sox1a_ka705Tg whole genome sequencing

The transcription factors sox1a and sox1b label a neuronal subtype in the developing spinal cord of zebrafish embryos. Analysis and mapping of sox1a/b gene expression revealed that both genes are necessary for the specification of a new V2 interneuron subtype, which we call V2s. A transgenic line created by Tol2-transgenesis to test a regulatory element of an unrelated gene, reliably labelled these V2s neurons. This led us to analyse this line in more detail. The eGFP expression of the transgene fully recapitulated sox1a expression throughout embryonic development. WGS revealed then that the transgene integrated into the 5´-UTR of the sox1a gene, indicating that it is under the control of the endogenous sox1a promoter. Subsequent analysis showed that the eGFP expression is not related to the initially used regulatory element, thus it is a gene trap line for the sox1a gene. With the help of this line we were able to show that V2s neurons grow long ipsilateral descending axonal projections and they are inhibitory interneurons. Unilateral ablation of V2s interneurons causes a delay in touch-provoked zebrafish escape behaviour, suggesting that V2s interneurons are involved in locomotion.转录因子sox1a与sox1b可标记斑马鱼胚胎发育阶段脊髓内的一种神经元亚型。针对sox1a/b基因表达的分析与定位研究表明，这两个基因对于一种新型V2中间神经元亚型的特化不可或缺，我们将该亚型命名为V2s神经元。本研究利用Tol2转基因技术（Tol2-transgenesis）构建了一条转基因品系，最初用于验证某一无关基因的调控元件，却可稳定标记上述V2s神经元，这推动我们对该品系开展更为深入的解析。该转基因元件携带的增强型绿色荧光蛋白（enhanced Green Fluorescent Protein, eGFP）的表达模式，在整个胚胎发育周期中均完全重现了sox1a的表达特征。全基因组测序（Whole Genome Sequencing, WGS）结果显示，该转基因片段整合至sox1a基因的5'非翻译区（5'-untranslated region, 5'-UTR），表明其表达受内源性sox1a启动子的调控。后续分析证实，该eGFP的表达与最初使用的调控元件并无关联，因此该品系属于sox1a基因的基因捕获品系（gene trap line）。借助该转基因品系，我们明确了V2s神经元具有长距离同侧下行轴突投射，且属于抑制性中间神经元。对V2s中间神经元进行单侧消融会导致斑马鱼触诱发逃逸行为出现延迟，这提示V2s中间神经元参与了运动调控过程。