/. Embryol. exp. Morph. Vol. 58, pp. 251-264, 1980 251 Printed in Great Britain © Company of Biologists Limited 1980 Tissue interactions and the initiation of osteogenesis and chondrogenesis in the neural crest-derived mandibular skeleton of the embryonic mouse as seen in isolated murine tissues and in recombinations of murine and avian tissues By BRIAN K. HALL1 From the Department of Biology, Life Sciences Centre, Dalhousie University, Halifax SUMMARY Mandibular processes from 9- to 13-day-old embryonic mice formed both bone and cartilage when grafted to the chorioallantoic membranes of host embryonic chicks. Isolated ectomesenchyme, taken from 9-day-old embryos did not form bone or cartilage, while older ectomesenchyme formed both. Recombination of the epithelial and ectomesenchymal com- ponents confirmed that the presence of the epithelium was a sufficient stimulus for the initiation of both chondro- and osteogenesis. Recombinations between components of mouse and chick mandibular processes showed that 9-day-old mouse ectomesenchyme could re- spond to chick epithelium but that, although older murine epithelia could initiate osteo- genesis from the avian ectomesenchyme, epithelia from 9-day-old embryos could not. These results indicated that an epithelial-ectomesenchymal interaction was responsible for the initiation of both osteo- and chondrogenesis within the mandibular arch of the mouse; that the interaction began at 10 days of gestation; that the ectomesenchyme was capable of responding at 9 days, but that the epithelium did not acquire its ability to act on the ecto- mesenchyme until 10 days of gestation. INTRODUCTION Both the cartilaginous and the bony elements in the mandibular skeletons of vertebrate embryos form from ectomesenchymal cells derived from the embryo- nic neural crest. These cells have their origin within the crests of the neural folds. During neurulation these neural crest cells migrate away from the neural folds. Those cells which originate in the cranial neural crest become preferentially localized within the primordia of the cranio-facial skeleton. The ability of these cells to form the cartilages and bones of the craniofacial skeleton depends upon 1 Author's address; Department of Biology, Life Sciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4J1. 17 EMB 58 252 B. K. HALL interactions which they undergo, either during or after, their migration. Thus, in the embryo of the lamprey, Lampetrafluviatilis L., contact with the endoderm is required before chondrogenesis can be initiated (Damas, 1944; Newth, 1951, 1956). While it is known that cranio-facial skeletal elements are of neural crest origin in the fishes (Jollie, 1971; Berkovitz & Moore, 1974; Schaeffer, 1977), the role of tissue interactions in their genesis has not been studied. Interaction of neural crest cells with pharyngeal endoderm is a prerequisite for the initiation of chondrogenesis in the visceral skeleton of the urodele amphibians (Wagner, 1949; Wilde, 1955; Cusimano-Carollo, 1963, 1972; Drews, Kocher-Becker & Drews, 1972; Epperlein, 1974; Corsin, 1975). However, although neural crest cells are known to form bone in the urodeles (de Beer, 1947; Cassin & Capuron, 1977), tissue interactions have not been studied. Nor is there any information for the anuran amphibians (see Horstadius, 1950 and Hall, 1978a for a discussion). The initiation of intramembranous osteogenesis within the scleral, maxillary and mandibular skeletons of the domestic fowl results from inductive inter- actions between scleral, maxillary, or mandibular epithelia and post-migratory ectomesenchymal cells from the neural crest (Murray, 1943; Tyler & Hall, 1977; Tyler, 1978; Hall, 1978c, b, 1980; Bradamante & Hall, 1980; Tyler & McCobb, 1980). Pre-migratory neural crest cells must interact, (a) with cranial epithelial ectoderm if they are to be able to form Meckel's cartilage later in development (Hall & Tremaine, 1979; Bee & Thorogood, 1980), or (b) with the pigmented retinal epithelium if they are to form scleral cartilage (Reinbold, 1968; Newsome, 1972, 1976; Hall, 1978a). Although the details vary, epithelial-mesenchymal interactions emerge as important prerequisites for the initiation of osteogenesis and chondrogenesis in the sub-mammalian vertebrates. The possible existence of similar interactions in the mammalian embryo has not been explored. Indeed, even the origin of the mammalian cranio-facial skeleton from neural crest cells has not been substan- tiated experimentally. There is histological evidence for spurs of ectodermal cells leading into the visceral arches (Bartelmez, 1952; Mulnard, 1955; O'Rahilly, 1965; Mayer, 1973); histochemical visualization of presumed neural crest cells (Milaire, 1959; 1974); indications that high doses of vitamin A both arrest migration of cranial neural crest cells (Morriss & Thorogood, 1978) and pro- duce first arch defects such as Treacher Collins syndrome (Poswillo, 1974), and the evidence that ectoderm or teratocarcinomas isolated from rat embryos form both cartilage and bone (Levak-Svajger & Svajger, 1971; Damjanov, Solter & Serman, 1973) - all of which provide circumstantial evidence for contri- butions to the facial skeleton from the neural crest. The only study known to the author which explores the role of tissue inter- actions in the development of the mammalian mandibular skeleton is that of Svajger and Levak-Svajger (1976). These workers separated first branchial arch mesenchyme of 13-day rat embryos from their epithelia, organ cultured the mesenchyme, and observed the subsequent development of bone and cartilage. Skeletogenesis in the murine mandible 253 If an epithelial-mesenchymal interaction was involved, it must have taken place before thirteen days of gestation. The aim of the present study was to determine whether an epithelial-mesen- chymal (ectomesenchymal) interaction was involved in the initiation ofchondro- genesis and/or osteogenesis within the mandibular skeleton of the embryonic mice. To this end, whole mandibular processes, and mandibular ectomesen- chyme, separated from the mandibular epithelium by treatment with proteo- lytic enzymes, were grafted to the chorioallantoic membranes of host embryonic chicks. (This vascularized site is preferred over organ culture when studying osteogenesis.) The separated components were also either recombined with one another or combined with components isolated from the mandibular processes of embryonic chicks and grafted to chorioallantoic membranes. Histological analysis was used to document the presence or absence of cartilage and bone in the recovered grafts. MATERIALS AND METHODS Animals ICR Swiss albino mice obtained from Bio-breeding Laboratories of Canada, Ottawa, Ontario, were used as the experimental animal. Mice were weaned at 4 weeks of age, housed with three or four of the same sex per cage, and fed Purina lab. chow and water ad Hbidum. Matings were carried out using animals which were at least 8 weeks old. Each male was placed with from one to three females overnight (16.00 to 08.00 hours), 08.00 hours was designated as the commencement of day zero of the pregnancy. Pregnancy was determined by presence of a vaginal plug and by weight gain, as measured on alternate days. Embryos Pregnant females were killed by an overdose of ether, the embryos removed by dissection under sterile conditions and aged using the morphological series of stages of Griineberg (1943). Embryos of 9-13 gestational days were used in this study. The 9-day-old embryos had well developed mandibular processes, lacked posterior limb buds, and had unsegmented posterior somitic mesoderm. By 13 days of gestation the embryos were recognizably murine, with five rows of whiskers containing hair follicles, footplates on the limbs and marked segmen- tation to the distal tips of the tails. Mandibular processes Mandibular processes were dissected from these embryos and either grafted intact, or separated into their epithelial and ectomesenchymal components and then grafted. Separation was achieved by placing the processes into a solution of trypsin and pancreatin in calcium- and magnesium-free Tyrode's solution (257 mg bovine pancreatic trypsin + 43 mg porcine pancreatic pancreatin/10 17-2 254 B. K. HALL ml. both obtained from BDH Chemicals, Toronto, Ontario) for 2 h at 4° C. The mandibular processes were then placed into a solution of the synthetic culture medium BGJb (GIBCO, Grand Islands, N.Y.) and Horse Serum (1:1) to stop the enzymic digestion and the epithelia separated from the ectomesenchyme by microdissection using sharpened hypodermic needles. A similar separation was performed on mandibular processes obtained from embryonic chicks which had been incubated for 4 days and attained Hamburger & Hamilton (1951) stage 22. Grafting Whole mandibular processes, recombined components from the mouse, or components combined with epithelia or ectomesenchyme from mandibular processes of the chick, were grafted to the chorio-allantoic membranes of 8- or 9-day-old embryonic chicks following the procedure outlined by Hall (1978 c). Briefly, the mandibular process or epithelium was placed onto a square of sterile, black, Millipore filter (0-45/*m porosity, 125-150/mi thick, Millipore Filter Corp., Montreal, Quebec), mandibular ectomesenchyme was placed in direct contact with the epithelium and the intact, or recombined processes, grafted for 7 days. Histology Grafts were recovered, dissected away from the chorioallantoic membranes, fixed in neutral buffered formal saline,