Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/18101
Title: CHARACTERIZATION OF NOVEL MOLECULAR TOOLS AND THEIR APPLICATION TO STUDIES OF HEART DEVELOPMENT
Authors: POON KAR LAI
Keywords: zebrafish, heart, cardiac conduction system, enhancer trap, pacemakers, fhf
Issue Date: 7-Jan-2010
Source: POON KAR LAI (2010-01-07). CHARACTERIZATION OF NOVEL MOLECULAR TOOLS AND THEIR APPLICATION TO STUDIES OF HEART DEVELOPMENT. ScholarBank@NUS Repository.
Abstract: Congenital heart disease and adult onset heart failure are amongst the main causes of death in the developed world. The understanding of these diseases requires a fundamental knowledge of heart development. However, many issues of heart physiology and development still remain elusive. This can be addressed by advanced technologies and new tools. This project began with the characterization of a collection of zebrafish Cardiac Enhancer Trap (CET) lines available in the lab. The aim is to extend the limited set of living cardiac markers available, hence enhancing the experimental utility of zebrafish for cardiovascular research. Our results show that one subset of CETs defines cell layers of the heart - endocardium, myocardium and epicardium; whereas another CET subset defines various structural elements of the heart, including its chambers ¿ the atrium, ventricle as well as the valve at the atrio-ventricular boundary and bulbus arteriosus. Furthermore, genomic information delineating the transposon insertion sites provided clues about genes whose expression was tagged by the insertions resulting in identification of genes that may function in the respective cell-types or regions of the heart. Among the CET lines, I focused my detailed analysis on CETs with unique EGFP expression in the sino-atrial (SA) junction of the heart. First, I showed that the EGFP-expressing cardiac cells in these SA lines are the heart¿s pacemaker, a component of the cardiac conduction system (CCS), which is pivotal for the initiation, maintenance and coordination of the rhythmic contraction of the heart. I then demonstrated that defects of hemodynamics, absence of endocardium as well as manipulation of Notch and Bmp signalling pathway affect pacemaker cells development. FACS-assisted microarray performed to identify novel CCS enriched genes in comparison to the VII Summary working myocardium yield a list of 706 up-regulated genes and 506 down-regulated genes, at p<0.05 and a fold change cut off >=2. These set of genes when thoroughly validated will shed light on the poorly understood development of CCS. The EGFP expression in the SA lines was found to recapitulate the endogenous expression of fhf2, which has three transcript variants. These were cloned and characterized, leading to the identification of fhf2(1S) as the specific transcript variant expressed in the SA junction. Morpholino knockdown of fhf2(1S) resulted in cardiac defects reminiscent of CCS defects. In addition, RNA-binding protein rbm24, identified in a microarray screen using human embryonic stem cell-derived cardiomyocytes, was studied in a collaboration project. I demonstrated that rbm24 is expressed in cardiomyocytes before the formation of the heart tube and may function in cardiac contraction since its knockdown resulted in cardiac defect leading to a lack of blood flow. Based on the well-established role of nitric oxide in the mammalian heart, we cloned, mapped and described the expression pattern of zebrafish nitric oxide synthase (nos) genes - nos1 and nos2b. Collectively, the data presented within demonstrate the potential power of transposon assisted transgenesis to illuminate the processes of cardiovascular development, and provides a valuable resource that may be further evaluated to elucidate the molecular development of CCS.
URI: http://scholarbank.nus.edu.sg/handle/10635/18101
Appears in Collections:Ph.D Theses (Open)

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