Regulation of Invasive Trophoblast Cell Lineage Development

侵袭性滋养层细胞谱系发育的调控

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要 /摘要子宫血管重塑在妊娠期间发生，以满足增加的胎儿营养需求。这重塑包括将子宫螺旋动脉的修饰为低电阻容器，以提供血液胎儿。子宫螺旋动脉重塑的中心是侵入性滋养细胞细胞，在人类中被称为额外的滋养细胞（EVT）。 EVT发育受损会导致胎儿的次要条件和逆境怀孕结局，包括怀孕丧失，先兆子痫，内脏生长限制和早产。我们确定了EVT谱系发展的关键和组成的调节剂，Achaete-Scute家族基本螺旋环螺旋转录因子2（ASCL2）。 ASCL2在人滋养细胞茎（HTS）细胞中的耗竭抑制EVT形成。同样，ASCL2体内的全球耗竭会破坏占地的发展和原因大鼠的胚胎致死性。但是，ASCL2指导EVT谱系的分子机制发展是未知的。我们已建立的HTS细胞系和用于生成突变大鼠模型的协议将允许我们直接检验我们的中心假设，即ASCL2控制EVT谱系的发展胎盘。研究ASCL2对EVT细胞的表观基因组景观的高阶动作血统，我们将确定ASCL2部署如何改变DNA甲基化，染色质的可及性和使用全基因组硫酸盐测序（WGB）的构象，转座酶可访问的染色质的测定分别使用HI-C进行测序（ATAC-SEQ）和染色质捕获（AIM 1A）。识别直接基因组 ASCL2的靶标我们将在EVT细胞中执行染色质免疫沉淀测序（CHIP-SEQ）（AIM 1b）。然后，将使用我们的验证产生大鼠肌电的技术（AIM 2A）。检查ASCL2阳性滋养细胞的开发正常和解散的大鼠斑点我们将进行单细胞RNA-sequest（SCRNA-SEQ）和单细胞 ATAC-SEQ（AIM 2B）。拟议的研究计划将为候选人提供一系列实验性工作对于R系列赠款的独立出版物和初步数据所必需的。候选人将利用堪萨斯大学医学中心和资源的专业知识以及资源儿童怜悯研究所的专业发展技能研究所。这些技能将是通过实习生的指导，数据演示和科学写作改进。在R00阶段候选人将通过识别ASCL2的目标并调查他们的导师来发展独立通过创新的大鼠模型对侵入性滋养细胞谱系发展的贡献。拟议的研究项目是候选人长期职业目标的基础动脉重塑导致从胎儿生长限制到先兆子痫的各种疾病。