PhD Graduate: Poon Kar Lai
Thesis Title: Characterization Of Novel Molecular Tools And Their Application To The Studies Of Heart Development And Diseases

Abstract
The causes of sudden cardiac death (SCD) are not fully understood, but they were linked to cardiac arrhythmia and cardiomyopathy, which often needs implantation of an artificial pacemaker. To be understood the molecular mechanisms behind these diseases require studies in model animals. We developed a collection of zebrafish Cardiac Enhancer Trap (CET) lines, generated by transposon-mediated insertional transgenesis, and establish these transgenics as tools for in vivo analysis of cardiac development and diseases. One of these transgenics expressed GFP in the sino-atrial node and I demonstrated that here the cardiac pacemaker, a component of the cardiac conduction system (CCS), is GFP tagged. The CCS is pivotal for the initiation, maintenance and coordination of the heart contraction. Abnormality of CCS causes arrhythmias and SCD. Hence, we developed the first zebrafish transgenics for the functional analysis of pacemaker. The EGFP expression in the SA lines recapitulates expression of fhf2 with one out of three transcript variants expressed in the pacemarker. Morpholino knockdown of fhf2 mimicked CCS defects. Further, function of a novel rbm-related gene encoding the RNA-binding protein from human ES cell-derived cardiomyocytes, a homologue of which is expressed in zebrafish cardiomyocytes prior to heart formation, was studied using zebrafish CET transgenics. In the rbm morphants, cardiac contractility is reduced and sarcomere assembly is affected illustrating cardiomyopathy. Collectively, the data proved usefulness of CET transgenics to study the molecular mechanisms behind cardiac arrhythmia and cardiomyopathy.

Diagram of zebrafish heart illustrating distribution of egfp expression domains in the cet lines.

A-C: The EGFP expression in ET33-1A, shown to be enveloped by a layer of cmlc2–dsRed-expressing myocardium (B), is expressed only in the endocardium and is absent from the rest of the vasculature. The dense vascularization of the adult ventricle is revealed by the EGFP expression, which is maintained into adulthood (C).
Click here to view the Endocardial CET movie.

D-F: The myocardial egfp expression (E) in ET33-mi3A is found to recapitulate the endogenous expression of fhla (F), which belongs to a family of FHL genes involved in myopathies.
Click here to view the Myocardial CET movie.

G-I: A thin layer of spindle shaped epicardial EGFP cells sparsely overlay the cmlc2–dsRed-expressing myocardium in the embryonic (G,H) and adult ET27 (I).

J-K: The EGFP expression in the BA of the embryonic heart (J) is restricted to the adult VB valves (K, K’)in GW2. L-N: The EGFP cells in ET31, found to be flanked by the cmlc2-expressing working myocardium (L,L’) and the endocardial cells (M) may represent the specialized myocardium of the AV conduction tissue. In the adult ET31 heart (N,N’), EGFP expression is localized to the AV valve and VB valves.
Click here to view the AV valve CET movie.

Abbreviations: A, atrium; AV, atrio-ventricular valve; BA, bulbus arteriosus; VB, ventriculo-bulbar valve; V–ventricle.

For more information on Vladimir KORZH’s lab, please click here.