/ . Embryol. exp' Morph. Vol. 47, pp. 137-148 1978 137Printed in Great Britain (g) Company of Biologists Limited 1978

On the origin of the ciliary ganglionin birds studied by the method of interspecific

transplantation of embryonic brain regionsbetween quail and chick

By C. H. NARAYANAN1 AND Y. NARAYANAN1

From the Department of Anatomy, Louisiana State University Schoolof Medicine

SUMMARYThe 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-, mid-and hindbrain regions, and by the method of transplantation of cranial neural crest fromspecific brain levels. In chimaerical embryos, quail cells originating from the graft end up inthe 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 ofchick cells only. The results indicate that the mesencephalon is the principal site for the pre-cursor cells of the ciliary ganglia and clearly rules out any contribution to the ganglia fromeither the forebrain or hindbrain levels.

In interspecific transplantation of cranial neural crest, quail cells originating from thegraft are observed consistently in the ciliary ganglion of the operated side when the graftedneural crest mateiial is derived from the mesencephalon of quail embryo donors. On thebasis of the evidence provided, it is concluded that the mesencephalon is the principal siteand the cranial neural crest of this level the source from which the precursor neurons of theciliary ganglia are derived.

INTRODUCTION

In trying to understand the site and cells of origin of cranial ganglia, theneuroembryologist is faced with problems of identifying those cells whichcontribute to the formation of the ganglion under investigation, the precisesite of origin of those cells, and their migration patterns. These problems havebeen overcome in recent years by the use of quail cells as natural biologicalcell markers in interspecific transplantation of tissues or organ primordiabetween quail and other avian embryos. Such studies already have foundimportant applications in problems dealing with migration patterns and thefate of neural crest cells in the development of cranial ganglia, autonomicganglia and other head structures (Le Douarin, 1973, 1975; Le Douarin &

1 Authors' address: Department of Anatomy, Louisiana State University School ofMedicine, 1100 Florida Avenue, New Orleans, Louisiana 70119, U.S.A.

138 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 ofcranial neural crest cells from various embryonic levels was investigated in orderto determine the site and cells of origin of the mesencephalic nucleus of thetrigeminal nerve (Narayanan & Narayanan, 1978). Although a few quail cellswere observed in the ciliary ganglion in some of the cases in the above experi-ments, it was impossible to assign them with certainty to any specific site orcells of origin. However, a cranial neural crest origin has been postulated forthe ciliary ganglion of the chick embryo by Hammond & Yntema (1958). Theirexperiments showed that removal of the neural crest and adjacent fold betweenthe optic vesicle and the first somite resulted in the absence or marked depletionof the neurons composing the ciliary ganglion in the chick. There is controversyon 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 forebrainbetween the optic stalks together with the midbrain resulted in the absence ofthe ciliary ganglion in the chick. This led him to conclude that the ciliaryganglion 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 and12 somites of development, removed the neural crest, head ectoderm andrudiments of the brain, singly and in various combinations, and concluded thatmesoderm, not ectoderm, gave rise to the neurons of the ciliary ganglion. In allof these studies the operative procedures resulted in abnormalities of headstructures including absence of other cranial ganglia, so that it was difficult toprecisely determine the source and the principal site of origin of the ciliaryganglion.

We have recently used the method of interspecific transplantation of cranialneural crest successfully between quail and duck embryos in our investigationson the developmental origin of the mesencephalic nucleus of the trigeminalnerve (Narayanan & Narayanan, 1978). This method, therefore, offers a newapproach to the investigation of the problems indicated above and shouldyield information on the precise localization and cells of origin of the ciliaryganglion. In the present study we have reinvestigated the origin of the ciliaryganglion in the chick embryo by the method of interspecific transplantation ofembryonic brain regions and presumptive neural crest material between quailembryo-donors and chick embryo-hosts. Our results indicate that the cranialneural crest constitute the source, and the mesencephalon is the principal siteof 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 camefrom the Babcock strain of fowls and the Japanese quail Coturnix coturnixjaponica, maintained in the animal care facility of this medical center. Theeggs were incubated in forced draft incubators maintained at 37-5 °C and rela-tive 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 developmentalstage 8 (Hamburger & Hamilton, 1951) and stage 7 (Zacchei, 1961) respectively,at the time of operation. The method of opening the eggs and preparing theembryos for transplantation experiments are procedures that we have routinelyfollowed in all of our experiments on avian embryos in this laboratory. Theseprocedures have been described in detail previously (Narayanan, 1970; Nara-yanan & Narayanan, 1978).

GRAFTING PROCEDURE

Appropriately-staged quail embryo-donors, and chick embryo-hosts, selectedfor each of the experiments below were placed in egg holders within a two-eggoperating carousel (Narayanan, 1970). The microsurgery was carried out understrict aseptic conditions. The vitelline membrane and the embryonic brainregion to be removed from the host embryo (chick) was lightly stained withneutral red impregnated in agar. The desired embryonic brain region of thedonor embryo (quail) to be manipulated was likewise stained lightly with Nileblue sulfate impregnated in agar as described in previous experiments (Nara-yanan & Hamburger, 1971). Each designated embryonic region demarcatedexternally by transverse constrictions was surgically removed from the chickembryo, by clean incisions using a vibrating needle with extremely sharp cuttingedges and fine point (Wenger, 1968). The corresponding embryonic brain levelfrom a donor embryo was then carefully excised, and was transferred by meansof a Spemann micropipette from donor to host embryo. In order to ensure aproper fit and good adhesion between host and graft tissues, excess fluid aroundthe grafted areas was carefully withdrawn with a micropipette. Using thisprocedure outlined above, three series of experiments as shown in Fig. 1 weremade:

(1) QFB series, graft consisting of the embryonic forebrain region derivedfrom 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 levelof the otic capsule posteriorly.

The experiments 1-3 were designed to determine the precise site of origin ofthe precursor cells of the ciliary ganglion. Based on the results obtained fromthese experiments, in a last series, interspecific transplantation of cranial neural

140 C.H.NARAYANAN AND Y.NARAYANAN

1

QFB series

QHB series

Donor-quail embryosStage 7 27-30 h QMB series

1, 2. 3: Host-chick embryosStage 8 30-33 h

Fig. 1. In the experiments shown schematically, embryonic fore-, mid- and hindbrainregions from quail embryo donors are excised and grafted in the place of embryonicbrain 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 carriedout as described by Narayanan & Narayanan 0978) in order to determine thecells of origin of the ciliary ganglion in the chick. After completion of theoperations, the egg was sealed and returned to the incubator for further de-velopment. The operated embryos were raised for periods ranging from 9 to16 days of incubation age. They were fixed in Zenker's fluid by immersion ortranscardiac perfusion. The heads were processed for paraffin embedding,serially sectioned at 12 /tm in a transverse plane, and stained according toFeulgen & Rossenbeck's procedure (1924), In all, 30 embryos were used in thisstudy. Of these eight belonged to QFB series, six to the QMB series, and sixwere of the QHB series. Ten embryos had transplanted neural crest from quailembryo-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, twowere 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 unionbetween 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 upperbeak derived from the quail graft were symmetrical and normal in externalappearance. The head structure of quail origin in the experimental animalsseemed to be in harmonious relationship to the rest of the host both in size andin position. However, with long survival times, as in animals fixed on day 13of incubation, quail features were very evident. The upper beak was shorterthan the lower (Fig. 2). The skin in this region of the head derived from grafttissue 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 relationswith other orbital structures. While all of the surrounding tissues of the headregion in this section are composed of quail cells, the ciliary ganglion alone isexclusively composed of chick cells. Histologic examination of sections throughthe head region in each case confirmed the fact that the neurons found in theciliary ganglia aie always derived from the chick embryo hosts. Since quail cellsare not found in ciliary ganglia in any of the chimaerical embryos in this seriesof experiments, it may be concluded that the forebrain graft of the quail hasmade no contributions per se to the ciliary ganglia under the conditions of theexperiment.

Quail midbrain grafts (QMB series). Six specimens have been studied; twowere fixed on day 9, two on day 11 and two on day 15 of incubation. The size ofthe optic tecta in all six cases is much smaller when compared to that of a normalchick embryo, but well within the range of development of the tecta in normal

10 EMB 47

142 C. H. NARAYANAN AND Y. NARAYANAN

Origin of the ciliary ganglion 143

quail embryos at corresponding stages of development. The transplantation didnot seem to affect the relative development of any of the head structures. Theeyes are normal in external appearance, and no deformity is observed in any ofthe specimens examined. The wound region had healed very well and wascovered with skin. As our findings based on histologic examination are essenti-ally the same in the six cases included for this report, only one, QMB 3 (9 daysold), will be described here. Figure 4 is a high-power photomicrograph of asection through the central part of the ciliary ganglion. Notice the absence ofchick cells in the ganglion. The majoiity of the cells in the ciliary ganglion havethe typical morphological characteristics of quail cells as revealed by the largeamount of DNA condensed in the form of a Feulgen positive heterochromaticmass when stained by the Feulgen-Rossenbeck's technique. Every experimentwith midbrain graft of the quail shows unequivocal presence of quail cells inthe ciliary ganglion, which suggests that the embryonic mesencephalon is thesite of origin for the neurons of the ciliary ganglia.

Quail rostral hindbrain grafts (QHB series). Of the six specimens belonging tothis series, three were fixed on the 11th day of incubation, and three on the13th day of incubation. The transplantation of the quail hindbrain was successfulin all cases, and no distortion of the head region or any of the related structureswas observed. Histologic examination of sections through the head region ofthe chimaerical embryos, show the presence of chick cells in the ciliary ganglia.Figure 5 is a high-power photomicrograph of a section through the ciliaryganglion of experimental case QHB 1 of 11 days of incubation age, and illu-strates very well the total absence of quail cells in the ciliary ganglion. Sincequail cells are not found in the ciliary ganglion in this group of animals, it maybe concluded that the hindbrain is not a source from which the neurons of theciliary ganglia are derived.

Effects of neural crest transplantation from quail embryo-donors on the develop-ment of the ciliary ganglion

Based on the results of the experiments involving the transplantation ofembryonic brain regions as described above, it became evident to us that theembryonic mesencephalon was indeed the only brain region from which theprecursor 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 excellentunion of graft and host tissues. Observe the short upper beak derived from the quailgraft and also the pattern of pigmentation on the dorsal aspect of the head typicalof the quail.Fig. 3. A high-power photomicrograph of a section through the ciliary ganglion of achimaerical embryo (QFB 2, 11 days), Feulgen and Rossenbeck's staining. Notethe absence of quail cells in the ciliary ganglion, while all of the surrounding tissuesare of quail origin. The lateral rectus (LR) is also composed of chick cells.

144 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 the field. Feulgen and Rossen-beck'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 prominentnucleoli (arrows) derived from the cranial neural crest graft are clearly recognizable.

of the orbit. Therefore in the next series of experiments, attempts were made totransplant orthotopically neural crest fragment of midbrain levels from quailembryo-donors to replace a similar fragment previously removed from chickembryo-hosts. The graft was made unilaterally and usually on the right side.Good incorporation of the neural crest graft from the mesencephalon wasobserved in all the ten cases. The operated region healed well and was coveredwith skin. The effect of neural crest transplantation was assessed in each case byhistologic examination. Quail cells were found in the ciliary ganglion of the rightside in all the specimens used in this study. Although no efforts were made tocount the number of quail cells, it is our impression that the ciliary ganglion ofthe experimental side was composed predominantly of quail cells. Incompleteoperation or regeneration or a combination of both could very well accountfor the presence of a few residual chick cells in the ciliary ganglion of theoperated side in our experimental animals. Apparently, a complete replacementof the neuial crest of mesencephalic region by microsurgery in early stages isperhaps next to impossible. However, qualitatively, it appears that in everyexperiment of this series, the presence of quail cells in the ciliary ganglion of theexperimental side is unmistakable. Photomicrograph of a section through the

146 C. H. NARAYANAN AND Y. NARAYANAN

ciliary ganglion in one case (NC 12; 16-day embryo) is shown in Fig. 6. Noticethat with the exception of a very few chick cells, the majority of the cells in theciliary 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 duringdevelopment remain controversial and invite explorations through the applica-tion of new, yet reliable methods such as the 'quail-chick marking system'devised by Le Douarin (1973). The reliability of the cell labeling techniqueusing quail cells as 'natural markers' in the study of various embryologicalproblems, particularly in the analysis of the developmental fate and migrationpattern of cranial neural crest cells, has been repeatedly demonstrated innumerous interspecific transplantation experiments between embryos of quailand 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 neuronsof the ciliary ganglion? Wheie do these precursor neurons come from (site)?In a previous study, Jones (1945) observed that removal of the forebrain betweenthe optic stalks together with the midbrain resulted in the absence of the ciliaryganglion and concluded that the ciliary ganglion develops from cells whichmigrate from the forebrain. This conclusion was subsequently refuted byLevi-Montalcini & Amprino (1947) who showed that removal of the eye andthe entire forebrain was quite ineffective, but that removal of the head at thelevel of the anterior end of the hindbrain in chick embryos of 7-12-somitestages almost invariably resulted in the absence of the ciliary ganglion. How-ever, their operation procedures were too drastic to implicate any otherembryonic brain region specifically.

In our experiments, neither the forebrain nor the hindbrain prove to bespecific sites for the origin of the precursor cells of the ciliary ganglion. This isborne out by the fact that in chimaerical embryos of our forebrain and hind-brain transplantation experiments between quail and chick embryos, the ciliaryganglia were exclusively composed of chick cells. This clearly rules out thepossibility of their origin from either of these sites. On the other hand, quailcells are found in the ciliary ganglion of chimaerical embryos when the graftis from the midbrain level of quail donors. These observations are consistent,and suggest that the precursor cells of the ciliary ganglion are located at thelevel of the embryonic midbrain (mesencephalon) from where they migrate totheir 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 amesodermal origin for the cells of the ciliary ganglion. However, their extirpa-tion experiments were too drastic, eliminating totally not only the head meso-derm but also the migrating neural crest material, thus making it difficult toascribe 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 opticvesicle and the first somite which included the embryonic midbrain and hind-brain levels, resulted in the absence or a marked depletion of the neuronscomposing the ciliary ganglion in the chick. Although they have implicated aneural crest origin for these neurons, a precise site of origin could not bespecified from their experiments.

Our own evidence on this point is based on transplantation of cranial neuralcrest from the level of the mesencephalon between quail embryo donors andchick embryo hosts. Histological examination of the ciliary ganglion of theoperated side (right) in the experimental animals showed consistently the pre-sence of quail cells which would validate the conclusion that they are indeedderived from neuial crest of that specific brain region. The view favored by theresults of the present experimental analysis, places the primary emphasis onthe mesencephalon as the principal site and the cranial neural crest of this levelas the cells of origin for the precursor neurons of the ciliary ganglia in the chickembryo.

We should like to thank Mr Thomas Lee for his technical assistance in the histologicalpart of this study, and Mrs Susan Orazio for her careful typing of the manuscript andsecretarial assistance. This investigation was supported by Research Grant DE 04258-02from the National Institute of Dental Research.

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{Received 13 March 1978, revised 17 May 1978)