Altered Epithelial-Mesenchymal Progenitor States Lead to Matrix Deposition, Tissue Inflammation, and Transitional Epithelial State in Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) lungs are characterized by pulmonary hypertension and lung hypoplasia. We have used single cell RNA sequencing (scRNA-seq) to show that mesenchyme is perturbed in CDH, leading to disrupted epithelial-mesenchymal transition (EMT) dynamics and inflammatory signaling. Normal and CDH fetal rat lungs were harvested at E17, E19 and E21 – which correlate to pseudoglandular, canalicular, and saccular stages, respectively - and dissociated into single cell suspension. Seurat was used for single cell analysis. Cell types were identified by canonical genes and differential expression of genes were then analyzed. Findings were confirmed by staining. Score for mesenchymal versus epithelial-like characteristics in EMT was calculated. During normal development, mesenchymal progenitors surround the developing airway undergoing EMT. At E17 in CDH, these cells downregulate Sox9, a plasticity marker, and upregulate extracellular matrix (ECM) proteins and TGFβ signaling molecules. CDH mesenchymal progenitors have an increased EMT score (<i>p</i> &lt; 0.001), meaning more mesenchymal characteristics compared to normal lung. At E21, CDH mesenchyme upregulates <i>TGFβ-2, TGFβR-2, and Smad2/3.</i> CDH alveolar type 1 (AT1) and AT2 cells upregulate <i>Krt8</i> and <i>Krt18</i>. CDH lung mesenchymal progenitors attain mesenchymal-like characteristics prematurely and there is upregulation of ECM proteins when compared to normal lung. Moreover, CDH distal epithelial cells (Krt8/18+) enter a transitional state that is seen in fibrotic lung diseases. These findings represent imbalance of EMT, and thus dysregulation of key molecular pathways, which leads to poorly developed mesenchymal and epithelial structures that we speculate causes the lung hypoplasia found in CDH.