Download Proliferation, differentiation and degeneration in the spinal ganglia

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 DEGENERATION I N THE SPl"4L G A N G L I S
O F THE C H I C K EMBRYO UNDER
XORNAL A S D E X P E R I N E X TAL COSDITIOSS
VIKTOR HAMBURGER AKD R I T A T,EVI-&fO?;TALCIKI
TTaslitngtor~O ? i x t i s i t y
N I N E 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 a n effort to
elucidate those aspects of iiormal development which a r e 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 r e
strongly emphasized and others a r e neglected. Special attention has been paid to the niodificatioiis which a r e 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 r o m 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 t o these phenomena. Cell
counts or volume determinations made at the end of an arbitrarily chosen observation period give no clues as to the
mechanisms which operate t o 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. Consequently, 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 t o 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 circumscribed units which contain within themselves all proliferating and differentiating cell elements. Futhermore,
regional differences in size exist between different ganglia
(cervical, brachial, ctc.) . Finally, considerable preliminary
~7orkhas 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 G A S G LI A
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 interplay of different components.
We wish t o 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 t o 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 Hamburger ( '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 t o the somites as possible t o 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 t o be used for mitotic and cell counts were
fixed in Bouin and stained in Heidenhain's Hematoxylin. The
embryos t o 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 t o 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. F o r most of
the observations on the latter material, ganglion 25, which
had been used in a previous study, was chosen (Levi-Montalcini and Levi, '43, '44).
The experimental production of hyperplastic ganglia is
difficult, f o r several reasons. I n tlie case of limb transplantations, it is not possible t o 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 t o be
intrinsic limitations t o 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 a s closelv t o the host wing as possible, in order t o
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.
I n most instances we used ganglia 16, 17, 18 in varying combinations. 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 t o 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 t o hatching was given by LeviMontalcini and Levi ( '43). W e shall give a brief summary
of their results t o 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 i n 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 i n 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. A t three and one-half days, their number is still small. They
iir~
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 tins of the fibers have reached the dermis. (See also
Visintini and Levi-Montalcini, '39).
From the beginnin?, the early differentiating cells are concentrated i n 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. Howr ~ 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. A t 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 i n 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 a t 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 acquire 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 a t 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 1 9 days (op. cit., fig. 5, p. 201) have shown
that both the originally smaller cells and the larger cells grow conspicuously 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 activity
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 o f them have
reached the size of the early differentiating ventrolateral
neurons, whereas others have remained small, and all intermediate 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
A X D 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 a r e very
scanty. No details a r e reported, except for the statement that
proliferation ends on the 7th day (Olivo, P o r t a 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 metaphases 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
t o differentiate and lose their proliferative capacity. Therefore, the ratio of mitoses t o 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 r e presented in columns 5, 11, 17 aiid 18 of table 1.
The “coiitrol” ganglia (coluniiis 17 ancl 18) a r e partly ganglia
of iiornial embryos (those with designation “ii ”) and partly
ganglia of wing extirpation (“ S ” ) and wing transplantation
( “ t r ”) cahes, which a r e 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 unilateral opemtion 011 tlie ganglia of the other side is unlikely.
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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.
250).
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 t o 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 11to 13 are 84, for both the 5th and
the 6th days. The averages f o r the brachial ganglia 14 to 16
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, t o differences in mitotic activity in different
ganglia. This does not imply that the mitotic index (that is
the ratio of dividing cells t o cells which are potentially capable
of dividing) is different in different ganglia. Once differences
TABLE 2
Xitotic
5 DAYS
0 INGT,.
NO.
11
12
13
14
15
16
17
18
19
20
21
actacrly of riomnul punglia
Number of mitoses
8.5
100,99; 71, 68
Sutnher of mitoses
85
66,66; 101,104
86,82
114,114; 156; 123
183,175; 164; 129
150,134; 147,188; 114,122
172
118,114 ; 1 3 i ; 103
112
75,715; 1 1 2 , 1 2 0
148,145
22
2.3
6 DAYS
Are
84
127 123;117
163 136,113, 141
144 196,201,157, 104,139,139; l i 8
1 7 2 109,104
118
112 120,117; 7 i
96 130,139
143
120
172
-~
A re
85
84
120
137
1.59
107
105
135
120
172
Averages :
11-13
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
V I I i T O B HBMBCRGER A N D R I T A L E V I - M O X T A L C I N I
affinity to silver and a r e 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 p a r t which is surrounded 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 i n other embryonic organs of Vertebrates
(Collin, ’06 ; Eriist, ’26 ; Glucksmann, ’30 ; Chang, ’40 ; a.0.).
They a r e usually referred t o 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 t o 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. I n order t o test this assumption,
the teclinique of vital staining with trypaii blue was applied
u;41icli is considered t o 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 LavdowEky (Williams and Frantz, ’48). They were dehydrated
in dioxane and sectioned a t 5 t o 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
macrophddoes.
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 6day 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
c
* 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 f o 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 time pattern. We have found, in agreement with
previous investigators (Ernst, ’26 ; a.o.), that the cellular
breakdown in the spinal ganglia is limited t o 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 f o r 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
471
D I F F E R E N T I A T I O N O F S P I N A L GANGLIA
statements made above concerning the time pattern. It shows,
in addition, that the degenerative process seems t o 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 -
P u t f e r n of cell degeneratiolz in spinal ganglza of chick embryos
GAXQLIA
NO.
5n 10
5 DAY 4
5n 1
41 DAYS
++
++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
1
2
3
4
7
6
7
8
9
10
11
12
-
-
15
++
+++
+++
+++
+++
+++
16
17
18
19
20
22
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 t o 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 t o 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 t o the brachial and lumbosacral ganglia.
( c ) Topographic distributioqa of “ d e g e n e r a t i n g 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 t o differentiate, beginning a t two and
one-half days, and that the outgrowth of their ncurites t o 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 degeiieration. I f one compares the cell density in a brachial with
that of a cervical ( o r 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. However, 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 a s they
are found in the adult.
IV. E X P E R I M E N T A L P A R T
A. Proliferation
Our findings concerning the mitotic pattern in normal ganglia (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, belonging 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 f o 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 e m b r y o s 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 x2 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 participate 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-
475
DIFFENEXTIATIOE O F SPINAL GASGLIA
( s 0 0 r i c 1 3 ( 3 o r i Q ,
0.1
01
a 0 1 u3 00 c CD Lo a 0.1 d cn .1
L2
m. 0.1
0
0.
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bi
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13
U
bi
0.1
ri
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ri
ri
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476
VIKTOR HAMBURGER AKD R IT A LEVI-MONTA4LCINI
centage increases range from 8.7% t o 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 n o 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 described 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 t o differentiate later, from 10 days on, and
proceeds t o differentiate and t o 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 advisable t o 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 peripliera1 area, and is differentiation of indifferent cells stimulated by a n increase in the peripheral a r e a ?
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 a n 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 i n its initial differentiation. There is, indeed, a
deficit of large, differentiated cells at 8 o r 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 r e
synchronous with the niajor phase of the differentiation of
the large neurons, and they mask any effect which the periphe r y 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 o r 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 pcI-iod, the small and large cells a r e topographically separate
w-liich facilitates the clistinctioii between them. Furthermore,
478
VIKTOlX H A M R I W G E B A K D RITA LEVI-MOKTATACIKI
the silver impregiiatioii makes a n identification of the large
neurons definite. The data are presented on table 4 (last 4
horizontal rows). I n all cases, there is an increase in the
absolute number of large, differentiated cells, ranging from
36% t o 80%. It was pointed out repeatedly that a considerable
range of variation in peripheral effects is t o be expected iii
all transplantation experiments, since tlie pattern of nerves
which supply a transplant varies from case t o 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 duplicated) 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 hdifferent 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 uiiderstandable 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 enlarged 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 (hypothetical) indifferent reserve cells.
C. D eg e nc ratio ?z
Few investigators have considered the possibility that a
l1~7poplasiafollowing 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 r a r e 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 i n 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 ) . Likewise, the topographic location of the degenerating cells is
the same in experimentally inciuced and in iiowmally occur-
481
D I F F E R E N T I A T I O N O F SPINAL GASGLIA
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
B
A
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
GANGLIA
K.
11
12
13
14
15
16
17
4$ DAYS
L
+
+
+
--
R
+
+
++
++
-
-
6 DAYS
5 DAYS
I,
K
++ ++
++ ++
++ +++
+++
+++
+ ++
++ ++
-
1,
7 DAYS
R
++ ++
++ ++
+i-++
+ +++
++++
+ ++
++ ++
L
+
+
+
+
+
+
+
R
+
+
+
++
++
+
+
I n order to check further the identity of normally occurring 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 progressing atrophy. I n 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
a s 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 t o 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. Prcparations 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 t o 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 yproceeding 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 cliffwentintiow, 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 y p e s in clecelopiiig s p i ~ i a lganglia
The study of the normal developmeiit of spinal ganglia
has revealed the existence of t x o cell types wliicli a r e clearly
distinguishable from early stages of illcubation on: First, a
group of early differentiating neurons, which grow and differentiate very rapidly and a r e 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 niediodorsal position aiid will be referred to a s “ M - D cells” (see
fig. 5).
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 a r e probably iiidifferent cells up to this stage. This is concluded from the
fact that they a r e 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 a r e not embryonic o r 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 surpass the size of the larger neurons; from then on, a distinction between the two classes is no longer possible.
Since the contention that we a r e dealing with two groups
supplying separate peripheral fields is of considerable im-
D I F F E R E N T I A T I O N 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.
1. The two cell types differ in their developmeiatal patterns. 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 ezteroceptive reflex; activity of
the limb at 7 days (Visintini and Levi-Montalcini, '39). Furthermore, 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 exterocept i v e 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 proprioceptive n e u r o w . However, both groups are
likely to contain cells of other functional assignments as well.
3. The V-L cells and the M-D cells in limb-innervating
ganglia give differential 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 degefzP 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. I n
cases of peripheral overloading, the V-L cells show consistently 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
486
VIKTOR HAMBURGER A K D RITA LEVI-MONTALCINI
innervate different peripheral structures arid that their differentiation and their niaintainaiice a r e controlled by their
respective teriniiial areas. Specific iiistaiices supporting this
interpretation a r e given 011 page 478.
4. A similar difference in the behavior of V-L and If-D
cells is found in the developmental process of cervical mid
t h o r a c i c 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 Bueker ('48) on the iiiiiei~ationof tumoi. implants. H e finds that
mouse sarcoma 180 when transplanted in tlie place of the
hind limb bud, or between the latter and the somites of threeday embryos, are exclusively innervated by sensory fibers,
whereas the lateral niotor columns a r e 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 n o increase in total
cell numbers or only a n 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 u p 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
D I F F E R E X T I A T I O N 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 :
1. 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
488
VIKTOR HAMBURGER A S D R I T S LEVI-MOXTBLCINI
can be produced iii larvae and even after metamorphosis, by
limb transplantation. This shows that undifferentiated cells
capable of mitosis a r e 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 u p 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. Detwiler ('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 a n 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.
3. Cowtintied differeiitintioia of m u r o b l a s t s , t h e i r growth
and nzaisatainance depend o n 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
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 t o
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 regenerated 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 generally n e g l e ~ t e d .Yet,
~ this effect seems t o 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-Montalcini, unpublished ; trochlear nucleus, Dnnnebacke, nnpublished).
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 induction of indifferent cells by adjacent neuroblasts. Their assumption is largely
based on their failure t o 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 a s rapidly a s 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. Apparently, the peripheral factors modify the conditions within
the ganglion by which indifferent cells are stimulated to divide.
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. I n n o 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 usefulness of the terms “hypo- and liyperpla~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. I n using them, one should at
least make a distiction between a “numerical h y p e r - and hypoplasia,” as determined by cell counts, and a “columetric
h y p e r - and ltypoplasiu,”4 as determined by area measurenients 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 t o establish for our material the factors which are responsible f o 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-Dcells 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, permitting a more dense packing of the remaining cells. There is
good evidence to show that the numerical hypoplasia followiiig limb extirpation, remains constant from the 8th day on
through incubation (Levi-Montalcini and Levi, ’44) and t o
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. If,
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 methodological 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 misunderstandings in embryology.
D. M e c h a n i s m of peripheral control
of ganglioul. d e v e l o p m e n t
(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 t o 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 “poiiztto-point ” effect.
The situation is different in the case of peripheral effects
on proliferatioiz and initial 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.”
HOWare 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 f o r the regressive
changes in their cell bodies. One could think of two alternatives. First, the inhibition of further outgrowth and of the
spinning out of neurite material in itself may upset the metabolism 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 alternatives.
A remarkable fact stands out: the high susceptibility of
the V-L cells t o 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 T A L C I N I
The mechaiiisni by which peripheral factors control prolifercrtion aiid iiiitiul d if f er e nt ia ti on is more difficult to unders t a i d because cells a r e affected which have no direct
coiiiiections with the periphery. It is very unlikely that we
a r e dealing with diffusible substances since closely adjacent
nerve centers respond differentially to the same peripheral
changes. I n 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 a r c 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 a s a, “field”
within which each unit would be affected by changes in other
units. More or less specific hypotheses based on this reasoning have been suggested by Barron ( ’43, ’44) and Hamburger
and Keefe ( ’44).
The peripheral control cannot be uiiderstood in terms of
deficient ( o r 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 Mas
~ ,was suggested by some of the earlier authors.
The present material gives new evidence against this point
of view. F o r 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 a r c 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 physical or chemical conditions a t the periphery a r e 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 G A K G L I A
495
E'. Cell clege,ieratioi$ as a size regulating factor
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.
Differential proliferntiogz is undoubtedly an important factor in producing the regional size differences. The observations reported above have shown, unexpectedly, that differential degeneration of neurons contributes significantly t o
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 t o 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 L E V I - N O K T A L C I K I
cells are highly susceptible to eiiviroiiniental conditions. Obviously, 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, h a r e iiot been examined in sufficient
detail to give ail aiiswer. F o 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 ~ d oi e cell degeneration of
individual cells, and large-scale, localized and pattertied dcgeneration 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~pliosisa r e 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. I n 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 coniparnble in all details t o 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 A N D 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 initial
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.
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BARRON,
DONALD
H. 1943 The early developnieiit of the motor cells and columns
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1944 The early development of the sensory and interiiuncial cells
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RUSSELL 1932 Spinal-ganglion respoiises to the transplantation of
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VIKTOR 1934 The effects of wing bud extirpation on the development of the central nervous system in chick embryos. J. Exp. Zool.,
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1942 A Manual of Experimental Embryology. Univ. Chicago Press.
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1948 The mitotic patterns in the spinal cord of the chick embryo
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AND E. KEEFE 1944 The effects of peripheral factors on
HAMBURGER,
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RITA 1949 The development of the acoustico-vestibular centers in the chick embryo in the absence of the afferent root fibers and
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1939 Relazione t r a differenziazione strutturale e funzionale dei centri e delle vie nervose nell’embrione d i
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500
TIICTOR H A M B U R G E R AKD RITA LEVI-MONTALCINI
WEISS, P. s m HELENHISCOE1948 Experinleiits 011 the mechanism of iierve
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347-557.
PLATE 1
bYPLAT4TION O F F I G L R F S
4 Xornial 3 - d eiiil~yo.
~ ~
Ceriical ganglion, no. 12. S o t e the numerous clegenerating 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 iieuroblasts. Silver inipiegiiation after Cxjal-DeCastro.
6 Six-day embryo, right wing bud removed (41539). Large veiitrolatrral
cells in left brachial ganglion (no. 1 6 ) .
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
i n figiiie 8.
PLATIC 1
501