Regulation of endothelin1 signaling during craniofacial development

How facial skeletons are patterned to obtain specialized characteristics across diverse vertebrates is an intriguing question. That some of the principles of facial patterning are shared, yet the outcome so different, speaks of the intricacies involved in the regulation. Genetic and pharmacological studies demonstrate that Endothelin1 (Edn1) signaling patterns the ventral facial skeleton in fish, chicks, and mice. When Edn1 function is reduced early in development the ventral lower jaw and supporting structures are reduced in size and often fused to their dorsal upper jaw counterparts. Through genetic analysis in zebrafish we show that FurinA and Plcb3 function within the Edn1 pathway. FurinA is a proprotein convertase which activates numerous proproteins. A loss of FurinA function results in facial skeletal phenotypes which resemble a partial loss of Edn1. The ventral structures are not strongly reduced in size but are fused to their dorsal counterparts. Furthermore, we show that edn1-dependent gene expression is compromised in furinA mutants, and that the combined loss of function of furinA and its co-ortholog furinB resembles the severe loss of Edn1. These results demonstrate the role of Furin in craniofacial patterning and its in vivo role in activating Edn1.; Similarly, plcb3 mutants have skeletal phenotypes resembling those of edn1 mutants, suggesting Plcb3 is also a component of the Edn1 pathway. The Edn1 ligand signals through a G protein-coupled receptor pathway that activates PLC*. PLC* are modular proteins that contain several structural domains. An N-terminal PH domain is critical for substrate binding and membrane localization. Through a morpholino-mediated deletion in the PH domain we demonstrate that two independent plcb3 catalytic domain mutations act as dominant negative regulators of Edn1-PLC* mediated signaling. As naturally occurring splice variants of PLC* and PLC-like genes have been identified which have intact PH domains but not intact catalytic domains, our results support a role for these genes as negative regulators of important developmental pathways, including the Edn1 pathway. Together, our study of furinA and plcb3 demonstrate complexities in regulating Edn1 signaling, allowing for fine control of facial patterning and helping to explain facial diversity among vertebrates. This dissertation includes my co-authored materials.