Regulation of Invasive Trophoblast Cell Lineage Development

项目摘要/摘要 子宫血管重塑发生在妊娠期间，以满足日益增长的胎儿营养需求。这 重塑包括将子宫螺旋动脉改造成供血的低阻力血管 胎儿。子宫螺旋动脉重建的中心是侵袭性滋养细胞，在人类被称为 绒毛外滋养细胞(EVT)。EVT发育受损导致胎儿状况不佳和不利 妊娠结局包括妊娠丢失、先兆子痫、宫内发育受限和早产。 我们确定了EVT谱系发育的一个关键和保守的调节因子，Achaete-scute Family Basic 螺旋-环-螺旋转录因子2(ASCL2)。人滋养层干细胞中ASCL2的耗竭 抑制EVT的形成。同样，体内ASCL2的全球耗尽会扰乱胎盘的发育，并导致 对大鼠的胚胎致死性。然而，ASCL2引导EVT谱系的分子机制 发展是未知的。我们已建立的HTS细胞系和用于产生突变大鼠模型的方案将 允许我们直接测试我们的中心假设，即ASCL2控制EVT谱系发育 胎盘摆放。研究ASCL2对EVT细胞表观基因组图谱的高阶作用 谱系，我们将确定ASCL2缺失如何改变DNA甲基化、染色质可及性和 利用全基因组亚硫酸氢盐测序(WGBS)，分析转座酶可及染色质的构象- 测序(ATAC-SEQ)和使用Hi-C捕获染色质(目标1A)。鉴定直接基因组 针对ASCL2，我们将在EVT细胞中进行染色质免疫沉淀测序(CHIP-SEQ)(AIM 1B)。ASCL2对滋养层细胞发育和侵袭的调节将在体内进行评估，使用我们已证实的 产生大鼠下胚层的技术(目标2 A)。检测ASCL2阳性滋养层细胞的发育 我们将对正常和疾病大鼠胎盘进行单细胞rna测序(scRNA-seq)和单细胞测序 ATAC-SEQ(目标2B)。拟议的研究计划将为候选人提供大量的实验工作。 对于独立出版物和R系列赠款的初步数据来说是必要的。候选人将利用 共同指导团队的专业知识以及堪萨斯大学医学中心和 儿童慈悲研究院培养专业发展技能。这些技能将是 通过实习生指导、数据演示和科学写作进行改进。在R00阶段期间 候选人将通过确定ASCL2的目标并调查他们的 通过创新的大鼠模型对侵袭性滋养细胞谱系发育的贡献。拟议的研究 项目是候选人长期职业目标的基础，目的是确定失调的螺旋 动脉重塑导致一系列疾病，从胎儿生长受限到先兆子痫。

Project Summary / Abstract Uterine vascular remodeling occurs during gestation to meet increasing fetal nutrient demands. This remodeling includes modification of the uterine spiral arteries into low resistance vessels for supplying blood to the fetus. Central to uterine spiral artery remodeling are invasive trophoblast cells, known in the human as extravillous trophoblast (EVT). Impaired EVT development leads to suboptimal fetal conditions and adverse pregnancy outcomes including pregnancy loss, preeclampsia, intrauterine growth restriction, and preterm birth. We identified a critical and conserved regulator of EVT lineage development, Achaete-Scute Family Basic Helix-Loop-Helix Transcription Factor 2 (ASCL2). Depletion of ASCL2 in human trophoblast stem (hTS) cells inhibits EVT formation. Similarly, global depletion of ASCL2 in vivo disrupts placental development and causes embryonic lethality in the rat. However, the molecular mechanisms by which ASCL2 directs EVT lineage development are unknown. Our established hTS cell lines and protocols for generating mutant rat models will allow us to directly test our central hypothesis that ASCL2 controls EVT lineage development during placentation. To investigate higher order actions of ASCL2 on the epigenomic landscape of the EVT cell lineage, we will identify how ASCL2 depletion alters DNA methylation, chromatin accessibility and conformation using whole genome bisulfite sequencing (WGBS), assay for transposase-accessible chromatin- sequencing (ATAC-seq), and chromatin capture using Hi-C, respectively (Aim 1A). To identify direct genomic targets of ASCL2 we will perform chromatin immunoprecipitation sequencing (ChIP-seq) in EVT cells (Aim 1B). ASCL2 regulation of trophoblast development and invasion will then be evaluated in vivo using our proven techniques to generate rat hypomorphs (Aim 2A). To examine ASCL2-positive trophoblast cell development in normal and diseased rat placentas we will conduct single cell RNA-sequencing (scRNA-seq) and single cell ATAC-seq (Aim 2B). The proposed research plan will provide the candidate with a body of experimental work necessary for independent publications and preliminary data for R-series grants. The candidate will utilize the expertise of the co-mentoring team as well as resources at the University of Kansas Medical Center and Children’s Mercy Research Institute for cultivation of professional development skills. These skills will be improved through trainee mentoring, data presentation, and scientific writing. During the R00 phase the candidate will develop independence from her mentors by identifying targets of ASCL2 and investigating their contributions to invasive trophoblast lineage development with innovative rat models. The proposed research project serves as the foundation for the candidate’s long-term career goal of identifying how dysregulated spiral artery remodeling leads to a spectrum of diseases ranging from fetal growth restriction to preeclampsia.