Regulation of gene expression in cultured embryonic mouse mandibular mesenchyme by serotonin antagonists

Abstract

 During murine embryogenesis, uptake sites for the neurotransmitter serotonin (5-HT) are transiently expressed in craniofacial epithelial structures. Based on malformations produced in cultured mouse embryos exposed to uptake inhibitors or receptor ligands, we have proposed that 5-HT acts as a dose-dependent morphogenetic signal during critical periods of craniofacial development. Several 5-HT receptor subtypes are co-distributed with tenascin and the calcium binding protein S-100β in developing craniofacial mesenchyme. Since these molecules are thought to be important for craniofacial development, their regulation by 5-HT could mediate some of its morphogenetic actions. Mandibular mesenchyme cells, from E12 mouse embryos (plug day=E1), grown in micromass cultures were used as an in vitro model to investigate whether 5-HT regulates expression of these molecules. Immunocytochemistry revealed expression of S-100β, tenascin, cartilage proteoglycan core protein (a component of the cartilage matrix) and a variety of 5-HT receptors in these cultures. To block the actions of 5-HT (from serum in the culture medium), cultures were exposed to one of these selective 5-HT receptor antagonists and effects on expression were investigated using quantitative immunobinding and in situ hybridization assays. These antagonists differentially regulated expression of cartilage core protein, S-100β and tenascin. Antagonism of 5-HT₃ receptors by Zofran or 5-HT_1A receptors by NAN-190 reduced the amount of core protein, whereas antagonism of 5-HT_2A-C receptors by mianserin had no significant effect. All three antagonists stimulated levels of tenascin mRNA and protein. Expression of S-100β mRNA and protein was inhibited by Zofran and stimulated by mianserin, whereas NAN-190 had no significant effect. The differential effects of antagonists suggest that in vivo, 5-HT could: (1) promote expression of cartilage core protein by activation of 5-HT₃ or 5-HT_1A receptors, (2) inhibit production of tenascin by activation of multiple receptors, (3) promote or inhibit synthesis of S-100β by activation of 5-HT₃ or 5-HT₂ receptors, respectively. These actions may be important components of the morphogenetic functions of 5-HT during craniofacial development.