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The Role of Sulf1 During Sonic Hedgehog Mediated Neural Patterning

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The Role of Sulf1 During Sonic Hedgehog Mediated Neural Patterning The role of Sulf1 during Sonic hedgehog mediated neural patterning Simon Alexander Ramsbottom PhD The University of York Department of Biology 2011 Abstract Heparan sulphate proteoglycans (HSPGs) are large molecules distributed ubiquitously, both at the cell surface and within the extracellular matrix. These molecules are known to play essential roles in developmental cell signalling, and the differential sulfation of HSPG chains gives rise to a high degree of variability in their binding specificity. Sulf1, an N-acetlyglucosamine O-6 endosulfatase, specifically removes sulphate groups from HSPG chains in regions of high sulfation, and removal of these groups by Sulf1 leads to the attenuation of both BMP and FGF signalling. The expression profile of Sulf1 within the neural tube of X. tropicalis is similar to that of Sonic Hedgehog (Shh) and work in both chick and Drosophila has shown that Sulf1 is able to modify the distribution of hedgehog proteins during development. Taken together, this suggests that Sulf1 may act within the ventral neural tube to modify the distribution and activity of Shh and so regulate vertebrate neural patterning. Using the paradigm of dorsoventral patterning within the vertebrate neural tube, this thesis establishes a role for Sulf1 in modulating the distribution and activity of Shh, and demonstrates that this regulation is an important factor during neural development. 2 Abstract .................................................................................................................... 2 List of figures ............................................................................................................ 8 List of Tables .......................................................................................................... 11 Acknowledgements ................................................................................................. 12 Author‟s declaration ................................................................................................ 13 1.0 Introduction ............................................................................................. 14 1.1 Morphogens in development ............................................................................. 15 1.2 The role of hedgehog during Drosophila development ..................................... 16 1.3 The role of hedgehog during vertebrate development ....................................... 16 1.3.1 Development of axial structures ................................................................. 17 1.3.2 Determination of cell lineage ...................................................................... 20 1.3.3 Development of the limb bud ...................................................................... 21 1.3.4 Axon guidance ........................................................................................... 21 1.3.5 Cell proliferation ......................................................................................... 22 1.4 Hedgehog synthesis ......................................................................................... 23 1.4.1 Controlled release of the hedgehog ligand ................................................. 25 1.5 Hedeghog signal transduction ........................................................................... 25 1.5.1 Patched ...................................................................................................... 25 1.5.2 Smoothened ............................................................................................... 26 1.5.3 Gas1 .......................................................................................................... 27 1.5.4 The ihog family ........................................................................................... 28 1.5.5 Hip ............................................................................................................. 29 1.5.6 Downstream of ptc and smo ....................................................................... 29 1.5.7 Role of cilium in vertebrate hedgehog signal transduction .......................... 33 1.5.8 Hedgehog interacts with the extracellular environment ............................... 36 1.6 Heparan sulphate proteoglycans ....................................................................... 37 1.6.1 Structure .................................................................................................... 37 1.6.2 Synthesis ................................................................................................... 40 1.6.3 Loss of HSPGs impacts on developmental signalling processes. ............... 44 1.6.4 Additions to HSPGs are critical for their function ........................................ 45 3 1.6.5 HSPG sulfation is required for developmental signalling pathways............. 46 1.7 Post-synthetic modification of HSPG structure; the Sulfs .................................. 47 1.7.1 Sulf1 ........................................................................................................... 48 1.7.2 Sulf2 ........................................................................................................... 50 1.7.3 Sulf effects on signalling pathways ............................................................. 51 1.7.4 The role of Sulf during development ........................................................... 52 1.7.5 Sulf and cancer .......................................................................................... 53 1.8 Aims of this study .............................................................................................. 54 2.0 Relationship between Shh and Sulf1 ...................................................... 55 2.1 Introduction ....................................................................................................... 56 2.2 Aims .................................................................................................................. 57 2.3 Methods ............................................................................................................ 58 2.3.1 Perturbation of Shh signalling ..................................................................... 58 2.3.2 Ptc2 as an indicator of Shh activity ............................................................. 59 2.3.3 Knockdown of Sulf1 ................................................................................... 60 2.3.4 Unilateral injections .................................................................................... 62 2.4 Results .............................................................................................................. 63 2.4.1 Shh and Sulf1 expression in Xenopus ........................................................ 63 2.4.2 Shh activity affects Sulf1 expression within the open neural plate .............. 68 2.4.3 Regulation of Shh expression by Sulf1 ....................................................... 72 2.4.4 .The activity of Sulf1 and 6-OST can modulate Shh signalling .................... 74 2.4.5 Sulf1 is required for correct levels of Shh signalling ................................... 75 2.5 Discussion ........................................................................................................ 77 2.5.1 Shh and Sulf1 show regions of co-expression ............................................ 77 2.5.2 Sulf1 is a Shh response gene in Xenopus .................................................. 77 2.5.3 Sulf1 regulates Shh activity but not Shh expression ................................... 79 2.5.4 Loss of Sulf1 leads to a reduction in Shh expression and activity within the floor plate ............................................................................................................ 80 3.0 Patterning the vertebrate neural tube ...................................................... 81 3.1 Introduction ....................................................................................................... 82 4 3.1.1 Shh and neural patterning .......................................................................... 82 3.1.2 Sulf1 and neuronal patterning .................................................................... 85 3.2 Aims .................................................................................................................. 87 3.3 Methods ............................................................................................................ 87 3.4 Results .............................................................................................................. 88 3.4.1 Neuronal transcription factors are conserved between vertebrates ............ 88 3.4.2 Shh signalling is required for floor plate and neuronal marker expression in Xenopus .............................................................................................................. 89 3.4.3 Sulf1 over expression promotes ventral neuronal markers ......................... 91 3.4.4 Sulf1 is required for correct neural patterning ............................................. 91 3.4.5 Expression levels of neuronal markers are reduced in response to Sulf knockdown .......................................................................................................... 96 3.4.6 Pax6 expression is increased within the neural tube following Sulf knockdown .......................................................................................................... 97 3.4.7 A microarray screen to find genes affected by Sulf1 ..................................
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