Resolving the heterogeneity of diaphragmatic mesenchyme: a novel mouse model of congenital diaphragmatic hernia

Abstract

Congenital diaphragmatic hernia (CDH) is a relatively common developmental defect with considerable mortality and morbidity. Diaphragm formation is a complex process, involving several cell types, each with different developmental origins. Due to this complexity, the aetiology of CDH is not well understood. The pleuroperitoneal folds (PPFs) and the post hepatic mesenchymal plate (PHMP) are transient structures that are essential during diaphragm development. Using several mouse models including lineage tracing, we demonstrate the heterogeneous nature of the cells that make up the PPFs. The conditional deletion of Wt1 (Wilms Tumour gene) in the non-muscle mesenchyme of the PPFs results in CDH. We show that the fusion of the PPFs and the PHMP to form a continuous band of tissue involves migration of cells from both sources. The PPFs of mutant mice fail to fuse with the PHMP and exhibit increased RALDH2 expression. However, no changes in the expression of genes implicated in epithelial-to-mesenchymal transition (EMT) are observed. Additionally, the mutant PPFs lack migrating myoblasts and muscle connective tissue fibroblasts (TCF4+/GATA4+), suggesting possible interactions between these cell types. Our study demonstrates the importance of the non-muscle mesenchyme in diaphragm development. Author SummaryCongenital diaphragmatic hernia (CDH) is a frequent developmental defect and it remains one of the most difficult problems of perinatology. The defect can be repaired by surgery but it is often associated with complications and total mortalities are still high (50-60%). The causes of CDH are largely unknown. Body cavity formation is a carefully regulated process, and diaphragm formation defines the thoracic and abdominal cavities. Connective tissue and muscle fibres are the known major players involved in diaphragm formation. Our current study emphasises another player, the mesenchymal cells. We manipulate the expression of an important developmental regulator, Wt1 (Wilms tumour gene), in mesenchymal cells in a tissue specific manner using transgenic mouse models. We found that mutant mice can survive till birth, develop diaphragmatic hernia, and die shortly after birth. Ablating Wt1 in the mesenchymal cells leads to ceased movement, and the failure to form a continuous band of tissue. In our model, disruption of mesenchymal cell movement leads to the attenuation of migration of connective tissue fibroblasts and myoblasts, suggesting possible interactions between these cell types.