Functional Analysis of Regional Gene Expression in Zebrafish Craniofacial Development

Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite their significance for craniofacial development, how genetic programs drive this regionalization remains poorly understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin1 (Edn1) signaling in the intermediate joint-forming region yet a surprisingly minor role in ventral-most regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of co-regulated genes, with in situ hybridization confirming the expression of novel genes with domain-specific expression. We then performed mutational analysis of a number of these genes, which uncovered antagonistic functions of two Edn1 targets, follistatin a (fsta) and emx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in arch CNCCs reveals complex regulation by Edn1 and points to novel candidates for craniofacial disorders. Overall design: mRNA profiles of 3 distinct populations of zebrafish pharyngeal arch cells isolated using FACS from 36 hpf wild type (WT) zebrafish. fli1a:GFP+;sox10:DsRed- and fli1a:GFP-;sox10:DsRed+ populations were also isolated at the same time and sequenced as controls (3 replicate each). Additionally there are mRNA profiles of zebrafish pharyngeal arches (all domains combined) isolated by FACS from 36 hpf zebrafish with the following genotypes: edn1-/- (2 replicates), jag1b-/-, jag1b+/+ (control), hsp70I:Gal4; UAS:Edn1, hsp70I:Gal4; UAS:Nicd, and hsp70I:Gal4 (control).

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Askary A, Xu P, Barske L, Bay M, Bump P, Balczerski B, Bonaguidi MA, Crump JG