Regulation of Invasive Trophoblast Cell Lineage Development

侵袭性滋养层细胞谱系发育的调控

• 批准号: 10525942
• 负责人: Kaela Margaret Varberg
• 金额: $ 13.07万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-02 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10525942
• 关键词: Abnormal placentation; Adult; Affect; BHLH Protein; Binding; Biological Assay; Cell Differentiation process; Cell Line; Cell Lineage; Cells; Cellular Assay; ChIP-seq; Child; Chromatin; Critical Pathways; DNA Methylation; Data; Development; Disease; Early Diagnosis; Embryo; Ensure; Environment; Epigenetic Process; Family; Fetal Growth Retardation; Fetus; Foundations; Genes; Genomics; Goals; Grant; Health; Hi-C; Homologous Gene; Human; Impairment; Kansas; Lead; Life; Mediating; Medical center; Mentors; Modeling; Modification; Molecular; Molecular Conformation; Morbidity - disease rate; Mothers; Nutrient; Phase; Physiological; Placenta; Placentation; Pre-Eclampsia; Predisposition; Pregnancy; Pregnancy Complications; Pregnancy loss; Premature Birth; Protocols documentation; Publications; Rattus; Regulation; Regulatory Element; Research; Research Institute; Research Project Grants; Resistance; Resources; Series; Signal Pathway; Spiral Artery of the Endometrium; Techniques; Testing; Training; Transposase; Universities; Uterus; Vascular blood supply; Vascular remodeling; Work; Writing; adverse outcome; adverse pregnancy outcome; bisulfite sequencing; career; chromosome conformation capture; early detection biomarkers; effective intervention; epigenomics; fetal; gene regulatory network; improved; in vivo; innovation; knock-down; lentiviral-mediated; methylation pattern; mortality; mutant; novel; postnatal; pregnancy disorder; progenitor; response; single-cell RNA sequencing; skill acquisition; skills; small hairpin RNA; transcription factor; trophoblast; trophoblast stem cell; whole genome

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.

项目总结/摘要 子宫血管重塑发生在妊娠期，以满足胎儿营养需求的增加。这 重塑包括将子宫螺旋动脉改变为低阻力血管， 胎儿子宫螺旋动脉重塑的中心是侵袭性滋养层细胞，在人类中称为 绒毛外滋养层细胞（EVT）。受损的EVT发育导致次优的胎儿状况和不利的 妊娠结局包括流产、先兆子痫、宫内生长受限和早产。 我们确定了EVT谱系发育的关键和保守的调节因子，Achaete-Scute家族基础 螺旋环抑制转录因子2（ASCL 2）。人滋养层干（hTS）细胞中ASCL 2的消耗 抑制EVT形成。类似地，体内ASCL 2的整体消耗破坏胎盘发育并导致 大鼠胚胎致死率然而，ASCL 2指导EVT谱系的分子机制 发展未知。我们建立的hTS细胞系和产生突变大鼠模型的方案将 这使得我们能够直接测试我们的中心假设，即ASCL 2控制EVT谱系的发展， 胎座式研究ASCL 2对EVT细胞表观基因组景观的高级作用 谱系，我们将确定ASCL 2缺失如何改变DNA甲基化，染色质可及性， 使用全基因组亚硫酸氢盐测序（WGBS）进行构象分析， 测序（ATAC-seq）和使用Hi-C的染色质捕获（Aim 1A）。为了识别直接基因组 我们将在EVT细胞中进行染色质免疫沉淀测序（ChIP-seq）（Aim 1B）。ASCL 2对滋养层发育和侵袭的调节将在体内进行评估，使用我们已经证实的方法。 技术来产生大鼠亚型（Aim 2A）。为了检查ASCL 2阳性滋养层细胞的发育， 正常和患病大鼠胎盘，我们将进行单细胞RNA测序（scRNA-seq）和单细胞 ATAC-seq（目标2B）。拟议的研究计划将为候选人提供一个实验工作的主体 独立出版物和R系列赠款的初步数据所必需的。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的资源， 儿童慈善研究所，培养专业发展技能。这些技能将 通过培训生指导、数据展示和科学写作得到改进。在R 00阶段， 候选人将通过确定ASCL 2的目标并调查他们的目标， 通过创新的大鼠模型，为侵入性滋养层细胞谱系的发展做出了贡献。拟议研究 项目作为候选人的长期职业目标的基础，确定如何失调的螺旋 动脉重塑导致从胎儿生长受限到先兆子痫的一系列疾病。