Elucidating the FOXF1 gene regulatory network in human alveologenesis

项目总结 这项拟议研究的首要目标是揭示FOXF1介导的分子机制 多种肺泡内皮细胞和上皮细胞亚型的时空协调性。FOXF1(叉头) Box F1)是一种转录因子，在肺血管发育、动态平衡和修复中发挥关键作用 受伤后。FOXF1杂合性缺失或点突变与肺泡毛细血管发育不良相关 肺静脉错位(ACD/MPV)，一种无法治愈的致命的肺部发育障碍。虽然 Foxf1突变小鼠模型是为研究ACD而建立的，在基因签名方面存在一定的变异 小鼠与人的肺泡化EC亚型和发育时序的比较，限制了其在 了解ACD的人类病因学。因此，FOXF1如何调控下游基因网络仍不清楚 促进肺泡内皮细胞亚型的形成，维持正常的肺泡内皮细胞 人类肺泡发育过程中的串扰。我们的初步研究表明，FOXF1突变导致不同的 两种新发现的肺泡内皮细胞亚型--肺泡内皮细胞(ACAP)和肺泡内皮细胞(ACAP)的细胞群和转录变化 普通毛细血管(GCap)，基于单核RNA测序，在对照和ACD人肺组织中。更多 有趣的是，尽管FOXF1在上皮细胞系中不表达，但肺泡I型(AT1)史诗种群 FOXF1中损伤相关的瞬时上皮祖细胞(DATPs)显著减少和增加 变种人的肺。配体-受体结合分析显示ACAP(TGFB2)-AT1细胞转化生长因子β信号受阻 (TGFBR2/3)相互作用。因此，我们假设FOXF1在ACAP和GCap中调节不同的通路 ACAP分泌依赖FOXF1的转化生长因子β-2对维持AT1的特性和功能至关重要。我们 建议用以下具体目标来检验这一假说：目标1：确定FOXF1在调节中的作用 肺泡内皮细胞亚型的分化及功能。目的2：确定ACAP细胞对维持的影响 AT1的身份和功能。目的3：通过IPSC衍生的有机化合物揭示FOXF1介导的早期肺发育 系统。对于目标3，我们建立了对照和ACD诱导的三维血管化肺泡器质模型 多能干细胞(IPSCs)，研究FOXF1基因调控和动态EC-EPIC相互作用 多个肺发育阶段。这三个目标的完成将提供对 FOXF1驱动的人肺细胞间信号通路的时空格局 为重建正常的肺泡-毛细血管界面提供了新的治疗靶点。 肺部发育障碍。

PROJECT SUMMARY The overarching goal of the proposed study is to uncover the molecular mechanism underlying FOXF1-mediated temporal and spatial coordination of multiple alveolar endothelial and epithelial cell subtypes. FOXF1 (Forkhead Box F1) is a transcription factor that plays a pivotal role in lung vascular development, homeostasis, and repair after injury. Heterozygous deletions or point mutations in FOXF1 are associated with alveolar-capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), a lethal lung developmental disorder with no cure. Although the Foxf1 mutant mouse model has been created to study ACD, there are certain variations in gene signatures of EC subtypes and developmental timing of the alveolarization in mouse vs. human, limiting its application in understanding ACD human etiology. Thus, it remains unclear how FOXF1 regulates downstream gene networks to drive the formation of alveolar endothelial cell (EC) subtypes and maintain normal EC-epithelial cell (EpiC) crosstalk during alveologenesis in human. Our preliminary studies showed that FOXF1 mutations lead to distinct cell population and transcriptomic changes in two newly identified alveolar EC subtypes- aerocyte (aCap) and general capillary (gCap), based on single nuclei RNA-sequencing in control and ACD human lung tissues. More interestingly, although FOXF1 does not express in epithelial lineage, alveolar type 1 (AT1) EpiC population was significantly reduced, and damage-associated transient epithelial progenitors (DATPs) were increased in FOXF1 mutant human lung. Ligand-receptor binding analysis revealed disrupted TGFβ signaling in aCap (TGFB2)-AT1 (TGFBR2/3) interaction. Therefore, we hypothesized that FOXF1 regulates distinct pathways in aCap and gCap cells, and FOXF1-dependent TGFβ2 secretion by aCap is critical in maintaining AT1 identity and function. We propose to test this hypothesis with the following specific aims: Aim 1: Determine the role of FOXF1 in regulating differentiation and function of alveolar EC subtypes. Aim 2: Determine the impact of aCap cells on maintaining AT1 identity and function. Aim 3: Uncover FOXF1 mediated early lung development using iPSC derived organoid systems. For Aim 3, we established 3D vascularized alveolar organoid models from control and ACD induced pluripotent stem cells (iPSCs), to study FOXF1 gene regulation and dynamic EC-EpiC interactions across multiple lung developmental stages. Completion of the three aims will provide granular mechanistic insights into the spatial and temporal patterning of intercellular signaling pathways driven by FOXF1 in human lung alveologenesis, and provide new therapeutic targets to re-establish normal alveolar-capillary interface in various lung developmental disorders.