/ . Embryo/, exp. Morph. Vol. 22, 1, pp. 1-14, August 1969
Printed in Great Britain
Differentiation capacities
of the prospective tail somite region of the neural
plate in the embryos of Ambystoma mexicanum
By I. A. NIAZI 1
From the Department of Zoology, University ofRajas than,
Jaipur, India
The hind part of the neural plate in amphibian embryos has a mesodermal
significance although it occupies an ectodermal position till late neurula stage.
In anurans it gives rise to the posterior tail somites (Smithberg, 1954) and in
urodeles the somites of the posterior trunk region and of the entire tail together
with several other mesodermal structures (Bijtel, 1931; Nakamura, 1938;
Aufsess, 1941; Spofford, 1945; Chuang, 1947; Ford, 1949). Presence of mesoderm in the neural plate is an interesting developmental problem. During normal
gastrulation this region is at first underlaid by the future anterior part of the
archenteric roof which exerts a neuralizing inductive influence. It is only later
that the posterior part of the notochord with its mesodermalizing influence
comes to lie under it. According to Eyal-Giladi (1954), who worked on gastrula
stages of the Axolotl, even a short and transient contact of the invaginating
archenteric roof with the overlying ectoderm produces archencephalic induction
in the latter. It is therefore relevant to ask whether the posterior part of
the neural plate does or does not pass through a phase of neuralization before
assuming mesodermal properties. This specific question has not been investigated although a number of workers have studied the determination, differentition capacities or inductive properties of the caudal portion of the neural plate
in amphibians (Bytinski-Salz, 1931; Bijtel, 1936, 1958; Chuang, 1947; Nakamura, 1947; Spofford, 1948; Smithberg, 1954). Spofford (1948) demonstrated
that in Ambystoma mexicanum neurulae the tail notochord induces the overlying ectoderm to acquire mesodermal properties. He replaced the posterior
part of the neural plate by a piece of competent ectoderm which itself had never
been subjected to the influence of a neural inductor. This technique could
demonstrate that, under experimental conditions, the so-called transformation
or mesodermalization need not be preceded by activation or neuralization.
These results, however, do not show whether this is also true during normal
embryogenesis.
1
Author's address: Department of Zoology, University of Rajasthan, Jaipur, India.
I
J E E M 22
2
I. A. NIAZI
According to Nakamura (1947), the presumptive tail somite region of the
neural plate even from an early neurula stage is able to differentiate into muscle
tissue if grafted together with prospective tail neural tube. This finds support
in the results obtained by Takaya (1959) who explanted as sandwiches the neural
plate as a whole or in parts at various stages of neurulation. He found that from
the beginning the neural plate possesses self-differentiation potencies which
manifest themselves even in the absence of the underlying mesoderm. These
potencies are of two kinds: (a) that for neural differentiation existing along the
whole length of the neural plate and (b) that for muscle differentiation restricted
to its caudal end. Takaya had worked on Triturus pyrrhogaster, Hynobius
nebulosus and Rana nigromaculata. Smitherberg (1954), on the contrary, has
emphasized the importance of the presence of the notochord to enable differentiation to proceed in somites from the posterior neural plate in Rana pipiens.
If it is correct that definitive cytological and histological determination of the
prospective tail somite material in the neural plate does not occur until late
neural stage and that it is caused by the underlying tail notochord, then it
appears surprising that a combination of the prospective tail somite and spinalcord materials from even the earliest neurula stage should enable the former
to differentiate into muscle tissue. The present investigation was therefore
designed to ascertain the differentiation capacities of the prospective tail somite
region of the neural plate from early to late neurula stages of Ambystoma
mexicanum when explanted alone or in combination with spinal cord and/or
notochord.
MATERIALS AND METHODS
Early, middle and late neurulae of Ambystoma mexicanum at Harrison's
stages 13, 14^ and 17-18 were used in this study. Chuang's (1947) maps of
presumptive regions of tail organs were followed to locate the areas to be isolated from the neural plate at various stages of neurulation. The general procedure consisted of cutting out the specific regions from the neural plate with the
help of glass needles and hair loops and explanting the isolated parts, sandwiched in pieces of ectoderm taken from the mesoderm-free anterior belly
region of stage 12-13 embryos of the same species. Seven different experiments
were performed; they were numbered as series I-VII. The details of procedure
followed in each series are described in the text and are also presented in Table 1.
Figure 1 shows the location of the presumptive regions of tail tissues isolated
from the three neurula stages used in this investigation. A 20 x 20 (lcm2) ocular
grid was used to cut out exactly the same region(s) of the neural plate in each
embryo. The figure also shows the manner in which each embryo was oriented
in relation to the grid during the operation.
The sandwiches were cultivated for 11-13 days in Holtfreter's solution on
agar underlay. Penicillin G (20000 i.u./l.) was added to prevent infection. The
medium and agar underlays were changed every 2 days and the explants cleaned
Differentiation of tail somite region
3
of extruded cells with a hair loop. At the end of cultivation the explants were
sketched by camera lucida and fixed in Smith's fluid. They were later sectioned
at 7-8 /<• thickness and stained with Mallory's triple stain or haematoxylin and
eosin.
The donors of the prospective tail materials were also reared for an equivalent
period after which they were sketched, fixed in Smith's fluid and their posterior
parts sectioned for microscopical examination to ascertain if removal of specific
tail primordia caused any corresponding deficiencies in them.
TS
Ambystoma
mexicanum embryos
(Harrison's stages)
NC
SC
Stage 17
Fig. 1. Semi-schematic drawings of Harrison's stage 13, 14£ and 17 embryos of
Ambystoma mexicanum. The outlines were drawn by camera lucida at x 25 magnification. The central part of an ocular grid is superimposed at the same magnification
to indicate the orientation of embryos and the parts isolated during operation.
TS, Presumptive tail somite; SC, spinal-cord regions of the neural plate; NC, tail
notochord.
4
I. A. NIAZI
RESULTS
The results of microscopical examination of the explants of all seven experimental series are presented in Table 1.
Series I. In this series the prospective tail somite region was cut out from
stage 13 embryos and explanted alone in an ectodermal sandwich. After cultivation for 11-13 days no muscle differentiation was found in any of the fourteen
surviving explants. On the contrary, neural tissue had differentiated in nine
cases. These included seven cases of good neural-tube formation, one of which
is shown in Fig. 2 A. In the other two explants there were many melanophores
Table 1. Tissue differentiation obtained in explants of the prospective tail somite
and spinal-cord regions of the neural plate o/Ambystoma mexicanum neurulae,
made alone or in combination with notochord
(TS = prospective tail-somite region of the neural plate; SC = prospective
spinal-cord region of the neural plate; NC = tail notochord.)
No. of series
I
11
111
IV
Stage of donor embryos
Composition of explants
13
TS
144TS
17-18
TS
V
VI
VII
14i
14414*
14i
TS
+
SC
TS
+ NC
SC TS + SC
+ NC
Number <3f cases
Tissue types found in explants
2
Neural tissue + connective
9
3
tissue + mesenchyme
Connective tissue+ undif8
5
9
ferentiated and/or
degenerating cells
—
Neural tissue + muscle
—
—
+ connective tissue
_
—
—
Neural tissue + muscle
+ notochord + connective
tissue
Muscle + connective tissue
5
0
No. of explants lost in cultiva- 4
tion or processing
15
12
18
Total no. of explants made
in each series
* Notochordal element was discarded
6
9
—
2
1
—
—
—
1*
—
—
—
7
2
4
6
5
1*
6
12
16
13
1*
—
10
in these explants.
together with some groups of cells whose nature could not be identified. Many
of these cells were degenerating and contained pycnotic nuclei. Since melanophores are of neural origin these two cases are also considered as those of neural
differentiation. In the remaining five cases connective tissue alone or with masses
of degenerating cells was found.
Differentiation of tail somite region
5
The examination of the donors of the explanted prospective somite region
confirmed that the isolated part of the neural plate did consist of the tail somite
material, for in thirteen out of eighteen donors the tail was either very small or
totally absent. The remaining five donors had somewhat better tails but all were
deficient in muscles. Several of the short tails had no muscles at all.
Series II. In this series twelve explants of isolated prospective tail somite
region from the neural plate of stage 14^ embryos were cultivated as sandwiches
for 12-13 days. All survived but muscle tissue did not develop in any of them.
In nine only connective tissue, with or without degenerating masses of an
unidentifiable type of cell, was found. Figure 2B shows a section of one of these
explants. Neural differentiation was found in three cases, including two of
neural-tube formation and one in which only melanophores were present.
Among the donors of tail somite material seven did not develop a tail of any
appreciable length. In the remaining five cases the tails were much smaller than
normal and had little muscle tissue.
Series III. In this experiment stage 17-18 embryos were used as donors of the
prospective tail somite material to be sandwiched alone in ectodermal pieces
and explanted. Out of fifteen sandwiches thirteen survived cultivation for 11-13
days, but in three of them the tail material was probably lost and only empty
epidermal bags resulted. Analysis of the remaining ten showed the presence of
melanophores and connective tissue in one case, a small neural tube in another
and only connective tissue with undifferentiated or degenerating cells in the rest.
The sandwich containing neural tube also contained a large vesicle (Fig. 2C),
whose lumen was continuous with that of the neural tube. This structure could
be an enlarged part of the neural tube. The cells of its walls did not look like
myoblasts. Cords of cells of an unidentifiable type and arranged in a similar
manner around a large cavity were also observed in a few other cases belonging
to series V, described below.
Nine donors of the explanted somite material did not develop tails at all or
possessed very small tails. Musculature was deficient in the tails of the remaining
six also; their tails were smaller than normal and also abnormal in shape.
Series IV. Stage 14^ embryos were used in this series. A strip of presumptive
tail spinal-cord material was isolated from the neural plate and sandwiched alone
between pieces of ectoderm. Out of twelve explants cultivated for 13 days eight
survived and in six of them neural tubes were formed. Only connective tissue
was present in the remaining two cases, in one of which capillaries and some
blood cells were also found.
The donors of the explanted spinal-cord material developed more or less
normal tails in nine out of twelve cases. The tails were often curved upwards
and the ventral fin was not well developed. The tail was very defective in three
cases; corresponding explants from two of these donors did not have neural
tubes. It is possible that in these cases the operation had not succeeded in isolating the spinal-cord region of the neural plate. Instead, some presumptive somite
6
I. A. NIAZI
material may have been cut out by mistake, which may explain the defective
tails in the donors and the absence of neural differentiation in the explants.
Series V. A total of sixteen explants of neural-plate material from stage 14£
embryos were cultivated for 13 days in the usual manner. In each case a rather
broad strip of the neural plate was cut out and sandwiched between ectodermal
pieces. The isolated strip consisted of presumptive spinal-cord and tail somite
regions. Out often survivors nine contained good neural tube formations. Their
lumen was generally narrow but enlarged at places. In five cases packed mesen-
Differentiation of tail somite region
1
chymal cells were also seen but there was no sign of either segmentation or
myofibril formation to indicate any degree of muscle differentiation. In two
cases cords of cells were found around large cavities as was seen in one case of
series III, described earlier. Sections of such examples from this series are
presented in Figs. 2D and 3 A. In both cases the neural tubes were continuous
with these vesicular structures. One explant contained only connective tissue
and degenerating cells with pycnotic nuclei.
Among the donors of the neural-plate strip the post-cloacal projections consisted largely of fins of abnormal shape. Four donors developed short tails with
small somites.
Series VI. In this series also a broad strip of the neural plate including
prospective tail spinal-cord and somite regions was cut out from stage 14£
embryos. This strip was combined with a piece of notochord from below this
region of the same embryo. A total of thirteen such combinations were sandwiched, each between two pieces of belly ectoderm from another neurula, and
cultivated for 12-13 days. Five explants were lost. In seven of the surviving
eight cases neural tube, somites and notochord differentiated and showed
definite tail-like organization. One of them is shown in Fig. 3B. The remaining
sandwich had broken into two pieces whose sections showed a neural tube in
one and groups of coalescing myoblasts in the other (Fig. 3C). It seems that
the notochord was extruded as the sandwich broke into two fragments.
Almost no tail developed in ten out of thirteen donors of the presumptive tail
tissues. In the remaining three the tail was small; one of these was sectioned and
contained sparse musculature.
Series VII. In these experiments only the prospective tail somite region of the
neural plate and a piece of underlying notochord were cut out from stage 14^
embryos and combined in a sandwich. The explants were cultivated for 12 days.
Out of ten explants six were lost. Two contained neural tubes, notochord and
somites and one explant had only neural tube with some mesenchyme and conFlGURE 2
Abbreviations: N, neural tube; NC, notochord; S, somites; V, vesicular
structure found in serial sections to be continuous with the neural tube.
A. A section of a series I sandwich, cultivated for 13 days. Presumptive tail-somite
region of the neural plate of stage 13 embryo was explanted alone. Only neural tube
differentiated, x 100.
B. A section of a series 111 sandwich, cultivated for 11 days. Presumptive tail-somite
region of the neural plate of a stage 17 embryo was explanted alone. Only connective tissue was formed, x 100.
C. A section of a series III sandwich, cultivated for 13 days. The operational
scheme was the same as in the case shown in the previous figure, x 100.
D. A section of a series V sandwich, cultivated for 12 days. A strip of the neural
plate of a stage 14^ embryo including presumptive tail spinal-cord and tail-somite
region was explanted. x 100.
8
I. A. NIAZI
nective tissue. In this case also the notochordal element perhaps slipped out of
the sandwich early during cultivation. The fourth surviving explant had broken
into two fragments, one of which was lost during processing and the other found
D
E
Differentiation of tail somite region
9
to contain coalescing groups of myoblasts close to the inner side of the epidermis. They appeared to be segmentally arranged (Fig. 3D, E).
With one exception all other donors of the explanted material lacked tails
of any appreciable length. A normal tail had developed in one case.
DISCUSSION
The results of the experimental series I—III clearly point to the conclusion
that the presumptive tail somite region of the neural plate in Ambystoma
mexicanum at even late neurula stage is incapable of self-differentiation into
muscle tissue if cultivated alone in an ectodermal sandwich. For Triton pyrrhogaster, Nakamura (1947) reported that whereas at early neurula stage the presumptive tail somite material is unable to form muscles it is determined enough
to follow its prospective histological fate by the middle of neurulation and
acquires the ability of stretching and segmentation at late neurula stage. Bijtel
(1936, 1958) also obtained good muscle differentiation of the hind part of the
neural plate when it was isolated from prominent neural fold (mid-neurula)
stage of A. mexicanum embryos. It should be noted that both these authors
transplanted the neural-plate strips into the flank or belly of host neurulae.
This technique does not permit the development of the part concerned in complete isolation from possible inductive influences of the host tissues. Both
Nakamura (1947) and Smithberg (1954) found that the ability to differentiate
into muscle tissue is not present in the posterior region of the neural plate in
early neurula and it is gradually acquired during later stages of neurulation.
Our results support this conclusion but with the qualification that in isolation
muscle differentiation capacities are not manifested even when the prospective
tail somite material is taken from late neurula stage.
The results of series I also suggest the conclusion that in early neurula stage
FIGURE 3
Abbreviations: N, neural tube; NC, notochord; S, somites; V, vesicular
structure found in serial sections to be continuous with the neural tube.
A. A section of a series V sandwich, cultivated for 13 days. The operational scheme
was the same as for Fig. 2D. x 100.
B. A section of a series VI sandwich, cultivated for 11 days. A strip of the neural plate
of a stage 14£ embryo including presumptive tail spinal-cord and somite regions
was explanted together with the tail notochord from the same embryo, x 100.
C. Sandwich no. VII (10) of series VI had broken into fragments which were cultivated for 13 days. A section of each is shown. The operational scheme was the same
as in B. x 35.
D. A section of one of the two fragments of sandwich no. VII(10) of series VJI,
cultivated for 13 days. Presumptive tail-somite region of the neural plate of a stage
14^ embryo together with its tail notochord was explanted. x 35.
E. A high-power photomicrograph of part of the section shown in D. x 400.
10
I. A. NIAZI
(no. 13) when neural plate is barely recognizable, the presumptive tail region of
the plate possesses definite neural differentiation capacities which are manifested
even when it is cultivated alone. Thus, neural tissue differentiated in nine out of
fourteen surviving sandwiched explants of this series. These included seven
cases of good neural-tube formation and two cases in which melanophores
together with some mesenchyme were found. Melanophores are of neural origin
and their differentiation would indicate a rather weak degree of neuralization.
Hence, these two cases should also be regarded as indicating neural tendencies
in the explanted material. However, it is possible that some prospective neuralcrest material may have been inadvertently included in the strip cut out from
the neural plate. At stage 13 neural folds are not present and the neural crest
region is not demarcated clearly.
The neural differentiation tendencies in the caudal part of the neural plate
are, however, sharply reduced, if not completely abolished, in the mid- and late
neurula stages. In series II and III, in which the donors of this region of the
neural plate were at stages 14| and 17-18, respectively, only five out of twentyone surviving explants showed neural differentiation and two of these contained
only melanophores.
Eyal-Giladi (1954) analysed the differentiation tendencies of the presumptive
neurectoderm of A. mexicanum at different gastrula stages as it came successively
under the influence of cranio-caudal regions of the archenteric roof. It was found
that these tendencies change from those of more cephalic to more caudal
structures as the particular area of the neurectoderm comes under the influence
of more caudal parts of the notochord. The posterior region of the neural plate
comes to overlie the tail notochord at the very end of gastrulation and, therefore,
inferring from the results of Eyal-Giladi, it can be expected that a change in
differentiation tendencies would similarly occur in this region of the neural
plate, and that this change will proceed to completion during post-gastrula
stages. Accordingly, neural differentiation of isolated prospective tail somite
region should be expected in early neurula stages. Loss of this tendency and
mesodermal properties should be manifested in later stages, as these additional
results confirm.
It is not essential that neuralization precede mesodermalization of the ectoderm. Spofford (1948) demonstrated this by transplanting un-induced, competent
ectoderm over the tail notochord in Ambystoma neurulae, and Toivonen
(1958) has shown the same in ectodermal sandwiches using guinea-pig bone
marrow as the inductor. As far as production of purely mesodermal structures
is concerned, the above is also accepted by Nieuwkoop (see footnote in Nieuwkoop & Grinten, 1961). In our opinion, although under experimental conditions
competent ectoderm can be made to develop into mesodermal structures directly
without passing through a phase of neuralization, this does not appear to occur
during normal embryogenesis as far as the tail somite material is concerned.
The particular dorsal and posterior position of this region of neurectoderm in
Differentiation of tail somite region
11
the gastrula and the presence in it of neural competence together with the path
of chordal invagination determine that it must pass through a phase of neuralization before acquiring properties for mesodermal differentiation. However, the
neural determination of the caudal part of the neural plate remains labile and is
ultimately abolished under the persistent inductive influence of the tail notochord during neurulation.
According to Nakamura (1947), the prospective tail somite portion of the
neural plate of even an early neurula forms well-differentiated muscle if transplanted in combination with prospective tail spinal cord and/or notochord.
Takaya's (1959) results would also support these observations. In his explants
of the neural plate, somites developed along with the spinal cord, even when no
notochord appeared in the sandwiches. The results obtained in our experiments
do not confirm these observations of Nakamura and Takaya made on other
species. Thus, in our series V only neural tubes were formed with some undifferentiated mesenchyme in nine out of ten surviving explants although the explanted material included both tail somite and spinal-cord regions of the neural
plate. It may be argued that in our experiments the isolated strip of the neural
plate may not have included the tail somite region. This is, however, highly
improbable because if this were so the donors of the neural-plate strip should
have been expected to develop more or less normal tails as the loss of the spinalcord material at this stage is normally regulated to a great extent. This was so in
the donors of our series IV, in which only prospective spinal-cord area was
removed; the great majority of donors in this series developed almost normal
tails. In series V, in which the removed neural-plate strip included both tail
somite and spinal-cord regions, the great majority of donors developed either
very small and deficient tails or no tails at all. However, neural-tube formations
occurred in the majority of explants of this series as for the explants of series IV.
It seems correct to conclude that the spinal cord did not support muscle differentiation from the presumptive somite material in the sandwiches of series V.
In Nakamura's experiments mesodermal inductive influences from the tissues
of the hosts may have caused muscle differentiation in the transplanted prospective somite material of the neural plate in combination with spinal cord from
donor neurulae. It is not possible to visualize such extraneous influences in
Takaya's experiments, at least for those cases in which somites developed along
with spinal cord even in the absence of notochord.
Smithberg (1954) emphasized the importance of the notochord for proper
differentiation of muscle tissue from the neural plate material in Rana pipiens
embryos. The results obtained in our series VI and VII confirm and support
Smithberg. In the former series the explanted material was a combination of
prospective spinal-cord, tail somite region and tail notochord. In the latter
series tail somite portion of the neural plate was combined with the notochord
alone. In both series somite developed along with spinal cord and notochord in
nine out of twelve cases. In one of the remaining three cases, belonging to series
12
I. A. NIAZI
VI, notochord was not found but neural tube and somite development had
occurred. Perhaps the notochord slipped out of the sandwich during cultivation
but stayed long enough to induce muscle differentiation in the presumptive
somite material. One of the other two explants, belonging to series VII, contained only neural tube and dense mesenchyme and the other had only a few
small somites. Notochord was absent in both. It should be noted, however, that
among all the series of the present investigation somite development occurred
only in those explants in which notochord had been deliberately included.
Yamada (1940) had found in Triturus that proximity of notochord was important for proper differentiation even of already invaginated trunk somitic mesoderm isolated from neurula stages and explanted. It should be even more
important at stages when the prospective mesoderm is still on the surface and
not yet completely determined.
It may be noted that in series VII, although the neural-plate strip cut out and
combined with the notochord did not include spinal-cord material, the neural
tube did appear in all four surviving explants. Smithberg (1954) found the same
in his material. The notochordal piece included in the sandwich is not a pure
mesodermal inductor and that may explain why both neural and mesodermal
structures were formed.
SUMMARY
1. Differentiation capacities of the presumptive tail somite region of the
neural plate in Ambystoma mexicanum embryos were investigated by culturing
this region sandwiched in ectoderm either alone or in combination with spinal
cord and/or notochord.
2. When the presumptive tail somite material was taken from stages 13, \A\
or 17-18 embryos and cultured alone it did not differentiate into muscle.
3. The prospective tail somite material from early stage 13 neurulae on being
explanted alone gave rise to neural differentiations in nine out of fourteen surviving explants. The neural tendency was considerably reduced in material taken
from stages 14^ and 17-18 neurulae and explanted similarly. Small neural
formations occurred in only five out of twenty-one cases from these older stages.
4. In explants of prospective tail somite material from stage 14£ embryos
combined with spinal-cord rudiment of the same embryos, good neural tubes
were formed in nine out of ten cases but muscle differentiation did not occur in
any case.
5. When explants contained a combination of tail somite and spinal-cord
regions of the neural plate and tail notochord or of the somite material and
notochord alone, taken from stage 14£ neurulae, somite development occurred
in eleven out of twelve cases; the notochord and spinal cord were also present
in nine of the eleven explants.
Differentiation of tail somite region
13
RESUME
Capacite de differentiation de la region des somites caudaux presomptifs
de la plaque neurale chez les embryons d'Ambystoma mexicanum
1. On a etudie la capacite de differentiation de la region des somites caudaux
presomptifs de la plaque neurale d'embryons cYA. mexicanum en cultivant cette
region, dans un sandwich d'ectoderme, soit isolee, soit en combinaison avec
de la moelle epiniere et (ou) de la notochorde.
2. Quand le materiel presomptif des somites caudaux a ete preleve sur des
embryons des stades 13, 14£ ou 17-18 et mis seul en sandwich, il ne s'est pas
differencie en muscles, sauf dans un cas.
3. Le materiel presomptif des somites caudaux de neurulas a partir du debut
du stade 13, explante isolement, a donne naissance a des differentiations neurales
dans neuf explants sur quatorze survivants. La tendance neurale etait considerablement reduite dans ce materiel pris sur des neurulas des stades 14£ et 17-18
et explante de la meme facon. De petites formations neurales sont apparues
dans cinq cas seulement sur vingt et un de ces stades plus ages.
4. Dans les explants de materiel somitique caudal presomptif d'embryons
du stade 14£ combine a une ebauche de moelle epiniere des memes embryons,
des tubes nerveaux convenables se sont formes dans neuf cas sur dix mais la
differentiation musculaire ne s'est jamais produite.
5. Quand les explants renfermaient une combinaison formee de regions des
somites caudaux et de la moelle epiniere de la plaque neurale avec de la notochorde caudale, ou de materiel somitique et de notochorde seule, preleves sur
des neurales du stade 14^, le developpement des somites a eu lieu dans onze
cas sur douze; la notochorde et la moelle epiniere etaient egalement presentes
dans neuf des onze explants.
I am grateful to Professor Dr P. D. Nieuwkoop, Director of Hubrecht Laboratory,
Utrecht (Holland), who suggested the problem and provided the necessary facilities during
my work at this laboratory with the 5th International Research Team in Embryology. The
experimental work was done at Utrecht and the data were processed and analysed at the
Zoology Department of the University of Rajasthan, Jaipur (India). I am also grateful to the
government of the Netherlands for a Fellowship which made this study possible.
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{Manuscript received 17 September 1968)