Deciphering the Endothelin Receptor Type A Dependent G Protein Code that Patterns the Mandibular First Pharyngeal Arch

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ABSTRACT Craniofacial disorders account for approximately 1/3 of birth defects in newborns and are a major cause of infant mortality. Preventing or mitigating craniofacial disorders requires a detailed understanding of normal developmental processes. One of the earliest events in craniofacial development is patterning of the first pharyngeal arch, from which the bone, cartilage and connective tissue of the face and neck arise. Much of this is mediated by Endothelin receptor type A (EDNRA) signaling, which establishes the ventral (future mandible) and intermediate (future middle ear/jaw joint) domains of the arch. This is an evolutionarily conserved mechanism, as loss of EDNRA signaling results in homeotic transformation of the lower jaw into upper jaw-like structures in zebrafish, mouse, and humans. Although EDNRA-regulated genes are well characterized, little is known about the mechanisms that link EDNRA signal transduction to gene regulation. This has human health implications because many facial birth defect syndromes have now been linked to aberrant EDNRA signaling. EDNRA is a G protein-coupled receptor that can activate multiple classes of G proteins; at least two of these are likely required to pattern the ventral and intermediate domains. This is illustrated in Gq-/-/G11-/- mice, in which only the proximal mandible and jaw joint (both intermediate derivatives) are affected. My preliminary data shows that zebrafish embryos exposed to YM-254890 (YM), a small molecule inhibitor for Gq/11, also only causes defects in intermediate-domain derived jaw structures. Based on these findings, I hypothesize that EDNRA-mediated patterning along the D-V axis of the first pharyngeal arch is implemented by a novel underlying G protein code. To begin dissecting this code, this proposal will investigate how Gq/11 mechanisms drive intermediate domain patterning and development in two Aims. In Aim 1, I will define the gene regulatory network controlled by Gq/11 in intermediate domain NCCs by performing single cell RNA- sequencing on YM-treated embryos. Identified genes will be functionally tested by transgenesis or gene inactivation in developing zebrafish. In Aim 2, I will determine whether Gq/11-mediated patterning coordinates joint and cartilage formation by preventing premature chondrocyte differentiation, as it has been shown that Gq/11 negatively regulates chondrocyte differentiation in other studies. This will be tested using time-lapse imaging and whole-mount immunohistochemistry assays in YM-treated embryos. Together these proposed Aims will elucidate the Gq/11-dependent mechanisms required for intermediate domain-derived jaw structure formation and provide insight into the novel G protein code of patterning. This mechanism is very likely conserved in all jawed vertebrates and would be a high value target in the quest for targeted pharmacological modulation of signaling in humans with craniofacial birth defects.