Embryonic palatal responses to teratogens in serum-free organ culture.

This study examines development of rat, mouse, and human embryonic palates in submerged, serum-free organ culture. The concentration-response profiles for retinoic acid (RA), triamcinolone (TRI), hydrocortisone (HC), dexamethasone (DEX), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined and the mechanisms of clefting in vitro were compared to observed in vivo responses. Craniofacial regions were dissected on gestational day (GD) 12 for mice and GD 14 for rat, and cultured for 3-4 days in Bigger's BGJb 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 mouse and rat palates at 5 x 10(-9) and 1 x 10(-10) M, respectively, with RA-induced clefting related to abnormal proliferation and differentiation of medial epithelia. In contrast, glucocorticoid-induced clefting was due to concentration-dependent inhibition of shelf growth. TRI significantly 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 response rate for DEX in the presence of 1% FBS was increased (42% unfused). TCDD clefting was due to altered medial epithelial differentiation and 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 of C57BL/6N embryos increased to 75%. This rate is similar to that reported for Trowell's-type cultures with IMEM:F12 medium and 1% FBS. The increased responsiveness to DEX or TCDD in the presence of serum suggests that an unknown factor in serum may be required for full activity. Three human embryonic palatal explants (GD 52 or 53) were cultured for 3-6 days and fused during culture. The present study demonstrates that serum-free organ culture supports development of mouse, rat, and human palatal explants. The present study demonstrates the capacity of this organ culture system to model palatogenesis for several species, and to distinguish between various mechanisms of clefting as presented through selected model compounds. This model should be useful for exploring mechanisms of activity at a cellular and molecular level.

Etiology of retinoic acid-induced cleft palate varies with the embryonic stage.

Retinoic acid (RA) has been shown to be teratogenic in many species, and 13-cis-RA is teratogenic in humans. Exposure to RA during embryonic morphogenesis produced a variety of malformations including limb defects and cleft palate. The type and severity of malformation depended on the stage of development exposed. The purpose of this study was to compare the effects of RA exposure in vivo on different stages of palate development. These results were compared to effects observed after exposure in organ culture. The vehicle used in RA dosing was also shown to be a major factor in the incidence of RA-induced cleft palate. For the in vivo studies, RA (100 mg/kg) in 10 ml corn oil/kg was given p.o. on gestation day (GD) 10 or 12, and the embryos were examined on GD 14 and 16. Exposure to RA in an oil:DMSO vehicle resulted in much higher incidences of cleft palate than were observed after dosing with RA in oil only. After exposure on GD 10, to RA, small palatal shelves formed which did not make contact and fuse on GD 14. The medial cells did not undergo programmed cell death. Instead, the medial cells differentiated into a stratified, squamous, oral-like epithelium. The RA-exposed medial cells did not incorporate 3H-TdR on GD 14 or 16, but the cells expressed EGF receptors and bound 125I-EGF. In contrast, RA-induced clefting after exposure on GD 12 did not involve growth inhibition. Shelves of normal size formed and made contact, but because of altered medial cell differentiation did not fuse. Medial cells differentiated into a pseudostratified, ciliated, nasal-like epithelium. This response was produced in vivo at exposure levels which produced cleft palate, and after exposure of palatal shelves to RA in vitro from GD 12-15. The medial cells exposed on GD 12 incorporated 3H-TdR on GD 14, expressed EGF receptors, and bound 125I-EGF. The responses to RA which lead to cleft palate differed after exposure on GD 10 or 12, and the pathways of differentiation which the medial cells followed depended on the developmental stage exposed.