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Embryonic intestinal development is a dynamic process where tissue undergoes drastic transitory changes from morphological and physiological changes, to chromatin restructuring for intestinal specification, to metabolic shifts for supporting growing tissue. My research focuses on two critical themes regulating embryonic intestinal development: Epigenomic and Metabolic. During early endoderm development, cells exhibit cellular plasticity with the ability to acquire cell fate of multiple endodermal lineages. Lineage-inducing transcription factors (TF) drive tissue-specific transcriptomes, leading to distinct cell types, with the loss of cellular plasticity as tissue matures. However, transcriptional mechanisms governing organ specification and cell fate are poorly understood. We provide evidence that loss of intestinal lineage-inducing TF CDX2 in the developing endoderm at E9.5, using Shh-cre, leads to underdeveloped intestine with cells exhibiting foregut-like cellular identity. This phenomenon is conserved across species with the induction of esophageal and stomach cell fates upon CDX2 loss in human intestinal organoid cultures (HIO). By temporal mapping of Cdx2 knockout in the developing intestine, we highlight the loss of intestine’s ability to transform in esophageal lineages by E9.5; between E9.5-E13.5 CDX2 loss leads to gastric lineage, while intestine’s window of CDX2-restricted plasticity is entirely lost by E15.5. Enhancers bound by CDX2 in the developing endoderm, identified using CDX2 ChIP-seq at E13 and E17, regulate genes involved in gut tube development and are enriched for patterning transcription factor motifs. In contrast, enhancers bound by CDX2 in adult epithelium regulate genes involved in metabolic processes and are enriched for mature intestinal transcription factors. This dynamic binding of CDX2 along development is conserved across species, as we identify different regions regulated by CDX2 in early specified hindgut when compared to adult human intestinal cultures, with similar relationships suggesting patterning roles for CDX2 early in development, and mature intestine-specific physiological functions in the adult tissue. In a temporal survey of developing intestinal chromatin assayed using ATAC-seq, we find that CDX2’s differential binding and intestine’s cellular plasticity coincide with dynamic chromatin restructuring during villus formation. Villogenesis is a transition point in intestinal development, when early embryonic enhancer chromatin condenses while mature intestinal enhancers gain accessibility. We highlight temporal CDX2 bound enhancers highly overlap with dynamic chromatin accessibility. Additionally, in human intestinal cultures, in presence of Wnt/FGF signaling CDX2 binds at enhancers specifying intestinal cell fate, however in absence of Wnt/FGF CDX2 fails to impart intestinal cell fate and loses its ability to bind at intestine-specifying enhancers. These results indicate that CDX2 requires additional factors to drive the intestinal transcriptome, and chromatin accessibility strongly correlates with dynamic CDX2 binding. Enhancer regions with transcriptionally active histone H3k27ac modifications, that become inactive after human hindgut specification, are enriched for foregut transcription factor motifs.This indicates that genomic regions where foregut transcription factors bind become inactive once intestinal cell fate is established. Furthermore, we analyze forestomach enhancer regions, identified using ATAC-seq from forestomach tissue at post-natal Day 1, for chromatin accessibility in temporally developing intestinal epithelium, assayed using ATAC-seq. We highlight that the foregut enhancer regions are accessible in the developing intestine at E11, which then progressively lose permissive chromatin and are mostly inaccessible by E16. However, in the absence of CDX2, foregut enhancers remain accessible in intestine at E16, permitting ectopic foregut cellular identity to establish in intestine. Our study highlights an unexplored mechanism of chromatin regulation of organ specification and cellular plasticity. To further decipher mechanisms regulating intestinal development, we investigate the role of transcription factor Yin-Yang1 (Yy1) and metabolism during intestinal development. While, CDX2 is a transcription factor that is appreciated to be involved in intestinal development, the role of YY1 in intestinal development has not been elucidated before. Although, we explore the mechanisms governed by CDX2 and YY1 independently, we highlight that during villogenesis both YY1 and CDX2 regulated processes are critical for intestinal development. During late gestation, structures called villi extend into the intestinal lumen, significantly increasing the surface area of the intestinal epithelium to prepare the gut for the neonatal diet. Incomplete development of the intestine is a most common gastrointestinal complication in neonates, but the causes remain unclear. We provide evidence that YY1 is critical for intestinal villus development. YY1 loss in the developing endoderm has no apparent consequences until late gestation, after which the intestine differentiates poorly and exhibits severely stunted villi. Transcriptome analysis revealed that YY1 is required for mitochondrial gene expression, and ultrastructural analysis confirmed compromised mitochondrial integrity in the mutant intestine. We found increased oxidative phosphorylation gene expression at the onset of villus elongation, suggesting that aerobic respiration may function as a regulator of villus growth. Mitochondrial inhibitors blocked villus growth in a fashion similar to Yy1 loss, thus further linking oxidative phosphorylation with late-gestation intestinal development. Interestingly, we find necrotizing enterocolitis patients also exhibit decreased expression of oxidative phosphorylation genes. Our study highlights the greatly unappreciated role of metabolic regulation during organogenesis, and suggests its possible contribution to the pathogenesis of neonatal gastrointestinal disorders.

ETD_7799

Ph.D.

Includes bibliographical references

by Namit Kumar

doi:10.7282/T32F7QW6

ETD doctoral

The author owns the copyright to this work.