Epithelial-to-Mesenchymal Transformation (EMT) is an important process in development, and occurs during key steps in both valvular and coronary vessel development. This dissertation uses transcriptional profiling strategies to identify novel regulators of EMT during cardiogenesis.
An early step in valvulogenesis occurs when endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo EMT to yield valvular interstitial cells. We developed an unbiased strategy to identify genes important in endocardial EMT using a spatial transcriptional profile. RNA-seq analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick (HH18) and mouse (E11.0) embryos at comparable stages of development was performed. EMT occurs in the AVC and OFT cushions but not the VEN at this time. We identified genes >2-fold enriched in chick and mouse cushions compared to VEN. Gene ontology and Gene Regulatory Network (GRN) analysis of cushion-enriched genes were consistent with cells undergoing EMT. Further analysis accurately identified and validated previously unrecognized novel candidate genes (Meis2, Id1, Hapln1, Foxp2) and the NF-κB pathway as regulators of endocardial cell EMT in vitro.
Epicardial EMT is a critical step in coronary vessel formation which is dysregulated in mice lacking TGFβR3. To elucidate the role of TGFβR3 in EMT we developed a strategy to identify genes downstream of TGFβR3 in cultured epicardial cells. Tgfbr3+/+ and Tgfbr3-/- immortalized epicardial cells were incubated with vehicle or ligands known to promote TGFβR3-dependent invasion (TGFβ1, TGFβ2, BMP2) and harvested for RNA-seq analysis. GRN analysis of genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells in each ligand incubation group revealed dysregulated NF-ĸB signaling. TGFβ2 or BMP2 incubation stimulated NF-ĸB activity in Tgfbr3+/+ but not Tgfbr3-/- epicardial cells. Inhibiting NF-ĸB signaling reduced TGFβ2- or BMP2-promoted invasion of Tgfbr3+/+ cell, further supporting a role for NF-ĸB signaling in promoting invasion downstream of TGFβR3. The genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFβR3 signaling.
These transcriptional profiling strategies identified and validated novel regulators of endocardial and epicardial EMT.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-05282013-123252 |
| Date | 10 June 2013 |
| Creators | DeLaughter, Daniel Morris |
| Contributors | Antonis Hatzopoulos, H. Scott Baldwin, Joey V. Barnett, Christopher V. E. Wright, Guoqiang Gu |
| 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-05282013-123252/ |
| Rights | restricted, 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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