EMBRYONIC PALATAL RESPONSES TO TERATOGENS IN SERUM-FREE ORGAN-CULTURE

This study examines development of rat, mouse, and human embryonic pal ates in submerged, serum-free organ culture. The concentration-respons e profiles for retinoic acid (RA), triamcinolone (TRI), hydrocortisone (HC), dexamethasone (DEX), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (T CDD) were examined and the mechanisms of clefting in vitro were compar ed to observed in vivo responses. Craniofacial regions were dissected on gestational day (GD) 12 for mice and GD 14 for rat, and cultured fo r 3-4 days in Bigger's BGJ(b) medium in flasks flushed with 50% O2, 45 % N2, 5% CO2. Growth and fusion of secondary palates were scored under a dissecting microscope. In serum-free control medium, mouse and rat palatal fusion occurred within the 4-day culture period. Supplementing with fetal bovine serum (FBS) in excess of 1% interfered with growth and fusion in control medium. RA significantly inhibited fusion of mou se and rat palates at 5 x 10(-9) and 1 x 10(-10) M, respectively, with RA-induced clefting related to abnormal proliferation and differentia tion of medial epithelia. In contrast, glucocorticoid-induced clefting was due to concentration-dependent inhibition of shelf growth. TRI si gnificantly inhibited fusion at 4 x 10(-5) M, and 1 X 10(-4) M DEX or HC, inhibited fusion of 19 and 42% of shelves, respectively. The respo nse rate for DEX in the presence of 1% FBS was increased (42% unfused) . TCDD clefting was due to altered medial epithelial differentiation a nd 1 x 10(-8) M TCDD affected 36% of CD-1 mouse, 23% of C57BL/6N mouse , and 47% of F344 rat palates. When the medium was supplemented with 1 % FBS, selenium, transferrin, and additional glutamine, the response o f C57BL/6N embryos increased to 75%. This rate is similar to that repo rted for Trowell's-type cultures with IMEM:Fl2 medium and 1% FBS. The increased responsiveness to DEX or TCDD in the presence of serum sugge sts that an unknown factor in serum may be required for full activity. Three human embryonic palatal explants (GD 52 or 53) were cultured fo r 3-6 days and fused during culture. The present study demonstrates th at serum-free organ culture supports development of mouse, rat, and hu man palatal explants. The present study demonstrates the capacity of t his organ culture system to model palatogenesis for several species, a nd to distinguish between various mechanisms of clefting as presented through selected model compounds. This model should be useful for expl oring mechanisms of activity at a cellular and molecular level.