Funding outcome

Background: Mouse models are an important tool for studying the pathogenesis and etiology of congenital diaphragmatic hernia (CDH). CDH has a prevalence of 1 in 3,000 live births, which poses a significant burden on the healthcare systems worldwide. Current treatments can facilitate breathing, but cannot address the root cause of morbidity and mortality: the developmental malformation of the diaphragm and lungs. The etiology of CDH is unknown; however, current research highlights the importance of the mesenchyme derived from the embryonic pleuroperitoneal folds (PPF) and retinoic acid (RA) signaling in this tissue during development. A better understanding of RA signaling in the PPF offers the possibility of preventing or rescuing the diaphragmatic defect in utero. Recently, we described a novel transgenic mouse model of CDH with a 100% penetrance of diaphragm defects (Prrx1Cre:Rardn; Garcia Rivas et al., 2025). In accord with the Retinoid Hypothesis of CDH, this model works by specifically blocking retinoic acid signaling in the mesenchyme of the developing diaphragm. Aims: To better understand RA’s role in the developing diaphragm, we have leveraged Prrx1Cre:Rardn mice. This study included three parts: 1) assessing the phenotypic differences in diaphragm and lung defects in Prrx1Cre:Rardn mice, 2) analyzing potential sex differences in the presentation of CDH in Prrx1Cre:Rardn mice, and 3) assessing gene-nutrient interactions in Prrx1Cre:Rardn mice. Methods: Mice expressing a dominant negative retinoic acid receptor (Rardn) were bred with Prrx1Cre mice, producing double transgenic (Prrx1Cre:Rardn) fetuses with conditionally blocked RA signaling in the developing diaphragm. Diaphragms from embryonic mice were collected between embryonic days (E)15-18, and assessed for the severity of CDH, including morphometric analysis and surface area measurement. Additionally, lungs were collected and analyzed, undergoing histology and gene expression analysis. Collected tissues were also analyzed using sex as a biological variable to determine its effect on CDH incidence and severity. To study gene-nutrient interactions in the phenotype of Prrx1Cre:Rardn mice, two approaches were taken. First, an RA-enriched diet was given to pregnant dams between embryonic days (E)8.5-13.5. Second, female mice were placed on diets with 0, 4, or 25 IU vitamin A/g throughout pregnancy. Results: Prrx1Cre:Rardn fetuses have 100% incidence of severe CDH, with defects in the diaphragm and lung. This model recreates the full spectrum of diaphragm defects seen clinically in CDH, ranging from eventration defects to complete hemidiaphragm agenesis. Moreover, similar to CDH in humans, we observe defects in the left hemidiaphragm more frequently. This is further supported by diaphragm surface area of Prrx1Cre:Rardn mice, which was significantly reduced for both the right and left hemidiaphragms compared to controls. The observed lung phenotype in Prrx1Cre:Rardn embryos indicate that the CDH lung experiences pulmonary hypoplasia as a result of the diaphragm defect being present. The hypoplastic lung demonstrates tissue compression, a significant increase in Mean Linear Intercept, reduced lung volume, and reduced alveolar air spaces in Prrx1Cre:Rardn embryos, recapitulating what is observed in human CDH. Initial results show that there is no significant difference between CDH incidence and severity between male and female embryos. When determining gene-nutrient interactions, our results indicate that there is a modest rescue effect in the CDH phenotype with retinoic acid administration and increased dietary vitamin A intake. Maternal RA supplementation reduces the severity of the CDH phenotype, and maternal vitamin A deficiency prior to pregnancy appears to exacerbate the herniation of abdominal organs into the chest compared to fetuses from a mother with sufficient vitamin A. Conclusion: Prrx1Cre:Rardn mice are a powerful tool to study the pathogenesis and etiology of CDH. While we observe no sex differences in the severity of this model, our results suggest it recapitulates human CDH and is sensitive to exogenous retinoids. Funding Acknowledgement: This project was supported by a Canadian Institute of Health Research (CIHR) Project Grant, Women's and Children's Health Research Institute (WCHRI) University of Alberta innovation grant, and CDH UK operating fund.