Tbx6 was shown to be a T-box transcription factor expressed in the primitive streak and presomitic mesoderm of the developing mouse embryo; null mutations in Tbx6 resulted in ectopic neural tube formation in place of posterior somites and embryonic lethality. However nothing was known about expression, transcriptional activity, or downstream targets of Tbx6 protein. Using antibodies generated against Tbx6, Tbx6 was confirmed to be a 58 kDa protein in vivo, and Tbx6 protein and mRNA have similar spatial and temporal expression patterns. T-box proteins were defined by the presence of the T-domain, the DNA binding region. Given the conservation of the T-domain, it was not surprising that many of the characterized T-box binding sites were also somewhat conserved. Therefore, I first demonstrated that Tbx6 recognized the T palindromic consensus sequence and half-site sequence in vitro and then determined that the Tbx6 consensus binding site was 5- AGGTGTBRNNN -3 using a PCR-based binding site selection assay. Using luciferase reporter assays in cell culture, Tbx6 was determined to be a weak transcriptional activator. Lastly, initial studies with T and Tbx6 suggested that these T-box proteins do not synergistically activate a luciferase reporter.
In other work, the spontaneous mutation rib-vertebrae was determined to be a Tbx6 hypomorphic mutation due to an insertion of 185 bp into a Tbx6 enhancer. Finally, I also sought to understand how the Notch signaling pathway and Tbx6 interacted. The Notch transcription factor, RBP-J?, was demonstrated to control the maintenance of Tbx6 in the PSM. Furthermore, Tbx6 bound to several putative binding sites in vitro within a previously known mesodermal Dll1 enhancer. These results suggested that Notch signaling functions upstream and downstream of Tbx6. The work described in this thesis used a variety of in vivo and in vitro approaches, molecular and biochemical, to gain further understanding into how Tbx6, a member of a large family of transcription factors, functions in the context of a developing embryo in the presence of other family members to regulate the expression of target genes.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-12012006-185706 |
| Date | 26 January 2007 |
| Creators | White, Phillip H |
| Contributors | Jeffrey L. Brodsky, Lewis A. Jacobson, Cynthia Lance-Jones, Deborah L. Chapman, Jeffrey D. Hildebrand |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-12012006-185706/ |
| 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 University of Pittsburgh 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. |
Page generated in 0.0024 seconds