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The Role of GDF5 in the Developing Vertebrate Nervous System

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The role of GDF5 in the developing vertebrate nervous system A thesis submitted to Cardiff University for the degree of PhD 2015 Christopher William Laurie Cardiff School of Biosciences Cardiff University Mae hwn yn ymroddedig I fy mhrydferth rhyfeddol, hebddi fyddai heb lwyddo. Dwi’n toddi, rwt ti wedi gwneud fi mor hapus. Declaration This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed ………………………………………… (candidate) Date ………………………… STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of …………………………(insert MCh, MD, MPhil, PhD etc, as appropriate) Signed ………………………………………… (candidate) Date ………………………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ………………………………………… (candidate) Date ………………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ………………………………………… (candidate) Date ………………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed ………………………………………… (candidate) Date ………………………… i Acknowledgements I would first and foremost like to thank Dr Sean Wyatt for giving me the opportunity to pursue my PhD. Thank you for being an amazing supervisor and friend by providing limitless advice and support during my time in the lab. I have been extremely lucky to have a supervisor who was so engaging and who appeared to have limitless patience and put up so courteously with my stupid questions! I do not think any PhD student could ask for a better supervisor. Thank you for everything. Thank you to Prof. Alun Davies for enabling me to tackle such an interesting research project and all the good advice along the way. Special thanks to Clara Jureky and Dr Laura Howard for making my time in lab so enjoyable and putting up with me in such a gracious manner! Thank you to Dr Catarina Osório for all her support, teaching of techniques, and general expertise as I began to embark on this research project. To all the other members of the Wyatt and Davies lab, both past and present, for you for creating such an excellent working environment and being such good friends. Thanks to all my friends, those from Cardiff, those from home and those who have moved on to new and exciting challenges in new locations. I would like to thank all my family for making it possible to complete my PhD, especially my Dad whose constant support made this all possible. Finally, to the most amazing wonderful friend any man could have. Your constant presence, love, friendship and unwavering support made this all possible. You were always happy to see me when I got home at stupid times of night, no matter how grumpy I was. I owe you more than I could ever repay. So this thesis is dedicated to you, the most amazing and wonderful dog in the world, Woody Laurie. And I should probably thank my wife, Becky Laurie. ii Abstract This thesis aimed to examine the roles of growth differentiation factor 5 (GDF5), a secreted member of the TGF-β superfamily of ligands with a well characterised role in limb morphogenesis, in the developing hippocampus and sympathetic nervous system. Previous studies have demonstrated that GDF5 promotes the growth and elaboration of dendrites from developing mouse hippocampal neurons in vitro and in vivo. As a first step to investigating whether GDF5 plays additional roles in the development of the mouse hippocampus, brains of P10 and adult Gdf5+/+, Gdf5+/bp and Gdf5bp/bp mice were analysed by anatomical MRI. The gross morphology and total volume of hippocampi were not significantly different between the three genotypes at either age, making it unlikely that GDF5 plays a significant role in modulating other aspects of hippocampal development in addition to promoting the growth and elaboration of dendrites. For this reason, no further time was spent on investigating whether GDF5 plays novel roles in regulating hippocampal development. Developing sympathetic neurons of the mouse superior cervical ganglion (SCG) require nerve growth factor (NGF) to promote their survival and target field innervation in vivo. Data in this thesis has revealed that GDF5 modulates NGF promoted survival and enhances NGF promoted process outgrowth in cultures of P0 SCG neurons. In addition, GDF5 promotes process outgrowth and branching from cultured perinatal SCG neurons in the absence of NGF. P10 Gdf5bp/bp mice, that lack functional GDF5 expression, display a marked deficit in sympathetic innervation of the iris, but not the submandibular gland, compared to P10 Gdf5+/+ mice. Whole-mount analysis of sympathetic innervation in P10 Gdf5bp/bp and Gdf5+/+ mice has revealed that GDF5 is required for correct innervation of the trachea and heart, but not the pineal gland. Further in vitro and in vivo investigations have suggested that the neurotrophic effects of GDF5 on developing SCG neurons are mediated by a receptor complex that includes the type 1 receptor, BMPR1A. These findings highlight a role for GDF5 in promoting the sympathetic innervation of selective target fields in vivo. iii Abbreviations Abbreviations Ab- Antibody Ach - Acetylcholine ACVR2 – Activin type 2 receptor ADAM - A disintegrin and metalloprotease domain Akt/PKB- Ak transforming/protein kinase B AP – Anterior-posterior axis APs- Apical progenitors AP-1- Activator protein 1 Apaf - apoptotic protease-activating factor APC - Adenomatous polyposis coli aPKC - atypical protein kinase C ADP - Adenosine diphosphate AIF - Apoptosis Inducing Factor APRIL- A proliferation-inducing ligand ATP- adenosine triphosphate ANS - Autonomic nervous system ARTN - Artemin BABB- 1 part benzyl alcohol: 2 parts benzyl benzoate Bad - BCL2-Associated Agonist Of Cell Death BAFF – B-cell-activator factor BAFFR – BAFF receptor Bak - Bcl2-Antagonist/Killer Bax - Bcl2-Associated X Protein Bcl-2 - B-cell lymphoma-2 BCMA – B-cell maturation antigen BDNF- Brain derived neurotrophic factor BH - Bcl-2 homology Bid – BH3 Interacting Domain Death Agonist Bim - Bcl-2 Interacting Mediator Of Cell Death BMP- Bone morphogenetic protein BMPR –Bone morphogenetic protein Bok - Bcl2-Related Ovarian Killer bp – Base pairs BPs – Basal progentors BSA- Bovine serum albumin °C- degree Celsius C domain – Central Domain Ca2+- Calcium CaCl2- Calcium chloride CAMs- Cell-adhesion molecules iv Abbreviations CaSR- Calcium-sensing receptor CD40- Cluster of differentiation 40 CDKs - Cyclin-dependent kinases cDNA- complementary DNA CDMP – cartilage-derived morphogenetic protein CG- Celiac ganglion cGKI - cGAMP-dependent protein kinase cGMP - Cyclic guanosine monophosphate CGT - Chondrodysplasia Grebe type CHO – Chinese hamster ovary CKI- Casein kinase I Co-Smad - Common mediator Smad CRD - Cysteine-rich domain cm- Centimetre CNP - C-type natriuretic peptide CNS- Central nervous system CNTF- Ciliary neurotrophic factor CO2- Carbon dioxide COS - CV-1 (simian) in Origin, and carrying the SV40 genetic materia CRD- Cysteine-rich domain CT-1- Cardiotrophin-1 Ct value – Cycle threshold value DAG - Diacyl-glycerol DAN – Differential screening-selected gene aberrative in neuroblastoma DAPI – 4’,6-diamidino-2-phenylindole dATP - Deoxyadenosine triphosphate DBH- Dopamine β-hydroxylase DCC - Deleted in Colorectal Carcinomas DcR- Decoy receptor DCX - Doublecortin DD- Death domain DMSO- Dimethyl sulfoxide DMEN – Dulbeccos’s modified Eagle’s mediumDNA-Deoxyribonucleic acid dNTPS- Deoxynucleotides triphosphates Dpp – Decapentaplegic DR3 – Death receptor 3 DREZ - Dorsal root entry zone DRG- Dorsal root ganglia DV – Dorsal-ventral axis DVR – Dpp and vegetal-1 (Vg1)-related E-cadherin – Epithelial cadherin EDA - Ectodysplasin A v Abbreviations E- Embryonic day 1 F-actin - Actin filaments EMT- Epithelial to mesenchymal transition Endothelin-3- End3 ENS - Enteric nervous system ER – Endoplasmic reticulum ERK- Ras/extracellular signal regulated kinase FADD- FAS-associated death domain FAK - Focal Adhesion Kinase FasL – Fas ligand FGF – Fibroblast growth factor FGFR - FGF receptor FOX – Forkhead box FRET- Fluorescence resonance energy transfer FRS2- Fibroblast receptor substrate-2 Fwd – Forward g – Gram G-actin -Globular actin monomers GAB1 - Grb2-associated-binding protein 1 GABA - Gamma-aminobutyric acid GAPDH- Glyceraldehyde 3-phosphate dehydrogenase GDNF- Glial cell derived neurotrophic factor GDP - Guanosine diphosphate GC base pairing – Guanine and Cytosine base pairing GDF – Growth differentiation factor Gdf5bp-J Growth differentiation factor 5, brachypodism-Jackson GFP- Green fluorescent protein GITR- Glucocorticoid-induced tumour necrosis factor-related protein GITRL- Glucocorticoid-induced tumour necrosis factor-related protein ligand GFAP- Glial fibrillary acidic protein GFL – GDNF family ligands GFR – GDNF family receptors GTP - Guanosine triphosphate GPI – Glycosylphosphatidylinositol Grb2 - Growth factor receptor-bound protein 2 GS region - Glycine-serine rich sequence h- Hour H2O2- Hydrogen peroxide
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  • Gene Pval Qval Log2 Fold Change AAMP 0.895690332 0.952598834

    Gene Pval Qval Log2 Fold Change AAMP 0.895690332 0.952598834

    BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut Gene pval qval Log2 Fold Change AAMP 0.895690332 0.952598834 -0.21 ABI3BP 0.002302151 0.020612283 0.465 ACHE 0.103542461 0.296385483 -0.16 ACTG2 2.99E-07 7.68E-05 3.195 ACVR1 0.071431098 0.224504378 0.19 ACVR1C 0.978209579 0.995008423 0.14 ACVRL1 0.006747504 0.042938663 0.235 ADAM15 0.158715519 0.380719469 0.285 ADAM17 0.978208929 0.995008423 -0.05 ADAM28 0.038932876 0.152174187 -0.62 ADAM8 0.622964796 0.790251882 0.085 ADAM9 0.122003358 0.329623107 0.25 ADAMTS1 0.180766659 0.414256926 0.23 ADAMTS12 0.009902195 0.05703885 0.425 ADAMTS8 4.60E-05 0.001169089 1.61 ADAP1 0.269811968 0.519388039 0.075 ADD1 0.233702809 0.487695826 0.11 ADM2 0.012213453 0.066227879 -0.36 ADRA2B 0.822777921 0.915518785 0.16 AEBP1 0.010738542 0.06035531 0.465 AGGF1 0.117946691 0.320915024 -0.095 AGR2 0.529860903 0.736120272 0.08 AGRN 0.85693743 0.928047568 -0.16 AGT 0.006849995 0.043233572 1.02 AHNAK 0.006519543 0.042542779 0.605 AKAP12 0.001747074 0.016405449 0.51 AKAP2 0.409929603 0.665919397 0.05 AKT1 0.95208288 0.985354963 -0.085 AKT2 0.367391504 0.620376005 0.055 AKT3 0.253556844 0.501934205 0.07 ALB 0.064833867 0.21195036 -0.315 ALDOA 0.83128831 0.918352939 0.08 ALOX5 0.029954404 0.125352668 -0.3 AMH 0.784746815 0.895196237 -0.03 ANG 0.050500474 0.181732067 0.255 ANGPT1 0.281853305 0.538528647 0.285 ANGPT2 0.43147281 0.675272487 -0.15 ANGPTL2 0.001368876 0.013688762 0.71 ANGPTL4 0.686032669 0.831882134 -0.175 ANPEP 0.019103243 0.089148466 -0.57 ANXA2P2 0.412553021 0.665966092 0.11 AP1M2 0.87843088 0.944681253 -0.045 APC 0.267444505 0.516134751 0.09 APOD 1.04E-05 0.000587404 0.985 APOE 0.023722987 0.104981036 -0.395 APOH 0.336334555 0.602273505 -0.065 Sundar R, et al.
  • Gene Standard Deviation MTOR 0.12553731 PRPF38A

    Gene Standard Deviation MTOR 0.12553731 PRPF38A

    BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut Gene Standard Deviation MTOR 0.12553731 PRPF38A 0.141472605 EIF2B4 0.154700091 DDX50 0.156333027 SMC3 0.161420017 NFAT5 0.166316903 MAP2K1 0.166585267 KDM1A 0.16904912 RPS6KB1 0.170330192 FCF1 0.170391706 MAP3K7 0.170660513 EIF4E2 0.171572093 TCEB1 0.175363093 CNOT10 0.178975095 SMAD1 0.179164705 NAA15 0.179904998 SETD2 0.180182498 HDAC3 0.183971158 AMMECR1L 0.184195031 CHD4 0.186678211 SF3A3 0.186697697 CNOT4 0.189434633 MTMR14 0.189734199 SMAD4 0.192451524 TLK2 0.192702667 DLG1 0.19336621 COG7 0.193422331 SP1 0.194364189 PPP3R1 0.196430217 ERBB2IP 0.201473001 RAF1 0.206887192 CUL1 0.207514271 VEZF1 0.207579584 SMAD3 0.208159809 TFDP1 0.208834504 VAV2 0.210269344 ADAM17 0.210687138 SMURF2 0.211437666 MRPS5 0.212428684 TMUB2 0.212560675 SRPK2 0.216217428 MAP2K4 0.216345366 VHL 0.219735582 SMURF1 0.221242495 PLCG1 0.221688351 EP300 0.221792349 Sundar R, et al. Gut 2020;0:1–10. doi: 10.1136/gutjnl-2020-320805 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut MGAT5 0.222050228 CDC42 0.2230598 DICER1 0.225358787 RBX1 0.228272533 ZFYVE16 0.22831803 PTEN 0.228595789 PDCD10 0.228799406 NF2 0.23091035 TP53 0.232683696 RB1 0.232729172 TCF20 0.2346075 PPP2CB 0.235117302 AGK 0.235416298
  • CD Markers Are Routinely Used for the Immunophenotyping of Cells

    CD Markers Are Routinely Used for the Immunophenotyping of Cells

    ptglab.com 1 CD MARKER ANTIBODIES www.ptglab.com Introduction The cluster of differentiation (abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules. So-called CD markers are routinely used for the immunophenotyping of cells. Despite this use, they are not limited to roles in the immune system and perform a variety of roles in cell differentiation, adhesion, migration, blood clotting, gamete fertilization, amino acid transport and apoptosis, among many others. As such, Proteintech’s mini catalog featuring its antibodies targeting CD markers is applicable to a wide range of research disciplines. PRODUCT FOCUS PECAM1 Platelet endothelial cell adhesion of blood vessels – making up a large portion molecule-1 (PECAM1), also known as cluster of its intracellular junctions. PECAM-1 is also CD Number of differentiation 31 (CD31), is a member of present on the surface of hematopoietic the immunoglobulin gene superfamily of cell cells and immune cells including platelets, CD31 adhesion molecules. It is highly expressed monocytes, neutrophils, natural killer cells, on the surface of the endothelium – the thin megakaryocytes and some types of T-cell. Catalog Number layer of endothelial cells lining the interior 11256-1-AP Type Rabbit Polyclonal Applications ELISA, FC, IF, IHC, IP, WB 16 Publications Immunohistochemical of paraffin-embedded Figure 1: Immunofluorescence staining human hepatocirrhosis using PECAM1, CD31 of PECAM1 (11256-1-AP), Alexa 488 goat antibody (11265-1-AP) at a dilution of 1:50 anti-rabbit (green), and smooth muscle KD/KO Validated (40x objective). alpha-actin (red), courtesy of Nicola Smart. PECAM1: Customer Testimonial Nicola Smart, a cardiovascular researcher “As you can see [the immunostaining] is and a group leader at the University of extremely clean and specific [and] displays Oxford, has said of the PECAM1 antibody strong intercellular junction expression, (11265-1-AP) that it “worked beautifully as expected for a cell adhesion molecule.” on every occasion I’ve tried it.” Proteintech thanks Dr.