The role of proteoglycans in the initiation of neural tube closure Oleksandr Nychyk Thesis submitted to UCL for the degree of Doctor of Philosophy 2017 Developmental Biology of Birth Defects Developmental Biology & Cancer Programme UCL Great Ormond Street Institute of Child Health Declaration of contribution I, Oleksandr Nychyk confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in my thesis. ___________________________________________ Oleksandr Nychyk 2 Abstract Neurulation is the embryonic process that gives rise to the neural tube (NT), the precursor of the brain and spinal cord. Recent work has emphasised the importance of proteoglycans in convergent extension movements and NT closure in lower vertebrates. The current study is focused on the role of proteoglycans in the initiation of NT closure in mammals, termed closure 1. In this project, the initial aim was to characterise the ‘matrisome’, or in vivo extracellular matrix (ECM) composition, during mammalian neurulation. Tissue site of mRNA expression and protein localisation of ECM components, including proteoglycans, were then investigated showing their distinct expression patterns prior to and after the onset of neural tube closure. The expression analysis raised various hypothesis that were subsequently tested, demonstrating that impaired sulfation of ECM proteoglycan chains worsens the phenotype of planar cell polarity (PCP) mutant loop tail (Vangl2Lp) predisposed to neural tube defects. Exposure of Vangl2Lp/+ embryos to chlorate, an inhibitor of glycosaminoglycan sulfation, during ex vivo whole embryo culture prevented NT closure, converting Vangl2Lp/+ to the mutant Vangl2Lp/Lp pathophenotype. The same result was obtained by exposure of Vangl2Lp/+ + embryos to chondroitinase or heparitinase. Taken together, it indicated that the PCP pathway functionally interacts with chondroitin and heparan sulfate proteoglycans during initiation of NT closure. In order to investigate the possible role of proteoglycans in mammalian convergent extension, the node of Vangl2Lp/+ embryos was labelled with DiO. The study revealed that the PCP-proteoglycan interaction is mediated independently of convergent extension. The failure of neural fold apposition and reduced Fgfr1 signalling was proposed as potential causative mechanism underlying failure of closure 1. In fish, the cilia motility is dependent on heparan sulfate chains, but this has not been studied in mammals. The present study identified a novel cellular localisation of cohesin/proteoglycan protein Smc3 and its GAG chains. Both Smc3 and CS-E are expressed in the midbody, primary and motile cilia. For the first time, this study showed the nuclear expression of CS chains in mouse embryo. The coordinated movement of Smc3 and CS-E chains during cytokinesis and ciliogenesis suggests conserved role of this protein in mouse cilia and cytokinetic apparatus. 3 Acknowledgements I would like to thank my supervisor, Andrew Copp, for excellent supervision and guidance over these four years. I have been extremely lucky to have a supervisor who cared so much about my work, and who responded to my questions and queries so promptly, and corrected my English. Thanks to my secondary supervisor Philip Stanier and a group leader Nick Greene for giving me a support and advice when I needed. I would like also to thank Paula and J.P. for providing me valuable suggestions and feedback. I am especially indebted to Gabe for being such a great mentor that always wheeling to help and support me. Many thanks to Matteo for being a good friend and a wonderful colleague from a Matrix field. I would also like to thank Sandrita and Dorothee for always finding a moment to talk and help me in the lab. Special thanks to Ana for giving me the SEM data and to Dale for being a great helper in the microscopy field. Also, I would like to thank all other members of Neural Tube Defects group. I am extremely thankful to my Mum and Dad for believing in me and giving me a chance to be what I am today. Finally, I am so thankful to my wife Jolita and my son Alexander for all their love, a huge patience and encouragement. I couldn’t have done this work without them. 4 Table of Contents Abstract………………………………………………………………………………………………………………………………………………3 Acknowledgments……………………………………………………………………………………………………………………………. 4 Table of Contents ………………………………………………………………………………………………………………………………5 List of Figures and Tables …………………………………………………………………………………………………………………10 Abbreviations …………………………………………………………………………………………………………………………………..13 1. General introduction ………………………………………………………………………………………………15 1.1. Neurulation ……………………………………………………………………………………………………15 1.1.1. The primary neurulation sequence: closure sites and timing …………………………..........………..15 1.1.2. Neural plate shaping and bending ……………………………………………………………………...........……17 1.1.3. Fusion of neural folds ……………………………………………………………………………….........……………...19 1.1.4. Neural tube defects ……………………………………………………………………………………………..........……20 1.2. The planar cell polarity pathway ……………………………………………………………………………………………21 1.2.1. Vertebrate PCP genes ……………………………………………………………………………………………………….21 1.2.2. PCP-dependent NT closure ……………………………………………………………………………………………….23 1.2.3. The loop-tail mouse model ……………………………………………………………………………………………….25 1.3. Glycosaminoglycans and proteoglycans ………………………………………………………………………………..28 1.3.1. Glycosaminoglycans ………………………………………………………………………………………………………….28 1.3.1.1. Biosynthesis of GAG chains …………………………………………………………………………29 1.3.1.2. Impairment of HS biosynthesis …………………………………………………………………...33 1.3.1.3. Impairment of CS biosynthesis …………………………………………………………………...33 1.3.2. Proteoglycans …………………………………………………………………………………………………………………..34 1.3.2.1. Syndecans and glypicans ……………………………………………………………………………34 1.3.2.2. Basement membrane proteoglycans ………………………………………………………….37 1.3.2.3. Versican, aggrecan, phosphocan and bamacan ..………………………………………..37 1.4. FGF/FGFR expression and signalling during development .........................................................39 1.5. Left-right asymmetry …………………………………………………………………………………………………………….41 1.6. Thesis hypothesis, aims and objectives ..............................………………………………...…………………45 2. Materials and Methods ……………………………………………………………………………………………………………..47 5 2.1. Mouse strains and genotyping ……………………………………………………………………………………………...47 2.1.1. Vangl2Lp ………………………………………………………………………………………………………………………….…47 2.1.2. Vangl2flox ……………………………………………………………………………………………………………………….....47 2.1.3. β-actincre …………………………………………………………………..………………………………………………………47 2.1.4. Wild type ..………………………………………………………………………………………………………………………..47 2.1.5. Genotyping ……………………………………………………………………………………………………………………….47 2.1.5.1. DNA extraction ……………………………………………………………………………………………47 2.1.5.2. Vangl2 genotyping assays ..…………………………………………………………………….…..48 2.1.5.3. Cre genotyping assays …………………………………………………………………………………50 2.1.5.4. Agarose gel electrophoresis …………………………………………………………………….….51 2.2. Mouse embryology ……………………………………………………..………………………………………………………..51 2.2.1. Embryo collection, dissection and storage .……………………………………………………………………...51 2.2.2. Preparation of rat serum for embryo culture ……………………………………………………………….……51 2.2.3. Whole embryo culture ……………………………………………………………………………………………….……..52 2.2.3.1. Assessment of embryos after the culture .……………….…………………………….……52 2.2.3.2. Chlorate treatment ………………………………………………………………………….............53 2.2.3.3. Chondroitinase ABC and HeparitinaseIII treatment ........................................53 2.2.3.4. DiO labelling of the node .……………………………………………………………………….....54 2.2.3.5. Embryonic measurements …………………………………………………………………………..54 2.3. RNA-seq ………………………………………………………………………….........................................................55 2.3.1. Tissue collection for RNA-seq, RNA extraction and quality control .......................................55 2.3.2. Generation of library and sequencing ...................................................................................55 2.3.3. Bioinformatics analysis ..........................................................................................................56 2.3.4. Analysis of the transcriptome of mid-gestation mouse embryos (MGME) from Werber 2014 study ………………………………………………………………………….........................................................57 2.4. Histology ………………………………………………………………………..........................................................57 2.4.1. Cryo-embedding ………………………………………………………………………..........................................57 2.4.2. Cryosectioning …………………………………………………………………...................................................57 2.5. 2.5 In situ hybridization ………………………………………………………………….........................................57 2.5.1. RNA isolation and cDNA synthesis ........................................................................................58 6 2.5.2. RNA probe preparation for in situ hybridization ..................................................................58 2.5.2.1. Probe design ...................................................................................................58 2.5.2.2. Ligation of the probe into the vector system .................................................61 2.5.2.3. Transformation of competent cells ................................................................61 2.5.2.4. Preparation of plasmid and transcription of DIG probe .................................61 2.5.3. Whole mount in situ hybridization ........................................................................................62