Abstract
BackgroundThe genetic cascades underpinning vertebrate early eye morphogenesis are poorly understood. One gene family essential for eye morphogenesis encodes the retinal homeobox (Rx) transcription factors. Mutations in the human retinal homeobox gene (RAX) can lead to gross morphological phenotypes ranging from microphthalmia to anophthalmia. Zebrafish rx3 null mutants produce a similar striking eyeless phenotype with an associated expanded forebrain. Thus, we used zebrafish rx3-/- mutants as a model to uncover an Rx3-regulated gene network during early eye morphogenesis.ResultsRx3-regulated genes were identified using whole transcriptomic sequencing (RNA-seq) of rx3-/- mutants and morphologically wild-type siblings during optic vesicle morphogenesis. A gene co-expression network was then constructed for the Rx3-regulated genes, identifying gene cross-talk during early eye development. Genes highly connected in the network are hub genes, which tend to exhibit higher expression changes between rx3-/- mutants and normal phenotype siblings. Hub genes down-regulated in rx3-/- mutants encompass homeodomain transcription factors and mediators of retinoid-signaling, both associated with eye development and known human eye disorders. In contrast, genes up-regulated in rx3-/- mutants are centered on Wnt signaling pathways, associated with brain development and disorders. The temporal expression pattern of Rx3-regulated genes was further profiled during early development from maternal stage until visual function is fully mature. Rx3-regulated genes exhibited synchronized expression patterns, and a transition of gene expression during the early segmentation stage when Rx3 was highly expressed. Furthermore, most of these deregulated genes are enriched with multiple RAX-binding motif sequences on the gene promoter.ConclusionsHere, we assembled a comprehensive model of Rx3-regulated genes during early eye morphogenesis. Rx3 promotes optic vesicle morphogenesis and represses brain development through a highly correlated and modulated network, exhibiting repression of genes mediating Wnt signaling and concomitant enhanced expression of homeodomain transcription factors and retinoid-signaling genes.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-825) contains supplementary material, which is available to authorized users.
Highlights
The genetic cascades underpinning vertebrate early eye morphogenesis are poorly understood
Whole transcriptome sequencing of 13 hpf Zebrafish rx3−/− mutants To identify genes regulated by Rx3 during optic vesicle morphogenesis, adult zebrafish carriers of a null rx3 mutation were mated
Offspring were phenotypically separated into pools consisting of mutants with an absence of optic vesicles or siblings exhibiting a wild-type phenotype at 13 hours post fertilization, the earliest time point at which optic vesicle evagination phenotypes can be reliably and quickly detected to ensure correct sampling at this timepoint
Summary
The genetic cascades underpinning vertebrate early eye morphogenesis are poorly understood. We used zebrafish rx3−/− mutants as a model to uncover an Rx3-regulated gene network during early eye morphogenesis. Results: Rx3-regulated genes were identified using whole transcriptomic sequencing (RNA-seq) of rx3−/− mutants and morphologically wild-type siblings during optic vesicle morphogenesis. Hub genes down-regulated in rx3−/− mutants encompass homeodomain transcription factors and mediators of retinoid-signaling, both associated with eye development and known human eye disorders. Rx3-regulated genes exhibited synchronized expression patterns, and a transition of gene expression during the early segmentation stage when Rx3 was highly expressed Most of these deregulated genes are enriched with multiple RAX-binding motif sequences on the gene promoter. The retinal homeobox transcription factor, RX ( known as retina and anterior neural fold homeobox, RAX) has an evolutionarily conserved role during early vertebrate eye development [2]. When expression of rx and rx are knocked-down in zebrafish, optic vesicles do form, but microphthalmia results [6]
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