Gene regulatory networks in early lung epithelial cell fate decisions

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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.