Nephronectin is required to maintain right lung lobar separation during embryonic development

Nephronectin (NPNT) is a basement membrane (BM) protein and high-affinity ligand of integrin α8β1 that is required for kidney morphogenesis in mice. In the lung, NPNT also localizes to BMs, but its potential role in pulmonary development has not been investigated. Mice with a floxed Npnt allele were used to generate global knockouts (KOs). Staged embryos were obtained by timed matings of heterozygotes and lungs were isolated for analysis. Although primary and secondary lung bud formation was normal in KO embryos, fusion of right lung lobes, primarily the medial and caudal, was first detected at E13.5 and persisted into adulthood. The lung parenchyma of KO mice was indistinguishable from wild-type (WT) and lobe fusion did not alter respiratory mechanics in adult KO mice. Interrogation of an existing single-cell RNA-seq atlas of embryonic and adult mouse lungs identified Npnt transcripts in mesothelial cells at E12.5 and into the early postnatal period, but not in adult lungs. KO embryonic lungs exhibited increased expression of laminin α5 and deposition of collagen IV in the mesothelial BM, accompanied by abnormalities in collagen fibrils in the adjacent stroma. Cranial and accessory lobes extracted from KO embryonic lungs fused ex vivo when cultured in juxtaposition, with the area of fusion showing loss of the mesothelial marker Wilms tumor 1. Because a similar pattern of lobe fusion was previously observed in integrin α8 KO embryos, our results suggest that NPNT signaling through integrin α8, likely in the visceral pleura, maintains right lung lobe separation during embryogenesis.