Calcif. Tissue Int. 31, 161-171 (1980) Calcified Tissue International ,g 1980 by Springer-Verlag Developmental Comparisons of Murine Secretory Amelogenesis in Vivo, as Xenografts on the Chick Chorio-ailantoic Membrane, and in Vitro Marie Yamada, Pablo Bringas. Jr., Martin Grodin, Mary MacDougall, and Harold C. Slavkin Laboratory for Developmental Biology, Graduate Program in Craniofacial Biology, and Department of Basic Sciences, Section of Biochemistry and Nutrition, School of Dentistry. University of Southern California, Los Angeles, California 90007, USA Summary. The temporal, spatial, and cytological characteristics of secretory amelogenesis in devel- oping mouse mandibular first molar tooth organs have been compared during in vivo odontogenesis (from the Cap Stage in Theiler stage 25 C57BL/6 embryos to 10-day-old postnatal mice), as xeno- grafts on the chick chorioallantoic membrane (CAM) for periods up to 7 days, and as explants in chemically defined medium without serum or antibi- otics for periods up to 2 l days in vitro. Tooth mor- phogenesis and cytodifferentiation proceeded in each environmental condition in the same sequence albeit at different rates of development. In vivo and CAM xenografts were remarkably comparable in their respective expressions of dentinogenesis and amelogenesis, whereas those explants cultured in a chemically defined medium ~ ithout serum or antibi- otics developed at a much slower rate (e.g., 0.5 days in vivo is equivalent to I day in vitro). In each experimental group, secretory amelogenesis was typically first detected along the mesial-buccal cusp of the molar organ independent of which environ- ment was evaluated. Tooth morphogenesis in vitro and as xenografts on the CAM was routinely small- er than in situ odontogenesis. In each environmen- tal condition a "'stippled" precursor ultrastructural form of enamel matrix preceded mineralization, ex- cept during in vitro cultures of tooth organs. In vitro secretory amelogenesis or dentinogenesis did not indicate morphological characteristics of mineral- ization: both dentine and enamel matrices did not mineralize under the permissive environmental con- ditions used in these experiments. Calcium hy- droxyapatite crystal formations within dentine and along the dentinoenamel junction during initial en- amel matrix tbrmation were not observed during in Send o lff[~rint requests t,, Dr. Harold C. Slavkin. Laboratory for Developmental Biology. Gerontology Center 314, University of Southern Calilkwnia. Los Angeles, CA 90007, USA. vitro tooth organogenesis, even for periods up to 21 days in vitro. We conclude that cap stage mandibu- lar first molar tooth organs (enamel organ epithe- lium and adjacent dental papilla mesenchyme) from Theiler stage 25 embryos contain all of the neces- sary developmental instructions to express morpho- genesis and cytodifferentiation except cues for the serum-containing factors for mineralization. Key words: Enamel matrix, in vitro, mouse -- Ame- logenesis -- Mineralization- CAM. Introduction The morphogenesis and cytodifferentiation of mam- malian odontogenesis results fl'om a series of recip- rocal epithelial-mesenchymal interactions which occur during embryogenesis [1-4]. The interactions associated with the initiation of secretory amelo- genesis have been described in vivo and in vitro [5]. One rather puzzling issue remains regarding those factors within the immediate microenvironment of the developing tooth organ, which are permissive for both dentine and enamel matrix synthesis and secretions, versus those factors which might be "'in- structive" or "'rate limiting" for the developmental processes of organic extracellular matrix formation and subsequent mineralization. Do embryonic tooth organs possess intrinsic factors which can promote morphogenesis and cytodifferentiation in vitro with- out external environmental cues, or are there unique humoral promoters (e.g., steroid and poly- peptide hormones and hormone-like molecules) re- quired for dentinogenesis and amelogenesis in vi- tro'? It has clearly been demonstrated that ameloblast cells in young postnatal mammalian tooth organs synthesize and secrete enamel matrix proteins both 0171-967X/80/0031-0161 $02.00