Analysis of wdr68 Function in Craniofacial Patterning in the Zebrafish
Authors:Greg Alvarado, Anish Bhandari, Andrew Martinez, Annie Pham, Shang Robin, Jessica Wang
Mentor:Robert Nissen, Professor of Biological Sciences, California State University, Los Angeles
Many craniofacial syndromes are caused by defects in signaling pathways that pattern the cranial neural crest cells (CNCCs) along the dorsal-ventral axis. For example, defects in Bone Morphogenetic Protein (BMP) signaling are associated with cleft lip/palate, auriculocondylar syndrome is caused by impaired Endothelin-1 (Edn1) signaling, and Alagille syndrome is caused by defects in Jagged-Notch signaling. The BMP, Edn1, and Jag1b pathways intersect because BMP signaling is required for ventral edn1 expression that, in turn, restricts jag1b to dorsal CNCC territory. Previously, we identified the wdr68 gene as essential for edn1 expression in the zebrafish. However, the temporal window during which wdr68 activity is required, and within which signaling pathway wdr68 functions, are unclear. Here, we show that wdr68 activity is required for jaw development between the 12-somites and 20-somites stages using immunohistochemistry (IHC) and an inducible Tg(hsp70l:GFP-Wdr68) zebrafish line. The identified developmental window overlaps with the known onset of edn1 expression at the 18-somites stage. Using in situ hybridization (ISH), we also report here that jag1b expression is expanded ventrally in wdr68 mutant embryos, while the expression of the BMP ligands bmp2b, bmp4, and bmp7a appear unchanged in wdr68 mutants. Thus, wdr68 appears to function in parallel or downstream of BMP signaling to induce edn1 and restrict jag1b. BMP signaling is mediated by the Smad1/5/8 transcription factors and partner co-Smad4. Previous reports detected Wdr68 in physical complexes with multiple Smads, suggesting potential roles for wdr68 within BMP signaling. Consistently, here we report synergistic reductions of the craniofacial cartilages in wdr68 mutant embryos co-treated with the BMP signaling inhibitor dorsomorphin. Thus, wdr68 may mediate edn1 expression for craniofacial development through modulating some aspect of BMP signaling. We will also present progress towards IHC analysis of pSmad1/5/8 and epistatic tests of wdr68 and edn1 function.