Diabetes is a worldwide health issue. In both type I and late stage type II diabetes, significant beta-cell loss causes insulin deficiency and hyperglycemia. Replenishing beta cells is a promising therapy and it requires enhancing beta-cell replication, activating a beta-cell neogenesis program or transplanting exogenous beta cells. This thesis investigates the in vivo pancreatic endocrine cell differentiation process and the regulation of endocrine lineage allocation by key transcription factors, which will be tremendously informative for regenerating beta cells and creating cell-based therapy for diabetes. In this thesis research, we designed a novel bipartite Cre cell lineage tracing technique to reconstitute Cre activity only in a subset of the Ngn3+ pro-endocrine progenitors and discovered that the Ngn3+Myt1+ subset favors beta-cell fate over alpha-cell fate. Transcriptional and epigenetic analysis of the Ngn3+ progenitors from different embryonic stages revealed that gene expression and DNA methylation of endocrine genes, including Myt1, undergo dynamic changes along the developmental timeline, which is correlated with the temporal change of the Ngn3+ progenitors' differentiation competence. Meanwhile, I designed a tamoxifen-inducible bipartite CreERT2 construct and characterized its recombination properties in cell lines. This inducible bipartite CreERT2 can be used to generate mouse models to further dissect the differentiation potential of the Ngn3+Myt1+ progenitors at various embryonic stages. In addition, we observed the variation of Cre reporter sensitivity and reported non-parallel recombination of floxed alleles in the same cell here as an integral part of the Cre technique. We also investigated the regulation of Ngn3 expression by Notch signaling and miRNAs and discovered that Ngn3 augments its own expression by a miRNA-mediated inhibition of Notch signaling. We further explored the possibility that miRNAs can translocate across the plasma membrane via gap junctions and exert their functions non-cell-autonomously to counteract the Notch lateral inhibition effect. With a better understanding of the in vivo beta-cell differentiation process, our research will shed light upon the development of cell replacement therapies for diabetes.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-11152015-162440 |
| Date | 17 November 2015 |
| Creators | Liu, Jing |
| Contributors | Mark Magnuson, Chris Wright, David Piston, Andrea Page-McCaw |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-11152015-162440/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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