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Cephalic ectodermal placodes and neurogenesis
TINS-April1986
175
Cephalic ectodermal placodes and neurogenesis Nlcole M. Le Douarin, Josiane Fontaine,-P(~rus and G(~rard Couly In the vertebrate embryo, the primary neural anlage, or neural plate, develops from the superficial ectoderm as a result of an inductive stimulus arising from the chordomesoderm. Although it is well established that the neural plate itself gives rise to CNS, the fate of those cells located at the junction of the neural and superficial ectoderm (also called neural ridges or neural folds), from which the neural crest and placodes are derived, has been the subject of controversy. Tracing these cells during ontogeny has been made possible by using the quail~chick chimaera system. Such studies have revealed the contribution of the neural ridge and the neurogenic placodes to various cephalic structures, and have allowed their relationships with the CNS to be followed from the early stages of neurulation throughout the whole period of morphogenesis. The term placode was coined by Von Kuppfer 1 to designate the epithelial thickenings appearing in the cephalic ectoderm of the early vertebrate embryo. They appear anteriorly and laterally with respect to the main neural primordium, i.e., the neural plate and its lateral ridges, the neural folds, from which both the central and peripheral nervous system respectively arise. Their fate is strikingly varied, since they give rise to such diverse structures as the anterior pituitary gland, the olfactory sensory epithelium and the lens. More caudally, the superficial cephalic epithelium also generates the 'neurogenic' placodes from which epithelial cells
detach and, after a migratory phase, end up in certain cranial peropheral ganglia. By the turn of the century, the neurogenic fate of these placodes had been proposed, and their participation, along with the neural crest, in the genesis of certain cranial sensory nerve ganglia was suggested by several authors (see Ref. 2 for references). These pioneering studies, concerning virtually all classes of vertebrates, were mainly based on careful observations of normally developing embryos. Later, experiments were performed in which the potential sources of ganglion cells (i.e., the placodal ectoderm or the neural crest) were selectively removed.
Careful and detailed though they were, studies using histological observations or extirpation techniques led to controversial views on what is a very complex morphogenetic process. Placodal and crest cells have to migrate to reach the sites where gangliogenesis takes place, and once they have left their sources they adopt a mesenchymal morphology and cannot be properly traced to their targets or recognized once they have differentiated. In addition, extirpation of early embryonic territories is generally followed by their more-or-less extensive replacement through proliferation of neighbouring cells, thus making the effect of extirpation experiments difficult to interpret. Labelling the putative cellular components of the ganglia finally clarified this problem, at least in the avian embryo, on which various types of cell-marking experiments have been performed. Isotopic labelling of the neurectodermal cells with tritiated thymidine, a technique introduced by Weston 3 and later applied to cranial ganglion ontogeny by Johnston and Hazelton 4,
B.
J.S V.A
m.v
G.~I
p.1Y
Fig. 1. (A) Distribution o f placodes in a 2-day-old
8 days
Neural
crest
Placodes
embryo. T, trigeminal placode; Ot, otic placode; PI, P2, P3, epibranchial placodes. (B) Sensory spinal (DRG) and cranial ganglia at 8 days, the neurons of which are either of crest (blue) or placodal (grey) origin. V, VII, VIII, I X and X indicate the number of the cranial nerve. The sensory ganglia are T, trigeminal; G, geniculate; J and S, jugular and superior; P and N, petrosal and nodose.
176
T I N S - A p r i l 1986
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chick host Fig. 2. Experimental designs used to trace the cells of neural crest and placodal origin during PNS ontogeny. Orthotopic and isochronic grafts ofplacodal ectoderm (A),cephalic neural crest OI) and neural primordium composed of the neural tube and the neural folds (C), between quail and chick embryos.
(Taken, with permission,from Ref. 2.)
NEURAL CREST CELLS
PLA C ODES
DERI W
IVATIVES
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Fig. 3. Schematic drawing of a stage 9.5 chick embryo (about 10 somites) indicating positions of neural crest and placodal anlagen for cranial sensory and autonomic ganglia. (Taken,withpermission,from
Ref. 2.)
and Noden 5x', certainly achieved progress in this field. However, it was only when the quail/chick chimaera system was developed7,s with its stable quail nuclear marker, that the respective roles of placodes and neural crest in the ontogeny of the cephalic peripheral nervous system could be accurately defined. Fate maps of neural crest and placodal derivatives could be constructed in the avian embryo and the early stages of crest and placodal cell migration followed with precision2'9. Recently, we have been interested in the relationships between the most anterior placodes (giving rise to the pituitary and the olfactory epithelium) and the neural primordium itself (neural plate and neural folds) during early neurogenesis~°. Interesting observations have been made regarding the spatial distribution of these territories with respect to the regions of the CNS to which they later become functionally connected. They will be briefly mentioned in this article, which will mainly review studies that have used the quail/chick chimaera system as a tool to investigate the fate and developmental potencies of placodal and neural crest cells in neurogenesis.
Contributions of neurogenic placodes and neural crest to cephalic gangliogenesis In the head as well as in the other parts of the body, the autonomic ganglia are derived entirely from the neural crest. In contrast, the ontogeny of the sensory ganglia is much more complex in the cephalic region than in the trunk, due to the participation of the neurogenic placodes. Fig, 1 illustrates the anatomical distribution of the cranial sensory nerves and ganglia in the avian embryo at the mid-point of its incubation. The contributionof the neural crest has been as~ssed by two slightly different experimental paradigms. The first consisted of exchanging fragments of the whole neural primordium between quail and chick embryos9,N (Fig. 2c), and the other of grafting fragments of the neural crest only2'12'13 (Fig. 2B). The derivation of neurons from placodal territories was carefully analysed by D'Amico-Martel and Noden 2, who replaced chick cephalic ectodermal areas with their quail counterparts (Fig. 2A), and could thus establish from which placodal regions neurons of the various ganglia arise (Fig. 3). As a general rule, sensory neurons
TINS-April1986 originate from both the neural crest and the ectodermal placodes, whereas glial cells of the head sensory ganglia and nerves are all derived from neural crest with no participation of the placodal ectoderm. Plaeode-derived neurons are present in the ganglia associated with nerves V (trigeminal), VII (facial), IX (glossopharyngeal) and X (vagal), and are always located distally to crest-derived neurons. The placodal neurons, generally larger in size than the neurons of crest origin, are found in the distal portions of both lobes of the trigeminal ganglion, and in the geniculate, petrosal and nodose ganglia, where in the latter they form the totality of the neuronal population (Fig. 1B). All the neurons in the vestibular and acoustic ganglia of cranial nerve VIII are derived from placodal ectoderm and their glial cells from neural crest, with the exception of a few neural crestderived neurons that correspond to the rudimentary proximal ganglion of nerve V I I - the latter fuses with the vestibular ganglion of nerve VIII during the course of development. Crest derived cells can be identified in this ganglion by the quail marker, after grafting a quail neural crest fragment to the rostrai myelencephalic area 2. The avian neurogenic ptacodes can be divided into three types according to their position along the dorsoventral axis14. The more dorsal (also designated dorsolateral) is the otic placode located lateral to the myelencephalon. It is clearly visible from stage 10 (of Hamburger and Hamilton 15) in the chick, and has formed the otic cup by stage 13. The neurons of the VIIIth nerve ganglia arise from cells shed off from the medioventral aspect of this placode from stage 14. The peak of cell migration takes place at about 60 h of incubation, and by stages 23-24, the acoustic and vestibular ganglia can be identified. The intermediate placode, from which the distal neurons of the trigeminal ganglia arise, is located more ventrally than the otic placode. The epibranchial placodes appear in the dorsal region of the branchial cleft (Fig. 1A). The first epibranchial placode (first branchial cleft) yields the geniculate ganglion (distal ganglion of the ViIth cranial nerve). The second and third epibranchial placodes (on the 2nd and 3rd branchial arches) are the sites of origin of the petrosal and nodose ganglion neurons, respectively.
177
A
B
Gr
. ,
2
C
These placodes become distinguishable after the pharyngeal pouches contact the overlying ectoderm. Cells budding off from the placodes, singly or in small clusters, show some signs of neuronal differentiation, such as high acetylcholinesterase activity and silverstained neurite-like extensionsz. When free from the epithelial surface, the cells of placodal origin meet those of neural crest origin5'6'9,16,17that have migrated earlier. It is very striking that the neural crest ceils that form the proximal ganglia of sensory nerves IX and X (jugular-superior ganglionic complex) as well as the proximal lobe of the trigeminal ganglion, aggregate soon after they have left the neural primordium and form ganglia that remain in close contact with the CNS. In contrast, those neural crest cells that will participate in the mixed placodalcrest ganglia must move appreciable distances from the CNS in order to meet their placode-derived partners.
Differentiating capadties of the cellular components of the cranial sensory ganglia Developmental potentialities* of the neurons (of crest or placodalorigin) and non-neuronalcells in the cranial sensory ganglia have been analysed under normal and experimental conditions. In an immunocytochemical study on proximal and distal cranial sensory ganglia in embryos, aimed at detecting substance P, it appeared that substance P-like immunoreactivity (SPLI) was regularly found in most small proximal neural crest-derived neurons, but not in placodal nerve cells. However, one exception was in the nodose ganglion, where a few bonafide placodal neurons showed SPLI. It is striking that in the dorsal root ganglion (DRG), the small *i.e., the phenotypes that a given cell is able to express if removed from its normal embryonic surroundings and submitted to a different microenvironment,
178 mediodorsal neurons that show many similarities to the crest-derived neurons of the cranial sensory ganglia also contain substance P, while the larger lateroventral nerve cells are, like those of placodal origin, substance P-negative. The developmental capacities of cells in the cranial sensory ganglia were investigated using an experimental system (described in Fig. 4) in which they are made to express phenotypes that they do not usually exhibit under normal conditions. This system consists of the retrotransplantationof a ganglion (of which all or some cells carry the quail nuclear marker) into the neural crest migration pathway of a younger chick host. The mixed neural crest and placodal origin of the distal ganglia of cranial nerves IX and X allow the construction of chimaeric petrosal and nodose ganglia in which either the neuronal or the non-neuronal cells are selectively labelled. In both cases, fragments (of about 2000 cells) of such ganglia are implanted into the neural crest migration pathway of 2-day-old chick host embryos. In such grafts, the piece of implanted ganglion rapidly loses its cohesiveness and its cell components become dispersed and migrate in the host's tissues. After a phase of dispersion, the ganglion cells aggregate to neural crest-derived structures in the host, where they become mixed up with chick crest-derived cells. Back-transplantation experiments of quail peripheral ganglia into chick embryos have been used extensively by our group9'18'22, and have provided relevant information about the state of commitment of the neural crest and peripheral ganglion cells23. If the graft involves chimaeric nodose and petrosal ganglia, the distributionof labelled cells is strikingly different according to the cell type carrying the quail marker. When neurons are labelled, practically no quail cells are subsequently found in the chick host, indicating that they are not able to survive graft conditions. In contrast, non-neuronalcells of the quail type show considerable invasion of the host, having proliferated abundantly. They can be found in the autonomic nervous system of the host, including the gut, where they differentiate into both neuronal and non-neuronal cells. The graft-derived neurons exhibit phenotypes in accordance with their position in the host, i.e., they differentiate into adrenergic neurons and chromaffin cells in the sympathetic ganglia and adrenal medulla, respec-
7 7 N 5 - A p r i l It)80 a QUAIL
CHICK
b
Fig. 5. (a and b) Substitution between quail and chick embryos (at stages of 0-3 somites) of the anterior neural ridge (150 lira long) in (a) and of the anterolateral neural ridge in (b). Fragments A and B (about 150 Wn long) are gra~ed either independently or as a single piece according to the experiment considered. (¢)Isotopic and isochronic transferof the anterior neural ridge and neural plate areas from a quail to a chick embryo at stages 0-3 somites. Here the embryos represented are at the 2somite stage. (d) Experiment in which the anterolateral neural ridge (Zone A in 5b) along with the lateral area of the neural plate was transplanted isotopically from a quail (Q) to a chick (Ch) embryo at stages of 0-3 somites. (e) Photograph of a 3-somite chick embryo in which the anterior neural ridge has been replaced by its quail counterpart. Arrows indicate the graft.
tively, and into enteric neurons, some containing neuropeptides, when they migrate into the gut wall. These results indicate that in the cranial sensory ganglia of mixed (crest and placode) origin, the non-neuronal cell population retains some of the developmental potentialities of the structure from which it is derived, the neural crest. These potentialities are not normally expressed during the ontogeny of these ganglia, since catecholamine containing cells are not detectable in either quail or chick petrosal and nodose ganglia at any of the developmental stages examined9.
When retrotransplanted into the crest cell migration pathway of a younger host, the quiescent neuronal precursors present in the non-neuronal cell population of the ganglia are stimulated to divide and differentiate. It is noteworthy that no graft-derived cells have ever been seen to develop into sensory neurons in the host DRG. It appears, therefore, that the developmental potentialities of the quiescent precursors in the distal sensory ganglia of cranial nerves IX and X are restricted to the autonomic (i.e., sympathetic, parasympathetic and the enteric net-
179
TINS-April 1986
vous system) differentiation pathway. They can therefore be called type ' A ' precursors according to the model proposed to account for sensory and autonomic cell lineage divergence during PNS differentiation2a. Recently, we obtained differentiation of type A precursors contained in D R G and nodose ganglia in vitro 24 and were able to show that a great many adrenergic cells developing in cultures of dissociated sensory ganglion cells arose from cycling progenitor cells, a property that allowed them to be distinguished from the pool of post-mitotic sensory neurons present in the culture. Development of the hypophyseal and the olfactory placodes We recently published experiments 1° that were primarily directed at investigating the early relationships between the CNS and PNS anlagen (i.e. the neural plate/neural fold complex) and the placodes. The hypothesis underlying this endeavour was that induction by the chordomesoderm of the whole nervous system in the ectodermal germ layer is a unique event eliciting the formation of a virtually continuous area of neural epithelium. According to this view, placodes would become secondarily detached from the neural primordium, which would then become segregated into the neural plate/neural fold complex on the one hand and the placode-derived organs on the other. It is of note that the spatial relationships between the neural plate and the placodal territories has attracted the attention of several workers. The observations from amphibians corroborate the notion that neural plate and placodes are closely related since the placodal ectoderm originates from a region termed the primitive placodai thickening25-28,located in the outer side of the neural fold itself. The future olfactory neurons, in particular, are localized in apposition to the primitive neural fold and close to the anlage of the pituitary29-33. Our own investigations were based on the substitution in chick embryos of small and defined regions of the anterior neural ridge and neural plate by their quail counterparts, as indicated in Fig. 5. The recipient embryos were examined by chimaerism analysis after 5 and 8 days of incubation, and the following conclusions were reached. At the early stages of neurogenesis in the avian embryo, the neural ridges limiting the neural plate anteriorly and laterally at the level of the forebrain, contain the
b
Fig. 6. Results o f the experiment (represented in Figs l a and l e) in which the anterior neural ridge of a quail embryo (1-somite stage) was grafted isotopically to a chick host. (a) Sagittal section o f the host at E5 showing, at low magnification, the areas represented in (b) and (c). (b) Tip o f Rathke's pouch shows the quail nuclear marker while the unfundibulum belongs to the host. (c) Mouth epithelium made up o f quail cells. Note that the adjacent mesenchymal cells are o f chick host type. (a) x 90; (b) x 1350; (c) x 1350.
(Taken, with permission, from Ref. 10.)
180
presumptive territories of the hypophyseal (Fig. 6) and olfactory ectodermal placodes. This part of the neural ridge also includes the precursor cells of large areas of superficial ectoderm corresponding to the nasal cavities and the ectoderm covering the premaxillary area and the upper beak (Fig. 7). This latter finding was unexpected since, until then, it had been thought that the neural ridge (apart from the mesectodermal cells that originate from the neural crest) only yielded neural structures. When areas of the neural plate adjacent to the presumptive hypophyseal and olfactory placodes were included in the graft (i.e., labelled by the quail nuclear marker) (Fig. 5), it was then found that the primordia of both hypophysis and hypothalamus were continuous, as were those of the olfactory epithelium and the olfactory bulb. It thus appears that at the early somitic stages, mapping of the neural primordium leads to the identification of territories from which central and peripheral structures of particular functional units (here the hypophysealhypothalamic complex and the olfactory system) are derived. Although the localization of the presumptive territories of the dorsolateral, intermediate and epibranchial placodes, has not been determined at the very earliest stages of neurogenesis, it is tempting to hypothesize that, like the hypophyseal and olfactory placodes, they also have their origin in the neural ridge. This would mean that the chordomesoderm's induction of the whole nervous system in the ectodermal germ layer is a unique event giving rise to the so-called neural plate from which the ectodermal placodes migrate secondarily. In addition, results showing the close relationships of the forebrain neural primordium to the adenohypophysis, olfactory organs and facial ectoderm are interesting to correlate with certain congenital pathologies in humans. This is the case in De Myer's mediofacial syndrome, in which malformations of the diencephalo-telencephalic regions are associated with naso-fronto-premaxillary hypoplasia 34. Other examples include adenohypophyseal deficiencies, revealed by an insufficient production of growth hormone, concomitant with naso-frontal malformation 35 and De Morsier's olfactogenital syndrome 36,
TINS--April 1986
i
E
Fig. 7. Fate map of the anterolateral neural ridge and anterior neural plate at stages of O--3somites in the avian embryo. (a) presumptive area of the nasal ectoderm, (b) neural ridge containing the hypo-
physeal placode, (e) presumptive region containing the olfactory placode, (d)presumptive territory o f the ectoderm of upper beak, (e) region where the anlage of the egg tooth is located, (f) presumptive territory of the diencephalon (i.e. infundibulum and hypothalamus).
associated with anosmia. This work will continue in an endeavour to analyse systematically, via the quail/chick marker (1) the patterning of the cephalic structures derived from the neural primordium and their relationships with the cells derived from the other germ layers, and (2) the sequential commitment and phenotypic expression of the neuroectodermal cells. One of the goals in view is also to provide relevant information on the genesis of certain congenital human malformations.
Selected references 1 Von Kuppfer, C. (18941 Lehmann Munchen 2 D'Amico-Martel, A. and Noden, D . M . (19831 Amer. J. Anat. 166, 445-468 3 Weston, J. A. (1963) Dev. Biol. 6, 279--310 4 Johnston, M. C. and Hazelton, R. D. (1972) in Third Symposium on Oral Sensation and Perception: The Mouth of the Infant (Bosma, J. S. and Thomas, H. C., eds), Springfield, II1., pp. 76-97 5 Noden, D. M. (1973) in Fourth Symposium on Oral Sensation and Perception: Development in the Fetus andlnfant (Bosma, J., ed.), pp. 933, US Department of Health, Education and Welfare 6 Noden, D. M. (1975) Dev. Biol. 42, 106-130 7 Le Douarin, N. M. (1969) Bull. Biol. Fr. Belg. 103, 435-452 8 Le Douarin, N. M. (1973) Dev. Biol. 30,217222
9 Ayer-Le Lic3vre, C. S. and Le Douarin, N. NI~ (19821 Dev. Biol. 94, 291-310 t0 Couly, G. F. and Le Douari~. N M. (1~;85) Dev. Biol. 110, 422--439 11 Fontaine-Prrus, J., Chanconie. M. and l,e Douarin, N. M. (1985) Dev. Biol. 107,227 238 12 Noden, D. M. (1978) Dev. Biol. 67,313-329 13 Narayanan, C. H. and Narayanan, Y. (19801 Anat. Ree. 196, 71-82 14 Ari~ns Kapper, .I. (19411 Ergeb. Anat. ~.ntwicklungsgesch 33, 370-412 15 Hamburger, V. andHamilton, H. L. ( 195t)J. Morphol, 88, 49-92 16 Noden, D. M. (1980) in Current Researth Trends in Prenatal Craniofacial Development (Pratt, R. M. and Christiansen, R. C. edsL pp. 1-25, Elsevier 17 Tosney, K. W. (1982) Dev. Biol. 89, 13-24 18 Le Douarin, N. M., Teillet, M-A., Zil[er, C. and Smith, J. (19781Proc. Natl Acad. Sci. USA 75, 2O30-2034 19 Le Dourarin, N. M., Le Lievre, C. S,, Schweizer, G. and Ziller, C. M. (1979) in Cell Lineage, Stem (?ellsand Cell Determination (Le Douarin, N. M., ed.), pp. 353-365, Elsevier 20 Le Li~vre, C. S., Schweizer, G. G., Ziller, C. M. and Le Douarin, N. M, (1980) Dev. Biol. 77,362-378 21 Schweizer, G., Ayer-Le Li~vre, C. and Le Douarin, N. M. (1983) Cell Differ. 13, 191 200 22 Le Douarin, N. M., Teillet, M . A . and Fontaine-Perus, J. (1984) in Chimaeras in Developmental Biology (Le Douarin, N. M. and McLaren, A., eds), pp. 313-352, Acacemic Press 23 Le Douarin, N. M. (1984) in Cellular and Molecular Biology of Neuronal Development (Black, 1., ed.), pp. 3-28, PLenum Press 24 Xue, Z. G., Smith, J. and Le Douarin, N. M (1985) C. R. Acad. Sci. Paris 300, 483-488 25 Brachet, A. (1907) Arch. Biol. 23, 165-257 26 Knouff, R. A. (19271 J. Comp. NeuroL 44, 250-361 27 Knouff, R. A. ( 1935)J. Comp. Neurol. 62, 1771 28 Platt, J. B. (1896) Q. J. Microsc. Sci. 38,485547 29 Jacobson, C. O. (19591 J. Lmbryol. Exp. MorphoL 7, 1-21 30 Rohlich, K. (1929) Arch. l£ntwicklungsmech 118, 164--199 31 Carpenter, E. (1937)J. Exp. ZooL 75, 103129 32 Van Oostrom, C. G. and Verwoerd, C. D. A. (1972) Aeta Morphol. Neerl. Stand. 9, 160 33 Klein, S. L. and Graziadei, P. P. C. (1983) J. Comp. Neurol. 217, 17-30 34 De Myer, I. (1967) Neurology 17, 961-972 35 Couly, G_ Rappaport, R., Brauner, R. and Rault, G. (1982) Pediatr. Res. 182, 886-906 36 De Morsier, G. (1967) in Etude sur les malformations du cerveau (De Morsier, G., ed.), M6decine et Hygiene de Geneve Pubt., pp. 101-153 Nicole M. Le Douarin, Josiane Fontaine-Perus and G~rard Couly are at the Instimt d' Embryologie du CNRS and the Coll~ge de France, 49bis A venue de la Belle-Gabrielle, 94130 Nogent-sur-Maine, France.
175
Cephalic ectodermal placodes and neurogenesis Nlcole M. Le Douarin, Josiane Fontaine,-P(~rus and G(~rard Couly In the vertebrate embryo, the primary neural anlage, or neural plate, develops from the superficial ectoderm as a result of an inductive stimulus arising from the chordomesoderm. Although it is well established that the neural plate itself gives rise to CNS, the fate of those cells located at the junction of the neural and superficial ectoderm (also called neural ridges or neural folds), from which the neural crest and placodes are derived, has been the subject of controversy. Tracing these cells during ontogeny has been made possible by using the quail~chick chimaera system. Such studies have revealed the contribution of the neural ridge and the neurogenic placodes to various cephalic structures, and have allowed their relationships with the CNS to be followed from the early stages of neurulation throughout the whole period of morphogenesis. The term placode was coined by Von Kuppfer 1 to designate the epithelial thickenings appearing in the cephalic ectoderm of the early vertebrate embryo. They appear anteriorly and laterally with respect to the main neural primordium, i.e., the neural plate and its lateral ridges, the neural folds, from which both the central and peripheral nervous system respectively arise. Their fate is strikingly varied, since they give rise to such diverse structures as the anterior pituitary gland, the olfactory sensory epithelium and the lens. More caudally, the superficial cephalic epithelium also generates the 'neurogenic' placodes from which epithelial cells
detach and, after a migratory phase, end up in certain cranial peropheral ganglia. By the turn of the century, the neurogenic fate of these placodes had been proposed, and their participation, along with the neural crest, in the genesis of certain cranial sensory nerve ganglia was suggested by several authors (see Ref. 2 for references). These pioneering studies, concerning virtually all classes of vertebrates, were mainly based on careful observations of normally developing embryos. Later, experiments were performed in which the potential sources of ganglion cells (i.e., the placodal ectoderm or the neural crest) were selectively removed.
Careful and detailed though they were, studies using histological observations or extirpation techniques led to controversial views on what is a very complex morphogenetic process. Placodal and crest cells have to migrate to reach the sites where gangliogenesis takes place, and once they have left their sources they adopt a mesenchymal morphology and cannot be properly traced to their targets or recognized once they have differentiated. In addition, extirpation of early embryonic territories is generally followed by their more-or-less extensive replacement through proliferation of neighbouring cells, thus making the effect of extirpation experiments difficult to interpret. Labelling the putative cellular components of the ganglia finally clarified this problem, at least in the avian embryo, on which various types of cell-marking experiments have been performed. Isotopic labelling of the neurectodermal cells with tritiated thymidine, a technique introduced by Weston 3 and later applied to cranial ganglion ontogeny by Johnston and Hazelton 4,
B.
J.S V.A
m.v
G.~I
p.1Y
Fig. 1. (A) Distribution o f placodes in a 2-day-old
8 days
Neural
crest
Placodes
embryo. T, trigeminal placode; Ot, otic placode; PI, P2, P3, epibranchial placodes. (B) Sensory spinal (DRG) and cranial ganglia at 8 days, the neurons of which are either of crest (blue) or placodal (grey) origin. V, VII, VIII, I X and X indicate the number of the cranial nerve. The sensory ganglia are T, trigeminal; G, geniculate; J and S, jugular and superior; P and N, petrosal and nodose.
176
T I N S - A p r i l 1986
~acode transplantation
C"Phrll/In¢~:~¢t~lt,or:S'
t
~
%% ~--==m===~#
~..........
~
" ~
chick host
chick host
o..,,0ooor
~tran|plant,tlon
chick host Fig. 2. Experimental designs used to trace the cells of neural crest and placodal origin during PNS ontogeny. Orthotopic and isochronic grafts ofplacodal ectoderm (A),cephalic neural crest OI) and neural primordium composed of the neural tube and the neural folds (C), between quail and chick embryos.
(Taken, with permission,from Ref. 2.)
NEURAL CREST CELLS
PLA C ODES
DERI W
IVATIVES
rninal
Ciliary Trigen
VII niculate) Proxim
Ethmo Sphen
ProxJrr:6,
IX
trosal) . . . . . .)ulo-acoustic
:^
(Superior) P r o x i m a l X ~-
t
700 oc
Fig. 3. Schematic drawing of a stage 9.5 chick embryo (about 10 somites) indicating positions of neural crest and placodal anlagen for cranial sensory and autonomic ganglia. (Taken,withpermission,from
Ref. 2.)
and Noden 5x', certainly achieved progress in this field. However, it was only when the quail/chick chimaera system was developed7,s with its stable quail nuclear marker, that the respective roles of placodes and neural crest in the ontogeny of the cephalic peripheral nervous system could be accurately defined. Fate maps of neural crest and placodal derivatives could be constructed in the avian embryo and the early stages of crest and placodal cell migration followed with precision2'9. Recently, we have been interested in the relationships between the most anterior placodes (giving rise to the pituitary and the olfactory epithelium) and the neural primordium itself (neural plate and neural folds) during early neurogenesis~°. Interesting observations have been made regarding the spatial distribution of these territories with respect to the regions of the CNS to which they later become functionally connected. They will be briefly mentioned in this article, which will mainly review studies that have used the quail/chick chimaera system as a tool to investigate the fate and developmental potencies of placodal and neural crest cells in neurogenesis.
Contributions of neurogenic placodes and neural crest to cephalic gangliogenesis In the head as well as in the other parts of the body, the autonomic ganglia are derived entirely from the neural crest. In contrast, the ontogeny of the sensory ganglia is much more complex in the cephalic region than in the trunk, due to the participation of the neurogenic placodes. Fig, 1 illustrates the anatomical distribution of the cranial sensory nerves and ganglia in the avian embryo at the mid-point of its incubation. The contributionof the neural crest has been as~ssed by two slightly different experimental paradigms. The first consisted of exchanging fragments of the whole neural primordium between quail and chick embryos9,N (Fig. 2c), and the other of grafting fragments of the neural crest only2'12'13 (Fig. 2B). The derivation of neurons from placodal territories was carefully analysed by D'Amico-Martel and Noden 2, who replaced chick cephalic ectodermal areas with their quail counterparts (Fig. 2A), and could thus establish from which placodal regions neurons of the various ganglia arise (Fig. 3). As a general rule, sensory neurons
TINS-April1986 originate from both the neural crest and the ectodermal placodes, whereas glial cells of the head sensory ganglia and nerves are all derived from neural crest with no participation of the placodal ectoderm. Plaeode-derived neurons are present in the ganglia associated with nerves V (trigeminal), VII (facial), IX (glossopharyngeal) and X (vagal), and are always located distally to crest-derived neurons. The placodal neurons, generally larger in size than the neurons of crest origin, are found in the distal portions of both lobes of the trigeminal ganglion, and in the geniculate, petrosal and nodose ganglia, where in the latter they form the totality of the neuronal population (Fig. 1B). All the neurons in the vestibular and acoustic ganglia of cranial nerve VIII are derived from placodal ectoderm and their glial cells from neural crest, with the exception of a few neural crestderived neurons that correspond to the rudimentary proximal ganglion of nerve V I I - the latter fuses with the vestibular ganglion of nerve VIII during the course of development. Crest derived cells can be identified in this ganglion by the quail marker, after grafting a quail neural crest fragment to the rostrai myelencephalic area 2. The avian neurogenic ptacodes can be divided into three types according to their position along the dorsoventral axis14. The more dorsal (also designated dorsolateral) is the otic placode located lateral to the myelencephalon. It is clearly visible from stage 10 (of Hamburger and Hamilton 15) in the chick, and has formed the otic cup by stage 13. The neurons of the VIIIth nerve ganglia arise from cells shed off from the medioventral aspect of this placode from stage 14. The peak of cell migration takes place at about 60 h of incubation, and by stages 23-24, the acoustic and vestibular ganglia can be identified. The intermediate placode, from which the distal neurons of the trigeminal ganglia arise, is located more ventrally than the otic placode. The epibranchial placodes appear in the dorsal region of the branchial cleft (Fig. 1A). The first epibranchial placode (first branchial cleft) yields the geniculate ganglion (distal ganglion of the ViIth cranial nerve). The second and third epibranchial placodes (on the 2nd and 3rd branchial arches) are the sites of origin of the petrosal and nodose ganglion neurons, respectively.
177
A
B
Gr
. ,
2
C
These placodes become distinguishable after the pharyngeal pouches contact the overlying ectoderm. Cells budding off from the placodes, singly or in small clusters, show some signs of neuronal differentiation, such as high acetylcholinesterase activity and silverstained neurite-like extensionsz. When free from the epithelial surface, the cells of placodal origin meet those of neural crest origin5'6'9,16,17that have migrated earlier. It is very striking that the neural crest ceils that form the proximal ganglia of sensory nerves IX and X (jugular-superior ganglionic complex) as well as the proximal lobe of the trigeminal ganglion, aggregate soon after they have left the neural primordium and form ganglia that remain in close contact with the CNS. In contrast, those neural crest cells that will participate in the mixed placodalcrest ganglia must move appreciable distances from the CNS in order to meet their placode-derived partners.
Differentiating capadties of the cellular components of the cranial sensory ganglia Developmental potentialities* of the neurons (of crest or placodalorigin) and non-neuronalcells in the cranial sensory ganglia have been analysed under normal and experimental conditions. In an immunocytochemical study on proximal and distal cranial sensory ganglia in embryos, aimed at detecting substance P, it appeared that substance P-like immunoreactivity (SPLI) was regularly found in most small proximal neural crest-derived neurons, but not in placodal nerve cells. However, one exception was in the nodose ganglion, where a few bonafide placodal neurons showed SPLI. It is striking that in the dorsal root ganglion (DRG), the small *i.e., the phenotypes that a given cell is able to express if removed from its normal embryonic surroundings and submitted to a different microenvironment,
178 mediodorsal neurons that show many similarities to the crest-derived neurons of the cranial sensory ganglia also contain substance P, while the larger lateroventral nerve cells are, like those of placodal origin, substance P-negative. The developmental capacities of cells in the cranial sensory ganglia were investigated using an experimental system (described in Fig. 4) in which they are made to express phenotypes that they do not usually exhibit under normal conditions. This system consists of the retrotransplantationof a ganglion (of which all or some cells carry the quail nuclear marker) into the neural crest migration pathway of a younger chick host. The mixed neural crest and placodal origin of the distal ganglia of cranial nerves IX and X allow the construction of chimaeric petrosal and nodose ganglia in which either the neuronal or the non-neuronal cells are selectively labelled. In both cases, fragments (of about 2000 cells) of such ganglia are implanted into the neural crest migration pathway of 2-day-old chick host embryos. In such grafts, the piece of implanted ganglion rapidly loses its cohesiveness and its cell components become dispersed and migrate in the host's tissues. After a phase of dispersion, the ganglion cells aggregate to neural crest-derived structures in the host, where they become mixed up with chick crest-derived cells. Back-transplantation experiments of quail peripheral ganglia into chick embryos have been used extensively by our group9'18'22, and have provided relevant information about the state of commitment of the neural crest and peripheral ganglion cells23. If the graft involves chimaeric nodose and petrosal ganglia, the distributionof labelled cells is strikingly different according to the cell type carrying the quail marker. When neurons are labelled, practically no quail cells are subsequently found in the chick host, indicating that they are not able to survive graft conditions. In contrast, non-neuronalcells of the quail type show considerable invasion of the host, having proliferated abundantly. They can be found in the autonomic nervous system of the host, including the gut, where they differentiate into both neuronal and non-neuronal cells. The graft-derived neurons exhibit phenotypes in accordance with their position in the host, i.e., they differentiate into adrenergic neurons and chromaffin cells in the sympathetic ganglia and adrenal medulla, respec-
7 7 N 5 - A p r i l It)80 a QUAIL
CHICK
b
Fig. 5. (a and b) Substitution between quail and chick embryos (at stages of 0-3 somites) of the anterior neural ridge (150 lira long) in (a) and of the anterolateral neural ridge in (b). Fragments A and B (about 150 Wn long) are gra~ed either independently or as a single piece according to the experiment considered. (¢)Isotopic and isochronic transferof the anterior neural ridge and neural plate areas from a quail to a chick embryo at stages 0-3 somites. Here the embryos represented are at the 2somite stage. (d) Experiment in which the anterolateral neural ridge (Zone A in 5b) along with the lateral area of the neural plate was transplanted isotopically from a quail (Q) to a chick (Ch) embryo at stages of 0-3 somites. (e) Photograph of a 3-somite chick embryo in which the anterior neural ridge has been replaced by its quail counterpart. Arrows indicate the graft.
tively, and into enteric neurons, some containing neuropeptides, when they migrate into the gut wall. These results indicate that in the cranial sensory ganglia of mixed (crest and placode) origin, the non-neuronal cell population retains some of the developmental potentialities of the structure from which it is derived, the neural crest. These potentialities are not normally expressed during the ontogeny of these ganglia, since catecholamine containing cells are not detectable in either quail or chick petrosal and nodose ganglia at any of the developmental stages examined9.
When retrotransplanted into the crest cell migration pathway of a younger host, the quiescent neuronal precursors present in the non-neuronal cell population of the ganglia are stimulated to divide and differentiate. It is noteworthy that no graft-derived cells have ever been seen to develop into sensory neurons in the host DRG. It appears, therefore, that the developmental potentialities of the quiescent precursors in the distal sensory ganglia of cranial nerves IX and X are restricted to the autonomic (i.e., sympathetic, parasympathetic and the enteric net-
179
TINS-April 1986
vous system) differentiation pathway. They can therefore be called type ' A ' precursors according to the model proposed to account for sensory and autonomic cell lineage divergence during PNS differentiation2a. Recently, we obtained differentiation of type A precursors contained in D R G and nodose ganglia in vitro 24 and were able to show that a great many adrenergic cells developing in cultures of dissociated sensory ganglion cells arose from cycling progenitor cells, a property that allowed them to be distinguished from the pool of post-mitotic sensory neurons present in the culture. Development of the hypophyseal and the olfactory placodes We recently published experiments 1° that were primarily directed at investigating the early relationships between the CNS and PNS anlagen (i.e. the neural plate/neural fold complex) and the placodes. The hypothesis underlying this endeavour was that induction by the chordomesoderm of the whole nervous system in the ectodermal germ layer is a unique event eliciting the formation of a virtually continuous area of neural epithelium. According to this view, placodes would become secondarily detached from the neural primordium, which would then become segregated into the neural plate/neural fold complex on the one hand and the placode-derived organs on the other. It is of note that the spatial relationships between the neural plate and the placodal territories has attracted the attention of several workers. The observations from amphibians corroborate the notion that neural plate and placodes are closely related since the placodal ectoderm originates from a region termed the primitive placodai thickening25-28,located in the outer side of the neural fold itself. The future olfactory neurons, in particular, are localized in apposition to the primitive neural fold and close to the anlage of the pituitary29-33. Our own investigations were based on the substitution in chick embryos of small and defined regions of the anterior neural ridge and neural plate by their quail counterparts, as indicated in Fig. 5. The recipient embryos were examined by chimaerism analysis after 5 and 8 days of incubation, and the following conclusions were reached. At the early stages of neurogenesis in the avian embryo, the neural ridges limiting the neural plate anteriorly and laterally at the level of the forebrain, contain the
b
Fig. 6. Results o f the experiment (represented in Figs l a and l e) in which the anterior neural ridge of a quail embryo (1-somite stage) was grafted isotopically to a chick host. (a) Sagittal section o f the host at E5 showing, at low magnification, the areas represented in (b) and (c). (b) Tip o f Rathke's pouch shows the quail nuclear marker while the unfundibulum belongs to the host. (c) Mouth epithelium made up o f quail cells. Note that the adjacent mesenchymal cells are o f chick host type. (a) x 90; (b) x 1350; (c) x 1350.
(Taken, with permission, from Ref. 10.)
180
presumptive territories of the hypophyseal (Fig. 6) and olfactory ectodermal placodes. This part of the neural ridge also includes the precursor cells of large areas of superficial ectoderm corresponding to the nasal cavities and the ectoderm covering the premaxillary area and the upper beak (Fig. 7). This latter finding was unexpected since, until then, it had been thought that the neural ridge (apart from the mesectodermal cells that originate from the neural crest) only yielded neural structures. When areas of the neural plate adjacent to the presumptive hypophyseal and olfactory placodes were included in the graft (i.e., labelled by the quail nuclear marker) (Fig. 5), it was then found that the primordia of both hypophysis and hypothalamus were continuous, as were those of the olfactory epithelium and the olfactory bulb. It thus appears that at the early somitic stages, mapping of the neural primordium leads to the identification of territories from which central and peripheral structures of particular functional units (here the hypophysealhypothalamic complex and the olfactory system) are derived. Although the localization of the presumptive territories of the dorsolateral, intermediate and epibranchial placodes, has not been determined at the very earliest stages of neurogenesis, it is tempting to hypothesize that, like the hypophyseal and olfactory placodes, they also have their origin in the neural ridge. This would mean that the chordomesoderm's induction of the whole nervous system in the ectodermal germ layer is a unique event giving rise to the so-called neural plate from which the ectodermal placodes migrate secondarily. In addition, results showing the close relationships of the forebrain neural primordium to the adenohypophysis, olfactory organs and facial ectoderm are interesting to correlate with certain congenital pathologies in humans. This is the case in De Myer's mediofacial syndrome, in which malformations of the diencephalo-telencephalic regions are associated with naso-fronto-premaxillary hypoplasia 34. Other examples include adenohypophyseal deficiencies, revealed by an insufficient production of growth hormone, concomitant with naso-frontal malformation 35 and De Morsier's olfactogenital syndrome 36,
TINS--April 1986
i
E
Fig. 7. Fate map of the anterolateral neural ridge and anterior neural plate at stages of O--3somites in the avian embryo. (a) presumptive area of the nasal ectoderm, (b) neural ridge containing the hypo-
physeal placode, (e) presumptive region containing the olfactory placode, (d)presumptive territory o f the ectoderm of upper beak, (e) region where the anlage of the egg tooth is located, (f) presumptive territory of the diencephalon (i.e. infundibulum and hypothalamus).
associated with anosmia. This work will continue in an endeavour to analyse systematically, via the quail/chick marker (1) the patterning of the cephalic structures derived from the neural primordium and their relationships with the cells derived from the other germ layers, and (2) the sequential commitment and phenotypic expression of the neuroectodermal cells. One of the goals in view is also to provide relevant information on the genesis of certain congenital human malformations.
Selected references 1 Von Kuppfer, C. (18941 Lehmann Munchen 2 D'Amico-Martel, A. and Noden, D . M . (19831 Amer. J. Anat. 166, 445-468 3 Weston, J. A. (1963) Dev. Biol. 6, 279--310 4 Johnston, M. C. and Hazelton, R. D. (1972) in Third Symposium on Oral Sensation and Perception: The Mouth of the Infant (Bosma, J. S. and Thomas, H. C., eds), Springfield, II1., pp. 76-97 5 Noden, D. M. (1973) in Fourth Symposium on Oral Sensation and Perception: Development in the Fetus andlnfant (Bosma, J., ed.), pp. 933, US Department of Health, Education and Welfare 6 Noden, D. M. (1975) Dev. Biol. 42, 106-130 7 Le Douarin, N. M. (1969) Bull. Biol. Fr. Belg. 103, 435-452 8 Le Douarin, N. M. (1973) Dev. Biol. 30,217222
9 Ayer-Le Lic3vre, C. S. and Le Douarin, N. NI~ (19821 Dev. Biol. 94, 291-310 t0 Couly, G. F. and Le Douari~. N M. (1~;85) Dev. Biol. 110, 422--439 11 Fontaine-Prrus, J., Chanconie. M. and l,e Douarin, N. M. (1985) Dev. Biol. 107,227 238 12 Noden, D. M. (1978) Dev. Biol. 67,313-329 13 Narayanan, C. H. and Narayanan, Y. (19801 Anat. Ree. 196, 71-82 14 Ari~ns Kapper, .I. (19411 Ergeb. Anat. ~.ntwicklungsgesch 33, 370-412 15 Hamburger, V. andHamilton, H. L. ( 195t)J. Morphol, 88, 49-92 16 Noden, D. M. (1980) in Current Researth Trends in Prenatal Craniofacial Development (Pratt, R. M. and Christiansen, R. C. edsL pp. 1-25, Elsevier 17 Tosney, K. W. (1982) Dev. Biol. 89, 13-24 18 Le Douarin, N. M., Teillet, M-A., Zil[er, C. and Smith, J. (19781Proc. Natl Acad. Sci. USA 75, 2O30-2034 19 Le Dourarin, N. M., Le Lievre, C. S,, Schweizer, G. and Ziller, C. M. (1979) in Cell Lineage, Stem (?ellsand Cell Determination (Le Douarin, N. M., ed.), pp. 353-365, Elsevier 20 Le Li~vre, C. S., Schweizer, G. G., Ziller, C. M. and Le Douarin, N. M, (1980) Dev. Biol. 77,362-378 21 Schweizer, G., Ayer-Le Li~vre, C. and Le Douarin, N. M. (1983) Cell Differ. 13, 191 200 22 Le Douarin, N. M., Teillet, M . A . and Fontaine-Perus, J. (1984) in Chimaeras in Developmental Biology (Le Douarin, N. M. and McLaren, A., eds), pp. 313-352, Acacemic Press 23 Le Douarin, N. M. (1984) in Cellular and Molecular Biology of Neuronal Development (Black, 1., ed.), pp. 3-28, PLenum Press 24 Xue, Z. G., Smith, J. and Le Douarin, N. M (1985) C. R. Acad. Sci. Paris 300, 483-488 25 Brachet, A. (1907) Arch. Biol. 23, 165-257 26 Knouff, R. A. (19271 J. Comp. NeuroL 44, 250-361 27 Knouff, R. A. ( 1935)J. Comp. Neurol. 62, 1771 28 Platt, J. B. (1896) Q. J. Microsc. Sci. 38,485547 29 Jacobson, C. O. (19591 J. Lmbryol. Exp. MorphoL 7, 1-21 30 Rohlich, K. (1929) Arch. l£ntwicklungsmech 118, 164--199 31 Carpenter, E. (1937)J. Exp. ZooL 75, 103129 32 Van Oostrom, C. G. and Verwoerd, C. D. A. (1972) Aeta Morphol. Neerl. Stand. 9, 160 33 Klein, S. L. and Graziadei, P. P. C. (1983) J. Comp. Neurol. 217, 17-30 34 De Myer, I. (1967) Neurology 17, 961-972 35 Couly, G_ Rappaport, R., Brauner, R. and Rault, G. (1982) Pediatr. Res. 182, 886-906 36 De Morsier, G. (1967) in Etude sur les malformations du cerveau (De Morsier, G., ed.), M6decine et Hygiene de Geneve Pubt., pp. 101-153 Nicole M. Le Douarin, Josiane Fontaine-Perus and G~rard Couly are at the Instimt d' Embryologie du CNRS and the Coll~ge de France, 49bis A venue de la Belle-Gabrielle, 94130 Nogent-sur-Maine, France.