Signal triangulation coordinates cell fate decisions in the developing jaw

Development of the vertebrate lower jaw depends on spatially precise cell fate decisions by cranial neural crest-derived cells (CNCCs) of the mandibular arch. From closer to the mouth (oral) to farther away (aboral), CNCCs adopt bone, cartilage, tendon, and stromal fates to shape the jaw skeleton and ensure proper connectivity to the musculature. How signaling pathways impact downstream transcriptional programs to generate distinct cell fates along the oral-aboral axis remains incompletely understood. Using photoconversion-based lineage tracing of CNCCs in zebrafish, we show that oral cells contribute to the lower jaw skeleton and aboral cells primarily to tendon, ligament, and stromal tissues. During embryogenesis, the oral domain is partitioned into lateral pitx1+ and medial foxf1+ subdomains distinct from an aboral nr5a2+, gsc+ domain. Using pharmacological inhibition and transgenic misexpression, we find that Bmp signaling establishes aboral nr5a2 and gsc expression, Fgf signaling oral-lateral pitx1 expression, and Hedgehog signaling oral-medial foxf1 expression. Analysis of mutants for pitx1, nr5a2, and gsc reveal that their oral-aboral expression domains are established independently of each other. We also identify enhancers regulating oral-aboral expression of pitx1 and nr5a2, with mutagenesis showing roles for Fgf-dependent ETS motifs in oral pitx1 and Bmp-dependent E-box motifs in aboral nr5a2 enhancer activity, consistent with dependence of nr5a2 expression on the Bmp-dependent E-box factor Hand2. These findings reveal how triangulation of three major signaling pathways converge on distinct gene regulatory modules to establish distinct oral-aboral cell fate decisions in the developing jaw.