A Rho-GTPase Activating Proten (Arhgap28) Regulates Palate Development in Zebrafish

Cleft palate is the most prevalent craniofacial malformation in humans, and it entails problems in neural crest (NC) derived cartilages and bones in the skull. The underlying molecular causes of cleft palate are largely unknown. Recently one form of human non-syndromic cleft lip with or without cleft palate has been linked to a Rho-GTPase activating protein (ARHGAP29), implicating Rho regulation of the actin cytoskeleton in the migration of NC cells that form the palate. Through a genetic screen for craniofacial defects in zebrafish, a recessive mutation in arhgap28 was identified that causes cleft palate, which we call splithead (spd). Skeletal staining in arhgap28^spd/spd embryos revealed abnormalities in the larval palate, such as a split ethmoid plate and parasphenoid bone, as well as increased separation of the trabeculae. Interestingly, other components of the skull were largely unaffected, suggesting that this mutation specifically affects the palate. In situ hybridization in wild-type embryos revealed that arhgap28 is expressed in the cranial mesoderm, which provides important signals to migrating NC, at 12 hours post-fertilization (hpf), and in NC precursors of the trabeculae at 48 hpf, the onset of palate morphogenesis. In addition, arhgap28^spd/spd embryos had wide heads and were shorter. The notochord often failed to ossify. Total RNA from wild-type embryos at two hours post-fertilization revealed that arhgap28 was also expressed maternally. Altogether, these data suggests that Arhgap28 may be initially required during gastrulation for convergence and extension movements, as well as later during NC migration. arhgap28^spd/spd mutant palates resemble the clefts seen in zebrafish deficient in Sonic Hedgehog (Shh) signaling, suggesting that one role for Arhgap28 may be to control NC migration downstream of Shh through regulation of its main target, RhoA. Future work will explore this possibility by cell tracing and live imaging of small GTPase activity and cytoskeleton dynamics in palate precursors.