A Single Trophoblast Layer Acts as the Gatekeeper at the Endothelial-Hematopoietic Crossroad in the Placenta

During embryonic development the placental vasculature acts as a major hematopoietic niche, where endolthelial to hematopoietic transition ensures emergence of hematopoietic stem cells (HSCs). However, the molecular mechanisms that regulate the placental hematoendothelial niche are poorly understood. Using a parietal trophoblast giant cell (TGC)-specific knockout mouse model and single-cell RNA-sequencing, we show that the paracrine factors secreted by this single layer of TGCs are critical in the development of this niche. Disruptions in the TGC specific paracrine signaling leads to the loss of HSC population and the concomitant expansion of a KDR+/DLL4+/PROM1+ hematoendothelial cell-population in the placenta. Combining single-cell transcriptomics and receptor-ligand pair analyses, we also define the parietal TGC-dependent paracrine signaling network and identify Integrin signaling as a fundamental regulator of this process. Our study elucidates novel mechanisms by which non autonomous signaling from the primary parietal TGCs maintains the delicate placental hematopoietic-angiogenic balance and ensures embryonic and extraembryonic development. Overall design: Gata2f/f;Gata3f/f; Pl1Cre +/- males were crossed to Gata2f/f;Gata3f/f females and the embryos were analyzed at E13.5. Single cell RNA sequencing of the placental samples were generated by deep sequencing using the 10x Genomics Chromium Single Cell Gene Expression Solution (10xgenomics.com).

在胚胎发育进程中，胎盘血管系统是核心造血微环境（hematopoietic niche），内皮细胞向造血细胞的转化（endothelial to hematopoietic transition）可保障造血干细胞（hematopoietic stem cells, HSCs）的生成。然而，当前调控胎盘造血内皮微环境的分子机制仍知之甚少。本研究借助壁滋养层巨细胞（parietal trophoblast giant cell, TGC）特异性敲除小鼠模型与单细胞RNA测序（single-cell RNA-sequencing）技术，证实该单层TGC分泌的旁分泌因子对该微环境的发育至关重要。TGC特异性旁分泌信号通路的紊乱会导致胎盘中造血干细胞群体的丢失，并伴随KDR+/DLL4+/PROM1+造血内皮细胞群体的异常扩增。结合单细胞转录组学与受体-配体配对分析，本研究还明确了依赖壁TGC的旁分泌信号网络，并鉴定出整合素信号通路（Integrin signaling）为该过程的核心调控因子。本研究阐明了原发性壁TGC介导的非自主信号如何维持胎盘造血-血管生成的精细平衡，进而保障胚胎及胚外正常发育的全新机制。
实验整体设计：将基因型为Gata2f/f;Gata3f/f; Pl1Cre +/-的雄性小鼠与Gata2f/f;Gata3f/f雌性小鼠交配，于胚胎发育第13.5天（E13.5）对胚胎进行分析。胎盘样本的单细胞RNA测序采用10x Genomics Chromium单细胞基因表达解决方案（10xgenomics.com）通过深度测序完成。