ROLES OF CUG-BP, ELAV-LIKE FAMILY MEMBER 1 (CELF1), AN RNA BINDING PROTEIN, DURING VERTEBRATE HEART DEVELOPMENT by YOTAM N. BLECH-HERMONI Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Advisor: Dr. Andrea N. Ladd Program in Cell Biology, Department of Molecular Biology and Microbiology, School of Medicine CASE WESTERN RESERVE UNIVERSITY January 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Yotam N. Blech-Hermoni Candidate for the degree of Doctor of Philosophy*. Committee Chair Dr. Donna M. Driscoll Committee Member Dr. Alan M. Tartakoff Committee Member Dr. Hua Lou Date of Defense November 20, 2014 *Published material is included in accordance with contracts and stipulations set forth by the publishers. i Table of Contents List of Tables ........................................................................................................................... vii List of Figures ........................................................................................................................ viii Acknowledgements ................................................................................................................. x List of Abbreviations ............................................................................................................. xi Abstract ................................................................................................................................... xiv Chapter 1: Introduction ........................................................................................................ 1 1.1. Post-transcriptional regulation of gene expression ............................................................ 1 1.2. The CUG-BP, Elav-like family (CELF) of proteins ................................................................. 2 1.2.1. Nomenclature ........................................................................................................................ 3 1.2.2. Structure and RNA binding .............................................................................................. 6 1.2.3. The ‘Divergent Domain’ .................................................................................................... 9 1.2.4. Expression patterns and roles ....................................................................................... 9 1.3. CELF1 ................................................................................................................................................... 10 1.3.1. Roles in the cytoplasm .................................................................................................... 11 1.3.2. Roles in the nucleus ......................................................................................................... 13 1.3.3. Biological functions of CELF1 ...................................................................................... 15 1.3.4. Mouse models for the study of CELF1 ..................................................................... 17 1.4. Model organisms ............................................................................................................................. 17 1.4.1. The mouse ............................................................................................................................ 18 1.4.2. The chicken .......................................................................................................................... 20 1.4.3. The frog ................................................................................................................................. 21 1.4.4. Comparative staging ........................................................................................................ 22 ii 1.5. The current work ............................................................................................................................ 22 Chapter 2: Materials and Methods .................................................................................. 24 2.1. Animal use .......................................................................................................................................... 24 2.2. Chickens ............................................................................................................................................... 24 2.3. Chicken embryonic tissue and embryo collection ............................................................. 24 2.4. Chicken embryonic primary cardiomyocyte isolation and culture ........................... 25 2.5. Transfection of primary embryonic cardiomyocytes with siRNAs ............................ 26 2.6. EdU staining of cultured primary cardiomyocytes .......................................................... 27 2.7. Mice ....................................................................................................................................................... 28 2.8. Mouse embryo collection ............................................................................................................. 29 2.9. Western blotting and nuclear-cytoplasmic fractionation of tissues ........................ 29 2.10. Immunofluorescence [IF] .......................................................................................................... 31 2.11. In situ hybridization .................................................................................................................... 32 2.12. Semi-quantitative RT-PCR ........................................................................................................ 33 2.13. Real-time RT-PCR ......................................................................................................................... 33 2.14. Cross-linking immunoprecipitation (CLIP) ....................................................................... 36 2.15. Analysis of CLIP tags ................................................................................................................... 38 2.16. Frog embryo production ........................................................................................................... 39 2.17. Frog embryo morpholino oligonucleotide (MO) microinjection ............................. 40 2.18. Whole-mount immunohistochemistry [IHC] and IF ...................................................... 41 2.19. Bright-field imaging .................................................................................................................... 42 2.20. Frog construct design ................................................................................................................. 42 2.21. Alternative Splicing Assay ........................................................................................................ 43 iii Chapter 3: RNA binding proteins in the regulation of heart development ....... 45 3.1. Introduction ...................................................................................................................................... 45 3.2. Cardiac cell fate, heart tube formation, and differentiation ........................................ 48 3.2.1. Formation of the heart tube ......................................................................................... 53 3.2.2. RBPs in cardiomyocyte differentiation and myofibril development .......... 56 3.3. Cardiac morphogenesis ................................................................................................................ 59 3.3.1. Endocardial cushion development ............................................................................ 60 3.3.2. Myocardial trabeculation and compaction ............................................................ 63 3.4. Postnatal maturation of the heart .......................................................................................... 64 3.4.1. CELF- and MBNL-mediated alternative splicing programs ............................ 65 3.4.2. Roles of the SR protein family in the maturing heart ........................................ 67 3.5. Developmental dysregulation and disease .......................................................................... 69 3.5.1. RNA binding proteins and congenital heart defects .......................................... 70 3.5.2. The recapitulation of fetal programs during adult heart disease ................ 71 3.6. Concluding remarks ....................................................................................................................... 73 3.7. Acknowledgements ......................................................................................................................... 76 Chapter 4: Diversity and conservation of CELF1 and CELF2 RNA and protein expression patterns during embryonic development .............................................. 77 4.1. Introduction ...................................................................................................................................... 77 4.2. Results and Discussion .................................................................................................................. 80 4.2.1. CELF1 and CELF2 undergo dynamic post-transcriptional regulation during early heart development in chicken ...................................................................... 80 4.2.2. Different CELF1 and CELF2 isoforms are expressed in different tissues . 88 iv 4.2.3. Regional distribution of CELF1 and CELF2 in the chicken embryo differ between tissues