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Characterisation of the ADAMTS Family in Xenopus Development

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Characterisation of the ADAMTS Family in Xenopus Development Characterisation of the ADAMTS family in Xenopus development Ines Desanlis Thesis submitted for the degree of Doctor of Philosophy University of East Anglia School of Biological Sciences Norwich, United Kingdom August 2017 Word count: 40,361 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognize that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution Abstract Extracellular matrix (ECM) remodeling by metalloproteinases is crucial during development. The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin type I motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling. The human family includes 19 members. In this study Xenopus was used as an in vivo model for studying the function of ADAMTSs during development. 19 members of the ADAMTS family were identified in Xenopus. A phylogenetic study and synteny analysis revealed strong conservation of the ADAMTS family, provided a view of the evolutionary history and contributed to a better annotation of the Xenopus genomes. The expression of the ADAMTS family was studied from early stages to tadpole stages of Xenopus with a focus on ADAMTS9 showing expression in neural crest (NC) derivative tissues and in the pronephros. ADAMTS9 function was investigated in these structures. ADAMTS9 knock-down does not affect NC migration but causes a phenotype in the pronephros defined by a delay of development from early tailbud stage of pronephric nephrostomes, tubules and duct. Versican, a matrix component substract of ADAMTS9, is expressed in the pronephros at tailbud stage but not at tadpole stage suggesting a transient role during development. The lack of its degradation by ADAMTS9 could be the cause of the delay of pronephros development. The expression of the ADAMTS family as well as other metalloproteinases, ECM components and tissue inhibitor of metalloproteinases was studied in the skin, the brain and the heart undergoing remodeling during Xenopus laevis metamorphosis, as well as in the heart after ventricular resection to look at regeneration during Xenopus laevis metamorphosis. The results showed specific gene regulation depending on the stage of development and in response to heart injury revealing an important role for the ECM and its remodeling during these processes. ii Table of contents Abstract .................................................................................................. ii Table of contents .................................................................................. iii List of figures ....................................................................................... vii List of tables .......................................................................................... x Abbreviations ........................................................................................ xi Acknowledgements ............................................................................ xiv 1 Chapter I: Introduction ...................................................................... 1 1.1 Xenopus as an animal model ....................................................... 2 1.1.1 Life cycle and advantages ........................................................... 2 1.1.2 Animal model to study ECM remodeling during organogenesis . 3 1.1.3 Animal model to study ECM remodeling during metamorphosis 4 1.1.4 Animal model to study ECM remodeling during regeneration ..... 6 1.2 ECM in development ..................................................................... 7 1.2.1 Collagen ...................................................................................... 8 1.2.2 Fibronectin ................................................................................ 10 1.2.3 Proteoglycans ........................................................................... 10 1.2.3.1 Hyalectans ............................................................................. 11 1.2.3.2 Syndecans ............................................................................. 13 1.2.4 Matricellular proteins ................................................................. 14 1.3 Metalloproteinases ...................................................................... 15 1.3.1 Matrix Metalloproteinases (MMP) ............................................. 16 1.3.2 ADAM ........................................................................................ 19 1.3.3 ADAMTSL ................................................................................. 21 1.3.4 ADAMTS ................................................................................... 21 1.3.4.1 Evolutionary history ................................................................ 21 1.3.4.2 Molecular structure ................................................................ 24 1.3.4.2.1 Pro-domain ........................................................................ 24 1.3.4.2.2 Catalytic domain ................................................................ 26 1.3.4.2.3 Ancillary domain ................................................................. 26 1.3.4.3 Post-translational modifications ............................................. 27 1.3.4.4 Functions in development ...................................................... 27 1.3.4.4.1 Aggrecanase/Hyalectanase family ..................................... 27 1.3.4.4.2 Procollagen N-propeptidase family .................................... 29 1.3.4.4.3 von-Willebrand Factor family ............................................. 29 1.3.4.4.4 COMP proteinases family .................................................. 30 1.3.4.4.5 “orphan” family ................................................................... 30 1.3.4.5 Different mode of actions ....................................................... 30 1.3.4.6 Functions in cancer and diseases .......................................... 31 1.3.5 TIMP .......................................................................................... 31 1.4 Aims of the project ........................................................................ 32 2 Chapter II: Materials and Methods ................................................. 35 2.1 Data source of ADAMTS gene sequences ................................ 36 iii 2.2 Alignment and phylogenetic analysis ....................................... 36 2.3 Obtaining Xenopus laevis and Xenopus tropicalis embryos .. 36 2.4 Fixing and storage of Xenopus embryos .................................. 37 2.5 Skin and brain dissection from stage 57 to stage 66 .............. 37 2.6 RNA extraction ............................................................................ 37 2.6.1 For Xenopus embryos from unfertilised egg to stage 45 .......... 37 2.6.2 For Xenopus embryos from stage 57 to stage 66 ..................... 38 2.7 DNase treatment .......................................................................... 38 2.8 cDNA synthesis ........................................................................... 39 2.8.1 For Xenopus embryos from unfertilised egg to stage 45 .......... 39 2.8.2 For Xenopus embryos from stage 57 to stage 66 ..................... 39 2.9 Pre-amplification of cDNA .......................................................... 39 2.10 Polymerase Chain Reaction (PCR) ........................................... 39 2.11 Quantitative PCR ........................................................................ 40 2.11.1 96 well plate ............................................................................ 40 2.11.2 384 well plate .......................................................................... 40 2.12 Microfluidics ................................................................................ 40 2.12.1 Primers validation .................................................................... 40 2.12.2 Reaction .................................................................................. 41 2.13 Statistical analysis ...................................................................... 42 2.14 Cloning into pGEM®-T Easy Vector .......................................... 42 2.15 Gateway cloning ......................................................................... 43 2.16 Transformation ........................................................................... 44 2.17 Mini and Midi Extraction of Plasmid DNA ................................ 45 2.18 Probe synthesis .......................................................................... 45 2.18.1 Template ................................................................................. 45 2.18.1.1 Linearized plasmid ............................................................... 45 2.18.1.2 PCR product ........................................................................ 46 2.18.2 Reaction .................................................................................. 46 2.19 Capped RNA synthesis .............................................................. 47 2.20 Microinjection, morpholinos and mRNA used, GFP detection.... .......................................................................................... 48 2.21 Wholemount in situ hybridization ............................................. 48 2.22 Wholemount antibody staining ................................................
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