C26-20 Foxf1 Drives Capillary Endothelial Cell Fate in the Developing Lung

Abstract Rationale The pulmonary microvasculature is composed of two transcriptionally and functionally distinct endothelial populations, CAP1 and CAP2 cells, that form the alveolar capillary network. CAP1 and CAP2 differ in their morphology, localization, and gene expression, but the transcriptional mechanisms that specify and maintain these endothelial fates remain poorly understood. The Forkhead transcription factor Foxf1 is known to regulate vascular development and is implicated in the neonatal disease alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). However, the role of Foxf1 in the specification of pulmonary capillary endothelial subtypes has not been defined. Methods To identify transcriptional regulators of endothelial subtype specification, we analyzed single cell accessibility (scATAC-seq) and transcriptomic (scRNA-seq) data from developing mouse lungs. Foxf1 expression and localization were validated by immunofluorescence across developmental stages. To test its function, Foxf1 was deleted from endothelial cells using a tamoxifen-inducible pan-endothelial driver (Cdh5-CreER), and lungs were analyzed at postnatal day (P)4 by histology, immunostaining, and single cell RNA-seq. Results scATAC-seq revealed preferential Foxf1 chromatin accessibility in CAP2 cells, and scRNA-seq showed broad endothelial expression with perinatal enrichment in CAP2. Endothelial deletion of Foxf1 disrupted lung architecture and reduced vascular density without loss of total endothelial cell number. Surprisingly, CAP2 and CAP1 marker genes were markedly decreased, while macrovascular genes were ectopically upregulated. scRNA-seq of mutant lungs demonstrated loss of mature CAP2 cells, transcriptional reprogramming of CAP1 cells, and emergence of stress-associated and macrovascular-like endothelial populations. Conclusion Foxf1 is a key transcriptional regulator of pulmonary capillary endothelial identity. It is required for the specification and maintenance of CAP2 cells and for preserving the capillary phenotype, potentially by repressing macrovascular gene expression. These findings identify Foxf1 as a central determinant of capillary endothelial cell fate during lung development and suggest a mechanistic link to human FOXF1-associated vascular disease. This abstract is funded by: NHLBI