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 TGFß signaling in aCap (TGFB2)-AT1
(TGFBR2/3) interaction. Therefore, we hypothesized that FOXF1 regulates distinct pathways in aCap and gCap
cells, and FOXF1-dependent TGFß2 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.