Fungiform papillae are epithelial specializations that develop in a linear pattern on the anterior mammalian tongue and differentiate to eventually contain taste buds. Little is known about morphogenetic and pattern regulation of these crucial taste organs. We used embryonic rat tongue, organ cultures to test roles for bone morphogenetic proteins, BMP2, 4 and 7, and antagonists noggin and follistatin, in development of papillae from a stage before morphological initiation (E13) or from a stage after the pre-papilla placodes have formed (E14). BMPs and noggin proteins become progressively restricted to papilla locations during tongue development. In E13 cultures, exogenous BMPs or noggin induce increased numbers of fungiform papillae, in a concentration-dependent manner, compared to standard tongue cultures; BMPs, but not noggin, lead to a decreased tongue size at this stage. In E14 cultures, however, exogenous BMP2, 4 or 7 each inhibits papilla formation so that there is a decrease in papilla number. Noggin substantially increases number of papillae in E14 cultures. Using beads for a highly localized protein delivery, papillae are inhibited in the surround of BMP-soaked beads and induced in large clusters around noggin-soaked beads. Follistatin, presented in culture medium or by bead, does not alter papilla formation or number. In all fungiform papillae that form under various culture conditions, the molecular marker, sonic hedgehog, is within each papilla. However, the BMP inhibitory effect on papillae is not prevented by disrupting sonic hedgehog signaling through addition of cyclopamine to cultures. BMPs and noggin alter cell proliferation in tongue epithelium in opposite ways, demonstrated with Ki67 immunostaining. We propose that the BMPs and noggin, colocalized within papilla placodes and the fungiform papillae per se, have opposing inhibitory and activating or inducing roles in papilla development in linear patterns. We present a model for these effects.

Fungiform papillae are epithelial taste organs that form on the tongue, requiring differentiation of papillae and inter-papilla epithelium. We tested roles of epidermal growth factor (EGF) and the receptor EGFR in papilla development. Developmentally, EGF was localized within and between papillae whereas EGFR was progressively restricted to inter-papilla epithelium. In tongue cultures, EGF decreased papillae and increased cell proliferation in inter-papilla epithelium in a concentration-dependent manner, whereas EGFR inhibitor increased and fused papillae. EGF preincubation could over-ride disruption of Shh signaling that ordinarily would effect a doubling of fungiform papillae. With EGF-induced activation of EGFR, we demonstrated phosphorylation in PI3K/Akt, MEK/ERK, and p38 MAPK pathways; with pathway inhibitors (LY294002, U0126, SB203580) the EGF-mediated decrease in papillae was reversed, and synergistic actions were shown. Thus, EGF/EGFR signaling by means of PI3K/Akt, MEK/ERK, and p38 MAPK contributes to epithelial cell proliferation between papillae; this biases against papilla differentiation and reduces numbers of papillae. Developmental Dynamics 237:2378 -2393, 2008.

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