Chao Chung-Hao, Wang Horng-Dar, Yuh Chiou-Hwa
Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
BMC Dev Biol. 2010 Mar 26;10:35. doi: 10.1186/1471-213X-10-35.
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.
To 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.
This 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.
Six3a属于同源异型域蛋白的Six家族，在多种物种神经发生开始时于最前端神经板中表达。尽管Six3a作为眼睛和前脑发育关键调节因子的功能已得到充分研究，但其转录调控机制尚不清楚。
为阐明six3a的转录调控机制，我们进行了体内报告基因检测。将斑马鱼six3a基因周围21 kb区域与不同物种的类似区域进行比对，鉴定出几个保守的非编码模块。斑马鱼转基因实验鉴定出两个增强子元件和一个抑制子。D模块在早期驱动前脑和眼睛中的绿色荧光蛋白报告基因表达，而A模块负责后期表达。A模块还作为抑制子抑制D模块的异位表达。突变分析进一步将A模块最小化为四个高度保守元件，将D模块最小化为三个元件。通过电泳迁移率变动分析，我们还证明了胚胎核提取物中存在DNA结合蛋白。还预测了可能占据这些高度保守元件的转录因子。
本研究全面阐述了six3a在脑和眼发育过程中的转录调控机制，并为建立斑马鱼神经发生的基因调控网络提供了契机。
