Targeted Deletion of Functionally Validated Enhancers Defines Their Role In Mouse Limb Development

<p>Transcriptional enhancers are genomic DNA sequences that contain clustered transcription factor (TF) binding sites. When combinations of TFs bind to enhancer sequences they act together with basal transcriptional machinery to regulate the timing, location and quantity of gene transcription. Elucidating the genetic mechanisms responsible for differential gene expression, including the role of enhancers, during embryological and postnatal development is essential to an understanding of evolutionary processes and disease etiology. Numerous methods are in use to identify and characterize enhancers. Several high-throughput methods generate large datasets of enhancer sequences with putative roles in embryonic development. However, few enhancers have been deleted from the genome to determine their roles in the development of specific structures, such as the limb. Manipulation of enhancers at their endogenous loci, such as the deletion of such elements, leads to a better understanding of the regulatory interactions, rules and complexities that contribute to faithful and variant gene transcription – the molecular genetic substrate of evolution and disease. To understand the endogenous roles of two distinct enhancers known to be active in the mouse embryo limb bud we deleted them from the mouse genome. I hypothesized that deletion of these enhancers would lead to aberrant limb development.</p> <p>The enhancers were selected because of their association with p300, a protein associated with active transcription, and because the human enhancer sequences drive distinct <em>lacZ</em> expression patterns in limb buds of embryonic day (E) 11.5 transgenic mice. To confirm that the orthologous mouse enhancers, mouse <em>280</em> and <em>1442 </em>(<em>M280</em> and <em>M1442</em>, respectively), regulate expression in the developing limb we generated stable transgenic lines, and examined <em>lacZ</em> expression. In <em>M280-lacZ </em>mice,<em> </em>expression was detected in E11.5 fore- and hindlimbs in a region that corresponds to digits II-IV. <em>M1442-lacZ </em>mice exhibited <em>lacZ</em> expression in posterior and anterior margins of the fore- and hindlimbs that overlapped with digits I and V and several wrist bones. We generated mice lacking the <em>M280</em> and <em>M1442 </em>enhancers by gene targeting. Intercrosses between <em>M280 </em>-/+ and <em>M1442 </em>-/+, respectively, generated <em>M280</em> and <em>M1442 </em>null mice, which are born at expected Mendelian ratios and manifest no gross limb malformations. Quantitative real-time PCR of mutant E11.5 limb buds indicated that significant changes in transcriptional output of enhancer-proximal genes accompanied the deletion of both <em>M280</em> and <em>M1442. </em>In neonatal null mice we observed that all limb bones are present in their expected positions, an observation also confirmed by histology of E18.5 distal limbs. Fine-scale measurement of E18.5 digit bone lengths found no differences between mutant and control embryos. Furthermore, when the developmental progression of cartilaginous elements was analyzed in <em>M280</em> and <em>M1442 </em>embryos from E13.5-E15.5, transient development defects were not detected.<em> </em>These results demonstrate that <em>M280</em> and <em>M1442 </em>are not required for mouse limb development. Though <em>M280 </em>is not required for embryonic limb development it is required for the development and/or maintenance of body size – adult <em>M280 </em>mice are significantly smaller than control littermates. These studies highlight the importance of experiments that manipulate enhancers <em>in situ</em> to understand their contribution to development.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2013

Author dc:creator

• Nolte, Mark J.

Contributors dc:contributor

• Richard R. Behringer, Ph.D.
• Michelle C. Barton, Ph.D.
• Yasuhide Furuta, Ph.D.

• enhancer
• limb
• mouse
• development
• p300
• functional genetics
• Medicine and Health Sciences

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/388

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1420