Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada; Graduate School of Biomedical Sciences, Tokushima University, Tokushima, Japan.
Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Dev Biol. 2019 Nov 15;455(2):382-392. doi: 10.1016/j.ydbio.2019.07.008. Epub 2019 Jul 14.
Estrogen related receptor beta (Esrrb) is an orphan nuclear receptor that is required for self-renewal and pluripotency in mouse embryonic stem (ES) cells. However, in the early post-implantation mouse embryo, Esrrb is specifically expressed in the extraembryonic ectoderm (ExE) and plays a crucial role in trophoblast development. Previous studies showed that Esrrb is also required to maintain trophoblast stem (TS) cells, the in vitro stem cell model of the early trophoblast lineage. In order to identify regulatory targets of Esrrb in vivo, we performed microarray analysis of Esrrb-null versus wild-type post-implantation ExE, and identified 30 genes down-regulated in Esrrb-mutants. Among them is Bmp4, which is produced by the ExE and known to be critical for primordial germ cell (PGC) specification in vivo. We further identified an enhancer region bound by Esrrb at the Bmp4 locus by performing Esrrb ChIP-seq and luciferase reporter assay using TS cells. Finally, we established a knockout mouse line in which the enhancer region was deleted using CRISPR/Cas9 technology. Both Esrrb-null embryos and enhancer knockout embryos expressed lower levels of Bmp4 in the ExE, and had reduced numbers of PGCs. These results suggested that Esrrb functions as an upstream factor of Bmp4 in the ExE, regulating proper PGC development in mice.
雌激素相关受体β（Esrrb）是一种孤儿核受体，对于维持小鼠胚胎干细胞（ES 细胞）的自我更新和多能性是必需的。然而，在早期胚胎植入后，Esrrb 特异性地在胚胎外胚层（ExE）中表达，并在滋养层发育中发挥关键作用。先前的研究表明，Esrrb 对于维持滋养层干细胞（TS 细胞）也是必需的，TS 细胞是早期滋养层谱系的体外干细胞模型。为了鉴定 Esrrb 在体内的调控靶标，我们对 Esrrb 缺失型与野生型胚胎植入后 ExE 进行了微阵列分析，发现 30 个基因在 Esrrb 突变体中下调。其中包括 Bmp4，它由 ExE 产生，已知在体内对原始生殖细胞（PGC）的特化至关重要。我们进一步通过使用 TS 细胞进行 Esrrb ChIP-seq 和荧光素酶报告基因检测，鉴定了 Bmp4 基因座上由 Esrrb 结合的增强子区域。最后，我们使用 CRISPR/Cas9 技术建立了一个缺失增强子区域的敲除小鼠品系。Esrrb 缺失型胚胎和增强子敲除胚胎的 ExE 中 Bmp4 表达水平较低，PGC 数量减少。这些结果表明，Esrrb 在 ExE 中作为 Bmp4 的上游因子发挥作用，调节小鼠中 PG 的正常发育。
