Browsing by Author "Uribe, Rosa A"

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    Investigation of Enteric Neural Crest Cell Differentiation, Proliferation Dynamics and Hirschsprung Disease Manifestation During Zebrafish Enteric Nervous System Development
    (2022-04-22) Baker, Phillip Austin; Uribe, Rosa A; Wagner, Dan S
    The enteric nervous system (ENS) is a vast intrinsic network of neurons and glia within the gastrointestinal tract that is largely derived from enteric neural crest cells (ENCCs) that emigrate into the gut during vertebrate embryonic development. Study of ENCC migration dynamics and their genetic regulators provides great insights into fundamentals of collective cell migration and nervous system formation and are a pertinent subject for study due to their relevance to the human congenital disease, Hirschsprung disease (HSCR). To accomplish these studies contained within my thesis, I used the zebrafish model to investigate early enteric nervous system development in its endogenous context. My first study set out to bridge our gap in understanding between zebrafish and mammalian enteric nervous system cell diversity and structure. For this investigation, I utilized immunohistochemical detection of axonal and glial cell profiles in the larval gut to reveal novel details about zebrafish ENS architecture and maturation. Specifically, I observed that the zebrafish larval enteric plexus is histologically distinct by 7 dpf and contains axon and glial cell profiles that encircle the intestinal epithelium, the details of which had yet to be described in zebrafish. My second study profiling the transcriptomes of migratory ENCCs uncovered evolutionarily conserved and novel transcriptional signatures of differentiating enteric neuron cell types, thereby expanding our knowledge of ENS development. I further detected the presence of opioid receptor transcripts within the ENS at 68–70 hpf, a time when immature enteric neurons are continuing to migrate and pattern within the developing embryo. This discovery of opioid transcripts, oprl1 and oprd1b in migrating enteric neuroblasts, represents the earliest stage in which they have been shown to be expressed within the early developing ENS. Lastly, my final study in which I performed in toto imaging of ENS development in heterozygotic deficient ret mutant zebrafish led to novel findings pertaining to ENCC migration and proliferation dynamics in a Hirschsprung Disease relevant context. Specifically, I discovered proliferation dependent migration behaviors downstream of Ret. These findings suggest that Ret signaling may function to regulate maintenance of a stem-state in enteric neural progenitors.
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    Posterior Hox Transcription Factors Drive Multiple Phases of Neural Crest Development
    (2022-12-02) Howard, Aubrey Gaylon Adam; Uribe, Rosa A
    In vertebrate embryos, neural crest cells (NCC) as a stem cell population demonstrate a remarkable capacity for both migratory potential as well as a high degree of plasticity. From early embryonic stages, these multipotent cells delaminate from the neural tube, migrate throughout the embryonic body, and differentiate in a wide host of cells lineages, including pigment lineages, corneal endothelium, bone, mesenchyme, glia, and various populations of neurons, such as the enteric nervous system. NCC differentiate into terminal tissues collinear with the anterior-posterior axis, dividing them into four groups: cranial, vagal, trunk, and sacral. Notably, the developmental span of these NCC subpopulations is coincident with the complex and overlapping expression of hox genes, which encode an ancient family of conserved transcription factors. Throughout each of the NCC developmental phases (specification, epithelial-to-mesenchymal transition (EMT), migration, and differentiation), the development of NCC is regulated by a complex and dynamic gene regulatory network, which remains to be full characterized, particularly among the vagal and trunk NCC. Further, the role for more posterior hox transcription factors are here unto unknown within the posterior NCC populations, unlike the cranial NCC. To this end, I have utilized the vertebrate model zebrafish (Danio rerio) to investigate the transcriptional landscape of NCC. I led a team to build and identify an atlas of sox10 cell lineages which includes thousands of transcriptomes from single cells representing stages spanning the lifetime of the fish. Using this transcriptional atlas, I have found a dynamic and diverse set of hox expression codes which define specific NCC and non-NCC lineages in the posterior embryo. Among these codes, hoxb5b was highly enriched and was selected for deeper characterization. Overexpression of hoxb5b revealed that it was sufficient to expand NCC localization domains, but was restricted in its function to early developmental spans. Further, suppression of single and overlapping members of the hox codes identified in the transcriptional atlas demonstrated a requirement for specific hox codes in NCC EMT. Cumulatively these findings support a model in which hox genes participate as potent and required drivers of vagal NCC patterning, expanding our fundamental knowledge of vertebrate development.