Head morphogenesis in embryonic avian chimeras: evidence for a segmental pattern in the ectoderm corresponding to the neuromeres

0 lnstitut d'Embryologie cellulaire el moliculaire du CNRS et du College de France , 49bis, Avenue de la Belle-Gabrielle, 94736 Nogent-sur-Marne Cedex ( France) Head morphogenesis in embryonic avian chimeras: evidence for a segmental pattern in the ectoderm corresponding to the neuromeres - Areas of the superficial cephalic ectoderm, including or excluding the neural fold at the same level, were surgically removed from 3-somite chick embryos and replaced by their counterparts excised from a quail embryo at the same developmental stage. Strips of ectoderm corresponding to the presumptive branchial arches were delineated, thus defining anteroposterior 'segments' (designated here as 'ectomeres') that coincided with the spatial distribution of neural crest cells arising from the adjacent levels of the neural fold. This discrete ectodermal metamerisation parallels the segThe development of the cephalic region of higher vertebrates involves extremely complicated morphogenetic processes. This part of the body has been the site of enormous evolutionary changes, most of which have involved the addition of structures related to the increasing complexity of the brain and associated sensory organs. The mechanisms involved in assembling the various pieces of the puzzle during ontogeny of the head are still imperfectly understood. This is why we thought that it would be informative to draw up a precise map of the cephalic region at the early stages of neurogenesis and to combine the cartographical study with a topological and dynamic analysis of development. The quail-chick chimera system is ideally suited to such an investigation. The work done in our laboratory and elsewhere concerning the fate of the cephalic neural crest (Le Lievre, 1974, 1978; Le Lievre and Le Douarin, 1974, 1975; Johnston, 1966; Johnston and Hazelton, 1972; Noden, 1975, 1984) has already shown that the facial skeleton and dermis do not originate in situ but develop after a phase of dorsoventral migration of cells from the cephalic neural crest. In our previous studies (Couly and Le Douarin, 1985,1987), we found that the anterior neural fold (located cranially with respect to the prospective neural crest) yields the superficial epidermis covering the forehead and nasofrontal regions. The morphogenetic movements affecting the more rostral mentation of the hindbrain into rhombomeres. It seems, therefore, that not only is the neural crest patterned according to its rhombomeric origin but that the superficial ectoderm covering the branchial arches may be part of a larger developmental unit that includes the entire neurectoderm, i.e., the neural tube and the neural crest. parts of the neural fold could be followed from the presomitic stage until the cells derived from this zone were definitively positioned in the head structures. The present work is essentially concerned with the development of the remaining cephalic ectoderm, from the neurula to the late embryonic stage, and with its relationships to cerebral and neural crest structures. We report here a striking regionalization of the presumptive facial and hypobranchial ectoderm in areas, arranged in a metameric-like fashion, that we call 'ectomeres' and which parallel the segmentation of the neural anlage into individual neuromeres. This metamerisation is reflected in neural crest cell migration to specific arch structures, since it is possible to correlate the origin of mesectodermal cells, superficial ectoderm and that of the cranial nerves specifying transverse levels of the head. These ectomeres are oriented cranially at a 45 angle with respect to the embryonic axis at these early stages of neurogenesis. Materials and methods Quail and chick eggs from commercial sources were used in these experiments. Microsurgery was performed on embryos at the 3-somite stage (after about 30 to 32 h of incubation in a humidified atmosphere at 38 C for chick and 28 to 30 h for quail eggs). The microsurgical technique involved the selective removal, by means of a microscalpel, of the ectodermal germ layer from defined areas of the presumptive cephalic territory, including, in some series, the neural fold at the same level (for details, see Fig. 1 and Table 1). Microscalpels are made by sharpening thin needles on an Arkansas stone. An ocular micrometer in the dissecting microscope enables the size of the fragments of neural fold or of ectoderm involved in the experiment to be precisely measured. Trypsin was not used to separate ectoderm from the underlying mesenchymal cells. However, at the stage considered, only a few of the latter were present in the areas included in the operations since the neural crest cells had not yet migrated. In all cases, quail embryos were used as donors and chick embryos, from which the corresponding ectodermal area had been removed, as recipients. The operated embryos were killed at embryonic day 3.5 to 8 (E3.5-E8), according to the experimental series, and fixed in Zenker's fluid. Chimerism was analysed at the head and neck level on 5 jum serial sections stained according to the Feulgen-Rossenbeck method, which allows quail cells to be distinguished from chick cells (Le Douarin, 1969, 1973). Description of the territories involved in microsurgery The neural fold was divided into anteroposterior segments 150 fun long. Zones A, B and C (Fig. la) were investigated previously in 0- to 3-somite stage embryos (Couly and Le Douarin, 1985, 1987). The experiments described in the present article were performed exclusively on 3-somite embryos and involved areas of the head as defined by zones D, E and F in Fig. 1. Preliminary experiments In a preliminary experimental series (experiments Pr), rectangular territories of lateral ectoderm perpendicular to the embryonic axis were delineated as indicated in Fig. 2a for levels D and E of the neural fold. Numerous types of grafts, involving topographically distinct ectodermal areas lateral to areas D, E and F, Fig. 1. (a) The neural fold anterior to the first somite is divided into six 150 /.im segments (A-F). (b) Dorsal view of a 3-somite chick embryo showing the areas of ectoderm involved in experiments II, III and IV. A-series experiments include the neural fold, B-series experiments exclude it. Note that experiment HB includes the strip of tissue that curls under the rostral extremity of the embryo. Bar=100jum. Fig. 2. Results of preliminary experiments. The host embryos were observed at E8. (a) The areas of quail ectoderm and neural folds substituted for their counterparts in chick embryos are in experiments PrI and PrII indicated by the dotted and cross-hatched zones, respectively, (b) The island of quail ectoderm corresponding to level D is located in a laterofacial region including the otic and posterior maxillomandibular areas. In PrII quail ectoderm covers a small laterocervical region caudal to that concerned by experiment PrI. (c and d) The neural crest from levels D and E (stipple) mig (...truncated)