ASCL2 reciprocally controls key trophoblast lineage decisions during hemochorial placenta development

Invasive trophoblast cells are critical to spiral artery remodeling in hemochorial placentation. Insufficient trophoblast invasion and vascular remodeling can lead to pregnancy disorders including preeclampsia, preterm birth, and intrauterine growth restriction. Previous studies in the mouse identified achaete-scute homolog 2 (ASCL2) as essential to extraembryonic development. We hypothesized that ASCL2 is a critical and conserved regulator of invasive trophoblast lineage development. In contrast to the mouse, the rat possesses deep intrauterine trophoblast cell invasion and spiral artery remodeling similar to human placentation. In this report, we investigated invasive/extravillous trophoblast (EVT) cell differentiation using human trophoblast stem (TS) cells and a loss-of-function mutant Ascl2 rat model. ASCL2 transcripts are expressed in the EVT column and junctional zone, which represent tissue sources of invasive trophoblast progenitor cells within human and rat placentation sites, respectively. Differentiation of human TS cells into EVT cells resulted in significant upregulation of ASCL2 and several other transcripts indicative of EVT cell differentiation. Disruption of ASCL2 impaired EVT cell differentiation as indicated by cell morphology and transcript profiles. RNA sequencing analysis of ASCL2-deficient trophoblast cells identified both downregulation of EVT cell-associated transcripts and upregulation of syncytiotrophoblast-associated transcripts, indicative of dual activating and repressing functions. ASCL2 deficiency in the rat impacted placental morphogenesis resulting in junctional zone dysgenesis and failed intrauterine trophoblast cell invasion. ASCL2 acts as a critical and conserved regulator of invasive trophoblast cell lineage development and a species-specific modulator of the syncytiotrophoblast lineage.侵袭性滋养层细胞（invasive trophoblast cells）在血绒毛膜型胎盘形成（hemochorial placentation）过程中对螺旋动脉重塑（spiral artery remodeling）发挥关键作用。滋养层侵袭不足与血管重塑异常可引发子痫前期（preeclampsia）、早产（preterm birth）及宫内生长受限（intrauterine growth restriction）等妊娠疾病。既往小鼠研究证实，achaete-scute同源盒2（achaete-scute homolog 2, ASCL2）对胚外发育至关重要。本研究提出假设：ASCL2是侵袭性滋养层细胞谱系发育的关键保守调控因子。与小鼠不同，大鼠具备与人类胎盘形成相似的深层宫内滋养层细胞侵袭及螺旋动脉重塑过程。本研究利用人类滋养层干细胞（trophoblast stem, TS）与Ascl2功能丧失突变大鼠模型，探究了侵袭性/绒毛外滋养层细胞（invasive/extravillous trophoblast, EVT）的分化过程。ASCL2转录本分别表达于EVT柱与胎盘交界区，二者分别为人类与大鼠胎盘形成部位中侵袭性滋养层祖细胞的组织来源。将人类TS细胞诱导分化为EVT细胞后，ASCL2及其他数个标志EVT细胞分化的转录本表达量显著上调。通过细胞形态与转录本谱分析可见，ASCL2功能缺失会损害EVT细胞的分化进程。对ASCL2缺陷型滋养层细胞的RNA测序分析显示，EVT细胞相关转录本表达下调的同时，合体滋养层细胞（syncytiotrophoblast）相关转录本表达上调，提示ASCL2兼具激活与抑制双重调控功能。大鼠体内ASCL2缺失会影响胎盘形态发生，导致胎盘交界区发育异常及宫内滋养层细胞侵袭失败。ASCL2作为侵袭性滋养层细胞谱系发育的关键保守调控因子，同时也是合体滋养层细胞谱系的物种特异性调控因子。