Patterning of avian craniofacial muscles

DEVELOPMENTAL

BIOLOGY

116,347-356

(1986)

Patterning of Avian Craniofacial Muscles DREW M. NODEN Department

of Anatomy,

New

York State College of Veterimzry

Medicine,

Received September 9, 1985; accepted in revised fm

Cornell University, February

Ithaca, New York 148X?

9, 1986

Vertebrate voluntary muscles are composed of myotubes and connective tissue cells. These two cell types have different embryonic origins: myogenie cells arise from paraxial mesoderm, while in the head many of the connective tissues are formed by neural crest cells. The objective of this research was to study interactions between heterotopically transplanted trunk myotomal cells and presumptive connective tissue-forming cephalic neural crest mesenchyme. Presumptive or newly formed cervical somites from quail embryos were implanted lateral to the midbrain of chick hosts prior to the onset of neural crest emigration. Hosts were sacrificed between 7 and 12 days of incubation, and sections examined for in situ, forming ectopic skeletal, connective, and muscle the presence of quail cells. Some grafted tissues differentiated tissues. However, many myotomal cells broke away from the implant, became integrated into adjacent neural crest mesenchyme, and subsequently formed normal extrinsic ocular or jaw muscles. In these muscles it was evident that only the myogenic populations were derived from grafted trunk cells. Ancillary findings were that grafted trunk paraxial mesoderm frequently interfered with the movement of neural crest cells which form the cornea1 posterior epithelial and stromal tissues, and that some grafted cells formed ectopic intramembranous bones adjacent to the eye. These results verify that presumptive connective tissue-forming mesenchyme derived from the neural crest imparts spatial patterning information upon myogenic cells that invade it. Moreover, interactions between myotomal cells and both lateral plate somatic mesoderm in the trunk and neural crest mesenchyme in the head appear to operate according to 0 1986 Academic PWS, 1~. similar mechanisms.

INTRODUCTION

Avian voluntary muscles consist of several cell types, with myogenic and connective tissue components having separate embryonic origins. Within cephalic voluntary muscles the myogenic subpopulations are largely of mesodermal origin, while most, but not all, of the connective tissues are derived from the neural crest (Noden, 1983a,b). The objectives of this study are to examine the role of neural crest-derived craniofacial mesenchyme in the patterning of cephalic voluntary muscles, and to establish whether the transmission of spatial programming from neural crest to cephalic myogenic mesoderm involves similar mechanisms as those that occur between appendicular lateral mesoderm and paraxial myogenic cells. Combined microscopic (Christ et al., 1977; Jacob et al., 1979) and transplantation (Chevallier, 1979) analyses proved that avian appendicular muscles develop from somitic myoepithelial cells that invade limb bud lateral mesoderm. The same is true in other amniote species (reviewed by Gumpel-Pinot, 1984). A similar process occurs in the establishment of craniofacial muscles (Noden, 1983b), but here the origins of connective tissue-forming mesenchyme are manifold. In the midfacial, periocular, glossal, and visceral arch regions, cells derived from neural crest constitute connective tissue precursors (Le

FIG. 1. Transplantation designs. In series 1 caudal paraxial mesoderm and overlying surface ectoderm from the segmental plate of donor embryos stages 9+ to 11 was implanted into cephalic paraxial mesoderm lateral to the mesencephalon. In series 2, somitic mesoderm and surface ectoderm from the cervical or thoracic somites of donor embryos stages 11-13 was grafted into a gap prepared by extirpating equivalent sized pieces of cephalic paraxial mesoderm and surface ectoderm. 0012-1606186 $3.00 Copyright All rights

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

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Lievre, 1974; Le Lievre and Le Douarin, 1975; Noden, 1978). Laryngeal and tracheal connective tissues are derived from lateral plate mesoderm originating at the level of the first two somites (Noden, 1986), while the connective tissues for head elevator and depressor muscles are somitic in origin (reviewed in Noden, 1984a,b; Noden and de Lahunta, 1985). The roles for each of the two precursor populations have been investigated in the limb. Heterotopic grafts of somites (Adelmann, 1938; Chevallier et al, 1977; Chevallier and Kieny, 1982; Jacob et ab, 1983) and trunk somatopleure (Jacob and Christ, 1980) clearly indicate that myotomal populations are not committed to the formation of particular muscles. Rather, it is the connective tissue mesenchyme derived from lateral plate somatic mesoderm that imparts spatial organization to appendicular myogenic populations (reviewed by Gumpel-Pinot, 1984). The situation for craniofacial muscles is less clear. When presumptive mandibular arch neural crest cells are grafted in place of second or third arch precursor populations, an ectopic jaw skeleton develops caudal to the normal mandibular arch skeleton (Noden, 1983a). In these host embryos the jaw opening (mandibular depressor-l) and a few superficial first arch muscles (mandibular epibranchial, intermandibular) were located in ectopic locations. However, it could not be determined whether these arose from presumptive second and third arch muscle precursors (somitomeres 6,7) or from normal first arch muscle precursors (somitomere 4) whose pattern of dispersal might have been disrupted by the transplantations. To resolve this issue, somites from the future cervical and thoracic levels of quail embryos were grafted into the head region of chick embryos. These species were selected to utilize the nucleolar heterochromatic marker found in quail cells (Le Douarin, 1973). Preliminary accounts of these results have been presented (Noden, 1985a,b). i Avian muscle nomenclature Baumel et aL (1979).

FIG. 2. Ectopic structures formed by grafted presumptive somitic mesoderm. (A) Several cartilages (C) and feather papillae (arrows) that are abnormal in this S-day (stage 33) embryo. This coronal section

according

to McClelland

(1968) and

is through the eye, with rostra1 (nasal) structures toward the right side of the figure. (B) A parasagittal section from the embryo illustrated in Fig. 3A, shows ectopic feathers, a cartilage nodule (C), and an ectopic intramembranous bone (arrow) located anterior to the temporal margin of the eye, adjacent to the sclera. (C) Illustrates the contribution of quail mesoderm to the antorbital dermis, where feathers do not normally form at this stage (10 days). The unique quail marker is clearly visible within the nuclei of dermal cells. Note the presence of quail peridermal cells (arrows), which have migrated from an adjacent area where the entire surface ectoderm is of donor origin. (A) 43X; (B) 67x; (C) 420X.

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FIG. 3. Two host embryos from series 1, showing the presence of ectopic, precocious feathers and interference with normal palpebral development. (A) stage 34 (8 days); (B) stage 38 (12 days). Scleral ossieles have failed to form over the temporal cornea-scleral junction in embryo 3B; mesenchyme underlying this region was derived from the mesodermal implant rather than the host neural crest. MATERIALS

AND

METHODS

Surgeries. The procedures used in my lab to gain access to and transplant tissues between quail (Coturnix coturnixjaponica) and chick (White Leghorn) embryos at stages 9 to 10 have been described in detail elsewhere (Noden, 1978, 1983a,b). Embryos of both species were staged using the Hamilton and Hamburger (1951) descriptions. Two series of heterotopic mesoderm transplants have been performed for this study. In the first (28 cases), pieces of unsegmented quail paraxial mesoderm and overlying surface ectoderm were excised from embryos at stages 9+ to 11. Surface ectoderm helps maintain the integrity as well as the orientation of the graft. The excision site (Fig. 1) was located caudal to the last formed somite, and equaled approximately two somite lengths. Part but not all of the presumptive sclerotome was included in the excised fragment. This tissue was then implanted with the same orientation through a longitudinal slit in the surface ectoderm into the paraxial mesoderm located adjacent to the mesencephalon. Host embryos ranged in age from stages 9+ to 94, which is prior to the onset of neural crest emigration. It is important to note that no tissue was removed from these host embryos; rather, the amount of paraxial mesoderm was augmented. In the second series (11 cases), pieces of paraxial mesoderm and overlying ectoderm equal in length to two somites were removed from beside the midbrain or metencephalon of chick host embryos at stages 9 to lo-. The deeper, skeletogenic portion of the head paraxial mesoderm was not excised; this permitted normal devel-

opment of the braincase and rostra1 calvarial structures. Into this gap was placed a pair of somites taken from a stage 11 to 14 quail donor. Depending upon the exact age of the donor, the paraxial mesoderm excised was located between somites 13 and 18, which is the future caudal cervical region from which some limb and pectoral myogenic cells originate (Chevallier, 1979). Grafts larger than two somites resulted in severe craniofacial malformations and early death of the hosts due to interference with neural crest dispersal and disruption of angiogenesis. Controls for these series consisted of orthotopic transplantations of paraxial mesoderm (n = 47) and cephalic neural crest (n = 60); these data have been reported in detail elsewhere (Noden, 19’78,1983a,b). Also, all muscles on the operated side were compared with homologous structures, if present, on the unoperated contralateral side. Analyses. The 39 embryos examined for this study were fixed in Carnoy’s or Zenker’s solutions, dehydrated in a graded butanol series, embedded in Paraplast and sectioned at 8 pm. The age at fixation was between 5 and 12 days of incubation. Adjacent sections were stained with the Feulgen reaction to identify the quail marker, and hematoxylin and eosin to aid in visualizing muscles and connective tissues. Whenever both chick and quail nuclei were present within the same muscle mass, careful attention was given to their distribution within multinucleated myotubes and connective tissue cells. The latter were identified based on their organization within fascial sheaths or attachment zones. The relative contributions of host

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of striated muscles were often associated with these skeletal structures. Also, large areas of quail dermal mesenchyme were typically present. With one exception the ectopic cartilages did not fuse with adjacent, normal cranial cartilages such as the sclera or post-orbital process. Even when donor and host cartilages became directly apposed, the perichondrial sheaths remained intact and distinct. The only exception was one animal in which a spur of quail chondrocytes projected from the anterior margin of a normal host sclera. Clusters of quail mesonephric epithelial tubules were present in four hosts, indicating that intermediate mesoderm had been inadvertantly included with the graft. Superficial mesenchyme derived from implanted quail tissue spread over the anterior margin of the dorsotemporal aspect of the eye. In this location it elicited the formation of feathers (Figs. 2 and 3) characteristic in both size and time of appearance of trunk dorsolateral feathers. All embryos contained ectopic clusters of striated muscle tissue in which both the myocyte and connective tissue components were labeled (Figs. 4 and 5). The size and exact location of these ectopic muscles varied greatly. Most typically they formed broad, thin, multifasciculated slips aligned parallel to the posterior or lateral curvature of the eye or lateral midbrain, as shown in Fig. 4. Although usually situated caudal to the eye and superficial to the level of the rectus muscles, ectopic muscles were occasionally located posterior to the eye. Ectopic voluntary muscles never developed anterior to the eye, even though graft-derived mesenchyme was often present in this region. These muscles showed no resemblance to the normal FIG. 4. Ectopic muscles derived from implanted quail myotomes. (A) musculature of the head. Most often their only attachshows several small, abnormal muscle slips located posterior to the ments were with ectopic donor skeletal tissues. In the sclera near the temporal margin of the eye, from the g-day (stage 33) rare case in which a slip from one of these muscles atembryo reconstructed in Fig. 5C. (B) illustrates the presence of quail tached to a normal host cartilage the tendons of attachnuclei in both myocytes (brackets) and connective tissue cells (between open arrows) in ectopic muscles. (A) 87X; (B) 485X. ment were of host origin. All periocular and jaw muscles were carefully examined with respect to their locations and attachments, and donor precursors to isolated, mononucleated cells and also to identify the cellular origins of their myogenic located between myotubes were noted. However, this and connective components. In 16 of these 23 embryos population includes presumptive secondary myocytes as quail myogenic cells formed part or all of one or more well as satellite, vascular endothelial and endomysial cells that are indistinguishable in the preparations used normal extrinsic ocular muscles, as illustrated in Figs. 5 through 7. In these muscles most or all of the myocytes for this study. Only cells in which both the distribution of nuclear heterochromatin and the phenotype were were labeled; however, few or no quail cells were found in the fascia, tendons, or aponeuroses. The dorsal clearly identified have been included in this analysis. oblique, lateral rectus, pyramidalis nictitans, and palRESULTS pebral muscles were most frequently labeled; in only Series 1 two hosts were quail nuclei found in other eye muscles. Neither the ventral oblique or rectus muscles were ever In all 28 hosts in this series parts of the implanted labeled, which is expected because implants were not presumptive somitic tissue underwent self-differentiation, forming ectopic masses of connective tissue, in- placed at the levels of their precursors, somitomeres 1 cluding cartilaginous nodules or rods (Fig. 2). Bundles and 2.

DREW

NO QUAIL QUAIL

M. NODEN

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351

CELLS

MYOCYTES

ONLY

QUAIL MYOCYTES and CONNECTIVE TISSUES FIG. 5. Schematic reconstructions illustrating the locations of normal and abnormal periocular muscles in series 1 hosts, all fixed between 8 and 10 days of incubation. In (B) and (C) only the outline of the eye is drawn, but the orientation is the same as in (A) and (D). Grafted trunk pre-somitic mesoderm contributed to the dorsal oblique in (A), medial reetus and pyramidalis in (B), medial rectus in (C), and lateral rectus and quadratus in (D). Ectopic intramembranous bones (solid black) are also shown in (A) and (D). Ectopic cartilages were present in these embryos, but only one small nodule (cross hatched in (D)) has been illustrated. Note in (A) the projection of the trochlear nerve (n.IV) to the transplant-derived dorsal oblique muscle. D.O., V.O., dorsal and ventral oblique muscles; D.R., L.R., M.R., V.R., dorsal, lateral, medial, and ventral rectus muscles; PYR., pyramidalis nictitans muscle; Q.N., quadratus nictitans muscle.

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FIG. 6. Transplant-derived myocytes in the dorsal oblique muscle of a P-day (stage 34) host embryo. (A) Low magnification H & E-stained section at the level where the dorsal oblique muscles attach to the interorbital septum (1.0.). (B) shows the presence of the quail marker (arrows) in myocytes cut perpendicular to their axis, but not in connective tissue cells (on the right side) near the muscle attachment site. In (C) the muscle fibers are tangential to the plane of sectioning, and the presence of quail nuclei within myocytes is more apparent. (A) 42X; (B) 580X; (C) 665X.

Not only were these chimeric muscles normal in size, location and attachments, but they appeared to be normally innervated, This was most easily seen for the dorsal oblique, which is the sole target tissue for the trochlear (IVth) nerve (Fig. 5A). In many host embryos there was evidence that the presence of implanted trunk mesoderm disrupted the normal dispersal of cephalic neural crest and paraxial mesodermal cells. Normally, neural crest cells emigrating from the mid-mesencephalic region migrate around the caudal surface of the optic vesicle and, later, over its anterior surface. Here they form the posterior epithelium (endothelium) and stromal cells of the cornea, the scleral ossicles, the scleral cartilage and dense connective tissue, the iris, and the mesenchyme of the eyelids and nictitating membrane (Johnston et al., 1979; Noden, 19X$1982; Meier, 1982; Nakano and Nakamura, 1985). All of these structures were abnormal or partially missing whenever the postorbital region was populated by graft-derived mesodermal cells. For example, grafted mesodermal cells contiguous with the pigmented retinal

epithelium never underwent scleral condensation and chondrogenesis, nor did they form scleral ossicles. In fact, their presence prevented (or failed to promote) the formation of scleral papillae (Fig. 3B). Similarly, the eyelids did not develop in regions where mesodermal mesenchyme had replaced that of neural crest derivation. Occasionally the lateral rectus muscle was also missing. This occurred only when the implanted tissue developed deep to the caudal aspect of the eye, in the location normally occupied by this muscle. The cornea was abnormal in 10 host embryos. In each case the caudal (temporal) part of the posterior epithelium was missing. The stroma overlying this region was hypertrophic and edematous, and it was vascularized by host angiogenic tissues (Fig. 8). All animals in which there was a lesion of the cornea could be recognized grossly by a pink or red hue to the cornea. Since the appearance was the same regardless of whether or not quail cells were present in this location, it appears that the implant blocked the normal movement of presumptive caudal cornea1 endothelial cells. In several cases

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emphasized that dermal bone is not normally formed in periocular regions of the embryo, nor is it a normal derivative of these somites. Series

2

In most cases these somites were grafted to a site caudal to that described in the preceding series, in the place of cephalic paraxial mesoderm that normally contributes to the jaw closing musculature. In all essential respects the results of this series parallel those of series 1. The grafted mesoderm formed both ectopic muscles, in which myogenic and connective tissues were derived from the transplant, and normal head muscles, in which only the myogenic population contained the quail marker. Figure 10 illustrates a broad, thin palpebral depressor muscle formed by quail trunk myotomal cells. This muscle is normally formed by somitomeric cells that invade neural crest mesenchyme of the lower eyelid on Days 5-6 of incubation. The jaw closing complex (mandibular adductor, pseudotemporal, quadratopterygoid protractor) from one of these hosts is shown in Fig. 11. In this large set of muscles nearly all nuclei in myotubes contain the quail marker, which is absent from connective tissues at the attachment sites and in the fasciae. Fewer abnormal skeletal tissues and no ectopic intramembranous bones were seen in this series.

FIG. 7. (A) Low (80X) H & E and (B) higher (420X) Feulgen-stained sections showing the attachment of the lateral rectus (L.R.) muscle to the interorbital cartilage (1.0.) in the lo-day (stage 35) embryo drawn in Fig. 5D. Grafted quail mesodermal cells have contributed to the myocytes (brackets); connective tissues are of host neural crest origin. CIL., ciliary ganglion; n. III oculomotor nerve.

quail mesodermal cells were present adjacent to the anterior cornea1 epithelium. Their presence caused no grossly visible abnormalities. The most unexpected result in this series was the formation of plates of quail intramembranous bone in 9 hosts (Figs. 2B and 9). These plates were always found parallel to and sometimes in contact with the surface of the pigmented retinal epithelium and were located either anteriorly and equatorially. They clearly are not aberrations of scleral ossicles because they varied considerably in size and location, were present several days prior to the normal formation of scleral ossicles in either chick or quail embryos, and often formed adjacent to the surface of the pigmented epithelium. It should be

FIG. 8. The cornea from a lo-day (stage 35) series 1 embryo. The nasal side of the cornea (overlying the bracket on the right) has an intact posterior cornea1 epithelium and normally compacted stroma, but the temporal region (overlying the lens and on the left) lacks this epithelium and is edematous. Most of the mesenchymal cells in the temporal region are derived from implanted quail mesodermal cells. Note the presence of many blood vessels and a feather papilla in the abnormal hypertrophic cornea1 region. 40x.

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FIG. 9. Ectopic mesoderm-derived intramembranous bone located adjacent to the pigmented retinal epithelium in a position normally occupied by the cartilaginous sclera. The view is anterior to the eye of a lo-day host embryo (reconstructed in Fig. 5D). 420X.

DISCUSSION

The principal finding of this study is that prospective myotomes from either nascent or newly formed trunk somites are capable of forming normal craniofacial muscles following heterotopic transplantation. This clearly demonstrates that neural crest-derived connective tissue-forming mesenchyme provides spatial cues necessary to direct the distribution and alignment of

VOLUME 116, 1986

myogenic precursors. Moreover, these data indicate that the mechanisms used to direct patterns of appendicular, body wall, and craniofacial voluntary muscle development are similar, despite the disparate embryonic origins of the connective tissue precursors in these regions. This is another of many instances in which mesenchymal populations derived from the neural crest exhibit properties once thought to be exclusive to mesodermal tissues. Preliminary results of head into trunk paraxial mesoderm transplants indicate that cephalic paraxial mesoderm similarly can give rise to appendicular muscles (Noden, unpublished data). In contrast, cartilages formed from grafted mesenthyme failed in most cases to fuse with either mesoderma1 or crest-derived cephalic cartilages. This was unexpected since crest and mesoderm normally participate together in chondrogenesis of the neurocranium, columella, otic capsule, and sclera (Noden, 1978, 1983a). Whether this represents a simple disparity in timing of chondrogenesis or a more fundamental difference in chondrogenic precursors as suggested by the results of Chiakulas (1957) and Fyfe and Hall (1979) is not known. Two ancillary findings are noteworthy. One is the disruption of normal neural crest migration as a result of transplanting trunk paraxial mesoderm and surface ectoderm. This was most noticeable when more than two somites were transplanted, in which cases the hosts usually did not survive beyond 2 days of development. Most of these embryos showed signs associated with abnormal development of head vasculature. Those that survived to slightly older stages had craniofacial dysmorphologies typically associated with failure of crest cell migration, such as unilateral maxillary or mandibular brachygnathia (Johnston, 1975; McKee and Fer-

FIG. 10. This illustrates the ability of transplanted quail trunk somitie cells to form myocytes muscle in the eyelid of a series 2, stage 38 (12-day) chick embryo. (A) 67X; (B) 580X.

(arrows

in (B)) within

the palpebral

depressor

DREW

M. NODEN

FIG. 11. The jaw closing complex from a 12-day (stage 38) series 2 embryo. (A) is a low-magnification (35X) H & E-stained section for orientation; boxes indicate the locations illustrated in (B) and (C) (both 665X), which are Feulgen-stained sections to reveal the quail marker. The mandibular nerve (n.V) is entering the mandibular adductor (M.A.) muscle complex. Nearly all the myocytes in these muscles have the

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guson, 1984; Been, 1984). These results are consistent with those of Lofberg et al. (1985), who demonstrated that the pattern of extracellular matrix deposition between somites and overlying ectoderm varies along the body axis, and regional differences are critical in controlling the early stages of trunk neural crest migration. Experiments to compare head and trunk paraxial mesoderm with respect to their abilities to support lateral movements of neural crest populations are now in progress. The structure most affected was the cornea. Implanted mesoderm prevented crest cells from forming the caudal part of the posterior epithelium. However, both crest cells and grafted mesodermal cells were able to populate the stromal region. The presence of blood vessels in the hypertrophic stromal regions, but not in the normal parts of the cornea, is consistent with the findings of Feinberg et al. (1983) that hyaluronate-producing epithelial tissues such as the posterior epithelium of the cornea normally inhibit angiogenesis. The sharp demarcation between normal and abnormal parts of the cornea indicates these angiogenic controls operate over very localized, well circumscribed areas of the developing cornea. The appearance of intramembranous bone adjacent to the pigmented retina is difficult to explain. Trunk somitic mesoderm normally does not form dermal bone in avian embryos, nor is the equatorial region of the optic cup an osteogenesis-promoting area. All of the implants that formed these ectopic bones were from the region of presumptive somites 10 through 16. Since lateral mesoderm from this level normally gives rise to the clavicle (Chevallier, 19’77),the possibility that the grafts included small amounts of lateral mesoderm must be considered. At the stages during which these transplants were performed there are no grossly visible landmarks that demarcate paraxial from lateral mesoderm in the area of the segmental plate. However, in none of these hosts did nephrogenic tissues develop. One would expect intermediate mesoderm to be present if lateral mesoderm was accidentally included in the graft. Experiments are currently in progress to test whether this lateral mesoderm, or the post-otic level lateral mesoderm that forms the median part of the clavicle (Noden, unpublished data) might be programmed to form intramembranous bone at these early stages. An intriguing, but untestable, possibility is that this osteogenic capability within trunk paraxial mesoderm is an atavistic trait, reflecting a feature present in reptilian and piscine ancestors (see Hall, 1984). quail marker (small arrows), indicating they are derived from trunk somitic cells. In contrast, the connective tissues and fasciae (between open arrows in (B)) are of host neural crest origin. P.P., pterygoquadrate protractor; Ps., pseudotemporal; Q., quadrate cartilage; SC., sclera.

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The expert assistance of A. McVey with the surgeries, L. Hill, J. Oyer, and C. A. Wojcicki with the histology, and B. Burton with manuscript preparation is gratefully acknowledged. Supported by Grant DE06632 from the National Institute of Dental Research. REFERENCES ADELMANN, H. B. (1938). An experimental analysis of the developmental properties of the somites of Amblysknna punctatum. Anut Rec. 70.2. BAUMEL, J. J., KING, A. S., LUCAS, A. M., BREAZILE, J. E., and EVANS, H. E. (1979). “Nomina Anatomica Avium.” Academic Press, New York. BEEN, W. (1984). Developmental anomalies of the lower face and the hyoid cartilage due to partial elimination of the posterior mesencephalic and anterior rhombencephalic neural crest in chick embryos. Acta NeerL Suwui. 22,265-278. CHEVALLIER, A. (1977). Origine des ceintures scapulaires et pelviennes chez l’embryon d’oiseau. J. EmbryoL Exp. Morph01 42, 275-292. CHEVALLIER, A. (1979). Role of somitic mesoderm in the development of the thorax in bird embryos. II. Origin of thoracic and appendicular musculature. J. EmlqoL Exp. MorphoL 49, ‘73-88. CHEVALLIER, A., and KIENY, M. (1982). On the role of the connective tissue in the patterning of the chick limb musculature. Roux’ Arch Dev. BioL 191.245-258. CHEVALLIER, A., KIENY, M., and MAUGER, A. (1977). Limb-somite relationship: Origin of the limb musculature. J. EmbryoL Exp. Morpha! 41,245-258. CHIAKULAS, J. J. (1957). The specificity and differential fusion of cartilage derived from mesoderm and mesectoderm. J. Exp. ZooL 136, 287-300. CHRIST, B., JACOB, H. J., and JACOB, M. (1977). Experimental analysis of the origin of the wing musculature in avian embryos. Anat. Emhoi. 150,171-186. FEINBERG, R. N., REPO, M. A., and SAUNDERS, J. W., JR. (1983). Ectodermal control of the avascular zone of the peripheral mesoderm in the chick embryo. J. Exp. ZboL 226,391-398. FYFE, D. M., and HALL, B. K. (1979). Lack of association between avian cartilages of different embryological origins when maintained in vitro. Am. J. Anat. 154,485-495. GUMPEL-PINOT, M. (1984). Muscle and skeleton of limbs and body wall. In “Chimeras in Developmental Biology” (A. McLaren and N. Le Douarin, eds.), pp. 281-310. Academic Press, London/New York. HALL, B. K. (1984). Developmental mechanisms underlying the formation of atavisms. Biol. Rev. 59,89-124. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. MorphoL 88,49-92. JACOB, M., CHRIST, B., and JACOB, H. J. (1979). The migration of myogenie cells from the somites into the leg region of avian embryos. Anat. EmbryoL 157,291-309. JACOB, H. J., and CHRIST, B. (1980). On the formation of muscular pattern in the chick limb. In “Teratology of the Limbs” (H.-J. Merker, H. Nau, and D. Neubert, eds.), pp. 89-97. de Gruyter, Berlin. JACOB, H. J., CHRIST, B., and GRIM, M. (1983). Problems of muscle pattern formation and of neuromuscular relations in avian limb development. In “Limb Development and Regeneration, Part B”

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(R. 0. Kelley, P. F. Goetinck, and J. A. MacCabe, eds.), pp. 333-341. Liss, New York. JOHNSTON, M. C. (1975). The neural crest in abnormalities of the face and brain. Birth Defects, @rig. Articles Ser. ll, l-18. JOHNSTON, M. C., NODEN, D. M., HAZELTON, R. D., COULOMBRE, J. L., and COULOMBRE, A. J. (1979). Origins of avian ocular and periocular tissues. Exp. Eye Res. 29,27-45. LE DOUARIN, N. (1973). A biological cell labeling technique and its use in experimental embryology. Dew. BioL 30,217-222. LE LI~VRE, C. (1974). Role des cellules mesectodermiques issues des cretes neurales cephaliques dans la formation des arcs branchiaux et du squelette visceral. J. Embryo1 Exp. MwphoL 31,453-477. LE LIBVRE, C. S., and LE DOUARIN, N. (1975). Mesenchymal derivatives of the neural crest: analysis of chimeric quail and chick embryos. J. EmbryoL Exp. Morph01 34,125-X4. L~FBERG, J., NYNAS-MCCOY, A., OLSSON, C., JONSSON, L., and PERRIS, R. (1985). Stimulation of initial neural crest cell migration in the Axolotl embryo by tissue grafts and extracellular matrix transplanted on microcarriers. Dev. BioL 107,442-459. MCCLELLAND, J. (1968). The hyoid muscles of Gallus gallus. Acta Anat. 69,81-86. MCKEE, G. J., and FERGUSON, M. W. J. (1984). The effects of mesencephalic neural crest extirpation on the development of chicken embryos. J. Anat. 139,491-512. MEIER, S. (1982). The distribution of cranial neural crest cells during ocular morphogenesis. In “Clinical, Structural and Biochemical Advances in Hereditary Eye Disorders” (D. L. Daentl, ed.), pp. l-15. Liss, New York. NAKANO, K. E., and NAKAMURA, H. (1985). Origin of the irideal striated muscle in birds. J. EmbryoL Exp. MwrphoL 88,1-14. NODEN, D. M. (1978). The control of avian cephalic neural crest cytodifferentiation. I. Skeletal and connective tissues. Dev. BioL 67,296312. NODEN, D. M. (1982). Periocular mesenchyme: Origins and control of development. In “Biomedical Foundation of Opthalmology” (F. A. Jacobiec, ed.), Sect. 3, pp. l-23. Harper & Row, New York. NODEN, D. M. (1983a). The role of the neural crest in patterning of avian cranial skeletal, connective and muscle tissues. Dev. BioL 96, 144-165. NODEN, D. M. (198313). The embryonic origins of avian cephalic and cervical muscles and associated connective tissues. Am J. Anat. 168, 257-276. NODEN, D. M. (1984a). Craniofacial development: New views on old problems. Anat. Rec. 208, l-13. NODEN, D. M. (1984b). The use of chimeras in analyses of craniofacial development. In “Chimeras in Developmental Biology” (A. McLaren and N. Le Douarin, eds.), pp. 241-280. Academic Press, New York. NODEN, D. M. (1985a). Embryonic patterning of craniofacial muscles. Anat. Ret 211,139-140A. NODEN, D. M. (1985b). Properties of craniofacial mesenchymal populations. Cell &$er. 16, 111s. NODEN, D. M. (1986). Origins and patterning of avian craniofacial mesenchymal tissues. J. Craniofac. Gen. Den BioL, in press. NODEN, D. M., and DE LAHUNTA, A. (1985). “The Embryology of Domestic Animals: Developmental Mechanisms and Malformations.” Williams & Wilkins, Baltimore.