Date of Award

January 2026

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

David H. Stitelman

Second Advisor

Micha S. Raredon

Abstract

Background: Congenital diaphragmatic hernia (CDH) is prenatal disease characterized by pulmonary hypoplasia, pulmonary hypertension, and high postnatal morbidity and mortality. Although postnatal surgical repair corrects the diaphragmatic defect, clinical outcomes are largely determined by antenatal lung development. Fetoscopic endoluminal tracheal occlusion (FETO) can improve survival in select severe cases by stimulating lung growth, but it does not address the intrinsic molecular abnormalities underlying hypoplastic lung development. The spatially organized transcriptional programs that drive delayed lung maturation in CDH remain incompletely defined. Research Aims: This study aimed to define the spatially and temporally resolved molecular programs underlying pulmonary hypoplasia in CDH and to identify specific molecular targets for fetal therapeutic intervention. Hypothesis: We hypothesized that lung pathology in CDH reflects delayed execution of normal developmental gene programs rather than irreversible arrest or premature activation of later-stage pathways, with disproportionate effects on epithelial differentiation and epithelial–mesenchymal coordination. Methods: A nitrofen-induced rat model of CDH was used to analyze fetal lung development at embryonic days 17, 19, and 21, corresponding to pseudoglandular, canalicular, and saccular stages. Formalin-fixed, paraffin-embedded lung tissue was assembled into a tissue microarray and analyzed using 10x Genomics Xenium spatial transcriptomics with a custom 480-gene panel informed by prior single-cell RNA sequencing data. Spatial gene expression was integrated with histological architecture. Cell segmentation, cell type annotation, differential expression, cell abundance analysis, and pseudobulk statistical modeling were performed to assess developmental trajectories and disease-associated transcriptional changes. Results: Spatial transcriptomics preserved lung architecture and revealed hypoplastic airway morphology in CDH lungs. Across development, CDH lungs exhibited a slower decline in transcriptional activity compared to controls, consistent with prolonged engagement of early developmental programs. Although broad cell class proportions converged by embryonic day 21, lineage-specific abnormalities persisted, including a reduced proportion of alveolar type I cells. Temporal analysis of genes with monotonic developmental expression demonstrated that transcriptional dysregulation in CDH overwhelmingly reflected developmental delay rather than acceleration. This delay was most pronounced in epithelial populations, particularly Sox9-positive distal epithelial progenitors, while mesenchymal populations showed fewer dysregulated genes with a similar directional bias. At embryonic day 19, CDH epithelial cells exhibited increased expression of morphogen signaling pathways and proliferation-associated genes. In contrast, mesenchymal cells demonstrated increased proliferation-associated signaling and reduced expression of extracellular matrix–related genes, suggesting delayed structural support of airway development. Spatial mapping revealed misalignment of key branching and fate-specification signals, including persistent expression of early developmental regulators near airway tips and reduced spatial initiation of alveolar differentiation programs. Scientific Impact and Relevance: These findings demonstrate that pulmonary hypoplasia in CDH arises from delayed lung maturation rather than global transcriptional failure. CDH lungs retain developmental plasticity and growth competence but fail to appropriately transition toward epithelial differentiation and structural consolidation prior to birth. By defining the spatial and temporal molecular landscape of hypoplastic lung development, this study identifies critical cell populations, signaling pathways, and developmental windows that may be targeted to improve prenatal lung maturation. These insights provide a mechanistic framework to inform next-generation fetal therapies that extend beyond FETO and toward targeted molecular and regenerative interventions for patients with CDH.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 07/14/2028

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