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A Neuro-Specific Hedgehog-Responsive Enhancer from Intron 1 of the Murine Laminin Alpha 1 Gene

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A Neuro-Specific Hedgehog-Responsive Enhancer from Intron 1 of the Murine Laminin Alpha 1 Gene A neuro-specific hedgehog-responsive enhancer from intron 1 of the murine laminin alpha 1 gene Thesis presented for the degree of PhD by Kalin Dimitrov Narov Department of Biomedical Science University of Sheffield September 2013 i Acknowledgements I thank my supervisors Anne-Gaëlle Borycki and Philip Ingham for all expert guidance, patience, and for the opportunity to study vertebrate development in their laboratories. I also thank my advisors Pen Rashbass and Vincent Cunliffe for the helpful advices and their critical analyses on my work, and our collaborator Norris Ray Dunn for advising me on mouse transgenics and providing me with mouse embryos. Many thanks also to Shantisree Rayagiri, Joseph B. Pickering, Claire Anderson, Ashish Maurya, Weixin Niah, Harriet Jackson, Raymond Lee, Xingang Wang, Yogavali Pooblan for helping me with text formatting and embryo harvesting, and for providing me with reagents and advices. I am also grateful to the whole D-floor community, as well as to Martin Zeidler’s and Marcelo Rivolta’s labs for letting me use their equipment. Last but not least, I thank my family for the constant support and encouragement, and especially my parents for nurturing in me the love to nature and knowledge. Therefore, I dedicate this work to the memory of my father. ii Abstract 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. iii Table of Contents Chapter 1 ................................................................................................................................................i Introduction......................................................................................................................................... 1 1. Introduction ................................................................................................................................. 2 1.1. Basement membranes and animal development................................................................................. 2 1.2. Laminin alpha 1 – gene organization, protein structure and assembly ............................................... 2 1.2.1. Laminin α1 is part of a heterotrimeric complex .......................................................................... 4 1.2.2. Interactions of Laminin-111 with other ECM components and cellular receptors ..................... 6 1.2.3. Laminin-111 and basement membrane assembly ....................................................................... 7 1.3. Expression of laminin α 1 .................................................................................................................. 7 1.3.1. Lama1 expression in the mouse and human ............................................................................... 8 1.3.2. Lama1 expression in zebrafish.................................................................................................... 8 1.4. Regulation of Lama1 expression ........................................................................................................ 9 1.4.1. Signalling pathways and control of Lama1 expression in vitro .................................................. 9 1.4.2. cis-regulatory elements in Lama1 transcription ........................................................................ 11 1.4.3. Regulation of Lama1 transcription in embryonic development ................................................ 14 1.5. Laminin expression in skeletal muscle development ....................................................................... 14 1.6. SHH is required for Lama1 expression and myotomal basement membrane assembly ................... 16 1.6.1. Shh-/- mutant mouse embryos fail to express Lama1 in the somites and neural tube ................ 16 1.6.2. Shh is required for lama1 expression in zebrafish .................................................................... 17 1.7. Functions of laminin α1 in development .......................................................................................... 17 1.7.1. Laminins and neural development ............................................................................................ 17 1.7.1.1. Laminins and neural stem cells ......................................................................................... 17 1.7.1.2. Laminins and cortical morphogenesis ............................................................................... 18 1.7.1.3. Laminin α1 in axonal growth and migration ..................................................................... 19 1.7.2. Laminins and neural crest cells ................................................................................................. 20 1.7.3. Laminin α1 in ocular development ........................................................................................... 21 1.7.4. Laminin α1 and kidney development ........................................................................................ 22 1.7.5. Laminin α1 in skeletal muscle development ............................................................................. 22 1.8. The SHH signaling pathway ............................................................................................................ 23 1.8.1.Expression, secretion and diffusion of Shh ................................................................................ 24 1.8.2. Transduction of the HH signal .................................................................................................. 25 1.8.3. The GLI transcription factors are mediators of HH signaling in vertebrates ............................ 26 1.8.4. SHH and dorso-ventral patterning of the neural tube ............................................................... 28 1.9. Transcription-regulatory elements…………………………………………………………………30 1.9.1. The core promoter……………………………………………………………………………..30 1.9.2. Transcriptional enhancers……………………………………………………………………..31 1.9.3. Transcriptional silencers………………………………………………………………………32 1.9.4. Insulators………………………………………………………………………………………32 1.10. Methods for the prediction of transcription-regulatory elements………………………………...33 1.10.1. Direct (biochemical) methods………………………………………………………………..33 1.10.1.1. Prediction of TREs based on transcription factor occupancy and/or histone modifications as revealed by ChIP-seq………………………………………………………………………………………….33 1.10.1.2. Prediction of TREs in DNaseI hypersensitive domains…………………………………35 1.10.2. Indirect methods……………………………………………………………………………...36 1.10.2.1. Computational prediction of TREs based on sequence organisation/pattern, irrespective of overall conservation…………………………………………………………………………………………...36 iv 1.10.2.2. Comparative genomics and TRE prediction…………………………………………….38 1.11. Functional validation of candidate transcription-regulatory elements……………………………39 1.11.1. Zebrafish as a tool for the validation
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