Mechanical Compression Causes Lung Hypoplasia in Congenital Diaphragmatic Hernia with GATA4 Genetic Variants

Congenital diaphragmatic hernia (CDH) is a common and severe structural malformation in which the high rate of morbidity and mortality is caused by lung hypoplasia and pulmonary hypertension. Genetic variants have been identified in 30% of CDH patients and are associated with increased morbidity and mortality but it is unclear how these variants impact lung and pulmonary vascular defect severity. Deletions of 8p23.1 account for 3-5% of cases and encompass GATA4, a transcription factor that directs gene expression throughout the developing embryo. CDH patients with GATA4 haploinsufficiency have high mortality and severe lung hypoplasia and pulmonary hypertension. Given this information, our aim was to characterize the role of GATA4 during lung and pulmonary vascular development. We generated mice with lung-specific deletion of Gata4 and found that GATA4 is not required during lung or pulmonary vascular development. However, mice with diaphragm-specific inactivation of Gata4 (Gata4 D-CKO) die after birth with abnormal diaphragm formation and lung hypoplasia thus we focused on the latest stage of embryonic development. We obtained whole lung RNA at embryonic day 18 (E18) at the latest stage prior to delivery due to early postnatal death of Gata4 D-CKO mice. We performed bulk RNA sequencing on control embryos (termed Gata4 D-control, Prx1Cre;Gata4F/+) and embryos with diaphragm-specific inactivation of Gata4 (termed Gata4 D-CKO, Prx1Cre;Gata4F/D). In this bulk RNA sequencing dataset are expression data from Gata4 D-control and D-CKO whole lungs at E18 implicating abnormal expression of genes involved in cell cycling and motor protein pathways. Mechanical compression of the embryonic lungs was also associated with increased phosphorylation of mechanosensory protein YAP1 resulting in decreased cell cycling. Our data suggest that the lung and pulmonary vascular phenotype of patients with CDH and GATA4 haploinsufficiency is due to mechanical compression. We performed bulk RNA sequencing on whole lung RNA collected at embryonic day 18 (E18) from 3 controls (Prx1Cre;Gata4F/+) and 3 Gata4 CKO (Prx1Cre;Gata4F/D) embryos. The diaphragm and lungs of Gata4 controls (Prx1Cre;Gata4F/+) develop normally. In contrast, Gata4 CKO (Prx1Cre;Gata4F/D) embryos lack Gata4 expression in the lateral plate mesoderm leading to deletion of Gata4 in the developing diaphragm and resulting in defects during diaphragm formation, lung hypoplasia, and decreased lung cell proliferation. This dataset was used to compare the gene expression in E18 Prx1Cre;Gata4F/+ control lungs with the expression in E18 Prx1Cre;Gata4F/D Gata4 CKO lungs and to investigate the mechanisms responsible for lung hypoplasia in Gata4 CKO embryos.