The obesity epidemic, and resulting co-morbidities, are estimated to result in direct medical costs (U.S.) of $75 billion per year, however there are few effective treatments available. Consequently, identification of obesity-related genes may be indispensable for the development of effective therapeutics. Study of monogenic obesity mutants in the mouse and candidate gene approaches have led to the identification of a few dozen genes that play important roles in aspects of energy homeostasis. Given the complexity of the process, there are likely to be hundreds. The recent discovery of the adipostatic hormone leptin (1994), and the putative hunger factor ghrelin (1999) underscore the early stage of the discovery process in this field. Whole genome forward genetic screening for obesity-related mutations in a vertebrate system would be a highly valuable approach since entire collections of genes involved in energy homeostasis could be identified in an unbiased fashion, allowing identification both of previously unknown steps in existing pathways as well as entirely new pathways. Certain elements of the adipostat appear functionally conserved in teleosts, since the zebrafish ortholog of agouti-related protein (AgRP), a component of the hypothalamic melanocortin system, stimulates feeding when administered in the teleost brain, is upregulated by fasting, and causes obesity when overexpressed in fish. Thus, it is possible to use the zebrafish to screen for mutations that effect the central melanocortin system, and many of these mutations are likely to be relevant to mammalian energy homeostasis. In order to identify and characterize these genes using zebrafish the work proposed in this grant will focus on two essential components: 1) characterization of a collection of unique mutations affecting hypothalamic development, identified by screening the Hopkins collection of zebrafish early developmental mutants for genes that alter the expression of zebrafish POMC and AgRP and 2) completion of a large scale forward genetic screen of random retroviral insertional mutations in the zebrafish for genes that alter expression of hypothalamic POMC and AgRP genes. The genes identified in these two screens are likely to include novel genes involved in hypothalamic development, in the expression of POMC and AgRP, and possibly even genes involved in the regulation of these proteins by metabolic state. Certain elements of the adipostat, characterized in mammals, appear functionally conserved in teleosts, such as the laboratory zebrafish, and thus, it is possible to use the zebrafish to screen for mutations that effect the control of energy homeostasis, by screening for defects in conserved elements of the system, such as central melanocortin system. In this application, we propose to screen random retroviral insertional mutants in the zebrafish to identify genes that alter expression of hypothalamic POMC and AgRP. The genes identified in these two screens are likely to include novel genes involved in hypothalamic development, in the expression of POMC and AgRP, and possibly even genes involved in the regulation of these proteins by metabolic state.
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