Gene regulatory networks in early lung epithelial cell fate decisions

项目总结/摘要 该项目的长期目标是开发以干细胞为基础的自体疗法，用于治疗影响人类健康的疾病。 肺上皮使用干细胞治疗肺部疾病的不同潜在方法包括 增强内源性干细胞分化或干细胞体外分化为肺谱系 然后进行细胞移植。这两种方法都要求肺的鉴别和分化途径 干细胞或祖细胞是已知的并被充分表征。诱导多能干细胞的发现 （iPSCs）为再生医学开辟了新的可能性，因为这些细胞很容易获得， 伦理问题，并提供患者特异性治疗的可能性。在有效的 将iPSC技术应用于肺再生医学是对细胞命运的不完全理解， 在肺分化期间表征各种祖细胞类型的决策库。这是特别 与原始肺祖细胞相关，在肺特化时出现的罕见细胞， 产生所有类型的肺上皮细胞。我们假设这一障碍与信息的缺乏直接相关 在基因调控网络（GRN）上，即转录调控因子及其 相互作用，这定义了原始肺祖细胞的身份。理解和操纵GRN 原始肺祖细胞是该提议的总体目标。 在目标1中，将研究假定的原始肺命运的新调节剂。我们将绘制出他们在 体内肺特化和早期肺上皮分化。这些基因在肺发育中的功能， 将通过它们在前前肠内胚层内的缺失来评估。在目标2中，将进行类似的研究。 在人NKX 2 -1报告细胞系中使用CRISPR干扰体外进行。我们将引入扰动 在体外肺特化过程中推定的调节因子表达（过表达或敲低）， 评价所得肺祖细胞产生远端和近端肺上皮细胞的能力 类型在目标3中，将表征早期上皮肺发育的表观基因组景观。我们将 联合收割机将单细胞RNA-Seq与ATAC-Seq（一种揭示染色质可及性的技术）结合起来， 潜在的调节区域，并提供早期肺细胞命运决定的见解。ChIP-Seq还将用于 揭示了我们假定的调节因子和Wnt信号效应子β-连环蛋白的全局DNA结合位点。最后， 在目标3中，我们将使用计算方法来构建一个假定GRN的原理证明模型 控制肺原始身份和肺特化信号（例如Wnt）与核心的整合 监管方案。在我们的研究结束时，我们将定义肺原基GRN，并获得 在早期肺发育过程中重新配置这个GRN的见解。

PROJECT SUMMARY/ABSTRACT The long-term goal of this project is to develop stem-cell-based, autologous therapies for diseases affecting the lung epithelium. Different potential approaches for the use of stem cells for lung disease treatment include enhancement of endogenous stem cell differentiation or in vitro differentiation of stem cells to lung lineages followed by cell transplantation. Both approaches require that the identity and pathways of differentiation of lung stem or progenitor cells be known and well characterized. The discovery of induced pluripotent stem cells (iPSCs) has opened new possibilities for regenerative medicine as these cells are easy to derive, are not fraught with ethical issues and offer the possibility of patient-specific therapies. A major roadblock in the effective application of the iPSC technology to lung regenerative medicine is the incomplete understanding of the cell fate decision repertoire that characterizes various progenitor cell types during lung differentiation. This is particularly relevant to the primordial lung progenitors, the rare cells that appear at the moment of lung specification and give rise to all lung epithelial cell types. We hypothesize that this hurdle is directly related to paucity of information on the gene regulatory network (GRN), i.e. the specific combination of transcriptional regulators and their interactions, that defines the identity of primordial lung progenitors. Understanding and manipulating the GRN of the primordial lung progenitors is the overall objective of this proposal. In Aim 1 putative new regulators of primordial lung fate will be studied. We will map their expression during in vivo lung specification and early lung epithelial differentiation. The function of these genes in lung development, will be evaluated by their deletion within the anterior foregut endoderm. In Aim 2, similar studies will be undertaken in vitro using CRISPR interference in a human NKX2-1 reporter line. We will introduce perturbations of putative regulator expression (either overexpression or knock-down) during in vitro lung specification and evaluate the competency of the resulting lung progenitors to give rise to distal and proximal lung epithelial cell types. In Aim 3, the epigenomic landscape of early epithelial lung development will be characterized. We will combine single-cell RNA-Seq with ATAC-Seq, a technique that reveals chromatin accessibility, to identify the potential regulatory regions and provide insights in early lung cell-fate decisions. ChIP-Seq will also be used to reveal the global DNA binding sites of our putative regulators and the Wnt signaling effector, β-catenin. Finally, in Aim 3 we will use computational methods to construct a proof-of-principle model of the putative GRNs governing lung primordial identity and the integration of lung specification signals, such as Wnt, with the core regulatory program. At the conclusion of our studies, we will have defined the lung primordium GRN and gained insights in the reconfiguration of this GRN during early lung development.