Barx1 is necessary for zebrafish cranial neural crest patterning and pharyngeal arch chondrogenesis

Abstract

BARX1, most prominently expressed in the pharyngeal arches is a candidate gene implicated in human craniofacial anomalies, and syndromic malformations; however, little is understood of its regulation or role in patterning the arches. We examined the zebrafish barx1 gene during craniofacial development and performed a functional analysis using morpholino oligonucleotides. Embryos microinjected with barx1 morpholinos exhibit reduced crestin and dlx2a expression. By 2.5 days post fertilization barx1 morphants exhibit poor facial outgrowth and micrognathia. Histological analysis reveals reductions in chondrocyte differentiation and condensation within the arches. Alcian blue stained morphants have small and dysmorphic arch cartilage elements, and chondrogenic marker col2a1 arch expression is perturbed. We investigated barx1 regulation within the arches by implanting beads imbued with growth factors FGF8 and BMP2. The bead experiments show the influence of the growth factors on barx1 expression, and its role in modulating chondrocyte condensation. Together these results suggest a necessary role for barx1 at early stages of cranial neural crest patterning and chondrogenesis within the developing zebrafish viscerocranium. Investigating the zebrafish barx1 provides new insights into the human BARX1 ortholog that may be implicated in agnathia, micrognathia or other craniofacial syndromes. To ascertain its role we utilized a morpholino mediated knock-down approach. Morphants exhibited micrognathia. Alcian blue staining indicates abnormal formation and/or complete loss of pharyngeal arch cartilage elements suggesting that barx1 is necessary for organization of the prechondrogenic ectomesenchyme. Examination of arch markers, sox9a, and dlx2a as well as fli1 transgenic zebrafish indicate that cranial neural crest differentiation and migration remains unperturbed. However, we observed a loss of collagen type II expression in the mandible.