and the present investigation has shown a considerable discrepancy between the two. As usual, the volumetric exceeds the numerical hypo- o r hyperplasia.

We are in a position to establish for our material the factors which are responsible fo r this discrepancy. I n the case of hypoplasia the cell degeneration affects differentially the large V-L cells but leaves the M-D cells intact. The slow atrophy of the &I-D cells during the later stages of incubation leads to a further disproportionate reduction in volume. Finally, the reduction of numbers of neurons results in a deficiency in nerve fibers and possibly in glia cells, permit- ting a more dense packing of the remaining cells. There is good evidence to show that the numerical hypoplasia follow- iiig limb extirpation, remains constant from the 8th day on through incubation (Levi-Montalcini and Levi, ’44) and to the third month after hatching (Rueker, ’47). Hence, the discrepancy between volumetric and numerical hypoplasia increases constantly from the 8th day on, and it is doubtful whether a fixed ratio between the two is ever established.

The situation is similar in hyperplasia. A numerical hyperplasia, due to an increase of mitotic activity, is definitely established f o r spinal ganglia (table 4). Under the conditions of our limb transplantation experiments, the majority of the excessive cells undergo differentiation into large V-L cells. As a result, the volumetric hyperplasia, determined by area

We suggest this term in prefcrenee t o “atrophy” aiid “hypertrophy” because the latter terms are inisleadiiig and ambiguous when applied to developmental processes, We hare used the terms “cellular atrophy” aiid ‘ I cellular hypertrophy” in the present paper t o designate changes in cell size, hut not size changes of entire nerve centers.

492 VIKTOR HAMBURGER AND RITA LEVI-MONTALCINI

measurements, is greater than the numerical hyperplasia. I f , in addition, a cellular hypertrophy should occur, then this disproportion would be accentuated. We have not made cell size measurements in our material to establish this point.

TTTe recognize that the two terms are based on methodo- logical and not on analytical considerations, and that each one covers again heterogeneous phenomena. For instance, “numerical hypoplasia” is partly the result of a reduction of mitotic activity, and partly of neuron degeneration. Nevertheless, they may be useful in avoiding elementary mis- understandings in embryology.

D. Mechanism of peripheral control of ganglioul. deve lopment

(Point-to-point effects and field-effects) Our observations contribute little to the problem of the

mechanisms by which changes at the periphery bring about developmental changes in the spinal ganglia. However, they bring into focus the necessity of distinguishing between two basically different mechanisms.

Two of the observed effects, namely the degeneratio.12 of the V-L cells, and the atrophy of the M-D cells, are regressive changes. They concern neuroblasts which are affected after they have established connections with the periphery. These changes are, in certain respects, comparable to the effects of nerve transection in adults not followed by regeneration. They indicate that adequate connections with the periphery are necessary for the maintainance of sensory neurons. We may designate the mechanism involved as a “poiizt- to-point ” effect.

The situation is different in the case of peripheral effects on proliferatioiz and init ial differeuztintioq?. These effects are exerted on undifferentiated cells which have no direct connections of their own with the periphery. We shall refer to them as “field effects.”

HOW are the regressive changes brought about? I t will be remembered that the V-L neuroblasts begin to send out their

DIFFERENTIATION O F SPINAL GAL-GLIA 493

lieurites at very early stages, beginning a t two and a half days, i.e. in early limb bud stages. The process of differentiation of V-L cells is practically completed at 5-6 days. The large-scale degeneration of V-L cells resulting from limb bud extirpation is a t its peak a t 5-6 days of incubation, which implies that the rieuroblasts break down shortly after their lieurites have grown out. The small number of surviving cells are probably the ones which innervate the dorsal regions that were not affected by tlie operation. The abnormal conditions to which tlie growing tips of the majority of V-L lieurites are subjected are responsible fo r the regressive changes in their cell bodies. One could think of two alterna- tives. First, the inhibition of further outgrowth and of the spinning out of neurite material in itself may upset the me- tabolism of the V-L cells sufficiently to result in their breakdown. This assumption is, in a sense, the extension to embryonic neurons of the concept developed by P. Weiss ( '44; see also Weiss and Hiscoe, '48), according to which the continuous synthesis of axoplasm by the pericaryon and its centrifugal transport in the neurite is a normal physiological activity of an adult neuron. An alternative mechanism would postulate a metabolic exchange between the growing neurite and tlie substrate in which it grows. Substances necessary f o r neurite and neuroblast growth and maintainance would not be provided in adequate quantities when the limb bud is removed. MTe have no way of deciding between these alterna- tives.

A remarkable fact stands out: the high susceptibility of the V-L cells to eiivironmental conditions during the 5th and 6th days of incubation, that is shortly after the fiber outgrowth; the large-scale degeneration of V-L cells in normal cervical and thoracic ganglia during the same period has been interpreted in the same way (see p. 495).

The slow atrophy of the M-D cells can be understood in similar terms of inadequate growth conditions for their neurites. The atrophy does not begin until after the lieurites have grown out.

494 VIKTOR HAMBURGER .4ND RITA LEVI->IOS TALCINI

The mechaiiisni by which peripheral factors control pro- lifercrtion aiid iiiitiul dif ferent iat ion is more difficult to under- s t a id because cells are affected which have no direct coiiiiections with the periphery. It is very unlikely that we are dealing with diffusible substances since closely adjacent nerve centers respond differentially to the same peripheral changes. In the present paper, we have described the occurrence of liypoplasia of M-D cells aiid hypevplasia of V-L cells within tlie same ganglion. Likewise, differential responses of seiisory and motor centers arc coinnion (Hamburger, ’34 ; Bucker, ’48; a.0.). It is more reasonable to assume that each center is controlled by its own peripheral area, and that the pioneer fibers which coniiect the two in early stages of development mediate the effects of the periphery on undifferentiated cells, as was suggested before (Hamburger, ’34). The developing nerve center would be coiisidered as a, “field” within which each unit would be affected by changes in other units. More or less specific hypotheses based on this reason- ing have been suggested by Barron ( ’43, ’44) and Hamburger and Keefe ( ’44).

The peripheral control cannot be uiiderstood in terms of deficient (or excessive) functional c o i m e c t i o m , or f u n c t i o m l ~ ~ W X Y M ~ , as was suggested by some of the earlier authors. The present material gives new evidence against this point of view. For instance, the degeneration of large numbers of V-L cells following limb extirpation, and the increase in proliferation and in early differentiation of V-L cells following limb transplantation occur at 5 and 6 days of incubation. At that stage, the limbs arc not far advanced in development, and tlieir functional activity is 011 a primitive level. The strongest argument against the role of functional connections was given by tlic observation of Bucker ( ’48)) that a high degree of hyperplasia in spinal ganglia can be brought about by mouse sarcoma implants. Not the functioiial but tlie physi- cal or chemical conditions a t the periphery are ultimately responsible for the “periplicral” effects on tlie development of nerve centers.

D I F F E R E N T I A T I O N O F SPINSL GAKGLIA 495

E'. Cell clege,ieratioi$ as a size regulat ing fac tor In tetrapod Vertebrates, particularly in the Amniota, the

limb innervating brachial and lumbosacral ganglia are larger than the cervical, thoracic and caudal ganglia. Little is known of the embryonic origin of these regional differences. Barron ( '44) found no size differences in the cervical and first thoracic gaiiglia of sheep embryos of 24 days. He observed the first indication of a size increase of the brachial ganglia in a 30-day embryo. This is attributed to an increase in cell number, though no cell counts were made. We have observed differences in the size of cervical and brachial ganglia as early as at 4 days of incubation. Differences in the mitotic activity were found in 5-day embryos by mitotic counts (see table 1). The earliest stages at which an equal size of all ganglia might be expected, were not investigated.

Different ial proliferntiogz is undoubtedly an important factor in producing the regional size differences. The observations reported above have shown, unexpectedly, that dif ferential degenerat ion of neurons contributes significantly to these differences. The large-scale degeneration of neurons described above is limited to cervical and thoracic ganglia.

A remarkable feature of this degeneration in normal ganglia is its striking resemblance to the degeneration which can be produced experimentally in brachial and lumbosacral ganglia by limb bud extirpation (compare figs. 3A and 4). I n both instances, the early differentiating V-L cells are affected after their neurites have reached the periphery, and in both instances the breakdown occurs between 5 and 7 days, with its peak at 5 and 6 days. The M-D cells are not involved in either case.

I n the experimental situation, the reduction of the peripheral area is definitely responsible f o r the process of degeneration. T t is possible that the same mechanism operates in the case of the normal cervical and thoracic ganglia. This would imply that in early stages cervical and thoracic V-L cells send out more neurites than the periphery can support. The excess of neurons would break down at the stage at which the V-L

496 VIKTOR HABZBURGER A F D RITA LEVI-NOKTALCIKI

cells are highly susceptible to eiiviroiiniental conditions. Ob- viously, the limbs grow out rapidly aiid allow for further growth of the iieurites cluriiig this critical period, whereas the cervical and thoracic regioiis, particularly the skin, expand to a lesser degree. The experiment of peripheral overloading of cervical or thoracic ganglia should show whether this idea is correct. Our cases of linib transplantations, fixed at 5 to 6 days of incubation, hare iiot been examined in sufficient detail to give ail aiiswer. Fo r several reasons it is very difficult to obtain quantitatively valid data to show that peripheral overloading can block degeneration.

Degenerating cells are of widespread occurrence in embryonic tissues (Gliicksiiiaiiii, '30 ; Chaiig, '40 and many others). One should distinguish between s ~ o d i e cell degeneration of individual cells, and large-scale, localized and pattertied dc- generation processes which result in rnorphogenotically significant changes. Tlie forination of gill slits, of mouth and anus, the resorption of tail buds in birds aiid man and of the tadpole tail at metamoi~pliosis are but a few examples of the latter category. Tlie present iiistaiice is a special caLe within this group. Cellular breakdown in some spiiial ganglia but not in others is responsible, in part, for quantitative regional differences wliich are permanent features of the topographic pattern. The same situation was observed in the lateral motor column of the spinal cord (unpublished). TYe are not aware of similar instances in other organ systems.

VI. SCblMAKY

Observations on normal spinal gaiiglia gave the following results :

1. I n the spinal ganglia of the chick embryo, two topographically separate groups of neurons are distinguished which differ in rate and mode of differentiation and in size. Indirect evidence is presented to show that early and rapidly differentiating neurons are, in part, exteroceptive (tactile) neurons, and that the belatedly differentiating neurons include among other types, proprioceptive neurons. The morpho-

DIFFERENTIATION OF S P I N A L GANGLIA 497

logical and size differences disappear towards the end of incubation.

2. The mitotic activity in spinal ganglia is at its peak at 5 and 6 days and practically terminated at 9 days of incubation. The number of mitoses is higher in the limb innervating brachial and lumbosacral ganglia than in the cervical and thoracic ganglia.

3. In the cervical and thoracic ganglia a large-scale degeneration of early differentiated neuroblasts was discovered ; this process is a t its peak at 5 and 6 days. The presence of macrophages during this period was established by appropriate staining methods. No such degeneration occurs in limb innervating ganglia. The regional size differences between limb innervating ganglia and adjacent ganglia are brought about, in part, by differences in mitotic activity and, in part, by selective degeneration.

Observations on ganglia affected by limb extirpation or limb transplantation, respectively, gave the following results : 4. Mitotic counts show that the mitotic activity is reduced

by limb extirpation and increased by peripheral overloading, tlie changes amounting to approsiiiiately 2076 in either direction.

5. Overloaded ganglia sliow- an increase in the number of early differentiating neurons which may amount to 80%. This proves that peripheral factors control tlic differentiation process of indifferent cells.

6. Limb extirpation results in a rapid degeneration and disappearance of numerous early differentiating neurons in limb ganglia, at 5 and 6 days of incubation, which is coniparn- ble in all details to that occurring in normal cervical arid thoracic ganglia. The late differentiating neurons undergo an a trophy.

7. The complex and heterogeneous nature of the phenomena covered by the terms “hypoplasia” arid “hyperplasia” is discussed, and it is suggested to distinguish between “numerical” and “volumetric” hypo- or hyperplasia.

498 VIKTOI; HAMBURGER AND RITA LEVI-MONTALCINI

8. I t is pointed out that two basically different mechanisms operate in the coiitrol of spinal ganglion development by peripheral factors :

( a ) The periphery controls the proZiferatiorz and init ial differentiation of undifferentiated cells which have no connections of their own with the periphery.

(b) The periphery provides for conditioiis necessary f o r coutinued growth and maiiztairmnce of neurons in stages following the first outgrowth of iieurites.

LITERATURE CITED

AMPRINO, RODOLFO 1943 Correlazioni quantitative f r a ceritri nervosi e territori d 'innen-azione periferica duraiite lo sviluppo. Ricerche sperimeiitali sul ganglio ciliare del pollo. Arch. I tal . Anat. Embr., 49: 1-40.

The early developnieiit of the motor cells and columns in the spinal cord of the sherp. J. Comp. Neur., 78: 1-26.

The early development of the sensory and interiiuncial cells in the spinal cord of the sheep. J. Comp. Neur., 81: 193-225.

BUEKER, ELMER D. 1947 Limb ablation experiments on the embryonic chick and its effect as observed on the mature nervous system. Anat. Rec.,

Implantation of tumors in the hind limb field of the embryonic chick aiid the developmental response of the lumbosacral nervous system. Anat. Rec., 102: 369-390.

CARPENTER, RUSSELL 1932 Spinal-ganglion respoiises to the transplantation of differentiated limbs in Aiiiblystoma larvae. J. Exp. Zool., 6 1 : 149-173.

1933 Spinal-ganglioii responses to the transplantation of limbs after metamorphosis in Amblgstoma panctatum. J. Exp. Zool., 64:

CHANG, TSO-KAN 1940 Cellular inclusioiis and phagocytosis in normal development of mouse embryos. Peking Xat. Hist. Bull., 1 4 : 159-170.

COLLIN, R. 1906 RBcherches cytologiqurs sur le developpement de la cellule nerreuse. Le NrTraxe, 8 : 181-307.

DETWILER, S. R. 1920 On the hyperplasia of nerve centers resulting from excessive peripheral loading. Proc. Nat. Acad. Sci., 6: 96-101.

1923 Experiments on the transplantation of the spinal cord in Amblystoma, aiid their bearing upon the stimuli involred in the differentiation of nerve cells. J. Exp. Zool., 3 7 : 339-393.

1936 Keuroemlri-yology. Thr Maemillan Company, New York. 218 PP.

ERNST, M. 1926 Ueber Untergaiig voii Zelleii mahrcnd der normalen Entwick- lung bei Wirbeltieren. Zeitsch. Anat. u. Eiitw., 79 : 228-262.

GL~CKSXANS, 4. 1930 Ueber die Bedentuug von Zellvorgangen f u r die Form- bildung epithelialer Organe. Zeitschr. f . Anat. 11. Entw., 95 : 35-93.

BARRON, DONALD H. 1943

1944

97: 157-174. 1948

287-301.

DIFFERENTIATION O F SPINAL GANGLIA 491)

HALL, E. K. AND M. A . SCHNEIDERHAW

HAXIBURGER, VIKTOR 1934

1945 Spinal ganglion hypoplasia a f te r limb amputation in the fetal rat. J. Comp. Neur., 82: 19-34.

The effects of wing bud extirpation on the development of the central nervous system in chick embryos. J. Exp. Zool.,

1939a The development and innervation of transplanted limb primordia of chick embryos. J. Exp. Zool., 80: 347-389.

1939b Motor and sensory hyperplasia following limb bud transplantations in chick embryos. Physiol. Zool., 13: 268-284.

1942 A Manual of Experimental Embryology. Univ. Chicago Press. 213 pp.

1948 The mitotic patterns in the spinal cord of the chick embryo and their relation t o histogenetic processes. J. Comp. Neur., 88: 221- 284.

The effects of peripheral factors on the proliferation and differentiation in the spinal cord of chick embryos.

LCVI-MONTALCINI, RITA 1949 The development of the acoustico-vestibular centers in the chick embryo in the absence of the afferent root fibers and of drscending fiber tracts. I n press J. Comp. Neur.

LEVI-MONTALCINI, RITA AND G. LEVI 1943 Rocherches quantitatives sur la niarche du processus de differenciation des neurones dans les ganglions spinaux de l’embryon de Poulet. Arch. de Biol., 54: 189-206.

Correlazioni ncllo sviluppo t r a varie par t i del sistema nervoso. I. Conseguenze della demolizione dell’abbozzo di un art0 sui centri nervosi nell ’embrione d i pollo. Commentationes, Pontif. Acad. Sci.,

OLIVO, 0. M., E. PORTA AND L. BARBERIS 1932 Riccrche sulla velocit& d i accrescimento delle cellule e degli organi. IV. Modalit& di accrescimento delle cellule dei gangli spinali nel pollo durante l a vita embri- onale e postnatale. Arch. Ital d Anat Embr., 30: 34-71.

68: 449-494.

HAMBURGER, VIKTOR AND E. KEEFE 1944

J. Exp. ZOO^., 96: 223-242.

1944

8: 527-568.

PIATT, JEAN 1948 Form and causality in neurogenesis. Biol. Rev., 23: 1-45. T~YLoR, A. CECIL 1943 Development of the innervation pattern in the limb

bud of the frog. Anat. Rec., 87: 379-413. TELLO, J. F. 1922 Die Entstehung der motorischen und sensiblcn Nervenendi-

gungen. I. I n dem lokomotorischen System der hohercn Wirbeltiere. Zeitsrhr. Anat. Entw., 64: 348440.

Sulla correlazione f r a ampiezza del territorio d i innervazi- one e grandezza delle cellule ganglionari. 11. Ricerchi sui gangli spinali che innervano la coda rigencrata, nei Sauri (Gongylus ocellatus) . Arch. I tal . Anat. Embriol., 17 : 507-543.

VISINTINI, F. AND R. LEVI-MONTALCINI 1939 Relazione t r a differenziazione strut- turale e funzionale dei centri e delle vie nervose nell’embrione d i pollo. Arch. Suisses Neur. et Psych., 44: 119-150. 1944 Damming of axoplasm in constricted nerve: a sign of perpetual

gro.lr-th in nerve fibers. Anat. Rec., 88: 48 (abstract).

TERNI, TULLIO 1920

WEISS, P.

500 TIICTOR HAMBURGER AKD RITA LEVI-MONTALCINI

WEISS, P. s m HELEN HISCOE

WII,LIAIIS, W. L. AND X~RTIIELLI FRANTZ

1948 Experinleiits 011 the mechanism of iierve

1948 Histological technics in the study of ritally stained normal and damaged cells. Anat. Rec., 100: 347-557.

grolvth. J. EX^. Zool., 107: 315-396.

PLATE 1

bYPLAT4TION O F F I G L R F S

4 Xornial 3 - d ~ ~ eii i l~yo. Ceriical ganglion, no. 12. S o t e the numerous cle- generating cells in the reiitrolateral part . Heidenham’s Hcmatouylin.

5 NoriiiaI 13-day embryo. Thoracic ganglion, showiiig the contrast between the large, early differentiating iiruroiis and the small, late differentiating iieuro- blasts. Silver inipiegiiation after Cxjal-DeCastro.

6 Six-day embryo, right wing bud removed (41539). Large veiitrolatrral cells in left brachial ganglion (no. 16) .

7 Samc region, in corresponding right ganglion (see fig. G ) , showing numerous degenerating cells (D) . Most of the other iieuroiis show signs of impairment.

8 Fifteen-day enibrjo, right leg remowd (265). Left ganglion no. 25. 9 Same as figure 8. Right ganglion no. 25. Note the disappearance of most

rentiolateial cells a n d the atroplij of the ieiiiaiiiing cells. Same magnification a s in figiiie 8.

PLATIC 1

501

proliferation, differentiation and ... -...

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