Chd1 is essential in the endothelial lineage for the emergence of definitive hematopoietic stem/progenitor cells

Lineage specification during development involves reprogramming of chromatin states, but little is known about how this is regulated in vivo. We previously showed that the chromatin remodeler Chd1 regulates transcriptional output and self-renewal of mouse embryonic stem cells, and is essential for epiblast development. These results raise the question of whether Chd1 regulates the development of other progenitor populations. Here we report that endothelial-specific deletion of Chd1 using Tie2-Cre leads to embryonic lethality by E15.5. Development of the vasculature and of primitive hematopoiesis appears to occur normally in the mutants. However, mutant embryos show signs of anemia as early as E11.5, are depleted of definitive hematopoietic stem /progenitor cells, and display a complete failure of fetal liver erythropoiesis. While mutants at E10.5 appear morphologically normal and can develop hemogenic clusters in the dorsal aorta, the E10.5 mutant endothelium fails to activate a transcriptional program associated with hematopoiesis. This transcriptional program may serve as a resource for the identification of novel markers or regulators of definitive hematopoiesis. Finally, hematopoietic-specific Chd1 deletion using Vav-Cre yields no apparent defects during development or adulthood. These results suggest that Chd1 regulates chromatin-remodeling events critical for a specific developmental window during the transition of endothelial cells to definitive blood progenitors. Analysis of CD31+ tdTomato+ cells sorted from E10.5 whole embryos with an endothelial-specific deletion of Chd1, using 4 biological replicates of 2 genotypes (CreHet controls vs mutants).发育过程中的细胞谱系特化（lineage specification）涉及染色质状态（chromatin states）的重编程，但目前对其体内（in vivo）调控机制的研究仍较为匮乏。此前我们的研究表明，染色质重塑因子（chromatin remodeler）Chd1可调控小鼠胚胎干细胞（mouse embryonic stem cells）的转录输出与自我更新能力，并对上胚层（epiblast）发育不可或缺。上述研究结果提出了一个科学问题：Chd1是否调控其他祖细胞群的发育。本研究报道，利用Tie2-Cre重组酶系统（Tie2-Cre）介导的内皮细胞特异性Chd1敲除会导致小鼠在胚胎第15.5天（E15.5）时出现胚胎致死。突变体小鼠的血管系统（vasculature）与原始造血（primitive hematopoiesis）过程看似发育正常。然而，突变体胚胎早在胚胎第11.5天（E11.5）便出现贫血迹象，其体内定型造血干/祖细胞（definitive hematopoietic stem/progenitor cells）数量显著减少，且胎肝红细胞生成（fetal liver erythropoiesis）完全受阻。尽管胚胎第10.5天（E10.5）的突变体胚胎在形态上并无异常，且可在背主动脉（dorsal aorta）中形成生血簇（hemogenic clusters），但突变体的内皮细胞无法激活与造血相关的转录程序（transcriptional program）。该转录程序或可作为鉴定定型造血新型标志物或调控因子的研究资源。最后，利用Vav-Cre重组酶系统（Vav-Cre）介导的造血细胞特异性Chd1敲除，并未在小鼠发育阶段或成年阶段（adulthood）引发明显的发育缺陷。上述研究结果表明，Chd1可调控内皮细胞向定型血液祖细胞转化过程中特定发育窗口期（developmental window）内的关键染色质重塑事件。本研究采用两组基因型（CreHet controls）与突变体）的4次生物学重复（biological replicates），对从胚胎第10.5天（E10.5）全胚胎中分选得到的、内皮细胞特异性Chd1敲除的CD31+ tdTomato+（CD31分子与tdTomato荧光蛋白）阳性细胞进行了分析。