Long-range enhancers modulate Foxf1 transcription in blood vessels of pulmonary vascular network

Intimate crosstalk occurs between the pulmonary epithelium and the vascular network during lung development. The transcription factor forkhead box f1 (Foxf1) is expressed in the lung mesenchyme and plays an indispensable role in pulmonary angiogenesis. Sonic hedgehog (Shh), a signalling molecule, is expressed in lung epithelium and is required to establish proper angiogenesis. It has been suggested that Foxf1, a downstream target of the Shh signalling pathway, mediates interaction between angiogenesis and the epithelium in lung. However, there has been no clear evidence showing the mechanism how Foxf1 is regulated by Shh signalling pathway during lung development. In this study, we investigated the lung-specific enhancers of Foxf1 and the Gli binding on the enhancers. At first, we found three evolutionarily conserved Foxf1 enhancers, two of which were long-range enhancers. Of the long-range enhancers, one demonstrated tissue-specific activity in the proximal and distal pulmonary blood vessels, while the other one demonstrated activity only in distal blood vessels. At analogous positions in human, these long-range enhancers were included in a regulatory region that was reportedly repeatedly deleted in alveolar capillary dysplasia with misalignment of pulmonary vein patients, which indicates the importance of these enhancers in pulmonary blood vessel formation. We also determined that Gli increased the activity of one of these long-range enhancers, which was specific to distal blood vessel, suggesting that Shh regulates Foxf1 transcription in pulmonary distal blood vessel formation.