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.

项目概要/摘要子宫血管重塑在妊娠期间发生，以满足胎儿不断增加的营养需求。重塑包括将子宫螺旋动脉改造为低阻力血管，为子宫供血子宫螺旋动脉重塑的核心是侵袭性滋养层细胞，在人类中被称为绒毛外滋养层 (EVT) 发育受损会导致胎儿状况不佳和不利。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 谱系发育的关键且保守的调节因子 Achaete-Scute Family Basic 人类滋养层干细胞 (hTS) 中的螺旋-环-螺旋转录因子 2 (ASCL2) 耗尽。类似地，体内 ASCL2 的整体耗尽会破坏胎盘发育并导致 EVT 形成。然而，ASCL2 指导 EVT 谱系的分子机制。我们建立的 hTS 细胞系和用于生成突变大鼠模型的方案尚不清楚。让我们能够直接检验我们的中心假设，即 ASCL2 在 EVT 谱系发育过程中控制着研究 ASCL2 对 EVT 细胞表观基因组景观的高级作用。谱系，我们将确定 ASCL2 耗竭如何改变 DNA 甲基化、染色质可及性和使用全基因组亚硫酸氢盐测序 (WGBS) 进行构象分析，转座酶可及染色质分析分别使用 Hi-C 测序 (ATAC-seq) 和染色质捕获 (目标 1A) 来鉴定直接基因组。 ASCL2 的靶标，我们将在 EVT 细胞中进行染色质免疫沉淀测序 (ChIP-seq)（目的然后将使用我们经过验证的体内评估 ASCL2 对滋养层发育和侵袭的调节。产生大鼠次形体的技术（目标 2A）检查 ASCL2 阳性滋养层细胞的发育。我们将对正常和患病的大鼠胎盘进行单细胞 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大量实验工作。独立出版物和 R 系列补助金的初步数据所必需的。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和儿童慈悲研究所将培养这些技能的专业发展。在 R00 阶段，通过学员指导、数据演示和科学写作进行了改进。候选人将通过确定 ASCL2 的目标并调查他们的目标来培养独立于导师的能力拟议的研究对创新大鼠模型的侵袭性滋养层谱系发育的贡献。该项目是候选人长期职业目标的基础，即确定失调的螺旋如何动脉重塑会导致一系列疾病，从胎儿生长受限到先兆子痫。