Molecular cloning and expression analysis of ADAMs and cadherins during chicken embryonic development Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller-Universität Jena von Juntang Lin Master of Science in Zoologie (M.Sc.) geboren am 08 August 1976 in der Provinz Henan, China Molecular cloning and expression analysis of ADAMs and cadherins during chicken embryonic development Dissertation for obtaining the degree of Doctor rerum naturalium (Dr. rer. nat.) at the Faculty of Biology and Pharmacy, Friedrich-Schiller-University Jena submitted by Juntang Lin Master of Science in Zoology (M.Sc.) Born on 08 August 1976 in Henan Province, China Reviewers: Prof. Dr. Dr. Christoph Redies Institute of Anatomy I Friedrich-Schiller-University Jena, School of Medicine Teichgraben 7 07740 Jena, Germany Prof. Dr. Jürgen Bolz Institute of General Zoology and Animal Physiology Faculty for Biology and Pharmaceutics Friedrich-Schiller-University Jena Erbert Str. 1 07743 Jena, Germany Prof. Dr. Karina Reiss Department of Dermatology and Allergology University Hospital Schleswig-Holstein Schittenhelm Str. 7 24105 Kiel, Germany Day of the public defence: 16.12.2008 Content I Contents ABBREVIATIONS………………………………………………………………………III 1. INTRODUCTION…………………………………………………………………..…1 1.1 The chicken as an experimental model organism…………………………….…….1 1.2 Developmental processes mediated by cell adhesion molecules (CAMs)………....2 1.3 ADAMs play versatile roles during embryonic development…………………..….3 1.3.1 Structure and potential functions of ADAMs………………………………...…3 1.3.2 Expression and role of ADAMs in embryonic development……………………5 1.3.3 ADAMs in the developing nervous system……………………………………..7 1.4 Cadherins are important to embryonic development……………………..………..9 1.4.1 Structure and functions of cadherins…………………………………………….9 1.4.2 Cadherins in embryonic development………………………………..………...11 1.4.3 Cadherins in the developing and adult CNS…………………………………...12 1.5 Interaction between ADAMs and cadherins…………………………………...…..13 1.6 Aims of the study and experimental approach………………………………….....15 2. PUBLICATION OVERVIEW……………………………………………………….17 3. PUBLICATIONS……………………………………………………………………..20 3.1 Juntang Lin, Christoph Redies, Jiankai Luo. Regionalized expression of ADAM13 during chicken embryonic development. Developmental Dynamics, 2007, 236:862- 870……………………………………………………………………………………20 3.2 Jiankai Luo, Hong Wang, Juntang Lin, Christoph Redies. Cadherin expression in the developing chicken cochlea. Developmental Dynamics, 2007, 236:2331-2337……..30 3.3 Juntang Lin, Jiankai Luo, Christoph Redies. Molecular cloning and expression analysis of three cadherin-8 isoforms in the embryonic chicken brain. Brain Research, 2008, 1201:1-14………………………………………………………………….…..38 3.4 Juntang Lin, Jiankai Luo, Christoph Redies. Differential expression of five members of the ADAM family in the developing chicken brain. Neuroscience, 2008 (in press)……………………………………………………………………………........53 Content II 4. ADDITIONAL RESULTS………………………………………………...…………95 Overexpression of ADAM17 remodels blood vessels in the developing chicken tectum 5. DISCUSSION…………………………………………………………………..……109 5.1 Expression of ADAMs in the developing chicken embryo………………………110 5.1.1 ADAM13 expression during chicken embryonic development…………....…110 5.1.2 ADAM expression in the CNS………………………………………………..112 5.1.3 ADAM17 overexpression enlarges the wall of blood vessel through increasing the number of pericytes in the developing optic tectum…………...…………113 5.2 Cadherins are markers to analyze functional pattern formation in the nervous system……………………………………………………………………………….115 5.2.1 Cadherin expression in the developing chicken cochlea……………………...117 5.2.2 Cadherin-8 expression in the embryonic chicken brain……………………....118 5.3 Interaction between ADAMs and cadherins……………………………………...120 6. SUMMARY…………………………………………………………………………..122 7. ZUSAMMENFASSUNG…………………………………………………………….124 8. REFERENCES………………………...…………………………………………….126 9. APPENDIX…………………………………………………………………………..142 GenBank information on the submitted sequences 10. ACKNOWLEDGEMENTS……………………………………………………….147 Abbreviations III ABBREVIATIONS ADAM A disintegrin and metalloprotease APP Amyloid precursor protein BBB Blood-brain barrier bp Base pair CAM Cell adhesion molecule CD Cluster of differentiation Cdh Cadherin cDNA Complementary deoxyribonucleic acid C. elegans Caenorhabditis elegans CHL1 Cell adhesion molecule with homology to L1CAM CNS Central nervous system cRNA Complementary ribonucleic acid CTF C-terminal fragment CX3CL CX3 chemokine ligand DNA Deoxyribonucleic acid dsRNA Double-stranded RNA E Embryonic day E-Cdh Epithelial cadherin EGF Epithelial growth factor EGFP Enhanced green fluorescence protein EGFR Epithelial growth factor receptor ER Endoplasmic reticulum Fig. Figure Fn Fibronectin GAPDH Glyceraldehyde-3-phosphate dehydrogenase GBSS Gey’s buffered salt solution GFP Green fluorescence protein Golgi Golgi compartment HB-EGF Heparin-binding epidermal growth factor-like growth factor HBSS Hepes-buffered saline solution IGFBP Insulin-like growth factor-binding protein Abbreviations IV IgG Immunoglubulin G IL Interleukin ISH In situ hybridization MBP Myelin basic peptide MDC Metalloprotease/disintegrin/cysteine-rich min Minute MMP Matrix metalloproteinase mRNA Message ribonucleic acid ms Milli-second N-Cdh Neural cadherin ne Neuroepithelium NTF N-terminal fragment ORF Open reading frame P-Cdh Placental cadherin PBS Phosphate-buffered saline Pcdh Protocadherin PCR Polymerase chain reaction Pe Periventricular stratum PFA Paraformaldehyde PK Protease K PKC Protein kinase C PSGL P-selectin glycoprotein ligand R-Cdh Retinal cadherin RACE Rapid amplification of cDNA ends RCAS Replication competent avian splice RNA Ribonucleic acid RNAi Ribonucleic acid interference RT-PCR Reverse transcriptase polymerase chain reaction SAC Stratum album centrale of the tectum sec Second S.E.M Standard error of the mean SGC Stratum griseum centrale of the tectum SGFS Stratum griseum et fibrosum superficiale of the tectum Abbreviations V siRNA Small interfering RNA SMA Alpha smooth muscle actin SSC Standard sodium citrate TACE TNF-alpha converting enzyme TBS Tris-buffered saline Tect Tectum TEM Transmission electron microscopy TIMP Tissue inhibitor of metalloproteinase TM Transmembrane domain TNF Tumor necrosis factor TNFR Tumor necrosis factor receptor VE-Cdh Vascular endothelial cadherin 1. Introduction 1 1. INTRODUCTION This present dissertation explores the expression patterns of members of the ADAM and cadherin gene families in the chicken embryo. Section 1.1 of the Introduction introduces the chicken embryo as an experimental system in developmental biology. Section 1.2 is an outline about the superfamily of cell adhesion molecules, which includes the ADAM and cadherin families. Sections 1.3 and 1.4 give an overview on the functions of ADAMs and cadherins in cell adhesion and related developmental processes, especially during nervous system development. Section 1.5 describes the molecular interactions between ADAMs and cadherins and underlines the motivation to study both families of genes in this work. Section 1.6 outlines the aim and experimental approach of the present dissertation. 1.1 The chicken as an experimental model organism The chicken (Gallus gallus) is an important model organism for biomedical research, embryonic development, and molecular biology (Hamburger and Hamilton, 1951; Bellairs and Osmond, 2005). The chicken embryo has served as one of the most widely used experimental animals for both teaching and research for more than a century, for the following reasons: (1) fertilized eggs are cheap and available in large numbers at any time; (2) chicken embryonic development is fast in comparison with most mammalian embryos, lasting only 21 days, which is similar to that of the mouse; (3) the physiology of the chicken embryo is not complicated by the presence of a placenta; (4) chicken genetics are known well, both for normal embryos and for different mutant forms; and (5) as an amniote, chicken development resembles that of mammals in its broad outline (Bellairs and Osmond, 2005). The recent completion of the sequencing of the chicken genome has enhanced the importance of this species in embryology and developmental biology (http://www.ncbi.nlm.nih.gov/projects/genome/guide/chicken/). More recently, the chicken embryo has also been used broadly in the field of molecular biology and has provided an important model system for studies of gene regulation. Although chicken gene “knock out” is impractical because embryonic stem cells are elusive and generation times in the hen’s reproductive organ are long (Brown et al., 2003), the application of plasmid-based RNA interference (RNAi) in conjunction with in vivo (in ovo or ex ovo) electroporation makes loss-of-function studies possible (Das et al., 2006). In conjunction with gene overexpression (gain-of-function) mediated by the in ovo or ex ovo electroporation of 1. Introduction 2 recombinant plasmids, the chicken embryo allows to fully explore gene function regionally and temporally (Muramatsu et al., 1997, 1998; Luo and Redies, 2004, 2005). The Redies laboratory has extensive experience in chicken embryonic development, especially in the development of the central nervous system (CNS), both in the context of descriptive studies (Arndt et al., 1998; Fushimi et al., 1997; Redies et al., 1997, 2001, 2002a, b; Arndt and Redies, 1996, 1998;