Cardiac Expression of CaMK-II 2 in

ZebrafishLudmila Francescatto

Dr. Robert Tombes' Lab

July 20, 2007

OutlineOutline

• The Zebrafish animal model in Research• CaMK-II in Zebrafish• CaMK-II 2• Summary• Questions

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

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

Advantages of Studying the Heart in Zebrafish

• In 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, 2001Dev. Dyn. 222:552-563, 2001..

Stages of Embryonic Development of the Zebrafish

CaM Kinase II (CaMK-II)

• Ubiquitous protein kinase• Ca2+/CaM dependent• Alters key substrates by phosphorylation

throughout the cell• Undergo an autophosphorylation process

that regulates its dependence on Ca2+/CaM

• Contains catalytic, variable and association domains

Structure of CaMK-II

Research Research in thein the

Tombes’ Tombes’ LabLab

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

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

• 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 CaMK-II Genetics (cont’d)(cont’d)

Rothschild, et al. Dev. Dyn. 236:295-305, 2007Rothschild, et al. Dev. Dyn. 236:295-305, 2007

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

Temporal Expression of CaMK-II 2 Isoforms

NoNoYesYes84 hpf

NoNoNoYes72 hpf

NoNoNoYes48 hpf

YesYesNoNo12 hpf

NoYesNoYesMaternal

2-e2-C2-G 2-B

In Situ HybridizationIn Situ Hybridization

• From the CaMK-II Beta2 sequence, a From the CaMK-II Beta2 sequence, a probe was made for in situ probe was made for in situ hybridization usehybridization use

• This technique analysed CaMK-II This technique analysed CaMK-II mRNA expression in the embryomRNA expression in the embryo

In Situ Hybridization

Gilbert, Scott F, Developmental Biology 8E (2006)

In Situ Hybridization In Situ Hybridization (cont'd)(cont'd)

The heart is located posterior to the The heart is located posterior to the head (seen at LEFT) and anterior to head (seen at LEFT) and anterior to the yolk.the yolk.

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

CaMK-II InhibitorsCaMK-II Inhibitors

SummarySummary• 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 which2 impacts heart looping is yet to be elucidated, but has implications for normal development of the anatomy and physiology of the cardiovascular system

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 Ca2+/CaM-dependent protein kinase genes.” Gene 322: 17-31, 2003.

Hudmon, A., Schulman, H. “Neuronal Ca2+/ 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 Ca2+/ calmodulin-dependent protein kinase II. Biochem. J. 364, 593-611, 2002.

Gilbert, Scott F. Developmental Biology. Sinauer Associates, Inc. 6th 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.

Acknowledgements

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

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