Elucidating the FOXF1 gene regulatory network in human alveologenesis

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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.