Consequences of neural tube and notochord excision on the development of the peripheral nervous system in the chick embryo

DEVELOPMENTAL

BIOLOGY

98,

192-211

(1983)

Consequences of Neural Tube and Notochord Excision on the Development of the Peripheral Nervous System in the Chick Embryo MARIE-AIM~E Institut

d %mlnyologie

du CNRS

et du Coll6ge

Received

December

TEILLET’ de France,

AND NICOLE M. LE DOUARIN 49bis, Avenue

3, 1982; accepted

de la Belle-Gabrielle,

in revised

fm

February

94130 Nogent-sur-Marne,

France

8, 1983

Notochordectomy and neuralectomy were carried out either in one- or in two-step experiments on the chick embryo. The aim of this operation was to study the influence of the axial organs (notochord and neural tube) on the development of the ganglia of the peripheral nervous system. The neural crest cells from which most peripheral ganglion cells arise were labeled through the quail-chick marker system and their fate was followed under various experimental conditions. It appeared that the development of the dorsal root and sympathetic ganglia depends on survival and differentiation of somite-derived structures. In the absence of neural tube and notochord, somitic cells die rapidly, and so do the neural crest cells that are present in the somitic mesenchyme at that time. In contrast, those crest cells which can reach the mesenchymal wall of the aorta, the suprarenal glands, or the gut survive and develop normally into nerve and paraganglion cells. Differentiation of the neural crest- and placode-derived sensory ganglia of the head which develop in the cephalic mesenchyme is not affected by removal of notochord and encephalic vesicles. These results show that the peripheral ganglia are differentially sensitive to the presence of the neural tube and the notochord. Among the various ganglia of the peripheral nervous system, spinal and sympathetic ganglia are the only ones which require the presence of these axial structures. The neural tube allows both the spinal and the sympathetic ganglia to develop in the absence of the notochord. In contrast, if the notochord is left in situ and the neural tube removed, the spinal ganglia fail to differentiate and only sympathetic ganglia can develop.

established that chondrogenesis and myogenesis in the somitic mesenchyme are dependent on neural tube and notochord since both processes can be either induced or hampered, respectively, by addition or removal of these structures (Holtzer, 1952a,b; Holtzer and Detwiler, 1953; Strudel 1952, 1955). The present work deals with the consequences of the precocious removal of the notochord and of the primordium of the central nervous system (CNS) on the ontogeny of neural crest derivatives in bird embryos in ova. The processes of both gangliogenesis and cytodifferentiation of the sensory and autonomic sympathetic and parasympathetic ganglia have been studied. The role of the somitic mesenchyme, of the notochord, and of the neural tube in the differentiation of adrenergic neurons and paraganglia, already documented (Cohen, 1972; Norr, 1973; Teillet et al, 1978; Fauquet et al., 1981), was also reexamined and it was shown that gangliogenesis of the enteric nervous system, in contrast to that of DRG and SG, proceeds normally despite complete removal of the neural tube and the notochord.

INTRODUCTION

Apart from some contribution of the placodal ectoderm to the sensory ganglia of certain cranial nerves, the peripheral nervous system (PNS)’ is entirely derived from the neural crest and the precise origin of its various components on the neural axis has been established through a variety of experimental methods (see Le Douarin, 1980, 1982, for reviews). The metameric distribution of the dorsal root (DRG) and sympathetic ganglia (SG) is clearly related to somitic segmentation of the dorsal mesoderm and the developmental relationships between these structures have already been underlined by several workers (Weston and Butler, 1966; Thiery et al., 1982). A way to evaluate the extent to which the development of the segmented dorsal mesoderm and that of the peripheral ganglia are linked is to perturb experimentally ontogeny of the one and examine how much disturbance is generated in the development of the other. The role of the axial structures, notochord, and neural tube, on the development of the somitic mesoderm has been thoroughly documented in the past in amphibians and birds. It has been clearly

MATERIALS

192 Copyright All rights

$3.00

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

METHODS

We used for this investigation chick and quail eggs from commercial sources. They were incubated in humidified hot rooms at 38 5 1°C. Interventions on the notochord and the neural tube were performed microsurgically on chick embryos in 0210. The operated em-

i To whom all correspondence should be addressed. * Abbreviations used: PNS, peripheral uervous system; DRG, dorsal root ganglia; SG, sympathetic ganglia; CNS, central nervous system; CA, catecholamines; FIF, Formol-induced fluorescence; AChE, acetylcholinesterase.

0012-1606/83

AND

TEILLE:T

AND

LE

DOUARIN

Ne-uralectomy,

bryos were sacrificed after a few hours or several according to the experimental series. 1. MICROSURGICAL

(a) Removal

of the Neural

days

PROCEDURES

Tube and the Notochord

One-step operation: series 1A (Fig. 1A). The chick embryos were at 15- to 25-somite stages when operated. The neural tube was first separated with a microscalpel from the somitic mesenchyme in the trunk and from the cephalic mesenchyme at the mid- and hindbrain levels. Second, the head was cut at the junction between anterior and midbrain vesicles, meaning that the eye primordia were removed along with the prosencephalon. Third, excision of both neural tube and notochord was completed. Since the neural tube was not completely formed at the caudal level at this stage, it could not be totally removed in this type of operation. At 15- to 25-somite stages, neural crest cell migration is completed down to the level of the posterior rhombencephalon, while it is only in progress in the anterior somitic area and has not yet started at the level of posterior somites and in the unsegmented region (Fig. 1A). Therefore, the operation resulted in removal of neural crest cells in the posterior region of the embryo, while at anterior and intermediate levels the crest cells were totally or partly left in situ. Two-step operation: series 1B (Fig. 1B). In order to remove the entire neural tube and notochord and to allow the neural crest cells to migrate at the mid- and hindtrunk levels, the excision was carried out in two steps. First, at 15- to ZO-somite stages, neural tube and notochord were extirpated down to the 20th somite level, then the embryo was put back into the incubator until it had reached the 30- to 35-somite stages. Then the remnant of the neural tube-notochord complex was excised, leaving in situ those crest cells which had already migrated in the dorsal and lumbar regions. (b) Labeling

of the Neural

Crest Cells

Two series of experiments were performed.

Labeling

of neural

crest cells in the cervical

Notochm-dectmy,

and

PNS

193

Ontogeny

Labeling of neural crest cells at the dorsal and lumbosacral levels: series 2B (Fig. 2B). This is done by (1) removal of the neural tube-notochord complex and decapitation in chick embryos at ZO- to 25-somite stages (cf. one-step operation in Fig. 1A); (2) isotopic and isochronic graft of a fragment of quail neural tube into the same chick embryos at the level where the somitic mesoderm is still unsegmented; (3) either fixation for observation at 30- to 35-somite stages, or removal of the grafted neural tube and of the posterior region of the chick neural tube and notochord (cf. description of the two-step operation, Fig. 1B) and observation several days later.

(c) Removal of Neural Backtransplantation series 3 (Fig. 3)

Tube and Notochord

and

of One of These Structures:

In these experiments, the embryos of chick and quail were used at the 15- to 25-somite stages. The neural tube-notochord complex was removed in chick or quail embryos as described above in the one-step experiment (Fig. 1A). Then they were separated from one another by enzymatic digestion with pancreatin (Gibco) diluted to 20% in Tyrode solution (10 min at room temperature) and washed in Tyrode solution. Two types of transplantation were performed. In series 3A, a notochord (taken either from the chick embryo host or from a quail embryo at the same stage) was back-transplanted in the cervical area into the groove left by the excision, as indicated in Fig. 3A. In series 3B, a quail neural tube was back-transplanted in all the somitic area of the chick host (Fig. 3B).

(d) Removal

of the Anterior

Cephalic Region

The anterior head was removed without excision of either the neural tube or the notochord in a control series. The level of the section was at the junction of fore- and midbrain, behind the eye primordia, as in the previously described experiments.

region:

sties 2A (Fig. 2A). It consists in (1) orthotopic grafting of a segment of quail neural tube at the level of somites 1 to 10 into a lo-somite chick embryo, according to a technique described earlier (Le Douarin, 1969,19’73), (2) either sacrifice and observation after Feulgen-Rossenbeck staining of 5-Km serial sections at ZO- to 25-somite stages or removal of the grafted and host neural tube and notochord at ZO- to 25-somite stages (cf. description of the one-step operation in Fig. 1A). This permits the fate of the labeled neural crest cells to be followed after neuralectomy and notochordectomy.

2. HISTOLOGICAL

AND HISTOCHEMICAL

(a) DNA and Catecholamine

TECHNIQUES

(CA) Staining

The Feulgen-Rossenbeck reaction for DNA (1924) was routinely used in this study, on 5-pm serial sections of embryos fixed with Carnoy fluid. This method is useful to distinguish quail and chick cells in the chimeric embryos. The Feulgen-Rossenbeck technique was also combined with the Formol-induced fluorescence (FIF) procedure to detect CA (Falck, 1962) as described elsewhere (Le Douarin and Teillet, 1974).

DEVELOPMENTAL

BIOLOGY

VOLUME

98, 1983

B

A

FIG. 1. Diagramatic representation of the excision of the neural tube and notochord in chick embryos. (A) Operation in one step (series 1A): chick embryos are at 15- to 25-somite stages. Migration of neural crest cells (z.:.:.:.:) is completed at the level of the cephalic vesicles, in progress at the cervical level and not started at the trunk level. (i) The neural primodium and the notochord are removed microsurgically down to the posterior region (dash). (ii) The forebrain along with the eye primordia are cut off (dashed arrows). (B) Operation in two steps (series 1B): (1) In chick embryos at 15- to 20-somite stages the neural tube and notochord are excised down to the level of the somite 20 (dash). Operated embryos are incubated again. (2) Previously operated embryos are at 30- to 35-somite stages. Trunk neural crest cells have migrated from the neural primordium. Then, the remnant of the neural tube-notochord complex is removed in the posterior region of the embryo (dash).

(b) Staining

of Neurons

For detection of differentiated neurons in the tissues, we used the method of silver impregnation described by Ungewitter (1951) or the thiocholine method of Karnovsky and Roots (1964) for detection of acetylcholinesterase (AChE) using acetylthiocholine as substrate. The silver impregnation was applied to tissues treated for the FIF technique. After observation in uv light,

sections of tissues embedded in Epon-Araldite and mounted on glass slides were treated by the technique of Mayor et al. (1961) to remove the embedding material, postfixed in Bouin’s fluid and then treated by the Ungewitter method. Detection of AChE was performed on tissues (parts of the gut) fixed overnight at 4°C in a solution of (4%) paraformaldehyde (1% ) calcium chloride saturated with calcium carbonate and cut into 20pm cryostat sections.

TEILI.ET AND LE DOUARIN

Neuralectmy,

Notochm-dectomy,

and

PNS

Ontogeny

B

A

FIG. 2. Labeling of neural crest cells before neuralectomy and notochordectomy. (A) Labeling at the cervical level: (1) Chick embryos are at lo-somite stage. A fragment of neural primordium (from somite 1 to somite 20) is extirpated and replaced by its quail counterpart (I). Operated embryos are put back in the incubator. (2) Chimeric embryos are at 15- to 25-somite stages. They are neuralectomized and notochordectomized as in series 1A. (B) Labeling at the dorsal and lumbosacral levels: (1) Chick embryos are at 20- to 25-somite stages. They are neuralectomized and notochordectomized as in series 1A. A fragment of quail neural tube is grafted isotopically from somite 20 downwards (I). (2) Host embryos have reached 30- to 35-somite stages. The grafted quail neural tube and the new formed host neural tube and notochord are removed as in series 1B.

(c) Staining of Skeletal

Structures

The skeletons were stained in embryos in toto by the method of Simon and Van Horn (1970) in experimental and control birds at similar developmental stages. RESULTS GENERAL

DEVELOPMENT OF NEURALECTOMIZED NOTOCHORDECTOMIZED EMBRYOS

AND

Although they involved partial decapitation, hypophysectomy, total or nearly total removal of the CNS

primordium and of the notochord, these operations were not incompatible with the survival of a certain number of embryos until close to hatching. Out of 243 operated embryos, 173 (70%) died at various ages and the 70 others were sacrificed between 2 and 16 days of incubation (Table 1, series 1A and B). At all stages considered, the experimental embryos were significantly smaller both compared to normal and to decapitated embryos (embryos in which the anterior brain, including hypophysis and eye primordia, had been removed) (Fig. 4a). In neuralectomized and notochordectomized

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II:A

IEB

FIG. 3. Diagramatic representation of back-transplantation of either the notochord (NC) (series 3A) or the neural tube (NT) (series 3B) after neuralectomy and notochordectomy in chick embryos. (I) Excision of NT and NC in chick and quail embryos. Transverse section of a chick or quail embryo operated as in Fig. 1. The NT-NC complex is enzymatically dissociated (a,b). (II) Back-transplantation of one of the dissociated organs. (A) The chick or quail NC (dark line) is grafted between the dermomyotomes (DM), sclerotomes (S) and aorta (Ao) at the cervical level of a neuralectomized and notochordectomized chick embryo. (B) A quail NT (I) is grafted all along the excised chick embryo from the level of the first somites. (1) Dorsal views of the operated embryos. (2) Transverse sections at the level of somite 5.

birds the neck was notably shorter and thinner than in controls. Lower jaw and tongue, as well as wings and legs were developed and the corresponding skeleton pieces looked normal in shape but were reduced in size with respect to controls, as was the whole body (Figs. 4c and d) (more detailed information on the consequences of partial neuralectomy and notochordectomy on limb morphogenesis can be found in Kieny et aC (1971). The skin did not differentiate normally: in the oldest embryos, it had a translucid appearance with oedematous and featherless areas, especially in the dorsolateral regions of the body. In embryos operated in one step (Fig. lA), the tail region generally developed with the posterior part of the spinal cord and the caudal vertebrae plus a variable number of sacral segments

(Fig. 4b) This did not occur in embryos neuralectomized and notochordectomized in two steps (Fig. 1B) since in TABLE NUMBER

1

OF EMBRYOS RECOVERED AND IN EACH EXPERIMENTAL

Experimental series Operated Dead Recovered at various stages Studied histologically

STUDIED SERIES

HISTOLOGICALLY

1A

1B

2A

2B

3A

3B

Total number

223 162

20 11

12 1

12 5

26 10

20 7

313 196

61

9

11

7

16

13

117

23

6

11

6

16

9

71

FIO. 4. General development of neuralectomized and notochordectomized chick embryos. (a) Middle: chick embryo, neuralectomized and notochordectomized in one step (as in Fig. IA); left: normal chick embryo; right: chick embryo in which the anterior cephalic region has been removed as a second control. Three embryos have been sacrificed at 9 days of incubation. The experimental embryo has a reduced size with a particular short neck. Lower jaw and limbs are developed. Note that the size of the partially decapitated and hypophysectomized embryo is close to normal. (b-d) Staining of the skeleton of the normal ((b) profile view) and of the neuralectomized and notochordectomized ((c) dorsal view and (d) proflle view) embryos. In the operated embryo, pieces of the lower jaw (c) and of the limbs (d) look reduced but normal in shape as compared with the skeleton of the normal control (h). There are no vertebrae, except in the tail where the neural tube and notochord were not entirely removed (Simon and Van I-Iorn technique (19’70)). 197

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the second operation, excision of neural tube and notochord was completed at the caudal level. DEVELOPMENTOFTHETRUNK STRUCTURESAND FATE OFTHE NEURALCREST CELLSAFTERNEURALECTOMY ANDNOTOCHORDECTOMY

Stage of Neural

Crest Cell Migration

at Operation

As demonstrated previously in this and other laboratories (see reviews of Le Douarin, 1980, 1982; AyerLe Lievre and Le Douarin, 1982; Duband and Thiery, 1982; Thiery et aZ., 1982), crest cell migration was already in progress at the midbrain, hindbrain, and vagal levels when the first step of the operation was performed (15- to 25-somite stages) (Fig. 5). The cells had reached lateroventral regions in the head and could be seen between the superficial ectoderm and the dermomyotome at the level of the first three somites (Fig. 5a). In addition, some cells were seen close to the pharyngeal wall from the 20-somite stage onward (Fig. 5b). In the somitic region down to about the level of somites 7 to 15 (according to the age of the embryos, i.e., from 15to 25-somite stages), the neural crest cells were moving within the three migration pathways available, i.e., between two consecutive somites, between the neural tube and the bulk of the somites and between the superficial ectoderm and the dermomyotome (Fig. 5~). However, posteriorly, at the level of the last 5 to 10 somites formed, at any of these developmental stages, the crest cells covered the dorsal half of the neural tube but had not yet penetrated the migration pathways (see Tosney, 19’78; Thiery et al, 1982). Therefore excision of the neural tube at 15- to 25-somite stages did not result in removal of neural crest cells at cephalic and anterior cervical levels while, in contrast, they were partly or totally excised along with the neural tube in the more caudal regions (Fig. 1A). In the two-step operations, when the caudal part of the neural tube and of the notochord was removed at somite stages 30 to 35, crest cell migration was in progress in the entire region of the embryo located caudally to the level of somite 20. At the level of somites 20 to 28, the cells were already fully engaged in the three pathways described above (Fig. 6a) and therefore were left in situ after the operation. But since the migration proceeds according to a craniocaudal gradient, the crest cells were either partly or totally removed in the lumbosacral and eaudal regions of the embryos (Figs. 1B and 6b).

Effect of Neurakctomy and Notochwdectomy Semitic and Migrating Crest Cells

on the

The effect of neuralectomy and notochordectomy was observed on serial transverse sections of 25 one-stepoperated chick and quail-chick chimeric embryos (se-

ries 1A and 2A), fixed immediately, 6 hr, 1,2, and 3 days after the operation. Quail-chick chimeras had the advantage of providing information on the neural crest cell population that had migrated before neuralectomy was completed. No differences were observed in the overall development of chick and quail-chick chimeric embryos at equivalent stages. The following description will first concern the level of the fourth and fifth somite pairs, at which neural crest cells had already left the neural primordium at the time of its excision (Fig. 5b). Immediately after the removal of the neural tubenotochord complex, in quail-chick chimeras (series 2A), single or grouped quail cells were seen within, or along the internal aspect of the somitic mesenchyme (Fig. 7a); they represented crest cells that had migrated from the grafted quail neural primordium. Six hours after the operation (Fig. ‘7b), the two contralateral somites had joined in the midline above the aortic primordium. No major change in the somitic structure could be noticed at that time, except for a few pycnotic figures in the sclerotomal cells. One day (20 to 24 hr) after the excision, considerable changes had occurred, compared both to the observation at 6 hr postoperation and to the control embryos (Fig. 8a). Recognizable dermomyotomal and sclerotomal cells were very scarce and numerous pycnotic figures affected the whole somitic mesenchyme. In chimeric embryos (series 2A), some surviving quail crest cells could be identified in the necrotic dorsal mesenchyme (Fig. 8b). In striking contrast to the somites, gut morphogenesis was apparently unaffected by neuralectomy and notochordectomy: at the same transverse level where the somites disappeared, both the epitheliomesenchymal tissue and the labeled neural crest cells which had migrated into the intestinal primordium appeared perfectly healthy. Two days (40 to 48 hr) after the operation, some loose mesenchymal cells and small pieces of dermomyotomal epithelium which persisted metamerically were the only surviving somitic elements. In chimeric embryos (series 2A), gilail cells were very rare or absent in the loose mesenchyme of the dorsal region (Fig. 8~). In contrast, they were found within the gut wall in apparently normal numbers and locations. At more caudal levels of the same embryos, operated in one step at the 15- to the 25-somite stages, somite segmentation took place in the absence of neural tube and notochord (i.e., after these structures had been surgically removed) but the same degeneration processes affected the dermomyotome and the sclerotome a few hours after they formed. Finally, 3 days after the operation (5 days of incubation), the aorta was in a quite dorsal position, underneath the superficial ectoderm, with only a loose mesenchymal sheet of cells inbetween (Fig. 8d). The

b t

FIG. 5. Migration of the cervical neural crest. Diagramatic representation of a 20-somite quail-chick chimera; the chick embryo has been grafted orthotopically with a quail neural primordium, at the level of somites 1 to 10, at the stage of 10 somites. The levels of the transverse sections corresponding to a-c are indicated. (a) Level corresponding to the first somite. Quail neural crest cells (arrows) have reached a laterodorsal position far from the grafted neural tube (NT). (b) Level corresponding to the fifth somite. Some quail neural crest cells (arrows) are in the vicinity of the splanchnopleura (Sp). (c) Level corresponding to the seventh somite. Labeled crest cells (arrows) are seen between the neural tube and the somite (So) and between the somite and the ectoderm (E). Some crest cells are present inside the sclerotome (S). Ao, aorta (Feulgen and Rossenbeck staining (1924)). 199

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FIG. 6. Migration of the trunk neural treat cells. Diagramatic representation of a 3%somite quail-chick chimera; the chick embryo haa been grafted orthotopically with a quail neural primordium and notochord at the level of somites 21 to 33 at 20-somite stage, The levels of the sections corresponding to a and b are indicated. (a) Transverse section at the level of the 28th somite. Quail crest cells (arrows) are visible between the neural tube (NT) and the somite (So) and within the sclerotome (S) up to the lateral edge of the aorta (Ao). (b) Oblique section at the level of somites 31 and 32. On the right side stream of quail crest cells (arrows) moving between two consecutive somites (somites 31 and 32). On the left side labeled crest cells beginning their migration between the neural tube and somite 32 (Feulgen and Rossenbeck staining (1924)).

trunk structures of the operated embryos were reduced to the derivatives of the intermediate cell mass (mesonephros, gonads, and adrenal rudiments), to the major blood vessels and to the gut, surrounded ventrolaterally by the somatopleural wall of the coelomic cavity.

Further Development of Neural Crest Derivatives Gangliogenesis &ter Newalectmg and Notochordectorny

and

In embryos operated in me step. Serial transverse sections of 11 embryos sacrificed at 5, 6, 8, and 9 days of

TEILLET

AND

LE DOUARIN

Neuralectomy,

Notochmdectmy,

and

PNS

Ontqmny

FIG. 7. Labeling of cervical neural crest cells followed by neuralectomy and notochordectomy (series 2A). Transverse sections at the level of somite 5. (a) Sacrifice immediately after the excision of the neural tube and notochord (20-somite stage). (al) Enlargement of the inset. Labeled crest cells (arrows) are visible at the internal edge of the sclerotome (S) and within the sclerotome itself. (b) Sacrifice 6 hr after the excision (ZS-somite stage). Sclerotomes have fused on the midline. Necrotic cells are visible within them, (b,) Quail crest cells (arrows) are found among the remnant of sclerotomal cells. Ao, aorta (Feulgen and Rossenbeck staining (1924)).

incubation and of the different parts of the gut taken at 16 days were examined after FIF treatment and staining by Feulgen-Rossenbeck or silver impregnation techniques. Cryostat sections of the 16-day embryo gut were also treated for detection of AChE activity. As expected, in successfully operated embryos (11 out of 11), central nervous tissue was totally absent all along the craniocaudal axis, except in the ultimate sacrocauda1 region. As mentioned above, this was because the

most posterior part of the neural tube was not removed because it was not formed at the time of excision (15 to 25-somite stages). Head and Neck As described before, the surgical excision of the neural primordium involved partial decapitation of the embryos including the prosencephalon and the prestomodeal area. Only from the midbrain downward were

FIG. 8. Progressive disappearance of dorsal structures and fate of the migrated neural crest cells after excision of neural tube and notochord. (a) Transverse section at the level of the posterior rhombencephalon of a 3-day chimeric quail-chick embryo. Insets 1 and 2 show, respectively, a dorsal root ganglion (DRG) and enteric neurons (double arrows) in the wall of the oesophagus (Oe) evidenced by the quail-chick labeling. (Feulgen and Rossenbeck staining (1924)). (b) Neuralectomized and notochordectomized chimeric embryo (series 2A) at the same stage and 202

TEILLE:T

AND

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DOUARIN

Neuralectomy,

the neural tube and the notochord selectively removed. The lower jaw and the tongue, the mesenchyme of which is of neural crest origin (see Le Libvre and Le Douarin, 1975), were developed and the inner ears were present as epithelial vesicles. In the cephalic mesenchyme, several ganglia were found along cranial nerves. They were not easily identifiable, except for the otic ganglia which were connected to the ear anlagen, and for the petrous and nodose ganglia located in the proximity of the carotid arteries (Fig. 8d). Curiously in some birds, petrous and nodose ganglia were sometimes found connected by homologous pairs or fused altogether into a single ganglionic mass. This is likely to be related to the great reduction in neck elongation observed in these embryos.

Trunk Sensory ganglia failed to develop in the trunk regions where the neural tube and notochord were effectively removed. The fact that they did not form in the dorsal region was predictable, since the neural crest cells had not yet migrated at this level when the neural primordium was removed. In contrast, at the cervical level (until about somites ‘7 to 15) crest cells had already left the neural tube before the operation and were numerous in the somitic area (Fig. 5~). In spite of this, no DRG developed, due to the progressive disappearance of the neural crest cells within a few hours after neuralectomy and notochordectomy (Figs. 8b and c). SYMPATHETIC GANGLIAANDPARAGANGLIA No FIF-positive cells or fibers could be seen anywhere in cephalic, cervical, and dorsal regions of 5-, 6-, and 8day-operated embryos. There were no sympathetic chains. The suprarenal glands did not contain any adrenomedullary cells and the aortic plexuses were totally absent. Absence of sympathetic elements in the dorsal region was not surprising, since the neural crest of the brachial level (“adrenomedullary” level of the neural crest, from somites 18 to 24 according to Le Douarin and Teillet (1973)) had been removed along with the neural tube (series lA, Fig. 1A). However, cervical crest cells, especially those corresponding to somites 5 to 10 (precursors of the superior cervical ganglion, according to our unpublished results) had migrated before the excision. These cells did not give rise to adrenergic cells

Notochmiectomy,

and

PNS

203

Ontogeny

(as revealed by the FIF technique) at 5, 6, and 8 days of incubation. However, one g-day embryo of this series (one-step-operated embryos) possessed some groups of fluorescent cells localized within the wall of the aortic trunks arising from the heart. According to our previous studies, the FIF-positive cells which are located in this area originate from the neural crest corresponding to the posterior rhombencephalon (our unpublished results and, Le Li&re and Le Douarin (1975)). ENTERICNERVOUS

SYSTEM

As early as at 5 days of incubation, silver-impregnated cells with processes were found in the gut wall down to the duodenum. At later stages, similar cells were seen at progressively more caudal levels and, in the 16-day embryo (Figs. 9a and b), well-developed silver-impregnated and AChE-positive enteric ganglia were present in all parts of the gut including the rectum. Sections of the anterior gut treated by the FIF technique failed to reveal any fluorescent sympathetic nerve fibers. In embryos operated in two steps. The two-step excision (Fig. 1B) permitted the removal of the whole neural tube. As for the crest cells, they were totally or partly left in situ in the cephalic, anterior cervical, dorsal, and lumbosacral levels. Posterior cervical and caudal neural crest cells were totally removed along with the neural tube of the same levels, during the first and the second excision, respectively (Fig. 10). Transverse serial sections of six operated chick embryos (series lB), taken at 6 and 8 days of incubation, and of six chimeras (series 2B), taken at 5 and 6 days, were examined. In the anterior region of these embryos the situation was the same as that observed in the onestep-operated embryos: cranial ganglia, including petrous and nodose and ganglia of enteric plexuses, were the only PNS structures encountered. In the trunk region, removal of both notochord and neural tube, performed at the 30- to 35-somite stages, precluded the development of sensory and sympathetic chain ganglia. However, at the level corresponding to somites 20 and 25 some small ganglia were observed dorsally to the aorta. Certain of these exhibited a faint fluorescence with the FIF technique, whereas others did not. Interestingly, the presence of these ganglionic structures could always be correlated with the differ-

the same level than the control (a). Numerous pycnotic figures are seen in the somitic tissue (So). A few labeled crest cells are visible (arrows). Quail crest cells are also found (double arrows) in the wall of the embryonic oesophagus as in the control (Feulgen and Rossenbeck staining (1924)). (c) Neuralectomized and notochordectomized chimeric embryo (series 2A) at the same level by 1 day later (4 days of incubation). Semitic tissues have disappeared in the dorsal region except for some dermomyotomal cells (DM) which are seen metamerically. Labeled crest cells (arrows) are very rare (Feulgen and Rossenbeck staining (1924)). (d) Transverse section of an embryo of series 1A (neuralectomy and notochordectomy in one step) at 5 days of incubation in the region of the neck. Dorsal structures deriving from the somitic mesenchyme are totally missing. Petrous ganglia (PG) are present. Ao, aorta; Ph, pharynx (silver impregnation according to Ungewitter (1951).

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DEVELOPMENTAL BIOLOGY

VOLUME 98, 1983

FIG. 9. Transverse sections of the duodenum of a 16-day-neuralectomi zed and -notochordectomized embryo (series 1A). Ganglia of Auerbach (A) and Meissner (M) plexuses are normally developed. (a) Silver imp regnation according to Ungewitter (1951). (b) AChE evidenced by the Karnovsky and Roots technique (1964).

entiation of small amounts of striated muscles and cartilage (Table 2). In all the embryos of this series, except one, brightly fluorescent cells were found in a lateroventral position with respect to the aorta (i.e., in the aortic and pelvic plexuses) and within the adrenal glands (Fig. 11). Some of the two-step-operated embryos had been subjected to the transplantation of a quail neural primordium in the region posterior to somite 20 (series 2B, Fig. 2B), Among the 7 (out of 12) chimeric embryos which survived the three consecutive operations and were sacrificed at 5 and 6 days of incubation, 6 were examined in serial sections. Only one s-day embryo was found to be absolutely devoid of CNS and notochord. In the cephalic and cervical regions, this embryo was identical to the chick embryos operated in one or two steps, described above. But in the region where the quail neural tube was implanted (22 to 30 somites), the fluorescent cells of the aortic and pelvic plexuses as well as of the

adrenal glands were of the quail type (Figs. llb and c). In contrast, the Remak ganglion, whose origin is more caudal (see Le Douarin and Teillet, 1973; Teillet, 1978), was of the host type. These results were expected in view of the state of neural crest cell migration at the various levels considered when the excision of the neural tube was finally performed. In the five other grafted embryos, fragments of quail neural tube were recovered in the host dorsal structures. Fluorescent and nonfluorescent ganglia of the quail type were also found in the vicinity of these fragments of CNS. Figure 10 summarizes the results obtained in the various experimental conditions of series 1B and 2B. SELECTIVE REMOVAL OF THE NEURAL TUBE OR THE NOTOCHORD

The experiments described above stress the close relationships existing between the development of the so-

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FIG. 10. Diagram showing on the left the levels of origin of the various ganglia (G) of the peripheral nervous system (PNS) in the neural crest (NC). These levels are referred to the otocyst (O), the somite numbers (1 to 44) and to the corresponding vertebrae (C, to C14,cervical; DI to D7, dorsal; Lsl to LQ, lumbosacral; Cal to Ca,, caudal). On the right the presence (+) or the absence (-) of ganglia in the cervical, dorsal, and lumbosacral regions are indicated in embryos neuralectomized and notochordectomized in two steps (series 1B). The state of migration of the neural crest cells at the time of operation is indicated C+:+:). 205

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Note. Presence (+) or absence (-) of the various structures. Remak ganglion neurons and CA-containing cells of the adrenal glands and of the aortic and pelvic plexuses differentiated in all cases except one (number 3). In this case where no trunk crest cell derivatives were found, crest cells had probably been removed at the time of operation. In the other cases there is a good correlation between local differentiation of somitic derivatives and appearance of dorsally isolated peripheral ganglia (sympathetic and possibly sensory).

mites and the process of gangliogenesis of DRG and SG. Since notochord or neural tube are independently able to allow somitic mesenchyme to differentiate (Strudel, 1955), it seemed of interest to investigate their respective effects on gangliogenesis in the dorsal trunk structures. The experiments were carried out as described in Fig. 3 (series 3). Serial sections of operated embryos were examined several hours to several days after the interventions.

Role of the Notochord A fragment of notochord was grafted at the cervical level, into the groove left by excision of the neural tube and notochord (Figs. 3A and 12a). Six hours later, the two contralateral somites had joined and surrounded the notochord, which had become incorporated into the host tissues (Fig. 12b). One day after the graft, no evident sign of cell death was visible in the somitic tissue. The sclerotomal cells had become organized around the notochord. Ten embryos of this series were observed in serial sections at 6, 8, and 10 days of incubation. A ring of cartilage was found surrounding the notochord and striated muscles differentiated dorsolaterally above it (Fig. 13a). Sympathetic gangliogenesis occurred in two locations: (i) in the proximity of the carotid arteries, i.e., about in the position of the superior cervical ganglia (Fig. 13b); (ii) as a ring of ganglion cell aggregates distributed around the cartilage surrounding the notochord. At other levels, where notochord was not back-transplanted, the fate of the somites was the same as de-

scribed in the case of excision in one step. Massive necrosis of the somitic tissue resulted in the absence of vertebral cartilage and muscles.

Role of the Neural

Tube

When a quail neural tube was back-transplanted into the neuralectomized and notochordectomized chick embryos (series 3B, Fig. 3B), the development of the axial structures was different from all the above described situations. One day after the operation, no significant somitic cell death was observed. Then both dermomyotomes and sclerotomes underwent differentiation into muscle and vertebral cartilage, respectively, as previously reported by Strudel (1955). DRG, SG (Figs. 13~ and d), aortic and pelvic plexuses, and adrenal medulla developed. They were made up of cells belonging to the grafted quail neural primordium, with or without a contribution of host neural crest cells, depending on the level considered and on the state of chick crest cell migration at the time of operation. The primary sympathetic chains exhibited CA fluorescence from 4 days onward as in normal development. Although the overall development of these embryos was usually abnormal, especially at the level of the neural tube, which often remained open in the mid dorsal line, this did not disturb significantly the pattern of crest cell dispersion, localization, and differentiation. CONCLUSIONS

AND

DISCUSSION

We report in this article that both the primordium of the CNS and the notochord can be completely re-

FIG. 11. Differentiation of CA-containing cells in the trunk region of operated embryos. (a) Section of the adrenal gland (AG) and the aortic region (Ao) in an embryo operated in two steps (series 1B) and sacrificed at 6 days of incubation. The FIF technique shows the presence of CA-containing cells in the adrenal medulla and the aortic plexus. (b,c) Two adjacent sections of the adrenal gland and of the aortic region in a chimeric quail-chick embryo operated in two steps (series 2B), at 5 days of incubation. (b) The FIF technique evidences CA-containing cells of the aortic plexus. (c) The Feulgen and Rossenbeck staining shows that the Ca-containing cells arise from the quail neural primordium grafted some hours before neuralectomy and notochordectomy (arrows).

DEVELOPMENTAL BIOLOGY

VOLUME 98, 1983

FIG. 12. Back-grafting of a notochord in neuralectomized and notochordectomized embryos (series 3A). (a) Transverse section at the level of somite 5 in an embryo sacrificed immediately after the operation. (b) Section at the same level in an embryo sacrificed 2 hr after the operation. The notochord (NC) is perfectly incorporated in the somitic tissues (So) which have joined above it. Ao, aorta (Feulgen and Rossenbeck staining (1924)).

moved from the early chick embryo and that such an intervention is compatible with survival to (or close to) hatching time. The only potential nerve cells which remain in the body of these birds are those deriving from the ecto-

dermal placodes of the head and neural crest cells which had already left the neural primordium and begun to migrate when the operation was performed. Three types of mesenchymal substrates were offered to these potential placode- and crest-derived neurons: the cephalic

FIG. 13. (a, b) Same experiment as in Fig. 12 (series 3A), at 5 days of incubation. (a) The of cartilage and muscles (Feulgen and Rossenbeck staining (1924)). The inset enlarged in neighboring the carotid artery (C). (c,d) Back-grafting of a quail neural primordium after Transverse sections at 5 days of incubation. DRG and SG develop in the absence of notochord. (1951). (d) FIF technique. 209

grafted notochord (NC) is surrounded by a ring b shows a well-developed FIF-positive ganglion neuralectomy and notochordectomy (series 3B). (c) Silver impregnation according to Ungewitter

210

DEVELOPMENTALBIOLOGY

mesenchyme (mostly mesectodermal, i.e., of neural crest origin) and, at the trunk level, the somitic and gut wall mesenchymes. We observed a strikingly different developmental behavior of the potential ganglion cells according to the mesenchymal tissue of the body in which they were localized when the CNS and notochord anlagen were removed. Expansion and terminal differentiation of ectomesenchyme into bones, cartilage, muscle, and connective tissues occurred in operated embryos devoid of encephalic vesicles and notochord as it does in controls. The ganglia (nodose and petrous), whose neurons are placoda1but glial cells are neural crest derived (Narayanan and Narayanan, 1980; Ayer-Le Lievre and Le Douarin, 1982), developed in the cephalic mesenchyme despite removal of neural tube and notochord. Similarly, the neural crest cells which had migrated into the gut wall before neural tube and notochord excision formed enteric ganglia and plexuses. Preliminary results (to be published by M.-A. Teillet, J. Smith, F. Fontaine-Perus, and N. M. Le Douarin) indicate that choline acetyltransferase activity and neuropeptide-containing neurons develop in the gut of CNS- and notochord-deprived embryos in an apparently normal fashion. In striking contrast, the neural crest cells which had migrated in the somites before the operation died along with somitic cells after removal of the neural tube and the notochord and no spinal or sympathetic chain gangliogenesis took place. However, the neural crest cells which reached the suprarenal glands and the wall of the dorsal aorta survived and differentiated into CAcontaining ganglia and paraganglia. The consequences of the removal of neural tube and notochord have already been analyzed in the chick embryo by Strudel (1955). These experiments, however, concerned only a limited part of the neural axis, and the author’s major concern was the failure of vertebrae and striated muscle development consecutive to the operation. In the present work, notochordectomy and neuralectomy were virtually or wholly complete and their effects were sequentially followed from the time of the operation onward. Labeling of neural crest cells through transient implantations of the quail neural tube indicated that the somitic mesenchyme and the associated neural crest cells were the site of a devastating wave of cell death reaching its maximum about 24 hr postoperation. Observation 2 and 3 days after the excision showed that no (or very few) cells of the dorsal segmented mesoderm, in either dermomyotomes or sclerotomes, were able to survive the removal of both neural tube and notochord. When in the somites, the crest cells can survive and undergo gangliogenesis only in the presence of either one or the other of the axial organs. A differential effect of either the neural tube or the

VOLUME 98.1983

notochord on the fate of somite-associated crest cells was demonstrated through selective reimplantation of each of these structures. A pattern of PNS development very close to normal took place in the presence of neural tube, despite the absence of notochord. In contrast, removal of the former and presence of the latter affected the pattern of crest cell development in the dorsal structures more profoundly. When the neural tube was present, DRG and SG developed and were segmentally distributed. Strikingly, as reported by Strudel (1955), the neurarcuals and the dorsal striated muscles not only developed in these birds but also were segmentally organized, as in normal animals. In contrast, the ventral striated vertebral muscles as well as the vertebral bodies, although present, were deficient and always nonsegmented. When the neural tube was removed but the notochord regrafted, most somitic cells survived, since the extensive wave of cell death following the excision of both axial organs was not seen, but no segmental neurarcuals or dorsal striated muscles developed. Only an unsegmented tube of cartilage was formed around the notochord remnant with also some ventral striated vertebral muscles. In such embryos, the DRG were totally absent but adrenergic structures developed without any segmentary distribution recalling the paravertebral SG chains. Only at the cervical level, could the ganglionic masses be considered as homologous to the superior cervical ganglia. Relationships between the development of the somites and the neural crest derivatives have been underlined previously in experiments where neural crest cells were associated in culture with the somites. In this case also, cartilage differentiation and ganglion cell development were positively related (Teillet et aL, 1978). In the work reported here, we observed, in the embryos operated in two steps, some small ganglia located at the dorsal aspect of the aorta in embryonic areas where the cartilaginous and muscular structures also developed (see Table 2). These areas corresponded to the brachial level, where neural tube and notochord were removed late enough to ensure survival and differentiation of some somitic and crest cells. The mechanisms through which the axial organs influence somite development have been the subject of intensive investigations. The effect of the axial organs on survival of somitic cells already stated by Holtzer (see Holtzer, 1968) seems to be mediated by extracellular matrix components among which collagen, proteoglycans, glycosaminoglycans (forming the perichordal material) appear to play a major role, as shown by in vitro culture experiments (Strudel, 1975; Lash and Vasav, 1978; Belsky et al., 1980). The question remains open as to whether survival and gangliogenesis of the neural crest cells are triggered by mechanisms similar to those which ensure phenotypic expression in somitic

TEILI.ET

AND

LE DOUARIN

Neurakctom

cells. It is, however, striking to see that those crest cells which have migrated into either the gut wall mesenthyme or the mesectodermal mesenchyme of the head survive and develop, while those which home to the somitic mesoderm die when the neural tube and notochord are removed. In other words, the dependence of the crest cells on the axial organs strictly parallels that of the mesenchymal substrate in which they develop.

This work was supported by the Centre National de la Recherche Scientifique, by grants from the Delegation Generale a la Recherche Scientifique et Technique, from the NIH Grant 2R 01 DE0 4257-05, and from the Ligue Francaise contre le Cancer et la Fondation pour la Recherche Medicale Francaise.

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