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Abstract

Non syndromic cleft lip with or without cleft palate (NSCL/P) are the among most prevalent craniofacial birth defects (1:700 live births). NSCL/P is a complex trait, caused by contributions from multiple genes variants (40 genes) and exposure to environmental factors. We hypothesize that asymmetric growth of the facial processes would lead to a fusion defect on one side and ultimately a unilateral cleft lip. Very small asymmetries in development could be present in all embryos and fortunately normal homeostatic mechanisms can compensate. One cellular signaling pathway that is active in all cells and controls morphology is the Rho GTPase pathway. The rationale for focusing on Rho GTPases is supported by numerous human genetic studies linking variants of ARHGAP29—which converts the Rho GTPase from GTP-bound (active) to GDP-bound (inactive)—to human clefting. In drug-treated, in vitro organ cultures we showed that RhoA signaling is sufficient to alter face shape and is required for facial morphogenesis. Next, we used an in vivo model to activate and inhibit RhoA signaling in the maxillary prominence using bead implants. CN03 agonist-soaked beads caused a dose-dependent form of cleft lip, mirroring the clinical spectrum in NSCL/P. The mechanisms were increased tension in the cytoskeleton causing rapid contraction of the maxillary mesenchyme and disorganization of the fusing epithelium. ARHGAP29 human variants were added to human HEK293 cells to test which variants underwent nonsense mediated decay, which were likely loss of function, and which may have new functions. ARHGAP29 Lys326* variant underwent nonsense mediated decay, whereas ARHGAP29 Ser492* variant entered the nucleus. Other variants appeared to increase or decrease cytoskeletal tension as shown by changes in cellular morphology. Using time-lapse imaging we breakdown the types of epithelial movements in the fusion zone for the first time. There are directional cellular movements, and these are reversed by activating RhoA, preventing epithelial seam formation. Overall, these studies provide new insights into the general role of RhoA signaling during upper lip development and a specific functional defect caused by gene variants of ARHGAP29. The foundational discoveries on the druggable GTPase signaling pathway, will inform future therapeutic interventions for orofacial clefting.