Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, 621 Charles E Young Dr South, Los Angeles, CA 90095-1606, USA.
Circ Res. 2012 Jan 20;110(2):220-9. doi: 10.1161/CIRCRESAHA.111.251298. Epub 2011 Dec 1.
Endothelial cells are developmentally derived from angioblasts specified in the mesodermal germ cell layer. The transcription factor etsrp/etv2 is at the top of the known genetic hierarchy for angioblast development. The transcriptional events that induce etsrp expression and angioblast specification are not well understood.
We generated etsrp:gfp transgenic zebrafish and used them to identify regulatory regions and transcription factors critical for etsrp expression and angioblast specification from mesoderm.
To investigate the mechanisms that initiate angioblast cell transcription during embryogenesis, we have performed promoter analysis of the etsrp locus in zebrafish. We describe three enhancer elements sufficient for endothelial gene expression when place in front of a heterologous promoter. The deletion of all 3 regulatory regions led to a near complete loss of endothelial expression from the etsrp promoter. One of the enhancers, located 2.3 kb upstream of etsrp contains a consensus FOX binding site that binds Foxc1a and Foxc1b in vitro by EMSA and in vivo using ChIP. Combined knockdown of foxc1a/b, using morpholinos, led to a significant decrease in etsrp expression at early developmental stages as measured by quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization. Decreased expression of primitive erythrocyte genes scl and gata1 was also observed, whereas pronephric gene pax2a was relatively normal in expression level and pattern.
These findings identify mesodermal foxc1a/b as a direct upstream regulator of etsrp in angioblasts. This establishes a new molecular link in the process of mesoderm specification into angioblast.
内皮细胞是从中胚层生殖细胞层中特化的血管母细胞发育而来的。转录因子 etsrp/etv2 位于已知的血管母细胞发育的遗传层次结构的顶端。诱导 etsrp 表达和血管母细胞特化的转录事件尚不清楚。
我们生成了 etsrp:gfp 转基因斑马鱼，并利用它们来鉴定调控区域和转录因子，这些区域和转录因子对于从中胚层诱导 etsrp 表达和血管母细胞特化至关重要。
为了研究在胚胎发生过程中启动血管母细胞转录的机制，我们对斑马鱼 etsrp 基因座的启动子进行了分析。我们描述了三个增强子元件，当它们位于异源启动子前面时，足以表达内皮基因。删除所有 3 个调控区导致内皮表达从 etsrp 启动子几乎完全丢失。位于 etsrp 上游 2.3 kb 的一个增强子包含一个共识 FOX 结合位点，通过 EMSA 和 ChIP 在体内与 Foxc1a 和 Foxc1b 结合。使用 morpholino 对 foxc1a/b 进行联合敲低，导致早期发育阶段 etsrp 表达的显著下降，这可以通过定量逆转录聚合酶链反应和原位杂交来测量。还观察到原始红细胞基因 scl 和 gata1 的表达减少，而肾原基因 pax2a 的表达水平和模式相对正常。
这些发现确定了中胚层 foxc1a/b 是血管母细胞中 etsrp 的直接上游调节因子。这在中胚层特化为血管母细胞的过程中建立了一个新的分子联系。
