/. Embryol. exp' Morph. Vol. 47, pp. 137-148 1978
Printed in Great Britain (g) Company of Biologists Limited 1978
137
On the origin of the ciliary ganglion
in birds studied by the method of interspecific
transplantation of embryonic brain regions
between quail and chick
By C. H. NARAYANAN 1 AND Y. NARAYANAN 1
From the Department of Anatomy, Louisiana State University School
of Medicine
SUMMARY
The development of the ciliary ganglion with reference to the site and cells of origin have
been investigated by the method of quail-to-chick transplantation of embryonic fore-, midand hindbrain regions, and by the method of transplantation of cranial neural crest from
specific brain levels. In chimaerical embryos, quail cells originating from the graft end up in
the ciliary ganglion, only when the graft is from the midbrain level of quail embryo donors.
In fore- and midbrain grafts the ciliary ganglia of chimaerical embryos are composed of
chick cells only. The results indicate that the mesencephalon is the principal site for the precursor cells of the ciliary ganglia and clearly rules out any contribution to the ganglia from
either the forebrain or hindbrain levels.
In interspecific transplantation of cranial neural crest, quail cells originating from the
graft are observed consistently in the ciliary ganglion of the operated side when the grafted
neural crest mateiial is derived from the mesencephalon of quail embryo donors. On the
basis of the evidence provided, it is concluded that the mesencephalon is the principal site
and the cranial neural crest of this level the source from which the precursor neurons of the
ciliary ganglia are derived.
INTRODUCTION
In trying to understand the site and cells of origin of cranial ganglia, the
neuroembryologist is faced with problems of identifying those cells which
contribute to the formation of the ganglion under investigation, the precise
site of origin of those cells, and their migration patterns. These problems have
been overcome in recent years by the use of quail cells as natural biological
cell markers in interspecific transplantation of tissues or organ primordia
between quail and other avian embryos. Such studies already have found
important applications in problems dealing with migration patterns and the
fate of neural crest cells in the development of cranial ganglia, autonomic
ganglia and other head structures (Le Douarin, 1973, 1975; Le Douarin &
1
Authors' address: Department of Anatomy, Louisiana State University School of
Medicine, 1100 Florida Avenue, New Orleans, Louisiana 70119, U.S.A.
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C.H.NARAYANAN AND Y.NARAYANAN
Teillet, 1973, 1974; Le Lievre & Le Douarin, 1975; Narayanan & Narayanan,
1977).
The present study was suggested by earlier work in which the contribution of
cranial neural crest cells from various embryonic levels was investigated in order
to determine the site and cells of origin of the mesencephalic nucleus of the
trigeminal nerve (Narayanan & Narayanan, 1978). Although a few quail cells
were observed in the ciliary ganglion in some of the cases in the above experiments, it was impossible to assign them with certainty to any specific site or
cells of origin. However, a cranial neural crest origin has been postulated for
the ciliary ganglion of the chick embryo by Hammond & Yntema (1958). Their
experiments showed that removal of the neural crest and adjacent fold between
the optic vesicle and the first somite resulted in the absence or marked depletion
of the neurons composing the ciliary ganglion in the chick. There is controversy
on the site of origin of the precursor cells of the ciliary ganglion, since Jones
(1945) observed in three of his experimental cases that removal of the forebrain
between the optic stalks together with the midbrain resulted in the absence of
the ciliary ganglion in the chick. This led him to conclude that the ciliary
ganglion in the chick develops from cells which migrate from the forebrain.
The evidence on the cells of origin is likewise conflicting since Levi-Montalcini &
Amprino (1947), in an extensive series of experiments on chicks, between 5 and
12 somites of development, removed the neural crest, head ectoderm and
rudiments of the brain, singly and in various combinations, and concluded that
mesoderm, not ectoderm, gave rise to the neurons of the ciliary ganglion. In all
of these studies the operative procedures resulted in abnormalities of head
structures including absence of other cranial ganglia, so that it was difficult to
precisely determine the source and the principal site of origin of the ciliary
ganglion.
We have recently used the method of interspecific transplantation of cranial
neural crest successfully between quail and duck embryos in our investigations
on the developmental origin of the mesencephalic nucleus of the trigeminal
nerve (Narayanan & Narayanan, 1978). This method, therefore, offers a new
approach to the investigation of the problems indicated above and should
yield information on the precise localization and cells of origin of the ciliary
ganglion. In the present study we have reinvestigated the origin of the ciliary
ganglion in the chick embryo by the method of interspecific transplantation of
embryonic brain regions and presumptive neural crest material between quail
embryo-donors and chick embryo-hosts. Our results indicate that the cranial
neural crest constitute the source, and the mesencephalon is the principal site
of origin for the precursor neurons of the avian ciliary ganglion.
Origin of the ciliary ganglion
139
MATERIALS AND METHODS
Fertile eggs from which the embryos were raised for this experiment came
from the Babcock strain of fowls and the Japanese quail Coturnix coturnix
japonica, maintained in the animal care facility of this medical center. The
eggs were incubated in forced draft incubators maintained at 37-5 °C and relative humidity of 65-70%. The eggs were set for incubation times as follows:
30-33 h for the chick, and 27-30 h for the quail, in order to attain developmental
stage 8 (Hamburger & Hamilton, 1951) and stage 7 (Zacchei, 1961) respectively,
at the time of operation. The method of opening the eggs and preparing the
embryos for transplantation experiments are procedures that we have routinely
followed in all of our experiments on avian embryos in this laboratory. These
procedures have been described in detail previously (Narayanan, 1970; Narayanan & Narayanan, 1978).
GRAFTING PROCEDURE
Appropriately-staged quail embryo-donors, and chick embryo-hosts, selected
for each of the experiments below were placed in egg holders within a two-egg
operating carousel (Narayanan, 1970). The microsurgery was carried out under
strict aseptic conditions. The vitelline membrane and the embryonic brain
region to be removed from the host embryo (chick) was lightly stained with
neutral red impregnated in agar. The desired embryonic brain region of the
donor embryo (quail) to be manipulated was likewise stained lightly with Nile
blue sulfate impregnated in agar as described in previous experiments (Narayanan & Hamburger, 1971). Each designated embryonic region demarcated
externally by transverse constrictions was surgically removed from the chick
embryo, by clean incisions using a vibrating needle with extremely sharp cutting
edges and fine point (Wenger, 1968). The corresponding embryonic brain level
from a donor embryo was then carefully excised, and was transferred by means
of a Spemann micropipette from donor to host embryo. In order to ensure a
proper fit and good adhesion between host and graft tissues, excess fluid around
the grafted areas was carefully withdrawn with a micropipette. Using this
procedure outlined above, three series of experiments as shown in Fig. 1 were
made:
(1) QFB series, graft consisting of the embryonic forebrain region derived
from quail embryo-donor.
(2) QMB series, graft of the mesencephalon of the quail embryo.
(3) QHB series, graft of the rostral hindbrain of the quail embryo, from about
the level of the caudal limits of the mesencephalon anteriorly to about the level
of the otic capsule posteriorly.
The experiments 1-3 were designed to determine the precise site of origin of
the precursor cells of the ciliary ganglion. Based on the results obtained from
these experiments, in a last series, interspecific transplantation of cranial neural
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C.H.NARAYANAN AND Y.NARAYANAN
1
QFB series
QHB series
Donor-quail embryos
Stage 7 27-30 h
QMB series
1, 2. 3: Host-chick embryos
Stage 8 30-33 h
Fig. 1. In the experiments shown schematically, embryonic fore-, mid- and hindbrain
regions from quail embryo donors are excised and grafted in the place of embryonic
brain regions of corresponding levels previously removed from chick embryo hosts.
The resulting chimaerical embryos are designated: QFB, with quail forebrain grafts;
QMB, with quail midbrain grafts; QHB, with quail hindbrain grafts.
Origin of the ciliary ganglion
141
crest alone between quail embryo-donors and chick embryo-hosts was carried
out as described by Narayanan & Narayanan 0978) in order to determine the
cells of origin of the ciliary ganglion in the chick. After completion of the
operations, the egg was sealed and returned to the incubator for further development. The operated embryos were raised for periods ranging from 9 to
16 days of incubation age. They were fixed in Zenker's fluid by immersion or
transcardiac perfusion. The heads were processed for paraffin embedding,
serially sectioned at 12 /tm in a transverse plane, and stained according to
Feulgen & Rossenbeck's procedure (1924), In all, 30 embryos were used in this
study. Of these eight belonged to QFB series, six to the QMB series, and six
were of the QHB series. Ten embryos had transplanted neural crest from quail
embryo-donors.
RESULTS
Effects of grafted embryonic brain regions on the development of the ciliary ganglion
Quail for ebrain grafts (QFB series). Of the eight specimens in this series, two
were fixed on day 9, two on day 11, and the remainder on day 13 of incubation.
In all the above experimental cases, healing was very good, and the union
between the quail forebrain graft and the rest of the host tissues was perfect.
In chimaerical embryos of 9 and 11 days of incubation ages, the eyes and upper
beak derived from the quail graft were symmetrical and normal in external
appearance. The head structure of quail origin in the experimental animals
seemed to be in harmonious relationship to the rest of the host both in size and
in position. However, with long survival times, as in animals fixed on day 13
of incubation, quail features were very evident. The upper beak was shorter
than the lower (Fig. 2). The skin in this region of the head derived from graft
tissue showed the typical black, brown and yellow color pattern of the quail.
Eyelids were well developed, feathered and the opening was reduced to a slit.
Figure 3 is a section through the head of an 11-day chimaerical embryo(QFB 2).
The ciliary ganglion is located at the apex of the orbit and is in proper relations
with other orbital structures. While all of the surrounding tissues of the head
region in this section are composed of quail cells, the ciliary ganglion alone is
exclusively composed of chick cells. Histologic examination of sections through
the head region in each case confirmed the fact that the neurons found in the
ciliary ganglia aie always derived from the chick embryo hosts. Since quail cells
are not found in ciliary ganglia in any of the chimaerical embryos in this series
of experiments, it may be concluded that the forebrain graft of the quail has
made no contributions per se to the ciliary ganglia under the conditions of the
experiment.
Quail midbrain grafts (QMB series). Six specimens have been studied; two
were fixed on day 9, two on day 11 and two on day 15 of incubation. The size of
the optic tecta in all six cases is much smaller when compared to that of a normal
chick embryo, but well within the range of development of the tecta in normal
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C. H. NARAYANAN AND Y. NARAYANAN
Origin of the ciliary ganglion
143
quail embryos at corresponding stages of development. The transplantation did
not seem to affect the relative development of any of the head structures. The
eyes are normal in external appearance, and no deformity is observed in any of
the specimens examined. The wound region had healed very well and was
covered with skin. As our findings based on histologic examination are essentially the same in the six cases included for this report, only one, QMB 3 (9 days
old), will be described here. Figure 4 is a high-power photomicrograph of a
section through the central part of the ciliary ganglion. Notice the absence of
chick cells in the ganglion. The majoiity of the cells in the ciliary ganglion have
the typical morphological characteristics of quail cells as revealed by the large
amount of DNA condensed in the form of a Feulgen positive heterochromatic
mass when stained by the Feulgen-Rossenbeck's technique. Every experiment
with midbrain graft of the quail shows unequivocal presence of quail cells in
the ciliary ganglion, which suggests that the embryonic mesencephalon is the
site of origin for the neurons of the ciliary ganglia.
Quail rostral hindbrain grafts (QHB series). Of the six specimens belonging to
this series, three were fixed on the 11th day of incubation, and three on the
13th day of incubation. The transplantation of the quail hindbrain was successful
in all cases, and no distortion of the head region or any of the related structures
was observed. Histologic examination of sections through the head region of
the chimaerical embryos, show the presence of chick cells in the ciliary ganglia.
Figure 5 is a high-power photomicrograph of a section through the ciliary
ganglion of experimental case QHB 1 of 11 days of incubation age, and illustrates very well the total absence of quail cells in the ciliary ganglion. Since
quail cells are not found in the ciliary ganglion in this group of animals, it may
be concluded that the hindbrain is not a source from which the neurons of the
ciliary ganglia are derived.
Effects of neural crest transplantation from quail embryo-donors on the development of the ciliary ganglion
Based on the results of the experiments involving the transplantation of
embryonic brain regions as described above, it became evident to us that the
embryonic mesencephalon was indeed the only brain region from which the
precursor cells of the ciliary ganglion migrated to their final location at the apex
FIGURES 2 AND 3
Fig. 2. A chimaerical embryo (QFB 15) of 13 days incubation age showing excellent
union of graft and host tissues. Observe the short upper beak derived from the quail
graft and also the pattern of pigmentation on the dorsal aspect of the head typical
of the quail.
Fig. 3. A high-power photomicrograph of a section through the ciliary ganglion of a
chimaerical embryo (QFB 2, 11 days), Feulgen and Rossenbeck's staining. Note
the absence of quail cells in the ciliary ganglion, while all of the surrounding tissues
are of quail origin. The lateral rectus (LR) is also composed of chick cells.
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C. H. NARAYANAN AND Y. NARAYANAN
Fig. 4. A high-power view of the ciliary ganglion of QMB 3,9 days of incubation age.
Quail cells with prominent nucleoli are clearly seen in thefield.Feulgen and Rossenbeck's stain.
Fig. 5. A high-power photomicrograph of the ciliary ganglion in case QHB 1,
11 days of incubation age. Observe that there are no quail cells in the ganglion.
Origin of the ciliary ganglion
145
Fig. 6. A high-power view of case NC 12, 16 days old. Quail cells with prominent
nucleoli (arrows) derived from the cranial neural crest graft are clearly recognizable.
of the orbit. Therefore in the next series of experiments, attempts were made to
transplant orthotopically neural crest fragment of midbrain levels from quail
embryo-donors to replace a similar fragment previously removed from chick
embryo-hosts. The graft was made unilaterally and usually on the right side.
Good incorporation of the neural crest graft from the mesencephalon was
observed in all the ten cases. The operated region healed well and was covered
with skin. The effect of neural crest transplantation was assessed in each case by
histologic examination. Quail cells were found in the ciliary ganglion of the right
side in all the specimens used in this study. Although no efforts were made to
count the number of quail cells, it is our impression that the ciliary ganglion of
the experimental side was composed predominantly of quail cells. Incomplete
operation or regeneration or a combination of both could very well account
for the presence of a few residual chick cells in the ciliary ganglion of the
operated side in our experimental animals. Apparently, a complete replacement
of the neuial crest of mesencephalic region by microsurgery in early stages is
perhaps next to impossible. However, qualitatively, it appears that in every
experiment of this series, the presence of quail cells in the ciliary ganglion of the
experimental side is unmistakable. Photomicrograph of a section through the
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C. H. NARAYANAN AND Y. NARAYANAN
ciliary ganglion in one case (NC 12; 16-day embryo) is shown in Fig. 6. Notice
that with the exception of a very few chick cells, the majority of the cells in the
ciliary ganglion in this case is of quail origin.
DISCUSSION
Previous studies, to which reference has been made in the Introduction,
indicate clearly that the site and cells of origin of the ciliary ganglion during
development remain controversial and invite explorations through the application of new, yet reliable methods such as the 'quail-chick marking system'
devised by Le Douarin (1973). The reliability of the cell labeling technique
using quail cells as 'natural markers' in the study of various embryological
problems, particularly in the analysis of the developmental fate and migration
pattern of cranial neural crest cells, has been repeatedly demonstrated in
numerous interspecific transplantation experiments between embryos of quail
and chick or duck (Le Douarin, 1973, 1974, 1975; Le Douarin &Teillet, 1974;
Le Lievre & Le Douarin 1975; Fontaine & Le Douarin, 1977; Narayanan &
Narayanan, 1978).
In the present study, we have employed the 'quail-chick marking system'
with a view to answer two pertinent questions. What is the source of the neurons
of the ciliary ganglion? Wheie do these precursor neurons come from (site)?
In a previous study, Jones (1945) observed that removal of the forebrain between
the optic stalks together with the midbrain resulted in the absence of the ciliary
ganglion and concluded that the ciliary ganglion develops from cells which
migrate from the forebrain. This conclusion was subsequently refuted by
Levi-Montalcini & Amprino (1947) who showed that removal of the eye and
the entire forebrain was quite ineffective, but that removal of the head at the
level of the anterior end of the hindbrain in chick embryos of 7-12-somite
stages almost invariably resulted in the absence of the ciliary ganglion. However, their operation procedures were too drastic to implicate any other
embryonic brain region specifically.
In our experiments, neither the forebrain nor the hindbrain prove to be
specific sites for the origin of the precursor cells of the ciliary ganglion. This is
borne out by the fact that in chimaerical embryos of our forebrain and hindbrain transplantation experiments between quail and chick embryos, the ciliary
ganglia were exclusively composed of chick cells. This clearly rules out the
possibility of their origin from either of these sites. On the other hand, quail
cells are found in the ciliary ganglion of chimaerical embryos when the graft
is from the midbrain level of quail donors. These observations are consistent,
and suggest that the precursor cells of the ciliary ganglion are located at the
level of the embryonic midbrain (mesencephalon) from where they migrate to
their final location in the orbit.
Finally, the most critical point of these results remains to be discussed,
namely the neural crest origin for the precursor cells of the ciliary ganglion. It
Origin of the ciliary ganglion
147
was pointed out earlier that Levi-Montalcini & Amprino (1947) proposed a
mesodermal origin for the cells of the ciliary ganglion. However, their extirpation experiments were too drastic, eliminating totally not only the head mesoderm but also the migrating neural crest material, thus making it difficult to
ascribe the origin of the cells specifically to either the mesoderm or neural crest.
Hammond & Yntema (1958), on the basis of their extirpation experiments,
demonstrated that removal of neural crest and adjacent fold between the optic
vesicle and the first somite which included the embryonic midbrain and hindbrain levels, resulted in the absence or a marked depletion of the neurons
composing the ciliary ganglion in the chick. Although they have implicated a
neural crest origin for these neurons, a precise site of origin could not be
specified from their experiments.
Our own evidence on this point is based on transplantation of cranial neural
crest from the level of the mesencephalon between quail embryo donors and
chick embryo hosts. Histological examination of the ciliary ganglion of the
operated side (right) in the experimental animals showed consistently the presence of quail cells which would validate the conclusion that they are indeed
derived from neuial crest of that specific brain region. The view favored by the
results of the present experimental analysis, places the primary emphasis on
the mesencephalon as the principal site and the cranial neural crest of this level
as the cells of origin for the precursor neurons of the ciliary ganglia in the chick
embryo.
We should like to thank Mr Thomas Lee for his technical assistance in the histological
part of this study, and Mrs Susan Orazio for her careful typing of the manuscript and
secretarial assistance. This investigation was supported by Research Grant DE 04258-02
from the National Institute of Dental Research.
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{Received 13 March 1978, revised 17 May 1978)