Opposing regulation of cell proliferation by retinoic acid and the serotonin_2B receptor in the mouse frontonasal mass

Development of the frontonasal mass (FNM), branchial arches, heart, and limbs depends on neural crest-mediated epithelial–mesenchymal (E–M) interactions. Teratogenesis by retinoic acid (RA) or blockade of serotonergic (5-HT) signaling by the pan-5-HT₂ receptor antagonist, ritanserin, perturbs development of these embryonic structures. In both cases, resulting phenotypes include forebrain and olfactory placode anomalies, malformations of the face, eye and lens, as well as posterior neural tube and cardiac defects. Similar sites of malformations, together with the presence of RA response elements in the 5-HT_2B receptor promoter, have led to the suggestion that a negative regulatory relationship may exist between RA and 5-HT₂-mediated 5-HT signaling at sites of E–M interaction (Choi et al. 1997); however, another possibility is that RA and 5-HT act independently as opposing signals to regulate development of common embryonic targets. Together with recent evidence for opposite effects on chondrogenic differentiation in hindlimb micromass cultures (Bhasin et al. 2003a), results of the present study raise the possibility that these pathways may act as opposing signals for common targets in the mouse embryo. The RA receptors, co-factors and metabolic enzymes, and 5-HT_2B receptors were found to be are coordinately expressed at sites of E–M interaction, including the FNM, in the embryonic day (E)10.5 mouse. Cell proliferation experiments using [³H]thymidine incorporation demonstrated that RA or activation of 5-HT_2B receptors caused opposite effects in FNM explants, namely stimulation or inhibition of cell proliferation, respectively, 5-HT_2B receptor activation did not appreciably alter patterning in FNM explants. While RA has been shown to regulate lateral patterning in the FNM (LaMantia et al. 2000), 5-HT_2B receptor activation did not alter patterning in FNM explants. Quantification of 5-HT_2B receptor transcripts by real-time PCR provided no evidence of negative regulation of 5-HT_2B receptor expression by RA in FNM explants, although preliminary studies using in situ hybridization had suggested that this was a possibility in both explants and RA teratogenized embryos. Future studies using quantitative PCR may still show this to be the case in teratogenized embryos. Together with the finding of coordinate expression of 5-HT_2B receptors and RA signaling molecules, results of the present study suggest that RA, and 5-HT mediated by 5-HT_2B receptors, may act as opposing signals to regulate cell proliferation during craniofacial development in the mouse embryo.