The developmental fate of the cephalic paraxial and prechordal mesoderm at the late neorula stage (3- somite) in the avian embryo has been investigated by using the isotopic, isochronic substitution technique between quail and chick embryos. The territories involved in the operation were especially tiny and the size of the transplants was of about 150 by 50 to 60 μm. At that stage, the neural crest cells have not yet started migrating and the fate of mesodermal cells exclusively was under scrutiny. The prechordal mesoderm was found to give rise to the following ocular muscles: musculus rectus ventraUs and medialis and musculus oblicus verttralis. The paraxial mesoderm was separated in two longitudinal bands: one median, lying upon the cephalic vesicles (median paraxial mesoderm - MPM); one lateral, lying upon the foregut (lateral paraxial mesoderm - LPM). The former yields the three other ocular muscles, contributes to mesencephalic meninges and has essentially skeletogenic potencies. It contributes to the corpus sphenoid bone, the orbitosphenoid bone and the otic capsules; the rest of the facial skeleton is of neural crest origin. At 3-somite stage, MPM is represented by a few cells only. The LPM is more abundant at that stage and has essentially myogenic potencies with also some contribution to connective tissue. However, most of the connective cells associated with the facial and hypobranchial muscles are of neural crest origin.

The more important result of this work was to show that the cephalic mesoderm does not form dermis. This function is taken over by neural crest cells, which form both the skeleton and dermis of the face. If one draws a parallel between the so-called “somitomeres” of the head and the trunk somites, it appears that skeletogenic potencies are reduced in the former, which in constrast have kept their myogenic capacities, whilst the forma­tion of skeleton and dermis has been essentially taken over by the neural crest in the course of evolution of the vertebrate head.

The developmental fate of the paraxial mesoderm in the vertebrate embryo has been amply documented. It becomes metamerised according to a craniocaudal gradient into pairs of mesenchymal aggregates, the somites. Somitic cells first form an epithelium surround­ing the somitic cavity and later segregate into three layers with well-defined developmental potentialities. The dermatome yields a population of cells that after expanding laterally underneath the superficial ecto­derm forms the dermis. The myotome gives rise to all the striated muscles of the body, including those of the limb (see Gumpel-Pinot, 1984 for a review) with the exception of those of the cephalic area. The more ventral part of the somites is the sclerotome, which produces vertebrae and intervertebral discs. Segmen­tation of the cranial mesoderm located on each side of the encephalic vesicle is not so clearly evident and has been the subject of a longlasting debate. Namely, the relationships between the neuromeres, transitory segments in the hindbrain (Vaage, 1969; Lumdsen and Keynes, 1989) and the discrete segmentation of the paraxial cephalic mesoderm have been discussed: Locy (1885) emphasized that neuromeres represent the primitive cranial segmentation whereas Neal (1918) considered that mesodermal somites are the more reliable criteria for metamerism in the vertebrate head and, in any case, are primary to the segmental divisions of the neural tube. In more recent studies, Meier (1979, 1981) rejuvenated this problem by means of stereoscan- ning electron microscopy (SEM) of the cephalic mesoderm. He describes the cranial paraxial mesoder­mal layer of the chick embryo as being patterned into segmental units that he called somitomeres. By stage 8 of Hamburger and Hamilton (1951), he could dis­tinguish seven pairs of segments represented by dome­shaped masses of mesoblasts concentrically arranged and separated by grooves rather than slits like those separating the somites (Fig. 1). According to Meier (1979, 1981; see also Jacobson and Meier, 1984), somitomeres originate at the tip of the primitive streak near the Hensen’s node and are established during gastrulation according to a craniocaudal sequence. The 8th somitomere becomes eventually the first cervical somite. In addition to the paraxial mesoderm, the cephalic mesenchyme comprises also the notochord, the rostral end of which is located at the presumptive level of the mesencephalon, and the prechordal meso­derm filling the space located between the notochordal tip and the anterior neural fold (i.e. the adenohypophy­sis presumptive territory, Couly and Le Douarin, 1985).

Fig. 1.

Redrawn after Meier (1981), showing a schematic picture of the “somitomere” concept of the cephalic mesoderm.

Fig. 1.

Redrawn after Meier (1981), showing a schematic picture of the “somitomere” concept of the cephalic mesoderm.

The developmental fate of the cephalic mesoderm has been investigated by Noden (1983a,b; 1986) using the quail-chick chimera technique (Le Douarin, 1969, 1973). Isotopic and isochronic transplantations of quail paraxial mesoderm associated with superficial ectoderm were carried out into chick embryos at 4- to 11-somite stages (Noden, 1983b). In other experimental series, truncal somites were heterotopically and heterochroni- cally transplanted into the cephalic area (Noden, 1986). The work reported here was undertaken in view of completing the fate map of the cephalic mesoderm at the 3-somite stage. This study, based on the use of quail-chick chimeras was carried out on the prechordal and the paraxial mesoderm. Several investigations, dealing with the mapping of the neurectoderm (Couly and Le Douarin, 1985, 1987) and the lateral head ectoderm (Couly and Le Douarin, 1990) have already been performed by us at this particular stage of development. The interest of the present approach is that (1) in contrast to all the previous attempts to map the cranial mesoderm, it is done when the neural crest cells have not yet started to leave the neural primor- dium, the onset of this process being visible as early as the 6- to 7-somite stage at the mesencephalic level (Cochard and Coltey, 1983). (2) Transplantations carried out in the present work involved selectively small aggregates of mesoblast cells with the exclusion of superficial ectoderm. Moreover, the spatial coordinates of the areas investigated by microsurgery were carefully defined, as in our previous investigations on the ectoderm (Couly and Le Douarin, 1985, 1987, 1990). We can now draw a precise fate map of the somitomeres and of the prechordal and paraxial head mesoderm. A comparison of the developmental fate of somites and head mesoderm is presented as well as the relationships between the cephalic mesoderm and the neural-crest- derived mesectoderm.

Quail (Coturnix coturnix japonica) and chick eggs (White Leghorn strain) were from a commercial source and incubated in a humidified atmosphere at 38°C.

The experiments have involved in situ substitution in 3- somite-stage chick embryos of defined areas of the mesoder­mal sheet of cells lying on the lateral surface of the neural tube (medial paraxial mesoderm: MPM) and of the foregut (lateral paraxial mesoderm: LPM) by their counterpart taken from stage-matched quail embryos. The parachordal mesoderm located medioventrally with respect to the neural tube was not involved in the operation. In contrast, the fate of the prechordal mesoderm, located between the notochord tip and the anterior end of the foregut on the one hand and the anterior neural fold on the other hand (see Fig. 2A and Zone 1 of Fig. 2B) was studied.

Microsurgery was carried out in the egg using fine microscalpels. Fig. 3A shows, on the right side of a 3-somite chick embryo, the mesodermal sheet as it appears after removal of the superficial ectoderm. Fig. 3B and C, shows the graft of a 150 μιη long lateral paraxial mesodermal territory located at the caudal level of the developing eye vesicle (LPM rostral type of graft, experiment 5 see below).

Fig. 2.

(A) SEM view of a 3-somite quail embryo in which the superficial ectoderm has been removed on the right side. The paraxial Schematic representation of the embryo: on the right, the 7 types of transplants ros are indicated from 1 to 7. On the left, the levels of the somitomeres

Fig. 2.

(A) SEM view of a 3-somite quail embryo in which the superficial ectoderm has been removed on the right side. The paraxial Schematic representation of the embryo: on the right, the 7 types of transplants ros are indicated from 1 to 7. On the left, the levels of the somitomeres

Experimental series

The fate of the mesodermal sheet was systematically explored through the replacement of territories of about 150 μm long and 50 to 60 μm large as indicated in Fig. 3 and Table 1. Experiment 1 involves the replacement of the prechordal mesoderm.

Fig. 3.

(A) Schematic drawing of the graft of the rostral part of the LPM from a quail (Q) to a chick (C) embryo. (B,C) SEM view of the graft five hours after implantation (B) with the corresponding drawing in C. G, graft; OV, optic vesicle. Scale bar = 100 μm.

Fig. 3.

(A) Schematic drawing of the graft of the rostral part of the LPM from a quail (Q) to a chick (C) embryo. (B,C) SEM view of the graft five hours after implantation (B) with the corresponding drawing in C. G, graft; OV, optic vesicle. Scale bar = 100 μm.

Table 1.

Experimental series and number of embryos examined

Experimental series and number of embryos examined
Experimental series and number of embryos examined

Experiments 2, 3 and 4 concern the MPM, which has been divided into three zones, the rostral one i.e. paraocular mesoderm (experiment 2); the median one i.e. paramesence- phalic mesoderm (experiment 3); the caudal one parameten- cephalic mesoderm (experiment 4).

Experiments 5, 6, 7 concern the LPM, also divided into rostral (experiment 5), median (experiment 6) and caudal (exper­iment 7) regions corresponding, respectively, to the paraocu­lar, paramesencephalic and parametencephalic zones defined above for the MPM.

Experiment 8. A large segment of the quail neural fold corresponding to the level of the posterior prosencephalon (Zone C as defined in Couly and Le Douarin, 1987), mesencephalon and rostral rhombencephalon was orthopi- cally grafted into chick embryos at 3-somite stage. The reverse experiment in which the quail is the host and the chick the donor was also performed (see Fig. 12B).

Chimerism analysis

195 embryos were operated upon. Only those with a normal morphology were analysed further for chimerism at stages ranging from 4 to 11 days of incubation (E4 to Ell). The head was fixed in Zenker fluid, embedded in wax and 5 μm sagittal, parasagittal and frontal sections were stained according to the Feulgen-Rossenbeck’s procedure specific for DNA. Quail and chick cells were identified by the structure of their interphase nucleus (Le Douarin, 1969, 1973).

Some embryos were also treated for immunocytochemistry to identify muscle cells by means of a muscle-specific monoclonal antibody (mAb) 13F4 prepared in our laboratory (Rong et al. 1987) in Bouin’s fluid and eirujeuueu m waA. occuinis wcic ucated successively with 13F4 mAh and a second layer antibody, a goat anti-mouse IgM coupled with alkaline phosphatase (Burstone, 1958 with modifications according to Van Rooijen and Claassen, 1986).

The muscles analyzed in this study were named according to the nomenclature of Nomina Anatomica Avium (Baumel, 1979).

Scanning electron microscopy (SEM)

For SEM study, the embryos were immersed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4°C. After rinsing in the same buffer, the specimens were postfixed in 1% osmium tetroxide in the same buffer for 2 to 3 hours at room temperature, 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 tetroxide in water for 1 hour. After washing in water, the specimens were dehydrated through a graded series of ethanol substituted with iso-amyl acetate and critical point dried from CO2. After mounting on stubs with silver conductive paint, the specimens were vacuum coated with gold and examined with a Jeol 1200 EX electron microscope at 20 kV accelerating voltage.

(1) Replacement of the prechordal plate mesoderm

(experiment 1): labelling of musculi (m.) bulbi oculi Ten embryos have been analysed (Table 1). Quail nuclear labelling was exclusively found in the following ocular muscles: m, rectus ventralis, m. rectus medialis and m. obliquus ventralis (Figs 4 and 5). It is noteworthy that replacement of the prechordal plate results in the labelling of these muscles bilaterally, i.e. for the two eyes (Table 2). The muscle tissue in the oldest embryos examined (E9) was mostly composed of quail nuclei with the characteristic alignment seen in myofibrils. Intersarcomeric connective cells were of chick type and derived from the pro-mesencephalic neural crest as observed in our previous experiments (Le Liévre and Le Douarin, 1975; Couly and Le Douarin, 1987). The other ocular muscles, i.e. m. rectus dorsalis, m. obliquus dorsalis and m. rectus lateralis, were exclusively of chick type. The sclerotic, a derivative of the neural crest, was of chick host type in these experiments. The muscular structures belonging to the first branchial arch (m. mandibulae) were not contaminated by grafted quail cells (Fig. 6 and Table 2). Results were the same in the 10 embryos observed.

Fig. 4.

Results of experiment 1. (A) A parasagittal section of chimeric embryo at E9 showing position of enlargements. In B, the m. rectus ventralis and in C, the m. obliquus ventralis are of quail origin. In contrast, the m. obliquus dorsalis (D), mandibularis (E) and the sclerotical cartilage (F) are of host origin. Scale bar = l mm (A) and 10 μm (B-F).

Fig. 4.

Results of experiment 1. (A) A parasagittal section of chimeric embryo at E9 showing position of enlargements. In B, the m. rectus ventralis and in C, the m. obliquus ventralis are of quail origin. In contrast, the m. obliquus dorsalis (D), mandibularis (E) and the sclerotical cartilage (F) are of host origin. Scale bar = l mm (A) and 10 μm (B-F).

Fig. 5.

Results of experiment 1. (A) Parasagittal section of the same chimeric embryo as in Fig. 4 including in the m. rectus medialis (framed area). In B, it can be seen that this muscle is of quail origin. Two other parasagittal sections (C, E) show, in contrast, that m. rectus lateralis (D) and dorsalis (F) are of host origin. Scale bar = l mm (A, C and E) and 10 μm (B, D and F).

Fig. 5.

Results of experiment 1. (A) Parasagittal section of the same chimeric embryo as in Fig. 4 including in the m. rectus medialis (framed area). In B, it can be seen that this muscle is of quail origin. Two other parasagittal sections (C, E) show, in contrast, that m. rectus lateralis (D) and dorsalis (F) are of host origin. Scale bar = l mm (A, C and E) and 10 μm (B, D and F).

Fig. 6.

Schematic drawing (redrawn after P.P. Grassé, Traité de Zoologie, Les Oiseaux, Tome XV, Fig. 20, P. 124, Masson ed. Paris) of m. mandibulae (A, C, D) and m. bulbi oculi labelled in experiment 1, 2, 6, 7. D, dentary bone; Q, quadrate cartilage; A, angular bone; S, squamosal bone. The numbers refer to muscles whose denominations and origins are indicated on Table 2. Muscle 7 located in the inferior beak is not represented on the drawing. Schemes A, C and D: right lateral views of the bird head. B: Extrinsic muscles of the right eye.

Fig. 6.

Schematic drawing (redrawn after P.P. Grassé, Traité de Zoologie, Les Oiseaux, Tome XV, Fig. 20, P. 124, Masson ed. Paris) of m. mandibulae (A, C, D) and m. bulbi oculi labelled in experiment 1, 2, 6, 7. D, dentary bone; Q, quadrate cartilage; A, angular bone; S, squamosal bone. The numbers refer to muscles whose denominations and origins are indicated on Table 2. Muscle 7 located in the inferior beak is not represented on the drawing. Schemes A, C and D: right lateral views of the bird head. B: Extrinsic muscles of the right eye.

Table 2.

Levels of origin in the prechordal and paraxial mesoderm of the various cephalic muscles

Levels of origin in the prechordal and paraxial mesoderm of the various cephalic muscles
Levels of origin in the prechordal and paraxial mesoderm of the various cephalic muscles

(2) Replacement of the MPM Experiment 2

Experiment 2

The substitution involved the more rostral third of the MPM. This part of the cephalic mesoderm yielded muscles and skeleton.

Twelve embryos were analysed (Table 1) and showed that the remaining 3 ocular muscles unlabelled in the previous experiment involving the prechordal meso­derm m. rectus dorsalis, m. obliquus dorsalis and m. rectus lateralis were made up of quail cells (Figs 4 and 7, Table 2). In addition, the anterolateral part of the corpus sphenoidalis where the m. rectus lateralis is inserted, still cartilaginous at the stage of observation, was made up of quail cells, hence derived from the grafted rostral paraxial mesoderm. The orbitosphenoid was also partly of graft origin. It is interesting to notice that these two bones were shown by Le Liévre (1978) not to be of neural crest origin.

Fig. 7.

Results of experiment 2. Transverse sections of the head of E7 chimeras (A, C, E) showing the quail label in m. obliquus dorsalis (B), m. rectus lateralis (D) and in the anterior part of the sphenoid bone, which is cartilaginous at this stage (F). Scale bar = l mm (A, C and E) and 10 μm (B, D and F).

Fig. 7.

Results of experiment 2. Transverse sections of the head of E7 chimeras (A, C, E) showing the quail label in m. obliquus dorsalis (B), m. rectus lateralis (D) and in the anterior part of the sphenoid bone, which is cartilaginous at this stage (F). Scale bar = l mm (A, C and E) and 10 μm (B, D and F).

Experiment 3

The substitution involved the para-mesencephalic MPM. In this experiment, no muscle was labelled. In the 8 embryos analysed, quail mesenchymal cells were found in the inferior eyelid and in the periocular area. Moreover the external part of the orbitosphenoid was derived from the graft (Figs 8 and 9). In addition, the meninges covering the dorsolateral aspect of the mesencephalon was labelled by quail cells (Figs 8 and 9), whereas the sclerotic was, as expected, of chick type (Figs 8 and 9).

Fig. 8.

Results of experiment 3. (A) Transverse section (at the level of the tectum) of an E9 chimera head showing the mesencephalic dura mata (meninx) labelled by the quai] marker (B). (C) Transverse section of the same embryo at the level of the cerebral hemisphere in which the chondro-orbito-sphenoid (D) and the median part of chondrosphenoid (E) are of quail origin. Note that the sclerotic here is made of chick cells (F). Scale bar = l mm (A and C) and 10 μm (B, D, E and F).

Fig. 8.

Results of experiment 3. (A) Transverse section (at the level of the tectum) of an E9 chimera head showing the mesencephalic dura mata (meninx) labelled by the quai] marker (B). (C) Transverse section of the same embryo at the level of the cerebral hemisphere in which the chondro-orbito-sphenoid (D) and the median part of chondrosphenoid (E) are of quail origin. Note that the sclerotic here is made of chick cells (F). Scale bar = l mm (A and C) and 10 μm (B, D, E and F).

Fig. 9.

Schematic drawing showing the participation of the MPM in the skull: it is restricted to the basisphenoid bone (Bs), the orbitosphenoid bone (Os) and the otic capsule (Otic) (cf experiments 1, 3, 4, 7). Abbreviations:

Cartilages   
Bb basibranchial Io Interorbital septum 
Bh basihyal Mk Meckel’s cartilage 
Cb ceratobranchial Nc nasal capsule 
Co columella Po postorbital 
Eb epibranchial Qd quadrate 
En entoglossum   
Bones    
Ang angular Pal palatine 
Den dentary Par parietal 
Eoc exoccipital Pmx premaxilla 
Eth ethmoid Ptr pterygoid 
Fm frontal Qju quadratojugal 
Jug jugal Scl scleral ossicles 
Max maxilla Soc supraoccipital 
Nas nasal Sqm squamosal 
Opr opercular Vom vomer 
Cartilages   
Bb basibranchial Io Interorbital septum 
Bh basihyal Mk Meckel’s cartilage 
Cb ceratobranchial Nc nasal capsule 
Co columella Po postorbital 
Eb epibranchial Qd quadrate 
En entoglossum   
Bones    
Ang angular Pal palatine 
Den dentary Par parietal 
Eoc exoccipital Pmx premaxilla 
Eth ethmoid Ptr pterygoid 
Fm frontal Qju quadratojugal 
Jug jugal Scl scleral ossicles 
Max maxilla Soc supraoccipital 
Nas nasal Sqm squamosal 
Opr opercular Vom vomer 

Fig. 9.

Schematic drawing showing the participation of the MPM in the skull: it is restricted to the basisphenoid bone (Bs), the orbitosphenoid bone (Os) and the otic capsule (Otic) (cf experiments 1, 3, 4, 7). Abbreviations:

Cartilages   
Bb basibranchial Io Interorbital septum 
Bh basihyal Mk Meckel’s cartilage 
Cb ceratobranchial Nc nasal capsule 
Co columella Po postorbital 
Eb epibranchial Qd quadrate 
En entoglossum   
Bones    
Ang angular Pal palatine 
Den dentary Par parietal 
Eoc exoccipital Pmx premaxilla 
Eth ethmoid Ptr pterygoid 
Fm frontal Qju quadratojugal 
Jug jugal Scl scleral ossicles 
Max maxilla Soc supraoccipital 
Nas nasal Sqm squamosal 
Opr opercular Vom vomer 
Cartilages   
Bb basibranchial Io Interorbital septum 
Bh basihyal Mk Meckel’s cartilage 
Cb ceratobranchial Nc nasal capsule 
Co columella Po postorbital 
Eb epibranchial Qd quadrate 
En entoglossum   
Bones    
Ang angular Pal palatine 
Den dentary Par parietal 
Eoc exoccipital Pmx premaxilla 
Eth ethmoid Ptr pterygoid 
Fm frontal Qju quadratojugal 
Jug jugal Scl scleral ossicles 
Max maxilla Soc supraoccipital 
Nas nasal Sqm squamosal 
Opr opercular Vom vomer 

Experiment 4

The caudal part of the MPM of chick was replaced by its quail counterpart. As in experiment 3, no muscles were labelled following this graft which resulted in skeletal structures, meninges and connective tissue. The otic capsule was mostly of quail type (Fig. 10) on all the 6 embryos observed. So was the posterior corpus sphenoi­dalis. In addition, this part of the paraxial mesoderm participated in meninges (i.e. the ventral part of mesencephalic meninges and the metencephalic men­inges) (Fig. 10). The mesenchymal cells located caudo- dorsally between the eye anlage and the mes-mentence- phalic vesicles were also of quail origin (Fig. 10).

Fig. 10.

Results of experiment 4. Transverse sections of the head of a E7 chimera in A, C, E showing that the meninx (B), the posterior part of the chondrosphenoid (D) and the otic capsule (F, G) are of quail origin. Scale bar = l mm (A, C and E) and 10 μm (B, D, F and G).

Fig. 10.

Results of experiment 4. Transverse sections of the head of a E7 chimera in A, C, E showing that the meninx (B), the posterior part of the chondrosphenoid (D) and the otic capsule (F, G) are of quail origin. Scale bar = l mm (A, C and E) and 10 μm (B, D, F and G).

(3) Replacement of the LPM

Experiment 5

The rostral LPM was involved in the operation and was found to have myogenic potencies. Quail cells were exclusively in the intermandibularis dorsalis and ventra­lis also called m. apparatus hypobranchialis. No quail cells were present in the hyoid cartilage, which has previously been shown to be entirely of neural crest origin (see Le Douarin, 1982 for references). The Ungual muscles were not labelled. Preliminary exper­iments involving somite transplantation confirmed that the Ungual muscles originate from the myotome of the first somites (see also Noden, 1982 and references therein).

Experiment 6

The median LPM of quail was grafted in situ. The muscles labelled were m. pterygoideus medialis and lateralis and m. adductor mandibulae externis (Fig. 11; see Fig. 7, Table 2).

Fig. 11.

Results of experiment 6. (A) Transverse section at the mouth level of a E9 chimeric showing quail cells in the following muscles: (B) m. pterygoideus, (C) m. adductor mandibulae, (D) m. pseudotemporalis and protractor. In E, the cells of the Meckel’s cartilage are of host origin (E). Scale bar = l mm (A) and 10 μm (B-E).

Fig. 11.

Results of experiment 6. (A) Transverse section at the mouth level of a E9 chimeric showing quail cells in the following muscles: (B) m. pterygoideus, (C) m. adductor mandibulae, (D) m. pseudotemporalis and protractor. In E, the cells of the Meckel’s cartilage are of host origin (E). Scale bar = l mm (A) and 10 μm (B-E).

Experiment 7

The caudal LPM exchange between quail and chick led to labelling of the m. depressor mandibulae and m. adductor mandibulae caudalis. The mesenchyme located caudally between the eye and the metencepha- lon is also of quail origin as well as some of the palpebral mesenchyme (Table 2).

(4) Replacement of the neural fold

Experiment 8

This experiment was devised to study the participation of neural crest cells in the histogenesis of the muscles whose origin from the prechordal, median and lateral paraxial mesoderm has just been described. The observations, which concerned the oculomotor com­plex, involved double staining of muscles either on the same or on two adjacent sections by 13F4 mAb for muscle-specific staining and Feulgen-Rossenbeck or Gill's hematoxylin to identify the quail nuclear marker.

The intermyofibrillar connective cells and the anlage of the tendons were labelled by the quail marker and hence were of neural crest origin, whereas the muscle cells, stained with 13F4 mAb, contained chick host nuclei thus confirming their origin from the mesoderm (Fig. 12 A).

Fig. 12.

Results of experiment 8. (A) Immunostaining of m. obliquus dorsalis by 13F4 mAb of a E9 chimera in which the quail neural crest has been implanted on a 3-somite chick. The Feulgen reaction shows that the nuclei are of chick type in the myofibrills and of quail type (arrows) in the connective tissue cells. The myofibrills appear in longitudinal section. (B) Same experiment in which the graft of neural crest is done in chick donor to quail host. The muscle belonging to the adductor muscle complex is cut transversally. The quail nuclei (stained with Gill's hematoxylin) are in the muscle fibrills (arrows). The connective cells, here of graft origin are of the chick type. Scale bar = 10 μm (A, B)

Fig. 12.

Results of experiment 8. (A) Immunostaining of m. obliquus dorsalis by 13F4 mAb of a E9 chimera in which the quail neural crest has been implanted on a 3-somite chick. The Feulgen reaction shows that the nuclei are of chick type in the myofibrills and of quail type (arrows) in the connective tissue cells. The myofibrills appear in longitudinal section. (B) Same experiment in which the graft of neural crest is done in chick donor to quail host. The muscle belonging to the adductor muscle complex is cut transversally. The quail nuclei (stained with Gill's hematoxylin) are in the muscle fibrills (arrows). The connective cells, here of graft origin are of the chick type. Scale bar = 10 μm (A, B)

The complementary experiment comprised staining chimeras in which the cephalic mesoderm from quail was orthotopically grafted into chick. In this case, the neural crest was of chick host origin while the muscles were of graft type. Chimera heads obtained in exper­iment 6 (Table 1), where the MPM was involved in the operation, were double stained with 13F4 and hematox­ylin as previously described. In this case, chimerism analysis was done on m. pterygoideus lateralis and on the adductor muscle complex (Fig.l2B). The myofi- brills contained both the quail nucleus and 13F4 specificity. In contrast, the connective cells located between the muscle fibers were of the chick type.

(5) The origin of the cephalic dermis

It is noteworthy that in none of the experiments involving the cephalic mesoderm the dermis of the host was labelled by the quail marker. In contrast, replace­ment of the neural fold was followed by the labelling of the dermis which turned out to be, in the cephalic area, entirely of neural crest origin (Couly and Le Douarin, in preparation).

The present study yields information on the fate of the mesoderm occupying a paraxial and prechordal position in the cephalic region of the avian embryo at the late neurula stage.

The narrow band of cells located on each side of the notochord was not involved in this investigation due to its inaccessibility. It has been proposed that the paraxial cephalic mesenchyme is like the somites in the trunk segmentally distributed. Meier (1979, 1981, see also Jacobson and Meier, 1984 and references therein) has coined the term of somitomeres to designate the row of segments separated by shallow grooves that he could distinguish by means of stereo SEM on each side of the encephalic vesicles in the E2 embryo of the chick and later on of other animals including amphibians (Jacob­son and Meier, 1984), reptiles (Meier and Packard, 1984) and mammals (Meier and Tam, 1982).

Although attempts to map the cephalic mesoderm had already been made by using the quail-chick chimera system in ovo, (Noden, 1983a,b, 1986), the most rostral part of it had not been subjected to specific scrutiny; moreover, in view of the stages at which the exper­iments were done, some neural crest cells had already left the neural fold and were therefore included in the operation. Hence the present study used the quail-chick tissue substitution technique at a stage when, in embryos of these species, no contamination of the mesoderm by neurectodermal cells could have oc­curred.

In addition to determining the exact origin of the various mesodermal components of the cephalic region of vertebrates, it was also of interest to see whether the cephalic “somites” have the same developmental potentials as their better defined truncal counterpart. Namely, whether they yield the three components, i.e. dermis and connective tissue, striated muscles and skeleton (cartilage - bone), that arise from the somites.

The level of origin of the muscles arising from the cephalic mesoderm is indicated in Fig. 6 and Table 2.

The prechordal mesoderm has been found to have an exclusively muscular fate. Three of the oculomotor muscles are formed bilaterally by this thin sheet of mesenchymal cells. It is interesting to notice that, due to the rostrocaudal rotation of the anterior cephalic area and also of the eye vesicles themselves (the eye vesicles undergo a 90° rotation with respect to the rostrocaudal axis during their individualisation from the neural plate: Couly and Le Douarin, unpublished data) this part of the eye musculature, although originating from the extreme rostral mesodermal territory, finally occupies a ventrolateral position. It is noteworthy that these muscles are innervated by the cranial nerve oculomotorius. The dorsal and anterior oculomotor muscles originate from the anterior region of the medial paraxial mesoderm, situated caudally with respect to the prechordal plate and corresponding to somitomere 1 and 2 of Meier. In contrast to the truncal paraxial mesoderm that appears as a narrow band of cells densely packed on each side of the neural tube at the level where the first somites are being individualized, the cephalic paraxial mesoderm is largely spread on the surface of the cephalic vesicles and of the developing foregut at this late neurula stage (3-somite stage). Neither in our light microscope nor in SEM obser­vations were we able to distinguish the segmentation of somitomeres at the 3-somite stage. However, we have transversally divided the paraxial mesoderm as indi­cated in Fig. 4 to establish a correspondence between the levels of our operations and those of the presump­tive somitomeres as they have been described by Meier (1981). Moreover, the paraxial mesoderm was further cut into a medial (corresponding to the region lying upon the cephalic vesicles) and lateral (corresponding to the region covering the foregut) parts. At 3-somite stage, the cell density is much higher in the LPM than in the MPM (Fig. 13), and the fate of these two bands of mesenchymal cells is significantly different. Apart from the more rostral region of the MPM, which yields 3 oculomotor muscles, this dorsal mesenchymal area gives rise to skeletal structures and connective tissues, including the mesencephalic and metencephalic men­inges, but no muscles. In contrast, the LPM is essentially devoted to a myogenic fate. Apart from the lateral rectus, which was considered by Noden (1983a) as derived from somitomere 5 but we localize its presumptive territory more rostrally (see Table 2), the transverse levels of origin of the mandibular muscu­lature in our study are congruent with Noden’s (Noden, 1983a) (Experiments 6 and 7).

Fig. 13.

(A) SEM view of the mesodermal layer as it stands at 3-somite stage with, in B, the corresponding schematic drawing showing the cellular density in the LPM and MPM, respectively. Scale bar = 100 μm, (C) At 8- somite stage, the cephalic neural crest of which the cell migration is pointed out by the arrowheads covers dorsally the paraxial mesoderm and will later yield the dermis and the skull. Scale bar = 100 μm.

Fig. 13.

(A) SEM view of the mesodermal layer as it stands at 3-somite stage with, in B, the corresponding schematic drawing showing the cellular density in the LPM and MPM, respectively. Scale bar = 100 μm, (C) At 8- somite stage, the cephalic neural crest of which the cell migration is pointed out by the arrowheads covers dorsally the paraxial mesoderm and will later yield the dermis and the skull. Scale bar = 100 μm.

Although the MPM gives rise to some connective elements (in the meninges and in the paraocular region), most of the connective tissue of the face originates from the neural crest (Le Liévre and Le Douarin, 1975; Le Douarin, 1982; Noden, 1982, 1983b, 1986). The experiments reported here (Experiment 8) show, at the single cell level, that, while myofibrills, labelled with the specific 13F4 mAb, are of mesodermal origin, the connective cells involved in the muscle tissue are derived from the neural crest.

One of the most striking results of the present investigation is that, neither dorsally nor ventrally, is the dermis labelled in any of the experiments carried out in this study. The dermis in fact originates entirely from the neural crest. Our results in this respect differ from those reported by Noden who claims that somitomeres yield dermis (Noden, 1983a, p 272). We believe that this discrepancy may have arisen from the fact that this author operates on embryos at later stages (4 to 11 somites, stage 9 to 10 of Hamburger and Hamilton, see Noden, 1983a, p 260), when a few neural crest cells are already adherent to the superficial ectoderm that was involved in these operations. According to our observations, neural crest cells start to leave the neural fold at the mesencephalic level as early as the 6- to 7-somite stage (Cochard and Coltey, 1983). Already at the 8- to 9-somite stage, neural crest entirely covers the mesoderm dorsally (Fig. 13C).

As far as the comparison of somitomeres and somites is concerned the following can therefore be stated.

  1. The developmental potentialities of the former are essentially myogenic; their skeletogenic fate is restricted to a limited contribution to the orbito­sphenoid and corpus sphenoidalis and to the otic capsule. The rest of the facial skeleton has actually been shown to be of neural crest origin (Le Liévre, 1978; Noden, 1978; Le Douarin, 1982 and references there­in).

  2. It is remarkable that the distribution of the skeletogenic cells of the paraxial cephalic mesoderm, corresponding to the sclerotomes of the somites, is exclusively median and represented, at the late neurula stage, by a small number of cells. The myogenic cells corresponding to the myotomes of the somites are more abundant and lie laterally.

  3. The equivalent of the somitic dermatome is absent in somitomeres suggesting that cephalic meso­derm during the course of evolution has lost the function of extending underneath the ectoderm to form the dermis.

Interestingly, the ectodermally derived cephalic der­mis has the capability to develop calcified structures i.e. membrane bones that constitute most of the skull, a property that is lacking or at least extremely reduced in the mesodermally derived dermis of higher vertebrates.

The authors are grateful to Pr. C. Ordahl for his critical reading of the manuscript. The authors wish to thank M. Le Thierry for her technical assistance, B. Henri, Y. Rantier, S. Goumet and E. Bourson for help in preparing the manu­script. This work was supported by the Centre National de la Recherche Scientifique and grants from the Association pour la Recherche contre le Cancer, the Fondation pour la Recherche Midicale Franfaise and the Ligue Nationale Franfaise contre le Cancer.

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