Back to Annual Meeting Posters

PURPOSE:
Cranial neural crest (CNC) cells migrate and elaborate into craniofacial structures, dysregulation of which leads to craniofacial malformations. Many of the intrinsic genetic programs that cooperatively regulate CNC fate commitment and craniofacial morphogenesis remain largely unknown. Using comparative transcriptome analysis between premigration and postmigration CNC cells, we aimed to segregate the genes regulating early crest migration from those regulating late pharyngeal morphogenesis. We utilized the zebrafish model to carry out high-throughput expression and functional analysis and identified novel genes that modulate CNC development.
METHODS:
Pre vs. post-migration CNC cells were isolated from lineage-restricted transgenic embryos Tg(sox10:egfp) via FACS (Dougherty and Liao, Journal of Craniofacial Surgery, 23(5); 1333-7, 2012). Gene expression profiling was analyzed by Affymetrix gene-chip. Spatiotemporal expression of genes of interest was triaged by high-throughput wholemount RNA in situ hybridization (WISH). Morpholino-mediated gene knockdown was performed to assess gene function in vivo.
RESULTS:
408 transcripts were identified to be differentially expressed ≥ 1.5-fold between pre-migratory and post-migratory CNC cells from microarray in triplicates. Of these, 237 (58%) exhibit enhanced expression in premigration CNC, and 171 (42%) are enriched in postmigration CNC. Candidate genes with pharyngeal expression were further screened by WISH. Developmentally upregulated genes include: icn, s100a10b, aqp3, which labels lower jaw structures including pharyngeal arches and ceratohyal; and ctgfa marks the extending maxillary prominence and trabecula in upper jaw. Meanwhile, irx7 expression was detected at high levels along the neural tube at early migration (10 somites); and was restricted to specific pharyngeal arch structures (ceratohyal and hyosymplectic) and more posterior pharyngeal arches during development. By double WISH labeling, we found that irx7 was co-expressed with CNC markers such as sox10, sox9a, dlx2a, hoxa2b and/or hoxb2a in the leading edge of the second pharyngeal arch during CNC migration. Moreover, in vivo functional study shows that impaired irx7 leads to hypoplastic hyosymplectic and abrogation of ceratobranchial skeleton in day 4 embryos.
CONCLUSION:
We took advantage of the ability to utilize FACS to identify lineage-specific cell population in the sox10:egfp transgenic zebrafish model to carry out transcriptome profiling of embryonic CNC cells, and to perform high throughput expression and functional analysis of candidate genes. Comparative analysis of the transcriptome between pre-migratory and post-migratory CNC revealed a highly dynamic transcriptional profile, and identified several key regulators that are differentially expressed between early crest migration and subsequent craniofacial morphogenesis. Notably irx7 transcription factor was identified as a key gene required in development of second pharyngeal arch derived structures.