Integrative analysis of the developing diaphragm provides a better understanding of congenital diaphragmatic hernia
Creators: Garcia Rivas, Juan F
Publisher: name: University of Alberta Library
Year: 2023

<p>Introduction: Congenital Diaphragmatic Hernia (CDH) is a condition that affects 2.3 births every 10,000 and is characterized by an incomplete formation of the diaphragm. As a result of the incomplete development of the diaphragm, the abdominal organs will protrude into the thoracic cavity, impeding lung growth and causing lung hypoplasia. Due to the complexity of this condition, there are still gaps in the understanding of the etiology of CDH. Two main explanations are currently used to explain the pathogenesis of CDH: genetic and environmental. The retinoid hypothesis has tried to explain the etiology of this condition, and it states that abnormal retinoic acid signaling leads to the formation of diaphragmatic hernias. The goal of this thesis was to better understand the etiology of CDH, by trying to better understand both the genetic and environmental explanations. To fill the gaps in the knowledge in the etiology of CDH, we assessed (i) the levels of expression of multiple CDH and retinoid-associated genes in the pleuroperitoneal folds (PPF), and (ii) if blocking the retinoic acid signaling cascade in the developing diaphragm will cause CDH.</p>

<p>Methods: To answer the first questions, we performed single cell transcriptomics analysis in the developing diaphragm at gestational day (GD)13.5. Clustering analysis was performed utilizing Seurat in R. Expression analysis of curated retinoid and CDH-associated genes was performed in our scRNA-seq sample to determine the levels of expression of these genes in the different clusters present in the developing diaphragm. To test for the second hypothesis, we generated a conditional mutant mouse model expressing a truncated version of the retinoic acid receptors in the developing diaphragm. We also examined if Dhrs3-/- fetuses develop diaphragmatic hernias. The fetuses of our Prrx1-Cre:Rardn and Dhrs3-/- mouse models were collected and dissected via light microscopy to assess the incidence of diaphragmatic defects. The lung volumes of the Prrx1-Cre:Rardn fetuses was also determine by utilizing contrast enhanced micro-Computing Tomography (micro-CT).</p>

<p>Results: Based on our single cell expression analysis, we were able to identify 18 different clusters and 10 distinct cell populations in the developing diaphragm. Expression analysis of our sample revealed that expression of both retinoid and CDH-associated genes is mostly seen in the mesenchymal and mesothelial components of the primordial diaphragm. A total of 215 CDH-associated genes and 30 retinoid-associated genes were analyzed. Our mutant mouse models gave us varying results. We found that the Prrx1-Cre:Rardn fetuses present CDH, with a penetrance of 100%. The herniations were seen mostly on the left side of the diaphragm, and they normally took more than 50% of that side. Our micro-CT analysis revealed that the Prrx1-Cre:Rardn fetuses have lower left lung volumes compared to control fetuses. This difference was not seen in the right lungs. In contrast, our Dhrs3-/- mouse model was not able to produce CDH in the fetuses.</p>

<p>Conclusion: To our knowledge, this is the first time scRNA-seq analysis has been performed in the developing diaphragm. We demonstrated that the non-muscular mesenchyme is the primary hotspot for expression of both CDH- and retinoid-associated genes. The retinoid hypothesis was also validated with our conditional animal model, as blocking the retinoic acid signaling cascade utilizing our Prrx1-Cre:Rardn model led to the formation of CDH in these fetuses.</p>