Defective mesothelium and limited physical space are drivers of dysregulated lung development in a genetic model of congenital diaphragmatic hernia

Abstract

Congenital diaphragmatic hernia (CDH) is a developmental disorder associated with diaphragm defects and lung hypoplasia. The etiology of CDH is complex and its clinical presentation is variable. We investigated the role of the pulmonary mesothelium in dysregulated lung growth noted in the Wt1 knockout mouse model of CDH. Loss of WT1 leads to intrafetal effusions, altered lung growth, and branching defects prior to normal closure of the diaphragm. We found significant differences in key genes; however, when Wt1 null lungs were cultured ex vivo, growth and branching were indistinguishable from wild-type littermates. Micro-CT imaging of embryos in situ within the uterus revealed a near absence of space in the dorsal chest cavity, but no difference in total chest cavity volume in Wt1 null embryos, indicating a redistribution of pleural space. The altered space and normal ex vivo growth suggest that physical constraints are contributing to the CDH lung phenotype observed in this mouse model. These studies emphasize the importance of examining the mesothelium and chest cavity as a whole, rather than focusing on single organs in isolation to understand early CDH etiology.

Cellular Origin(s) of Congenital Diaphragmatic Hernia

“…A recent study reported similar findings and also reported an aberrant lung branching architecture already before closure of the diaphragm, when WT1 is expressed (32). When Wt1 −/− lungs were cultured ex vivo, lung branching was normal and any hypoplasia that had originated in vivo, was restored within 24 h ex vivo (32). Additional analyses showed that the space in the chest cavity-that is usually present for the lungs to grow-was nearly absent, explaining why culturing the lungs ex vivo without physical constraints recovered branching (32).…”

“…Cano et al (24) showed that homozygous Wt1 knockout (Wt1 −/− ) mice had a CDH-like phenotype, abnormally fused lung lobes and reduced immunoreactivity for FGF9 in the pulmonary mesenchyme and mesothelium. A recent study reported similar findings and also reported an aberrant lung branching architecture already before closure of the diaphragm, when WT1 is expressed (32). When Wt1 −/− lungs were cultured ex vivo, lung branching was normal and any hypoplasia that had originated in vivo, was restored within 24 h ex vivo (32).…”

“…To mimic limited chest space and compression, lung organoids have been subjected to mechanical pressure, which altered their development (44). These results show that extrinsic factors play a significant role in CDH pathogenesis and the influence of limited space and compression should be studied further, since Wt1 knock-out lungs still had the capacity to develop normally ex vivo, which is promising for potential treatment strategies (32).…”

“…All can contribute to a CDH phenotype, but also extrinsic factors play a role. Because pulmonary defects and alterations in the space of the chest cavity occurred prior to diaphragm closure and may even be the cause of a diaphragm defect ( 32 ), it is interesting to study the space in the chest cavity in other CDH models and potentially in human fetuses to serve as an early predictor for CDH. To mimic limited chest space and compression, lung organoids have been subjected to mechanical pressure, which altered their development ( 44 ).…”

“…When Wt1 −/− lungs were cultured ex vivo , lung branching was normal and any hypoplasia that had originated in vivo , was restored within 24 h ex viv o ( 32 ). Additional analyses showed that the space in the chest cavity—that is usually present for the lungs to grow—was nearly absent, explaining why culturing the lungs ex vivo without physical constraints recovered branching ( 32 ). Aberrant WT1 expression in the lung mesothelium results in defective lung development and CDH as result of limited space in the chest cavity and potentially by defective signaling and migration of mesothelial cells.…”