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Title: A neuro-specific hedgehog-responsive enhancer from intron 1 of the murine laminin alpha 1 gene
Author: Narov, Kalin
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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Laminin alpha 1 (LAMA1) is a major component of the earliest basement membranes in the mammalian embryo. Disruption of the murine Lama1 gene result in lethal failure of germ layer differentiation and extraembryonic membrane formation at gastrulation stages, while conditional deletion of Lama1 leads to aberrant organization of retinal neurons and vasculature, and defects in cerebellar glia and granule cell precursors later in development. Similarly, inactivation of lama1 in zebrafish affects lens, retina and anterior notochord development. This diverse range of phenotypes in Lama1-deficient animals reflects the complexity of its expression pattern during embryogenesis, which is largely conserved among vertebrates. Major sites of Lama1 transcription in the mouse embryo are the neural tube, presomitic mesoderm, somites, nephrogenic mesoderm, head mesenchyme and the lens. However, little is known about the signaling mechanisms governing the spatio-temporal control of Lama1 transcription. Previous studies in our lab revealed a requirement for SHH signaling in the transcription of Lama1 in the somites and neural tube of mouse embryos. Therefore, I hypothesized that SHH might directly modulate Lama1 expression via the binding of GLI transcription factors to regulatory regions in the Lama1 locus. In this study, I identified a cis-regulatory element that may be involved in the SHH-dependent control of Lama1 expression in the murine embryo. I began my study with a phylogenetic footprinting approach that uncovered 25 conserved non-coding elements upstream of the murine Lama1 locus, some of which contained GLI binding motifs. Subsequent luciferase reporter-based analysis in cell culture with a subset of the CNEs did not provide convincing evidence for enhancer- and/or silencer-like properties of the elements, except for CNE7. The CNEs were further characterised using an in vivo transgenesis reporter screen in zebrafish, which uncovered a skeletal-muscle specific regulatory region. In parallel, a detailed survey of the existing literature revealed the presence of a non-conserved GLI-occupied region in intron 1 of the murine Lama1 gene. Subsequently, I showed that this element behaves as a tissue-specific enhancer driving reporter expression in the neural tube of mouse and zebrafish embryos. I provided evidence that active Hh signaling is required and sufficient for the activity of this enhancer. Finally, I demonstrated that the GLI binding motifs within the element are essential for its function. Altogether, these results suggest that SHH may directly control Lama1 transcription in the mouse neural tube via an intronic enhancer, and also provide further insight in the relationship between cell signaling and the regulated expression of extracellular matrix components in development and disease.
Supervisor: Borycki, Anne-Gaelle Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available