INTRODUCTION

The skull of vertebrates consists of bones of membranousand cartilaginous origin, which have the double role of pro-tecting the brain and sense organs and of providing theanimal with a masticatory apparatus, which can also func-tion as a prehensile system for capturing preys.

The participation of the ectomesenchyme (also calledmesectoderm), derived from the neural crest, in the facialand visceral skeleton was first recognized at the end of thelast century by the pioneer works of Kastschenko (1888),Goronowitsch (1892, 1893) and Platt (1893, 1897) and waslater well documented in lower vertebrates (see Hörstadius,1950 for a review). The quail-chick marker system in theavian model eventually allowed the demonstration that inhigher vertebrates the skeleton is entirely of neural crestorigin (reviewed in Le Douarin, 1982).

Two points, however, remained unclear about vertebratehead skeletogenesis. One concerned the origin of the vaultof the skull, composed of the parietal and frontal bones,which were considered by Le Lièvre (1978, see also LeDouarin, 1982) as essentially derived from the cephalic

mesoderm. The second is the extent of the participation ofthe vertebrae to the occipital region of the skull.

The first problem clearly rose when, in one of our ear-lier works on the embryology of the vertebrate head (Coulyand Le Douarin, 1985, 1987, 1988), we scrutinized the fateof the cephalic paraxial mesoderm (Couly et al., 1992) fromthe early somitic stages onward. We realized that the parax-ial mesoderm essentially yielded muscle structures whereasits contribution to the skull was reduced to the sphenoidaland otic regions.

No cells of paraxial cephalic mesoderm were found toparticipate in the skull vault i.e. to the frontal and parietalbones. We then decided to reinvestigate this question byusing the classical substitution technique of defined embry-onic territories between quail and chick embryos (LeDouarin, 1969, 1973). The idea was to study the fate of thecephalic neural folds from an earlier stage of developmentthan envisaged in previous works and to follow carefullythe development of the dermis and membrane bones form-ing the vault of the skull. The experiments were done atthe late neurula stage (3-somite-stage) and showed an earlymigration of the cephalic neural crest cells, which had so

409Development 117, 409-429 (1993)Printed in Great Britain © The Company of Biologists Limited 1993

We have used the quail-chick chimera technique tostudy the origin of the bones of the skull in the avianembryo. Although the contribution of the neural crestto the facial and visceral skeleton had been establishedpreviously, the origin of the vault of the skull (i.e. frontaland parietal bones) remained uncertain. Moreover for-mation of the occipito-otic region from either the somiticor the cephalic paraxial mesoderm had not been exper-imentally investigated. The data obtained in the presentand previous works now allow us to assign a preciseembryonic origin from either the mesectoderm, theparaxial cephalic mesoderm or the five first somites, toall the bones forming the avian skull. We distinguish askull located in front of the extreme tip of the notochordwhich reaches the sella turcica and a skull locatedcaudally to this boundary. The former (‘prechordalskull’) is derived entirely from the neural crest, thelatter from the mesoderm (cephalic or somitic) in itsventromedial part (‘chordal skull’) and from the crest

for the parietal bone and for part of the otic region. Animportant point enlighten in this work concerns thedouble origin of the corpus of the sphenoid in whichbasipresphenoid is of neural crest origin and the basi-postsphenoid is formed by the cephalic mesoderm. For-mation of the occipito-otic region of the skeleton is par-ticularly complex and involves the cooperation of thefive first somites and the paraxial mesoderm at the hind-brain level. The morphogenetic movements leading tothe initial puzzle assembly could be visualized in areproducible way by means of small grafts of quailmesodermal areas into chick embryos.

The data reported here are discussed in the evolu-tionary context of the ‘New Head’ hypothesis of Gansand Northcutt (1983, Science, 220, 268-274).

Key words: neural crest, somites, cephalic mesenchyme, facialskeleton, origins of frontal, parietal, occipital bones, sphenoidbones

SUMMARY

The triple origin of skull in higher vertebrates: a study in quail-chick

chimeras

Gérard F. Couly, Pierre M. Coltey and Nicole M. Le Douarin

Institut d’Embryologie cellulaire et moléculaire du CNRS et du Collège de France, 49bis, Avenue de la Belle-Gabrielle, 94736Nogent-sur-Marne Cedex, France

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far remained unnoticed and which yields the dermis andmembrane bones of the entire vault of the skull includingthe frontal and the parietal bones and their sutures.

The remaining question of the participation of somites inhead morphogenesis is an ancient one. This idea was firstexpressed in the XIXth century by Goethe (1791) and Oken(1807) (see Jeffs and Keynes (1990) and references therein)and was formulated in an extreme form in the vertebratetheory of the skull, according to which the skull is madeup of one (or three) somehow modified vertebrae. Progressin embryology and comparative anatomy however, showedthat only the occipital region of the skull could be consid-ered of vertebral origin (reviewed in Augier, 1931). More-over, it is thought that no incorporation of vertebrae intothe skull exists in the skull of the agnathes and that, duringevolution of vertebrates, the enlargement of the headinvolved the contribution of an increasing number ofcephalic vertebrae to form the occipital bone of the gnathos-tomes. No experimental analysis, however, had so far beenconducted to investigate the embryonic origin of the occip-ito-otic region of the skull.

This old problem was rejuvenated by the discovery invertebrates of genes characterized by a highly conservedsequence of nucleotides, the homeobox (Hox genes), encod-ing proteins that regulate the expression of other genes (seeGehring, 1987 ; De Robertis et al., 1990 ; Kessel and Gruss,1991 for reviews and references therein).

The possible involvement of Hox genes of vertebrates inpatterning the segmental structures of the axial organs(paraxial mesoderm and neural tube) was suggested by theirstrictly regulated spatiotemporal pattern of expression in themajor segmented structures, the hindbrain and the paraxialmesoderm, and in the non-segmented neural tube. The classI Hox genes are distributed in four clusters located on fourchromosomes in mouse and man. They form 13 paraloggroups based on the similarity of their highly conservedhomeobox. The demonstration that Hox genes actuallydefine axial level specification in the vertebral column wasfirst brought about by Kessel and Gruss (1991) who demon-strated that retinoic acid (RA) administered to the embryosat defined developmental stages, while altering the patternof Hox gene expression in the somites, also modifies thecombinatorial arrangement of Hox gene expression at eachaxial level, thus generating homeotic transformations in thevertebrae. Targeted mutation of the Hox-3.1 gene in themouse also generated homeotic transformation in the ver-tebral column (Le Mouellic et al., 1992). Another hintpointing to the same assumption was the observation thattransgenic mice expressing ectopically the Hox-1.1 geneexhibited anomalies of the cervical region of the vertebralcolumn (Kessel et al., 1990). The anomalies observed inthe genetic manipulations of the embryo cited above mostlyaffected the base of the skull : absence of the basisphenoidand supraoccipital, malformations and/or fusions involvingthe exo- and basioccipital bones. Most interestingly, inabout one third of the embryos that received RA at E7,“additional ossifications between, and fused to, the occipi-tal bones and the atlas indicated the manifestation of a par-tial occipital vertebra, a proatlas” (quoted from Kessel andGrüss, 1991, p. 93).

It seemed therefore timely to reinvestigate, using classi-

cal methods of experimental embryology, what is, in normaldevelopment, the participation of the somites to the skull.This was done by substituting successively the six firstsomites of the chick by their quail counterpart in 3- to 6-somite-stage embryos.

By combining the data obtained in previous works (John-ston, 1966 ; Le Lièvre and Le Douarin, 1975 ; Le Lièvre,1978 ; Noden, 1982) and in the present investigation, wenow provide a complete picture of the embryogeny of theskull in birds and we assign to each bone a precise embry-onic origin from either the ectomesenchyme, the paraxialcephalic mesoderm or the somites.

We also show that the boundary between the mesoder-mal and the mesectodermal parts of the skull correspondsto the middle of the sella turcica, which is the cranialmostlevel reached by the notochord.

MATERIAL AND METHODS

Quail and chick (White Leghorn strain) eggs were from a com-mercial source and incubated in a humidified atmosphere at 38°C.

Quail to chick chimeras were constructed by isotopic andisochronic substitution of defined regions of the neural folds (i.e.the presumptive neural crest), of the paraxial cephalic and rostralsomitic mesoderm in the chick embryo by their counterparts fromthe quail at 3-somite-stage. In a second experimental series, thefates of somites 4, 5 and 6 were investigated in 4-, 5- and 6-somite-stage embryos. The grafts were always performed unilat-erally. The details of the experiments are recorded in Table 1.

Experimental seriesExperiment I. The level for the operation was a 450 µm lengthpiece of neural fold of the presumptive diencephalon and anteriorhalf of the mesencephalon according to the fate map of the neuralprimordium that we have previously established (Couly and LeDouarin, 1985, 1987, 1988). Fig. 1 shows the quail graft in situ2 1/2 hours after implantation into the stage-matched chickembryo.

Experiment II involved the graft of the neural fold corre-sponding to the posterior half of the mesencephalon and meten-cephalon down to the level of the 1st somite.

Experiment III involved the graft of the neural fold corre-sponding to the level of the three first somites (i.e., the rostralmyelencephalon).

Experiment IV involved the graft of the median paraxial meso-derm (MPM) at the mesencephalic level as defined in our previ-ous work on the fate of the paraxial mesoderm (Couly et al., 1992).

Experiment V involved the caudal region of the MPM corre-sponding to the metencephalon.

Experiments VI, VII, VIII, IX, X and XI correspond, respec-tively, to grafts of the 1st, 2nd, the 3rd right somite in 3-somite-stage embryos, the 4th, 5th, 6th somites in, respectively, 4-, 5-and 6-somite-stage embryos.

Chimerism analysis 145 embryos were operated upon and 73 developed normally andwere further analyzed at stages varying from 8 to 14 days of incu-bation (E8 to E14) (Table 1). It was necessary to keep chimerasalive until E14 since identification of certain bones of the skull(e.g. frontal, parietal, supraoccipital) can be reliably identified onlyduring the second half of the incubation period. The head wasfixed in Zenker’s fluid, embedded in wax and cut in 5-6 µm coro-nal sections. Identification of normal and chimera bones was doneon sections that were serially examined. The shape of each bone

G. F. Couly, P. M. Coltey and N. M. Le Douarin

411Embryogeny of skull in vertebrates

Fig. 1. (A) Dorsal view in SEM of the head of a 3-somite-stagechick embryo. (B)At the same stage, transverse section at thepresumptive level of the mesencephalon. No cephalic mesodermalcells are present dorsally within the neural fold. (C) 2.5 hours afterthe graft, SEM view of a chick embryo in which the rightprosencephalo-mesencephalic neural fold (arrows) has beenreplaced at 3-somite-stage by its counterpart taken from a stage-matched quail embryo. Note the perfect incorporation of thetransplant into the host neural fold. Scale bar, 100 µm.

Table 1. Experimental series and number of embryosexamined

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and its position with respect to the different brain regions werealso studied on dissected skulls, namely for identification of theroof and occipital regions. Quail and chick cells were identifiedby the structure of their nucleus after Feulgen-Rossenbeck’s stain-ing. Bone and cartilage were evidenced with alcian blue and chlo-rantine fast red (Lison, 1954). Alcian blue stains the extracellularmatrix of cartilage in blue and chlorantine fast red stains the bonematrix in red.

Scanning electron microscopy (SEM) For SEM study, the embryos were immersed in 2.5% glutaralde-hyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4°C. Afterrinsing in the same buffer, the specimens were postfixed in 1%osmium tetroxide in the same buffer for 2 to 3 hours at room tem-perature, thoroughly rinsed and incubated in thiocarbohydrazide(saturated aqueous solution) for 10 minutes (Kelley et al., 1973).After rinsing in water, they were returned into 1% osmium tetrox-ide in water for 1 hour. After washing in water, the specimenswere dehydrated through a graded series of ethanol substitutedwith iso-amyl acetate and critical point dried from CO2. Aftermounting on stubs with silver conductive paint, the specimenswere vacuum coated with gold and examined with a Jeol 1200EX electron microscope at 20 kV accelerating voltage.

The nomenclature of the bones used in the present work hasbeen adopted from Romanoff (1960) for the early stages of devel-opment, De Beer (1937) for the chondrocranium and from Baumel(1979) and Nickel et al. (1977) for the late embryonic stages andadult birds.

RESULTS

I - Grafts of cephalic neural crestGrafts of the diencephalon and anteriormesencephalic neural crest (experiment I) The results were examined in ten embryos ranging from E8to E14 (Table 1). In the facial area, the premaxillary, max-illary, nasal and vomer (i.e. membrane bones) and interor-bital septum (still cartilaginous at E14), the sclerotic andthe supraorbital bone were made up of quail cells. Inaddition, we found that the dermis of the scalp, themeninges of the forebrain (see Couly and Le Douarin, 1987)and the totality of the frontal bone, together with the ante-rior region of the squamosal, the basipresphenoid, the ros-trum of the parasphenoid and the pterygoid were of graftorigin. The sutural spaces between the membranous bones(the two frontal bones particularly) were also made up ofquail connective tissue (Figs 2, 3). It is noticeable that thisexperiment shows the caudal boundary of the participationof the neural crest in the floor of the skull. This limit lieswithin the sella turcica and strikingly corresponds to theanterior tip of the notochord (Fig. 4A,B).

Graft of the posterior mesencephalic, themetencephalic and the myelencephalic neural crest(experiments II and III) Experiment II concerns the posterior mesencephalic and themetencephalic level of the crest. Chimerism analysis wasperformed on ten embryos from E8 to E14 (Table 1).

The quail cells were localized in the parietal bones anddermis at the same level, the squamosal, the jugal, thequadrate, the pterygoid, the palatine, the skeleton of the

lower jaw (Meckel’s cartilage, dentary, angular, opercular)(see also Johnston et al., 1974 and Le Lièvre and LeDouarin, 1975) (Fig. 5). In addition, an overlap of labellingwas observed in experiments I and II (compare Figs 2B and5B). This is likely due to the mixing of crest cells in thisregion of the head where morphogenetic movements areactive. In all cases, the cells of the sutural space, still incom-pletely ossified at E14, were of donor type. In addition, partof the cochlearis area of the otic capsule (pro-otic area)originated from the grafted neural crest cells. The hyoidcartilage was also entirely of donor type (Fig. 5). Fig. 4A,Bshow the contribution of the neural crest to the lower jawand second branchial arch region.

Experiment III involved grafting the myelencephalicneural crest. Analysis of chimera was performed on fourembryos (E8 to E14) (Table 1). Migration of crest cellsfrom this level did not involve the cephalic area. The cellscolonized the 3rd, 4th branchial arches and their derivativesin the anterior thoracic region as previously established (seeLe Douarin, 1982 for a review).

II - Grafts of cephalic mesodermGrafts of the MPM at the mesencephalic level(experiment IV)Chimerism analysis was performed on eight embryos(Table 1). The graft does not yield quail dermis but onlythat part of the chondrocranium corresponding to the sphe-noid bone complex (basi-postsphenoid, alipostsphenoid alsocalled pleurosphenoid), the postorbital bone and a smallcontribution to the pars cochlearis of the otic capsule, asshown in Couly et al., (1992) (illustrated in figure 9 ofCouly et al., 1992 and in Fig. 4A,B herein). In addition,this graft gives rise to vascular endothelial cells of quailtype.

Graft of the MPM at the metencephalic level(experiment V)Eight embryos have been examined (Table 1). In that case,the supraoccipital bone and the margin of the medial por-tion of the vestibular region of the otic capsule are of quailorigin (Fig. 6). Moreover, the mesoderm of the meten-cephalic MPM participates in the formation of the wall ofthe semicircular canal in the following way : quails cellsare found in the lateral part and the ventral bend of theupper semicircular canal, the rest of it originates from meso-dermal cells of somitic origin (see below) (Fig. 4A, B). Inaddition, the grafted cephalic mesoderm gives rise to vas-cular endothelial cells of quail type located in the laterofa-cial structure of the chick host.

III - Grafts of the six first somitesAll these grafts give rise to occipital, dorso-cervical dermisand also to the meninx of myelencephalon and rostralmedulla. Details of experimental series are given in Table1.

Grafts of the 1st somite (experiment VI) The first somite is smaller than the following ones, inaddition, it loses its individuality by 7- to 10-somite-stage,in fusing with MPM of the metencephalic level. Then the

G. F. Couly, P. M. Coltey and N. M. Le Douarin

413Embryogeny of skull in vertebrates

second somite formed becomes the most cranial of theseries. Since the cells of the 1st somite disperse, their exactfate was difficult to assess with precision. They can be fol-lowed throughout development by the quail nuclear marker.They form the exoccipital part of the chondro-occipital arch(Figs 7, 8 and 9), and a narrow cartilaginous stripe in the

basioccipital between the 1st and 2nd roots of the hypoglos-sus nerve (Fig. 8). In addition, the 1st somite yields mostof the pars canalicularis of the otic capsule (Figs 7, 8 and9). The muscles of the neck and of the larynx were alsopartly labelled (Fig. 9, Table 2). The dermatome of the 1stsomite forms the dermis in the occipital region of the head

Fig. 2. Results of experiment I. Graft of prosencephalic and anterior mesencephalic neural crest. (A) Transverse section of the head of achimeric embryo at E14 and (B) the corresponding schematic drawing showing the localization of quail cells in the cephalic skeleton ofthis chimera. The totality of the frontal bone (C,D), the supra-orbital bone (E) and metopic (interfrontal) suture (F) are of quail origin (Fr,frontal bone ; S.O, supra-orbital). Scale bar, 1 mm (A) and 10 µm (C-F).

414 G. F. Couly, P. M. Coltey and N. M. Le Douarin

Fig. 3. Results of experiment I. Graft of prosencephalic and anterior mesencephalic neural crest. (A) Transverse section of the samechimeric embryo as in Fig. 2 at the level of the sella turcica (d and v indicate the dorsoventral axis). The dermis of the lateral region of theskull (squamosal) (B) and the masticatory aponevrosis (C) are of the quail origin. The chondrocytes of the rostral half of the sella turcica(basipresphenoid) (D), the pterygoid (E) and parasphenoid bone (F) are of quail origin. (G) High magnification of the sutural spacebetween pterygoid and palatine bones. (H) Enlargement of the central part of A (framed) corresponding to the sella turcica. (I) Boundarybetween the neural crest (quail cells) and the mesodermal (chick cells top) contribution to the cartilage of the sella turcica. The boundarycorresponds to the limit between the basipresphenoid (quail) and the basipostsphenoid (chick). (C, chick; Q, quail; Ah, adenohypophysis;Rh, rhombencephalon). Scale bar, 1 mm (A,H) and 10 µm (B-G and I).

415Embryogeny of skull in vertebrates

(Fig. 7B), thus, showing the boundary between the dermisof neural crest and of mesodermal somitic origin in thedorsal head region.

Graft of the 2nd somite (experiment VII)The contribution of the graft is a stripe of cartilaginous cellslocated between the 2nd and 3rd roots of the hypoglossusnerve caudally to the area derived from the 1st somite (Fig.10). Tongue and cervical muscles were also partly of graftorigin (Table 2). Notable is the fact that the tongue andmuscles are innervated by the hypoglossus nerve (N. XII),

Fig. 4. Chondrocranium of a bird at E9 (after De Beer, 1937)showing the origin of the different capsules. (A) Lateral viewincluding the lower jaw and hyoid complex. (B) Dorsal viewwithout the lower jaw and hyoid complex. Note that theparticipation of somites (in green) to the occipital and oticcapsules is not detailed here; see Fig. 8 for a more preciseanalysis. Red, cartilage of neural crest origin; blue, cartilage ofcephalic mesoderm origin; green, cartilage of somitic origin. Theblack line indicates the notochord.

1 nasal capsule2 orbital capsule 3 otic capsule (pars ampullaris)4 otic capsule (pars choclearis)5 occipital arch6 exo-occipital7 basi-occipital8 supra-occipital9 interorbital septum10 hyoid cartilage11 Meckel’s cartilage12 quadrato-articular cartilage13 basisphenoid (a, basi-post sphenoid; b, basi-presphenoid).

Fig. 3H,I

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which originates from the myelencephalon (i.e. rhom-bomere 8th) (Fig. 8).

Graft of the third somite (experiment VIII)The contribution of the 3rd somite is a specific zone of thecorpus occipitalis located between the 3rd and 4th rostralroots of the hypoglossus nerves (Fig. 8). In this case also,tongue and cervical muscles are partly derived from themyotome of the 3rd grafted quail somite (Table 2).

Graft of the fourth somite (experiment IX) This somite gives rise to the region of the chondro-occip-ital arch located caudally to the 4th hypoglossus root (Figs8 and 11). Tongue and cervical muscles are also partlylabelled by this graft (Table 2).

Graft of the fifth somite (experiment X) The 5th somite yields the distal part of the chondro-occip-ital arch, corresponding to the occipital condyles, the ante-rior arch of atlas and the odontoid apophysis of axis (therest of the axis is of chick host origin in this experiment,Fig. 12G). Therefore the 5th somite contributes to three dif-ferent skeletic structures which form the occipito-atlanto-

odontoid articulation (Fig. 8). In addition, some tongue andcervical muscles are of quail origin in this experiment (Fig12, Table 2).

Graft of the sixth somite (experiment XI) The dorsal arch of the atlas vertebra and the rostral half ofthe axis are of quail origin whereas its caudal half belongto the host. No tongue muscles were labelled. The myotomeof somite 6 participates in cervical muscles (Figs 8, 13,Table 2).

CONCLUSIONS AND DISCUSSION

The question raised at the initiation of this work concernedthe embryonic origin of the skull from the ectoderm via theneural crest and from the mesoderm via the somites and thecephalic paraxial mesoderm. The origin of the variousbones constituting the avian skull was investigated by usingthe quail/chick chimera system. Chimeras in which definiteterritories of the chick embryo were replaced isotopicallyby their counterpart coming from stage-matched quailswere constructed first in 3-somite-stage embryos when clo-

G. F. Couly, P. M. Coltey and N. M. Le Douarin

Table 2. Dermomyotomal and sclerotomal derivatives of the six first somites

*The bars indicate the contribution of each somite to muscles and bones.The insert represents the tongue and laryngeal muscles inserted on the hyoid cartilage. A to G refer to the muscles whose names are on the left of the

table.

417Embryogeny of skull in vertebrates

sure of the neural tube had not yet started. In a secondexperimental series (experiments IX to XI), the fate ofsomites 4 to 6 was investigated in older embryos. At 3-somite-stage, the head neural fold can be removed andreplaced selectively while no emigration of neural crest

cells has happened yet. Therefore, the paraxial mesoderm,which is accessible to surgical manipulation (see Couly etal., 1992), is not possibly contaminated by ectomesenchymecoming from the crest. This makes the experimentalapproach presented here different from the previous ones

Fig. 5. Results of experiment II. Graft of the posterior mesencephalic and anterior rhombencephalic neural crest. (A) Transverse sectionof the head of a E14 chimera and (B) the corresponding schematic drawing showing the localization of the quail cells in the cephalicskeleton. The parietal bone (C) and membranous fronto-parietal suture (D) are of quail origin (P, parietal bone; F, frontal bone; S,squamosal). Scale bar, 1 mm (A) and 10 µm (C,D).

418 G. F. Couly, P. M. Coltey and N. M. Le Douarin

Fig. 6. Results of experiment V. Graft of MPM at the metencephalic level. (A) Transverse section of the head of a E-13 chimera and (B)high magnification of the supra-occipital bone where the quail marker is located (d and v indicate the dorsoventral axis). Scale bar, 1 mm(A) and 10 µm (B).

Table 3. Origin of cephalic skeleton

419Embryogeny of skull in vertebrates

(e.g. Le Lièvre, 1978) that were based essentially on exper-iments carried out at later developmental stages. Moreover,the developmental stage of the chimeras analysed in the

present work ranged from E8 to E14 i.e. when the differ-ent components of the skull and associated dermis can beclearly identified.

Fig. 7. Results of experiment VI. Graft of the 1st somite. (A,C) Transverse sections of the head of a E9 chimera respectively at the levelof the occipital bone and of the laryngeal muscles. The dermis of the occipital region (B), the pars ampullaris of the otic capsule (D), them. ceratoglossus (E), the rostral basi chondro-occipital (F) and the m. dilatator glottidis (G) contained quail cells (see Table 2).Connective cells located between the quail muscle cells (E and G) originate from the neural crest of the chick host (see Couly et al.,1992). Scale bar, 1 mm (A,C) and 10 µm (B,D-G).

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Contribution of the neural crest to the skull anddermis The results obtained are indicated in Fig. 14A, B and inTable 3. In our earlier work (Couly et al., 1992), it clearlyappeared that the frontal and parietal bones were not meso-dermal in origin. This is why we decided to undertake ar e fined study on the mapping of the anteriormost part of

the neural crest by operating on embryos early and byallowing the chimeras to develop up to a stage when theskull is formed and when every bone can be clearly iden-t i fied (for the parietal bone for example from E13 and forthe supra-occipital from E12, see Romanoff, 1960). Theresults presented here show without any doubt that the pari-etal and frontal bones and the associated sutures arederived from the neural fold corresponding to the pre-sumptive level of the diencephalon, mesencephalon andmetencephalon. They also demonstrate that the dermis cor-responding to these bones as well as the totality of thedermis of the face and ventral part of the neck (as previ-ously stated, Le Lièvre and Le Douarin, 1975) originatesfrom the neurectoderm.

In addition to the facial and visceral skeleton (Le Lièvre,1978), the neural crest contributes entirely to the roof ofthe skull. The extent of this contribution was not perceivedin previous experiments (Le Lièvre, 1978) in which themajor part of the frontal and the parietal bones were con-sidered as derived from the mesoderm. In the present work,we have analysed in a very detailed manner the origin ofthe floor of the skull.

Origin of the chondrocranium

(1) The occipital and otic regionsThe chondrocranium includes certain elements originatingfrom mesoderm and others from neural crest (Fig. 14A,B;Tables 3, 4). The five first somites contribute to the occip-ital bone: the first somite yielding the exo-occipital and thesecond, third, fourth and rostral part of the fifth forming thebasi-occipital and the condyles (Fig. 8). The paraxial meso-derm of the metencephalic level (MPM of Couly et al.,1992) forms the supra-occipital. Therefore, the occipital asa whole can be considered as a giant vertebra enlarged toform a cupula in which the brain rests and in which theneural arch is represented by the exo-occipital and the

G. F. Couly, P. M. Coltey and N. M. Le Douarin

Fig. 8. Schematic drawing of a dorsal view of the E9 avianchondrocranium (after De Beer, 1937) and respective contributionof the 6 first somites. 1, The otic capsule; 2, the basi-occipital; 3,the exo-occipital; 4, roots of hypoglossus nerves; 5, supra-occipital; 6, anterior arch and 7, posterior arch of altas; 8,odontoid apophysis axis and 9, axis. The black median linerepresents the notochord (d and v indicate the dorsoventral axis).

*The respective situations of the latero-mesencephalic and latero-metencephalic medial paraxial mesoderm (MPM) are indicated in Table 1 (experimentIV and V) and in Couly et al., 1992).

Table 4. Contribution of the cephalic medial paraxial mesoderm (MPM)* to the genesis of the chondrocranium

421Embryogeny of skull in vertebrates

supra-occipital. The corpus of the vertebra is representedby the basi-occipital. We can use these embryonic originsto extrapolate the morphogenetic movements of mesoderm.The metencephalic MPM migrates dorsally (to form thesupra-occipital); the sclerotome of the 1st somite remainslaterally, while the corpus of the ‘occipital vertebra’ is

formed by the concentration around the notochord of scle-rotomal cells belonging to somites 2, 3, 4, 5 and a verysmall part of the 1st somite (Fig. 8).

Notable is the fact that the region of fusion between thebasi-post-sphenoid of MPM origin and the rostral part ofthe occipital bones of somitic origin correspond to the

Fig. 9. Results of experiment VI. Graft of the 1st somite. (A) Cephalic transverse section of a chimeric embryo at E13 crossing the oticregion ; the exo-occipital bone (B) and the cartilaginous pars canalicularis of otic capsule (C) are of quail origin. The m. cucullariscervicis is also of quail origin (D). Scale bar, 1 mm (A) and 10 µm (B, C and D).

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human spheno-occipital synchrondrosis, a zone which inhumans is the site of growth activity up to 20 years ofa g e .

In this conception of the construction of the occipitalarea, it is interesting to notice that the paraxial cephalicmesoderm (MPM) replaces, in the ‘giant occipital verte-

bra’, the sclerotomal somitic cells which normally form theneural arch (Bagnall et al., 1988, 1989).

It has been clearly established by the use of the quail-chick chimera system that, in the trunk, each vertebra arisesfrom the joint development of the posterior and anteriorhalves of two consecutive somites (Bagnall et al., 1988)

G. F. Couly, P. M. Coltey and N. M. Le Douarin

Fig. 10. Results of experiment VII. Graft of the second somite. (A,D) Transverse sections of the head of a E9 chimera at two differentlevels of the rhombencephalon showing the quail label in the basichondro-occipital (B,C) between the 2nd and 3rd roots of thehypoglossus nerve (HN 2-3) and in m. hypoglossus obliquus (E). Scale bar, 1 mm (A,D) and 10 µm (B,C,E).

423Embryogeny of skull in vertebrates

thus confirming the ancient view put forward by Remak(1855). Moreover, the rostral part of each somite is the sitewhere the dorsal root sensory ganglia (DRG) develop andthe motoneurone fibers penetrate (Keynes and Stern,1984 ; Teillet et al., 1987). It is important to notice that the

behaviour of the five first somites differs strikingly fromthat of the more posterior ones. With the exception of thecaudal part of the 5th somite, they all fuse to form thebasioccipital bone. The remaining evidence for the originof this bone from the five first segments is the foramens by

Fig. 11. Results of experiment IX. Graft of the fourth somite. (A) Transverse section of the head of a E9 chimera showing the quail labelin the basi-chondro-occipital (B) and in m. dilatator and constrictor glottidis (C and D, respectively). Scale bar, 1 mm (A) and 10 µm (B-D).

424

which the roots of the XIIth nerve (hypoglossus) exit fromthe myelencephalon.

No sensory ganglia arise from the five first somites (Teil-let et al., 1987 ; Le Douarin et al., 1992) whereas a largeamount of neural crest cells from this level reach the gut

to form the enteric ganglia (Le Douarin and Teillet, 1973)and the parasympathetic ganglia of the heart (Kirby et al.,1983).

In contrast to the cephalic paraxial mesoderm, whichdoes not yield dermis (Couly et al., 1992), the six fir s t

G. F. Couly, P. M. Coltey and N. M. Le Douarin

Fig. 12. Results of experiment X. Graft of the fifth somite. (A,B) Transverse sections of the head of a E9 chimera showing the quail labelin the posterior part of the m. hypoglossus lateralis (C), in occipital condyle (D), anterior arch of atlas (E) and odontoid apophysis of axis(F). In contrast, the axis vertebrae is of host origin (G). Scale bar, 1 mm (A,B) and 10 µm (C-G).

425Embryogeny of skull in vertebrates

Fig. 13. Results of experiment XI. Graft of the sixth somite. (A,F) Transverse sections of the head of a E9 chimera. The corpus of axis(B), dorsal arch of atlas (G) and m. rectus capitis (E) consist of quail cells. In contrast, the odontoid apophysis of axis (D) and the anteriorarch of atlas (C) are of host origin. Scale bar, 1 mm (A,F) and 10 µm (B-E,G).

426

somites generate the dermis covering the occipital and dor-socervical region of the head (see Table 2). As for theirmyotomal components, these somites give rise to thetongue muscles innervated by the hypoglossus nervesoriginating from the level of rhombomere 8 (see Noden,1983 ; Lumsden and Keynes, 1989 ; Keynes and Lums-den, 1990).

The otic capsule is formed by the cooperation ofosteochrondrogenic cells from three different sources. Themain one is the 1st somite from which most of the parscanalicularis of the otic capsule arises (Table 2). The meten-cephalic and mesencephalic median paraxial mesoderm(MPM) also contributes to the pars canalicularis and formsmost of the pars cochlearis of the otic capsule (see Coulyet al., 1992) (Table 4). Finally the neural crest participatesin the formation of the dorsolateral part of the parscochlearis (pro-otic process) close to the squamosal. Theneural crest is also involved in the constitution of the col-umella (see Le Lièvre, 1978 ; Noden, 1983). Therefore, aspreviously stated (Le Lièvre, 1978), the otic capsule resultsfrom the cooperation of mesodermal and mesectodermalcells.

(2) The sphenoid bone complex and the doubleorigin of the sella turcica

The sphenoid bone complex is formed by the mesen-

cephalic MPM, which generates the cartilaginous rudimentof the orbitosphenoid from which the basipostsphenoid andthe pleurosphenoid will arise (Fig. 4A,B) (Couly et al.,1992). The contribution of the neural crest to the sphenoidbone complex comprises the basipresphenoid (Fig. 14A,B),the rostrum of the parasphenoid and the pterygoid. The sellaturcica is therefore of mesodermal origin caudally and ofcrest origin rostrally. The contribution of the neural crestto this structure had not been reported before. It is inter-esting to notice that the anterior tip of the notochord reachesthe limit of the basipostsphenoid and therefore lies at aboutthe midpart of the sella turcica as already described in thehuman embryo (see figure 121, p. 167, Augier, 1931).

The mesenchyme participating in adenohypophysis his-togenesis (to form the connective tissue located betweenthe glandular cords and the blood vessel walls with theexception of the endothelia) is derived from the neural crest(Le Lièvre and Le Douarin, 1975 ; Couly and Le Douarin,1985, 1987). Therefore, the basisphenoid including the sellaturcica are of mesodermal and ectodermal (via the neuralcrest) origin. Thus this bone crosses the limit between the‘chordal’ and the ‘prechordal’ chondrocranium (Table 3).The anterior limit of the notochord then corresponds to thelimit where the mesodermal skeleton ends and the neuralcrest derived skeleton begins. The latter will be called the‘prechordal’ skeleton.

G. F. Couly, P. M. Coltey and N. M. Le Douarin

Fig. 14. Schematic drawing of cephalic skeleton of bird. (A) Rightexternal view ; (B) right internal view. Red, skeleton of neuralcrest origin; blue, skeleton of cephalic mesoderm origin; green,skeleton of somitic origin. 1 angular2 basibranchial3 basihyal4 ceratobranchial5 columella (a) and otic capsule (b)6 dentary7 epibranchial8 entoglossum9 ethmoid10 exoccipital11 frontal12 interorbital septum13 jugal14 maxilla15 Meckel’s cartilage16 nasal capsule17 nasal18 occipital (basi)19 postorbital20 quadrate21 palatine22 parietal23 premaxilla24 pterygoid25 quadratojugal26 scleral ossicles27 sphenoid (a, orbito-sphenoid; b, pleurosphenoid; c, basi-

presphenoid; d, basi-postsphenoid)28 supraoccipital29 squamosal30 temporal31 vomer

427Embryogeny of skull in vertebrates

(3) The ‘prechordal’ chondrocraniumThe interorbital septum and the ethmoidal complex are,like the basipresphenoid of neural crest origin. Therefore,all the prechordal chondrocranium originates from theneural crest and does not derive from the prechordal meso-derm, the fate of which is to yield three pairs of ocularmuscles (rectus medialis, rectus ventralis, obliquus ven -tralis) (Couly et al., 1992) (see also Jacob et al., 1984 ;Wachtler et al., 1984) and endothelial vascular cells (Colteyet al., unpublished data). The latter will be called the ‘pre-chordal’ skeleton.

Origin of the membrane bones of the skull One of the most important results of this work was to revealthat, in addition to the facial and visceral skeleton (LeLièvre, 1974), the neural crest is also at the origin of theroof of the skull comprising the frontal, parietal andsquamosal bones. Moreover, in the late embryos, the suturesof the calvarian and facial bones, forming the so-called sec-ondary cartilage (De Beer, 1937), which allow the growthof the skull, are made up of neural crest cells.

The extent of this contribution was not perceived in pre-vious experiments (Le Lièvre, 1978) in which the majorpart of the frontal and the parietal bones were consideredas derived from the mesoderm. This discrepancy comesfrom the fact that the experiments reported by Le Lièvre(1978) were done at later stages of development and thatthe host embryos were observed at early stages when thesebones were not completely individualized.

Origin of the skull and the new head hypothesis ofGans and Northcutt (1983)Gans and Northcutt (1983) and Northcutt and Gans (1983)put forward the hypothesis that the more anterior (or ros-tral) part of the head including sense organs, prosen-cephalon and mesencephalon together with the corre-sponding region of the skull, is derived from theneurectoderm. The results presented here and in our pre-vious article (Couly et al., 1992) support this view byshowing that the entire vault of the skull (including thefrontal and parietal bones) and what we call the ‘pre-chordal’ skull are actually of neural crest origin. Moreoverthey also demonstrate that the contribution of the meso-derm to the ‘new head’ is reduced essentially to musclesand a limited and posterior (or caudal) part of the skull(see Fig. 14A,B).

In addition, it should be noted that the vertebrate headnot only is built up from the neural crest in its more ros-tral region but also results from the incorporation in itscaudal area of an increasing number of vertebrae from theagnathes (where no vertebra participates in the skull(Augier, 1931, page 164)) to the higher vertebrates where,as shown here, up to 5 somites participate in the occipitalbone complex (Fig. 15).

In our first investigations on the ontogeny of the earlyneural primordium (Couly and Le Douarin, 1987, 1988),we constructed a fate map (Fig. 16) showing that themediodorsal and anterior region of the neural plate corre-sponds to the territory of the hypothalamo-adenohypoph-ysis, whereas the ectoderm of the first branchial arch islocated laterally to the adenohypophyseal placode and thepresumptive nasal ectoderm (Couly and Le Douarin,1990a,b). The rostral part of the head (i.e. most of the so-called ‘new head’) is formed by the lateral regions of theanterior neural plate from which originate the prosen-cephalon (including the cerebral hemispheres), part of themesencephalon and the neural crest that forms the vault ofthe skull, the ‘prechordal’ chondrocranium and the maxil-lary and mandibulary region of the face (Fig. 16).

The development of sense organs and the increase of thebrain volume have therefore involved the construction ofthe anterior skull from the neurectoderm and also the incor-poration of more and more paraxial mesoderm in the occip-ital and otic region of the head (Fig. 15).

The Hox-code and the specification of theoccipito-otic region of the skull The notion that the specification of the skull along theanteroposterior body axis is determined by the combinato-rial expression of various Hox genes, thus generating aHox-code, was clearly formulated by Kessel and Gruss(1991).

Fig. 15. Redrawn after Augier (1931, page 164). Evolution of theskull in vertebrates showing the progressive incorporation ofsomites to the occipital (‘occipitalization’ of the 1st mesodermalsegment) together with the increasing development in front of theanterior tip of the notochord of the rostral skull, which is shown inthis work to originate from the neurectoderm via the neural crestof the prosencephalon and mesencephalon. The increase in thevolume of the skull parallels that of forebrain and hindbrain.

428

It was also supported by several experimental evidencesbased either on the use of retinoic acid or by altering theHox-code by transgenes. Retinoic acid administered to thepregnant mice at defined stages of fetal development aswell as hyperexpression of H o x - 1 . 1 (Kessel et al., 1990)in transgenic mice or targeted mutation of H o x - 1 . 6 ( L u f k i net al., 1991 ; Chisaka et al., 1992) and H o x - 1 . 5 g e n e s(Chisaka and Capecchi, 1991) result in malformations ofvertebral morphogenesis. It is striking to see that many ofthe dysgeneses so induced are localized in the occipitalregion. The experiments reported here reveal that theoccipito-otic region of the skull is the site of very com-plex morphogenetic events involving fusions of embryonicstructures that in the vertebral column develop indepen-dently. The Hox-code therefore seems to exert a stringentcontrol on the movements of cells and other morphogeneticmechanisms leading to the incorporation to the skull ofelements of the cervical column. Differential expression ofHox genes at the hindbrain and first somites levels hastherefore been involved in the evolutionary selectionprocess that led to the construction of the skull in highervertebrates.

The authors are grateful to Dr F. Dieterlen for her reading ofthe manuscript. The authors wish to thank M. Le Thierry for hertechnical assistance, B. Henri, S. Gournet and E. Bourson for helpin preparing the manuscript. This work was supported by theCentre National de la Recherche Scientifique and grants from theAssociation pour la Recherche contre le Cancer, The Fondationpour la Recherche Médicale Française and the Ligue Nationalecontre le Cancer.

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(Accepted 23 October 1992)