Abstract
BackgroundSix3a belongs to the SIX family of homeodomain proteins and is expressed in the most anterior neural plate at the beginning of neurogenesis in various species. Though the function of Six3a as a crucial regulator of eye and forebrain development has been thoroughly investigated, the transcriptional regulation of six3a is not well understood.ResultsTo elucidate the transcriptional regulation of six3a, we performed an in vivo reporter assay. Alignment of the 21-kb region surrounding the zebrafish six3a gene with the analogous region from different species identified several conserved non-coding modules. Transgenesis in zebrafish identified two enhancer elements and one suppressor. The D module drives the GFP reporter in the forebrain and eyes at an early stage, while the A module is responsible for the later expression. The A module also works as a repressor suppressing ectopic expression from the D module. Mutational analysis further minimized the A module to four highly conserved elements and the D module to three elements. Using electrophoresis mobility shift assays, we also provided evidence for the presence of DNA-binding proteins in embryonic nuclear extracts. The transcription factors that may occupy those highly conserved elements were also predicted.ConclusionThis study provides a comprehensive view of six3a transcription regulation during brain and eye development and offers an opportunity to establish the gene regulatory networks underlying neurogenesis in zebrafish.
Highlights
Six3a belongs to the Sine Oculis Homeobox (SIX) family of homeodomain proteins and is expressed in the most anterior neural plate at the beginning of neurogenesis in various species
From the protein sequence comparison, we found that zebrafish Six3a is more similar to medaka Six3.2 (85% identity) than to Six3.1 (75% identity)
Box E and G of olsix3.2 matched to the sequence of zsix3a, but were not among our conserved modules; Box C and H of olsix3.2 did not match the zsix3a sequence
Summary
Six3a belongs to the SIX family of homeodomain proteins and is expressed in the most anterior neural plate at the beginning of neurogenesis in various species. Though the function of Six3a as a crucial regulator of eye and forebrain development has been thoroughly investigated, the transcriptional regulation of six3a is not well understood. Zebrafish (Danio rerio) has long been an excellent vertebrate model organism for developmental biology [1,2,3] and is used to study the mechanisms of axis formation [4], endoderm differentiation [3,5] and muscle development [6]. Development is controlled by the hierarchical regulation between signaling pathways and transcription factors. Disrupting the SIX domain or the homeodomain abolishes the ability of Six3a to induce rostral forebrain enlargement in zebrafish, implying that these domains are essential for six gene function [14]. It has been suggested that part of the biochemical and functional specificity between members of the SIX protein family is due to their non-conserved C-terminal segments [15]
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