1. Cardiac Expression of CaMK-II  2 in Zebrafish Ludmila Francescatto Dr. Robert Tombes' Lab July 20, 2007

2. Outline The Zebrafish animal model in Research CaMK-II in Zebrafish CaMK-II  2 Summary Questions

3. Why Zebrafish? Dooley and Zon (2000) External development (not in utero) and optical clarity during embryogenesis  visual analysis of early processes High fertility and Short generation  facilitate genetic analyses Processes in Murine models are in utero, therefore less accessible. Also, space requirements, maintenance, breeding = more $$. Curr. Opin. Genet. Dev. 10(3):252-256

4. Zebrafish as a Model for Research Nematode and fruitflies aren’t good models for vertebrate development. Zebrafish are valid models for corresponding pathophysiology in Homer, er, Homo sapiens Hematopoietic, Cardiovascular, Renal, Neurologic/Ophthalmic conditions Curr. Opin. Genet. Dev. 10(3):252-256

5. Advantages of Studying the Heart in Zebrafish I n vertebrate embryos, the heart is the first organ to form and function Diffusion is inadequate as multicellularity increases & metabolic demands increase Due to the clarity of the embryo, the heart can be visualized during development. Zebrafish do not depend on the cardiovascular circulation in order to survive during the first days of development. Therefore, it is possible to study cardiac mutation and also the recovery of mutants. Dev. Dyn. 222:552-563, 2001.

6. Stages of Embryonic Development of the Zebrafish

7. CaM Kinase II (CaMK-II) Ubiquitous protein kinase Ca 2+ /CaM dependent Alters key substrates by phosphorylation throughout the cell Undergo an autophosphorylation process that regulates its dependence on Ca 2+ /CaM Contains catalytic, variable and association domains

8. Structure of CaMK-II

9. Research in the Tombes’ Lab

10. Why Study CaMK-II in the Zebrafish Heart? Developmental defects in the heart rate and circulation were seen with use of a CaMK-II inhibitor, KN93 KN93 binds competitively to the calmodulin-binding domain of CaMK-II This naturally leads to the question of the role of CaMK-II in the development of the heart in vertebrates

11. CaMK-II Genetics There are different forms of CaMK-II, called splice variants which explain the variety of this enzyme Mammals & birds have four different genes (α, β, γ, δ) Possibly due to a duplication event, zebrafish has at least seven genes (α1, β1, β2, γ1, γ2, δ1, δ2) CaMK-II  2 was the most recently discovered among the seven & is currently our focus

12. Earlier studies on CamK-II genes (not including  2) revealed CamK-II expression in many parts of the zebrafish embryo For example,  1 in pectoral fins, midbrain, forebrain, gut, retina, & somites CamK-II inhibitors eg KN93 cause developmental defects in the cardiovascular system of the zebrafish. How do we explore which CamK-II genes are involved in cardiogenesis, and what their specific roles are? CaMK-II Genetics (cont’d) Rothschild, et al. Dev. Dyn. 236:295-305, 2007

13. CaMK-II  2 Designing the morpholino and probe for in situ hybridization requires knowing the Genetic sequence of  2 Genetic sequence of  2 was procured via BLAST (Basic Local Alignment Search Tool) Primers were designed for CaMK-II  2 and gene was amplified Gene cloned, screened and sequenced Analysis of splice variants demonstrated that four isoforms of  2 had been found (B, G, C, e) Within these four isoforms, none contain nuclear domains; rather, they are all cytosolic

14. Temporal Expression of CaMK-II  2 Isoforms No Yes 84 hpf No Yes72 hpf No Yes48 hpf Yes No 12 hpf NoYesNoYesMaternal  2-e  2-C  2-G  2-B

15. In Situ Hybridization From the CaMK-II Beta2 sequence, a probe was made for in situ hybridization use From the CaMK-II Beta2 sequence, a probe was made for in situ hybridization use This technique analysed CaMK-II mRNA expression in the embryo This technique analysed CaMK-II mRNA expression in the embryo

16. In Situ Hybridization Gilbert, Scott F, Developmental Biology 8E (2006)

17. In Situ Hybridization (cont'd) The heart is located posterior to the head (seen at LEFT) and anterior to the yolk.

18. In Situ Hybridization (cont'd) ● ● Results obtained from the in situ hybridization demonstrates that CaMK-II  2 is in fact expressed in the heart. ● ● Temporally, it is known that the looping of the zebrafish heart occurs at 36 hours post fertilization (hpf) ● ● Experimentally,  2 expression is seen at 36hpf ● ● At 37hpf  2 is expressed weakly. ● ● By 38hpf, the  2 mRNA is no longer detectable ● ● Thus the expression of mRNA  2 coincides temporally with the looping event of the zebrafish heart

19. CaMK-II Inhibitors

20. Summary Zebrafish is a valid model for studying human development CaMK-II inhibitors eg KN93 reveal a critical role of CaMK-II in cardiovascular development in the embryo Previous studies on CaMK-II genes did not illustrate expression in the heart Sequencing the CaMK-II gene demonstrated the presence of mRNA at certain time points In situ hybridization visually confirmed temporal expression of  2 during the heart looping event The mechanism by which  2 impacts heart looping is yet to be elucidated, but has implications for normal development of the anatomy and physiology of the cardiovascular system

21. References Rothschild, Sarah C., Lister, James A., Tombes, Robert M. “Differential Expression of CaMK-II During Early Zebrafish Embryogenesis.” Dev. Dyn. 236:295-305, 2007. Tombes, Robert M., Faison, M. O., Turbeville, J. M. “Organization and evolution of multifunctional Ca 2+ /CaM-dependent protein kinase genes.” Gene 322: 17-31, 2003. Hudmon, A., Schulman, H. “Neuronal Ca 2+ / Calmodulin-Dependent Protein Kinase II: The Role of Structure and Autoregulation in Cellular Function.” Ann. Rev. Biochem 71: 473-510, 2002. Hudmon, A., Schulman, H. “Structure-function of the multifunctional Ca 2+ / calmodulin-dependent protein kinase II. Biochem. J. 364, 593-611, 2002. Gilbert, Scott F. Developmental Biology. Sinauer Associates, Inc. 6 th 2000 Dooley, K., Zon, L. “Zebrafish: a model for the study of human disease.” Curr Opin Genet Dev. 10: 252-6, 2000 Yelon, D. “Cardiac Patterning and Morphogenesis in Zebrafish.” Dev. Dyn. 222:552-563, 2001.

22. Acknowledgements Dr. Tombes' Lab Robert Tombes Sarah Rothschild Chas Easley Jamie McLeod Colleen M Simon

23. CaMK-II Genetics (cont’d) Morpholinos were designed and employed to prevent cells from making different CaMK-II isoforms Morpholinos are synthetic nucleic acid molecules used to explore mRNA transcripts in embryos Complementarily bind to specifically designated sequences of RNA for ~25 base pairs Mechanism of action (“how Morpholinos work”) Prevent the Ribosomal initiation complex from advancing, thereby precluding translation of the coding region Insert picture of morpholino HERE