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Retinoids and Mitochondrial Effects on Enteric Nervous System Development, Structure, and Function
Date of Award
Doctor of Philosophy (PhD)
The enteric nervous system (ENS) is a complex network of neurons and glial cells that is formed from enteric neural crest-derived cells (ENCDC). Previous studies suggested that retinoic acid (RA), the active metabolite of vitamin A, is required for normal ENS development. However, the mechanisms through which RA supports ENS development remain poorly understood.
We investigated the role of retinaldehyde dehydrogenases (RALDH1, RALDH2 and RALDH3) in ENS development by quantitative anatomical comparisons in an allelic series of mice with mutations in Raldh1, Raldh2, and Raldh3. We showed that Raldh1-/-, Raldh2+/-, Raldh3+/- (R1KOR2HetR3Het) mutant mice had a reduced colon myenteric neuron density, reduced colon myenteric neuron to glia ratio, reduced colon submucosal neuron density, and increased colon myenteric fibers per neuron when compared to wild type (WT; Raldh1WT, Raldh2WT, Raldh3WT) mice. These defects are unlikely to be due to defective ENS precursor migration since R1KOR2HetR3KO mice had enhanced enteric neuron progenitor migration into the distal colon compared to WT during development. We also showed that RALDH mutant mice have reduced colon contractility compared to WT mice. Together, these results suggest that reduced local RA levels, as might be expected to occur in RALDH mutant mice or in the setting of maternal vitamin A deficiency, resulted in colonic hypoganglionosis with functional consequences.
We next sought to determine if RA acts directly on ENS precursor cells or indirectly via effects on neighboring cells using mice expressing a conditional dominant negative retinoic acid receptor (RARDN) within neural crest-derived cells. We showed that cell-autonomous RA signaling is required for intestinal colonization by ENCDC since mice expressing RARDN have total intestinal aganglionosis. We also showed that cell autonomous RA signaling is required for RET expression in ENS precursor cells. To identify the transcriptional targets of RA signaling in ENS precursor cells, we generated a new ENCDC bacTRAP transgenic mouse that facilitates the rapid isolation of ribosome-associated RNA from ENCDCs without prior separation of ENCDC from surrounding cells. We used this mouse to evaluate how gene expression within ENCDC changes after chemical inhibition of RAR signaling during fetal development.
We also investigated the effect of mitochondrial dysfunction on the ENS since gastrointestinal symptoms are common in people with mitochondrial disease yet the mechanisms underlying these symptoms are not well understood. Mice with impaired mitochondrial metabolism in enteric neurons and glia were generated through the targeted deletion of the mitochondrial transcription factor A gene (Tfam; called Tfam-ENSKO mice). We showed that Tfam-ENSKO mice develop intestinal pseudo-obstruction resulting in premature death associated with extensive and progressive degeneration of the ENS involving both neurons and glia. We further demonstrated that mitochondrial defects differentially affected specific subpopulations of enteric neurons and regions of the gastrointestinal tract. This regional and subtype-specific variability in susceptibility to mitochondrial defects resulted in an imbalance of inhibitory and excitatory neurons that likely accounted for the phenotype in Tfam-ENSKO mice. Mitochondrial dysfunction, therefore, may be an important contributor to neurodegeneration in the ENS and may cause gastrointestinal symptoms in people with neurodegenerative disorders.
These studies provide new insight into the role of vitamin A and retinoid metabolism in ENS development and the effect of mitochondrial dysfunction in the ENS. Moreover, we have developed a novel tool (ENCDC bacTRAP mouse) for elucidating transcriptional activity in the developing ENS.
Chair and Committee
Robert O Heuckeroth
Joseph Dougherty, Sanjay Jain, Kelly Monk, Lilianna Solnica-Krezel
Wright-Jin, Elizabeth Copeland, "Retinoids and Mitochondrial Effects on Enteric Nervous System Development, Structure, and Function" (2015). Arts & Sciences Electronic Theses and Dissertations. 463.