wnt9b Morphants Reveal Conservation of Ancient Pharyngeal Cartilage Patterning Pathway

Introduction: Oral-facial clefting has a prevalence of approximately 1-2/1000 live births in different populations, and as such is among the most common birth disorders worldwide. Our laboratory in the UAB SOD-IOHR utilizes the zebrafish model to study the role of Wnt signaling in oral-facial cleft development. Objectives: The purpose is to demonstrate clinical utility of the zebrafish model system by demonstrating conservation of ancient pharyngeal arch gene regulatory networks (GRNs) important in oral-facial patterning. To this end, we plan to establish a wnt9b morphant (wnt9b-MO) phenotype relative to wnt9a morphant and wildtype (WT) craniofacial cartilage phenotypes through time. Methods: Dilution-controlled antisense morpholino (MO) injection into zebrafish early staged embryos allows precise and consistent gene knockdown. Alcian blue staining allows easy classification and quantification of cartilages from zebrafish embryos. Morphants were compared between WT, wnt9a and wnt9b MO knockdowns, over time (3 to 4 to 5 Days Post Fertilization (DPF)), across a MO dosage range (from 1:7 to 1:10 dilution). Results: 1) A range of MO dilutions (from 1:10, 1:8, and 1:7) for wnt9a and wnt9b MOs produce anterior/first pharyngeal arch cartilage defects ranging from mild to severe. 2) wnt9b-MO does produce an observable, albeit more subtle craniofacial and cartilage phenotype, compared to wnt9a-MO. 3) wnt9b-MO palates (ethmoids) exhibit a phenotype that is potentially homologous to human cleft palates. 4) wnt9b-MO meckels and palatoquadrates are foreshortened in an anterior-posterior dimension, regressing towards a dorsal-ventral orientation. Conclusion: These data implicate wnt9a and wnt9b in dorsal-ventral signaling pathways, with wnt9b potentially upstream of the Endothelin/suc1 ventral signal. We demonstrate that wnt9b-MOs do have a consistent phenotype within jaw cartilages. We hypothesize that wnt9a and wnt9b act upstream of a highly conserved dorsal-ventral GRN responsible for patterning of oral-facial cartilage elements. These experiments may improve our understanding of human oral-facial clefting dysmorphic patterns.