Mesenchymal Derivatives from the Neural Crest

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1 REVIEW Arch. histol. jap., Vol. 45, No. 2 (1982) p Mesenchymal Derivatives from the Neural Crest Harukazu Department Japan NAKAMURA of Anatomy (Prof. M YASUDA), Hiroshima University School of Medicine, Hiroshima, Received February 12, 1982 Summary. 1. Quail cells and chick cells can be distinguished by light and electron microscopy. In this paper, the derivation of mesenchymal tissues from the neural crest is discussed, based mainly on the quail-chick chimera technique of using quail cells as biological cell markers. 2. Neural crest cells down to the level of the 4th somite have the capacity to become mesectoderm in the normal development of birds. The mesectodermal cells differentiate into the bones and cartilages of the visceral skeleton, dermis of the face and ventral part of the neck, connective tissue of the salivary gland and pharyngeal derivatives such as the thyroid, parathyroid, and thymus. Stromal and endothelial cells of the cornea, odontoblasts, and smooth muscle cells of the branchial arch arteries are also mesectodermal derivatives. 3. Though the fate of neural crest cells in normal development is precisely dependent on their original site in the neural axis, environmental cues are also very important in determining their fate. The neural crest appears in early embryonic development as a thickening of the ectoderm around the neural plate. Neural crest cells migrate long distances before differentiation, providing a model for the investigation of cell differentiation, cell migration and cell-to-cell interaction. The neural crest cells follow a precise region-specific pathway, and they differentiate into many kinds of cells: sensory, sympathetic and parasympathetic neurons, Schwann cells, glial cells of the peripheral nervous system, melanocytes, and cartilages, bones and connective tissues of the head and neck, and some endocrine cells (reviewed by WESTON, 1970; NODEN, 1978; LE DOUARIN, 1980). Soon after leaving the neural primordium, neural crest cells become indistinguishable from the cells of the neighboring tissues through which they migrate. Various methods have been developed to follow the crest cell migration and differentiation. The extirpation of neural crest before migration (HAMMOND and YNTEMA, 1953, 1964), and the grafting of 3H-thymidine labelled neural primordium (JoHNSTON, 1966) are among these. There are, however, some problems because, in the former, repair takes place from the neighboring neural crest cells, and sometimes the operated embryo does not survive long enough to be analysed, and in the latter, isotope dilution at each step of mitosis makes it difficult to identify the grafted cells from the host cells at a late stage of development. Analysis of neural crest migration and differentiation has made rapid progress since LE DoUARIN (1969, 1971, 1973) found that the quail nucleus is distinguishable from 127

2 128 H. NAKAMURA: that of the chick, and used the difference as a biological cell marker neural crest cell migration and differentiation. THE TECHNIQUE in the study of OF THE GRAFT The principle of the quail-chick chimera technique is based on the difference in nuclear structure between the Japanese quail and chick. A large condensation of nucleolusassociated heterochromatin is present in the quail. In the chick, by contrast, heterochromatin is dispersed in small chromocenters, and the amount of nucleolus-associated chromatin is small (LE DoUARIN, 1969, 1971, 1973). Owing to this disposition of heterochromatin, quail and chick cells can easily be distinguished by a light microscope after FEULGEN-RossENBECK's(1924) staining (Fig. 1, 2). They can also be distinguished through careful examination of an electron micrograph. As this difference in heterochromatin disposition exists in all embryonic and adult cell types, the quail-chick marker system has advantages over the isotope marker system and has become a valuable method in solving current embryological problems. LE DoUARINand her colleagues (reviewed, 1980) applied this marker system to the study of neural crest migration and differentiation. For the study of mesectoderm, the neural tube of prosencephalon, mesencephalon and/or rhombencephalon of the host chick embryo is excised using a microscalpel made of a needle. The stages of these embryos are 7-11 of HAMBURGER and HAMILTON(1951). The homologous fragment of neural primordium is isolated from a quail embryo at the same stage (if the graft is made orthotopically) as the chick embryo, and the tissues adhering to the neural tube are taken off after incubation in 0.5 solution of trypsin in Mg2+ and Ca2+ free buffered solution. Contamination of mesodermal cells in the graft is avoided by trypsinization. Then the fragment is inserted into the groove of the host embryo (Fig. 3). Though either the graft of quail neural primordium into the chick or the reverse procedure Fig. 1. and 2. Neuroblasts in the spinal cords of the chick (Fig. 1) and quail (Fig. 2), 7th day of incubation. Note the large mass of heterochromatic DNA in the quail cells. One can easily distinguish quail cells from chick ones. Feulgen-Rossenbeck staining.

3 Mesenchymal Derivatives from the Neural Crest 129 Fig. 3. A diagram showing the experimental procedure of the graft of a fragment of a quail neural primordium (neural tube +neural crest) into a chick embryo. A. Excision of the neural primordium (nt+nc) of the chick before the onset of neural crest cell migration by a microscalpel made of a needle. a Dorsal aorta, c notochord, s somite. B. Homologous fragment of neural primordium is isolated from a quail embryo. C. Tissues adhering to the quail neural tube are taken off after tripsinization (Trp). D. The cleaned fragment of neural tube is grafted isotopically into the chick. takes well, the chick is routinely used as a host because one can more easily detect quail cells in the background of chick cells than vice versa. The earlier stages of embryos are used for the transplantation of the more anterior part of neural primordium because the more differentiated state of neural crest cells can be obtained from the more cephalic part of the neural primordium. The operated embryos are fixed at the desired stage and the difference in nuclear pattern between the quail and the chick becomes clear by Feulgen-Rossenbeck of sections prepared from specimens fixed in Zenker's fluid. BONES AND CARTILAGES staining OF THE CEPHALIC REGION By using the quail-chick chimera technique, many of the problems of neural crest migration and differentiation have been elucidated. Fate maps along the cephalocaudal direction of neural crest were made (LE DoUARINet al., 1977). These workers showed that the mesectodermal capacities of the neural crest are limited to the cephalic region down to the 4th somite. The bones and cartilages of the cephalic region are mesodermal and/or of neural crest origin. Generally, neural crest cells migrate lateroventrally and occupy the more rostral part (the facial area), while most of the vault of the skull and cervical skeleton are derived from mesoderm. The intermediate part is a mixture of neural crest and mesoderm (LE LIEVRE,1978; NODEN,1978; THOROGOOD, 1981). Prosencephalic neural crest cells intermingle with mesencephalic neural crest cells and migrate into the orbital, nasal and pituitary region. Prefrontal and nasal bones differentiate in their entirety from the prosencephalic and mesencephalic neural crest. On the other hand, the orbital and sclerotic cartilages consist of a mixture of mesodermal and neural crest derived cells. The bones of the orbital skeleton, as well as the cartilages which are to be replaced by bone, are made up of a mixture of mesodermal and prosencephalic and mesencephalic neural crest derived cells. In addition, mesencephalic neural crest cells migrate into the first branchial arch. They differentiate not only into Meckel's cartilage but also into maxillary and mandibular bones (LE LIEVRE,1974, 1978).

4 130 H. NAKAMURA: Fig Photomicrographs taken from a chimeric embryo on the 19th day of incubation. A chick embryo at stage 11 was transplanted with rhombencephalic neural primordium of a quail embryo. Feulgen-Rossenbeck staining. Fig. 4. A part of the mandible. Many but not all cells show the chromatin pattern of the Fig. 5. Fig. 6. Fig. 7. quail indicating origin from the graft. High magnification of Figure 4. The osteocyte indicated by the arrow is derived from the grafted quail neural crest. x 1,800 Feather follicle in an anterior region of the neck. Note the connective tissue sheath of the feather follicle as well as the papilla possess the quail type nuclei indicating their neural crest origin. Pharyngeal wall. Note that connective tissue cells of the pharyngeal wall come from grafted neural crest (quail cells).

5 Mesenchymal Derivatives from the Neural 131 Crest A part of the rhombencephalic neural crest cells migrate into the otic capsule and they, along with the mesodermal cells, make up the otic capsule cartilage cells. Around the otic region, squamosal bones and some other small bones are entirely made up of neural crest derived cells from the mesencephalic and rhombencephalic regions. The parietal and occipital bones are of mesodermal origin. The rhombencephalic neural crest mainly migrates into the mesenchyme of the second, third, and fourth branchial arches and into the regions caudal to the branchial arch (LE LIEVEE,1974). The skeletal tissue from these mesenchymal cells are hyoid and a part of the mandible (Fig. 4, 5). Fig. 8. Thyroid gland. The come from the grafted Fig. Thymus. 9. Here again follicular cells are host cells neural crest (arrows). the connective tissue while the connective cells are of neural Fig. 10. Parathyroid gland. Parenchymal cells are host are of neural crest origin. cells while crest origin the connective Fig. 11. The wall of common carotid artery. Note that the endothelial origin while the smooth muscle cells are of neural crest origin. tissue cells (arrows). tissue cells cells are of mesodermal

6 132 H. NAKAMURA : CONNECTIVE TISSUES AND DERMIS FROM NEURAL CREST CELLS Cephalic neural crest cells follow a precise region-specific pathway as mentioned in the previous section, and most of the mesenchyme of the face and neck derives from the neural crest. Mesectoderm cells differentiate into dermis in the head and neck (LE LIEVRE and LE DOUARIN, 1975). Figure 6 shows that the feather papilla cells derive from grafted neural crest cells, while the epidermal cells belong to the host. The connective tissue cells of the mandible and the neck ventral to the notochord come mainly from the neural crest. The loose connective tissue in the tongue and the floor of the mouth are of neural crest origin. Therefore, the connective tissues of the salivary gland and the connective tissues of the tongue and the lower jaw muscle are of neural crest origin (LE LIEVRE and LE DOUARIN,1975). The mesenchymal components of the pharyngeal walls derive from neural crest (Fig. 7). The connective tissue of the thyroid gland (Fig. 8), thymus (Fig. 9), and parathyroid gland (Fig. 10) are also of neural crest origin (LE LIEVRE and LE DoUARIN, 1975). In addition, neural crest cells differentiate into Type I and II cells of the carotid body (PEARSE et al., 1973) and the calcitonin producing cells in the ultimobranchial body (LE DOUARIN et al., 1974). The mesenchymal component of the esophagus and the trachea is made up of mesodermal cells. The contribution of the neural crest derived cells to the walls of the esophagus is limited to the enteric parasympathetic ganglion cells. Prosencephalic and mesencephalic neural crest cells migrate into the cornea. A quail-chick chimera experiment revealed that the neural crest cells differentiate into the corneal mesenchymal stromal cells and the corneal endothelial cells (NODEN,1978), although the latter had been thought to be of mesodermal origin. OTHER MESENCHYMAL DERIVATIVES FROM THE NEURAL CREST According to LE LIEVRE and LE DOUARIN (1975), smooth muscle cells in the third (brachiocephalic trunks and common carotid arteries), the fourth (systemic aorta), and the sixth (pulmonary arteries) branchial arch arteries derive entirely from rhombencephalic neural crest (Fig. 11). Smooth muscle cells between the aortic trunk and bulbus arteriosus consist of the mixture of mesodermal cells and neural crest derived cells. The distal part of the aortic arch and the distal part of the sixth arches are also transitional areas and the neural crest derived cells and mesodermal cells together form the smooth muscle cells in these areas. After the sixth day of incubation, the neural crest derived cells take up positions as smooth muscle cells and elaborate elastin. However, endothelial cells are of mesodermal origin. The chromatin pattern of the quail smooth muscle cells in the arterial wall is modified during their differentiation, which means that the two or three Feulgen-positive patches attach to the nuclear membrane in the fully differentiated quail smooth muscle cells (Fig. 11). LE LIEVRE and LE DoUARIN (1975) used light microscopy to determine whether the endothelial cells of the capillaries in various pharyngeal structures, whose connective tissues differentiate from neural crest cells, are of crest or of mesodermal origin. They found that the endothelial cells in the dermis, thymus, thyroid were always of meso-

7 Mesenchymal Derivatives from the Neural Crest 133 dermal origin, though the pericytes are of neural crest origin. Similarly, in the large vesseles whose muscular walls are of neural crest origin, the endothelial cells are always of mesodermal origin. From these results, LE LIEVRE and LE DoUARIN concluded that the organs deriving from mesectodermal rudiments are invaded by mesodermal capillary buds during their histogenesis. Neural crest derived mesenchymal cells can also differentiate into the striated muscle cells to a certain extent in the tongue, face and neck region, though they mainly derive from somitic mesoderm (LE LIEVRE and LE DOUARIN, 1975). These workers showed, on the other hand, that the connective tissue cells in muscle are derived from the neural crest. Dental mesenchymal cells may also come from the neural crest (WESTON, 1970; THESLEFF and HURMERITA, 1981). They differentiate as odontoblasts through interaction with oral epithelial cells. Mesectodermal cells differentiate into leptomeningeal cells (PIATT, 1951; NODEN, 1978). ENVIRONMENTAL EFFECTS ON DETERMINATION AND DIFFERENTIATION OF MESECTODERMAL CELLS Normally, the fate of neural crest cells is dependent on their position along the cephalocaudal direction. Thus, the cephalic neural crest down to the 4th somite has the capacity to form mesenchymal cells. The autonomic ganglion cells of the sympathetic and parasympathetic systems originate from well-defined regions of the neural axis. The neural crest can give rise to melanoblasts along its whole length. A problem arises as to whether the fate of neural crest cells is determined before migration or through interaction with the environment during migration or after reaching their final positions. When the `adrenomedullary level' of the neural crest is transplanted into the `vagal' region, the crest cells colonize in the gut and differentiate into enteric ganglia of Auerbach's and Meissner's plexuses (LE DOUARIN and TEILLET, 1974). Even when cholinergic ciliary ganglion of 4-6 days of incubation is inserted into the groove between the neural tube and the somite of (adrenomedullary level), the ganglion cells detach and migrate into the adrenomedulla (LE DOUARIN et al., 1978; LE LIEVRE et al., 1980). Similar observations were made for clone cultured neural crest cells (BRONNER- FRASER and COHEN, 1980) and for homogenous populations of cultured melanogenous neural crest cells (ERICKSON et al., 1980) which were implanted into the neural crest pathways of younger chick embryos. These results show that the phenotypic expression of neural crest cells as adrenergic, cholinergic or sensory neurons is determined by environmental cues rather than by the site of their origin. On the other hand, the cephalic neural crest cells give rise to cartilages and connective tissues when the cephalic neural crest is heterotopically transplanted in the trunk (LE DOUARIN and TEILLET, 1974). However, the graft of trunk neural crest at the head level resulted in severe malformations like those observed after excision of cephalic neural crest due to the absence of facial and/or branchial mesectoderm (LE DOUARIN et al., 1977). NEWGREEN and THIERY (1980) showed that cranial and sacral neural crest cells of the earliest migrating population from these levels synthesized fibronectin in vitro, though cervico-lumbar crest cells did not. They concluded that this ability may be the first expression of mesenchymal differentiation in these crest cells. These results might indicate that the mesectodermal stem cells are segregated

8 NAKAMURA: as such from other cell lines before migration (HALLand TREMAINE,1979; THOROGOOD, 1981). NODEN(1978) carried out experiments to examine whether the crest cells of mesectodermal capacity were restricted to specific developmental pathways. He replaced chick hindbrain neural crest cells with quail forebrain crest cells, and found that the treated embryos were indistinguishable from those of homotopic transplantation. His results show that cytodifferentiation, growth and histogenesis of mesectodermal cells are directed by environmental influences encountered by crest cells after they leave their points of origin. One might wonder whether any trunk neural crest cell has the capacity to become mesectoderm under appropriate conditions. NAKAMURAand AYER-LE LIEVRE (1982) carried out interesting experiments to answer this question. They heterotopically transplanted unilateral trunk neural primordium, which was taken in early development at the level of unsegmented plate mesoderm just anterior to Hensen's node, into the level of rhombencephalon. Mesectodermal derivatives (connective tissues, dermis and muscle excluding cartilage and bone) developed from the heterotopically grafted trunk neural crest, but these graft derived cells mixed with the host cells. These host cells might have come from the contralateral neural crest (Fig. 12). When the bilateral trunk neural primordia are grafted heterotopically to the cephalic region, the operated embryo showed face and neck abnormalities because of a deficiency of mesectoderm. However, careful examination showed a few mesectodermal cells derived from the graft at the fringe areas (Fig. 12). The fringe areas mean the most cephalic and caudal limits of the migration of neural crest cells when the graft is made orthotopically at the same level (NAKAMURAand AYER-LE LIEVRE,1982). As the trunk neural crest does not produce mesectoderm in the orthotopic graft, the trunk region environment (provided by the neural tube, notochord, somites, and ectoderm) may be inappropriate for the migration and early differentiation of neural crest cells which have mesectodermal capacities in birds. Trunk neural crest cells with mesectodermal capacities grafted unilaterally to the cephalic region might find an appropriate environment for their migration and differentiation with host crest cells from the other side of the embryo. When trunk neural primordia are grafted bilaterally to the cephalic region, neural crest cells with mesecto- Fig. 12. A diagram showing the results of heterotopic transplantation of trunk neural primordia into the region of rhombencephalon, redrawn after NAKAMURA and AYER-LF. Lievee (1982). Orthotopic transplantation showed that rhombencephalic neural crest cells migrate into the mesenchyme of the 2nd, 3rd, 4th branchial arches and into the regions caudal to branchial arch when unilateral trunk neural primordium is grafted to the rhombencephalon, mesectodermal cells are found in the same regions as the orthotopic graft. However, mesectodermal cells from the graft always mix with the host ones. When bilateral trunk neural primordia are grafted to the same level, mesectodermal cells and their derivatives are found in the fringe areas of the rhombencephalic neural crest, that is, grafted neural crest cells migrate into the most cephalic and most caudal areas of the normal migration areas of the rhombencephalic neural crest

9 Mesenchymal Derivatives from the Neural Crest 135 Fig. 13. Schematic drawing of the neural crest differentiation. Pluripotent stem cells (P) may exist along the whole length of the neural axis during the early stages of development. Segregation of the mesenchymal cell lines (P,,,) from other cell lines (P_,,,) may occur earliest. P,, and P_,,, may express their phenotype through interaction with the environment. Environmental factors play a very differentiation important role in the of neural crest cells. dermal capacities from only the fringe areas (the most cephalic and most caudal areas) can find appropriate conditions for mesectodermal expression with nearby host cells. On the contrary, cephalic neural primordium might be able to modify the environment to be suitable for mesectodermal expression because mesectodermal derivatives were obtained when it was grafted to the trunk region (LE DOUARINand TEILLET, 1974). Do mesodermal cells, which have the capacity to give rise to mesenchyme, behave like neural crest cells when they are inserted into the crest pathway? NAKAMURAand AYER-LE LIEVRE (1982) inserted unsegmented plate mesoderm into the crest pathway at the level of rhombencephalon. Cells from the grafted mesoderm did not migrate ventrally in branchial region. Their derivatives and mesectodermal ones ended up in distinct locations. Similar results were obtained by BRONNER-FRASER and COHEN(1980) and by ERICKSONet al. (1980). These results show that the migration abilities of the mesoderm and mesectoderm vary quite early though their differentiational capacities are somewhat similar. The requirements of tissue interaction for chondrogenesis and osteogenesis of cranial neural crest were tested in vitro (BEE and THOROGOOD, 1980), or on the chorioallantoic grafting (HALL, 1981). BEE and THOROGOODcultured the neural crest cells alone or with the epithelium which neural crest cells encounter during migration or at their definitive sites. They isolated premigratory mesencephalic neural crest from stage 9 chick embryos and cultured it for 12 days. They found that neural crest grown alone in an organ culture consisted of large masses of predominantly undifferentiated mesenchyme cells. On the other hand, when the neural crest was combined with retinal pigmented epithelium or with maxillary ectoderm, chondrogenesis was observed in the culture of neural crest cells. Skeletogenesis in the neural crest culture was obtained when the neural crest was combined only with maxillary ectoderm. HATA and SLAVKIN (1978) found that heterotopic association of dental papilla mesenchyme from mouse tooth germ with chick limb ectoderm resulted in a switch from an odontogenic fate to a chondrogenic one. These results indicate that interaction with specific epithelia is required for the commitment of neural crest cells as osteogenic cells. Disturbance of tissue interactions may lead to different phenotypic expressions of neural crest cells. The author and his colleagues found skin-like changes as an islet in

10 136 H. NAKAMURA : the guinea pig cornea (NAKAMURA et al., unpublished data). Hair follicles were continuous to the corneal epithelium; hair papillae and sebaceous glands were found in the dermis-like structure which was continuous to the corneal stroma. Even hypodermal structures were observed. The mesectodermal cells which are directed to corneal stroma in normal development may be altered in their fate by environmental cues and become dermal structures, though the nature of the change in environmental cues is not clear in this case. CONCLUSIONS The neural crest cells follow a precise region-specific pathway, differentiating into many kinds of tissues according to their original sites. Thus, the capacity to become mesectoderm is limited to the cranial neural crest down to the level of the 4th somite in normal development. On the other hand, the data reported above indicate that the premigratory avian neural crest is not a mosaic of cells which are committed to specific ontogenetic pathways. Rather, their subsequent cytodiff erentiation is the result of interaction with environmental factors (summarised in Fig. 13). Interaction with other cell types (cells of the neural tube, notochord, somite, surface epithelium etc.), and with extracellular matrices and interaction among crest cells during migration and after arriving at the final site may be included in interaction with environmental factors. In the neural crest cells, segregation of mesenchymal precursor cells from other cell lines are postulated to take place at the earliest stage, but pluripotent stem cells are believed to remain throughout the whole length of the neural axis during the early stages of development (Fig. 13). The phenotypic expression of mesenchymal cells as chondrocyte, osteocyte or connective tissue cell etc. is determined through interactions with environmental factors. The importance of environmental cues to the expression of mesectodermal cells is confirmed by the experimental works in birds of NAKAMURA and AVER-LE LIEVRE (1982), HALL (1981), and BEE and THOROGOOD (1980) and by the observation of skin-like changes in the cornea in the guinea pig (NAKAMURA et al., unpublished data). REFERENCES Bee, J. and P. Thorogood : The role of tissue interactions in skeletogenic differentiation of avian neural crest cells. Devel. Biol. 78: (1980). Bronner-Fraser, M. and A. M. Cohen : Analysis of the neural crest central pathway using injected tracer cells. Devel. Biol. 77: (1980). Erickson, C. A., K. W. Tosney and J. A. Weston : Analysis of migratory behavior of neural crest and fibroblastic cells in embryonic tissues. Devel. Biol (1980). Feulgen, R. and H. Rossenbeck : Mikroskopisch-chemischer Nachweis einer Nucleinsaure vom Typus der Thymonucleinsaure and die darauf beruhende elektive Farbung von Zellkernen der mikroskopischen Praparaten. Hoppe-Seyler's Z. Physiol. Chem. 135: (1924). Hall, B. K.: The induction of neural crest-derived cartilage and bone by embryonic epithelia: an analysis of the mode of action of an epithelial-mesenchymal interaction. J. Embryol. exp. Morphol. 64: (1981).

11 Mesenchymal Derivatives from the Neural Crest 137 Hall, B. K. and R. Tremaine : Ability of neural crest cells from the embryonic chick to differentiate into cartilage before their migration away from the neural tube. Anat. Rec. 194: (1979). Hamburger, V. and H. L. Hamilton : A series of normal stages in the development of the chick embryo. J. Morphol. 88: (1951). Hammond, W. S. and C. L. Yntema : Deficiencies in visceral skeleton of the chick after removal of cranial neural crest. Anat. Rec. 115: (1953). Depletions of pharyngeal arch cartilages following extirpation of cranial neural crest in chick embryos. Acta anat. 56: (1964). Hata, R. I. and H. C. Slavkin : De novo induction of gene product during heterologous epithelial mesenchymal interactions in vitro. Proc. Nat. Acad. Sci. USA 75: (1978). Johnston, M. C.: A radioautographic study of the migration and fate of cranial neural crest cells in the chick embryo. Anat. Rec. 156: (1966). Le Douarin, N.: Particularites du noyau interphasique chez la caille japonaise (Coturnix coturnix japonica). Utilisation de ces particularites commes "marquage biologique" Bans des recherches sur les interactions tissulaires et les migrations cellulaires au cours de l'ontogenese. Bull. Biol. Fr. Belg.103: (1969). -: Caracteristique ultrastructurales du noyau interphasique chez la caille et chez le poulet et utilization de cellules de caille comme "marqueurs biologiques" en embryologie experimentale, Ann. Embryol. Morphol. 4: (1971). -: A biological cell labeling technique and its use in experimental embryology. Devel. Biol. 30: (1973). - : Migration and differentiation of neural crest. Curr. Topics level. Biol. 16: (1980). Le Douarin, N., J. Fontaine and C. Le Lievre : New studies on the neural crest origin of the avian ultimobranchial glandular cells-interspecific combinations and cytochemical characterization of C cells based on the uptake of biogenic amine precursors. Histochemistry 38: (1974). Le Douarin, N. M. and M.-A. M. Teillet: Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neuroectodermal mesenchymal derivatives, using a biological cell marking technique. Devel. Biol. 41: (1974). Le Douarin, N., M.-A. Teillet and C. Le Lievre : Influence of the tissue environment on the differentiation of neural crest cells. In: (ed. by) J. W. Lash and M. M. Burger: Cell and tissue interactions. Raven Press, New York, 1977 (p ). Le Douarin, N., M.-A. Teillet, C. Ziller and J. Smith: Adrenergic differentiation of cells of the cholinergic ciliary and Remak ganglia in avian embryo following in vivo transplantation. Proc. Nat. Acad. Sci. USA 75: (1978). Le Lievre, C.: Role des cellules mesectodermique issues des crates neurales cephaliques Bans la formation des arcs branchiaux et du squelette visceral. J. Embryol. exp. Morphol. 31: (1974). -: Participation of neural crest-derived cells in the genesis of the skull in birds. J. Embryol. exp. Morphol. 47:17-37 (1978). Le Lievre, C. and N. M. Le Douarin : Mesenchymal derivatives of the neural crest : Analysis of chimeric quail and chick embryos. J. Embryol. exp. Morphol. 34: (1975). Le Lievre, C. S., G. G. Schweizer, C. M. Ziller and N. M. Le Douarin : Restoration of developmental capabilities in neural crest cell derivatives as tested in vivo transplantation experiments. Devel. Biol. 77: (1980). Nakamura, H. and C. S. Ayer-Le Lievre : Mesectodermal capabilities of the trunk neural crest of birds. J. Embryol. exp. Morphol. (1982, in press) Newgreen, D. and J.-P. Thiery : Fibronectin in early avian embryo : synthesis and distribution along the migration pathways of neural crest cells. Cell Tiss. Res. 211: (1980). Noden, D. M.: The control of avian cephalic neural crest cytodifferentiation. 1. Skeletal connective tissues. Devel. Biol. 67: (1978). -: Interactions directing the migration and cytodifferentiation of avian neural crest cells, In: (ed. by) D. Garrod: The specificity of embryological interactions. Chapman and Hall, London, 1978 (p. 4-49).

12 138 H. NAKAMURA Pearse, A. G. E., J. M. Polak, F. W. D. Rost, J. Fontaine, C. Le Lievre and N. Le Douarin : Demonstration of the neural crest origin of Type 1(APUD) cells in the avian carotid body, using a cytochemical marker system. Histochemie 34: (1973). Piatt, J.: Transplantation experiments between pigmentless and pigmented eggs of ambystoma punctatum. J, exp. Zool. 118: (1951). Thesleff, I, and K. Hurmerita : Tissue interactions in tooth development. Differentiation 18: (1981). Thorogood, P.: Neural crest cells and skeletogenesis in vertebrate embryos. Histochem. J. 13: (1981). Weston, J. A.: The migration and differentiation of neural crest cells. Adv. Morphogen. 8: (1970). Dr. Harukazu NAKAMURA Department of Anatomy Hiroshima University School of Medicine Kasumi 1-2-3, Minami-ku Hiroshima, 734 Japan