Foxf1 is required for the specification and maintenance of pulmonary capillary identity.

The pulmonary microvasculature consists of two transcriptionally distinct capillary (CAP) endothelial populations, CAP1 and CAP2 cells, that form the alveolar capillary network. While CAP1 and CAP2 are functionally distinct, the transcriptional mechanisms that specify and maintain these endothelial fates remain poorly understood. The Forkhead transcription factor Foxf1 regulates vascular development and is implicated in the neonatal disease alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). However, its role in the specification of pulmonary endothelial subtypes has not been defined. Using single cell ATAC-sequencing and RNA-sequencing of developing mouse lung endothelial cells (ECs), we found that Foxf1 is broadly expressed across pulmonary ECs but exhibits preferential chromatin accessibility in CAP2 ECs. Endothelial deletion of Foxf1 impaired CAP2 specification and disrupted CAP1 identity, unexpectedly leading to the emergence of a mutant CAP1-like population with a macrovascular transcriptional signature. Adult endothelial deletion of Foxf1 similarly resulted in loss of capillary identity, demonstrating that Foxf1 is required for capillary fate maintenance. Mutant CAP1 ECs upregulated matrix remodeling genes and exhibited altered communication with neighboring mesenchymal populations, suggesting a potential role in disease development. Consistent with these findings, this mutant CAP1 transcriptional signature was present in human lungs with ACDMPV. This work identifies Foxf1 as a key transcriptional regulator for the specification and active maintenance of pulmonary capillary EC fate across the lifespan, and positions capillary identity loss as a driver of vascular remodeling.