RESUMO
Forward genetics is an unbiased methodology to discover new genes or functions of genes. At the present, the zebrafish is one of the few vertebrate systems where large-scale forward genetic studies are practical. Fluorescent lipid labeling of zebrafish larvae derived from families created from ENU-mutagenized fish enabled us to perform a large scale in vivo screen to identify mutants with perturbed lipid processing. With the aid of the zebrafish genome project, positional cloning of mutated genes with abnormal lipid metabolism can be accelerated. MO- and gripNA-based transient gene silencing is feasible in zebrafish embryos and provides a reverse genetic screening strategy to search for important lipid regulators. The advantages of using zebrafish as a vertebrate model to study lipid metabolism include its rapid external development and its optical clarity that enables the monitoring of biological processes. Large scale, high-throughput drug screening in vivo, especially for drugs that inhibit lipid absorption, can be easily achieved in this model. These zebrafish-based assays are important tools to understand aspects of lipid biology with significant clinical implications.
Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Colesterol/análogos & derivados , Metabolismo dos Lipídeos , Peixe-Zebra/metabolismo , 4-Cloro-7-nitrobenzofurazano/metabolismo , Alquil e Aril Transferases/antagonistas & inibidores , Animais , Anexina A2/metabolismo , Atorvastatina , Azetidinas/farmacologia , Compostos de Boro/química , Caveolina 1 , Caveolinas/metabolismo , Movimento Celular/efeitos dos fármacos , Colesterol/metabolismo , DNA/efeitos dos fármacos , DNA/genética , Diterpenos/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Etilnitrosoureia/farmacologia , Ezetimiba , Farneseno Álcool/farmacologia , Farnesiltranstransferase , Geraniltranstransferase , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Ácidos Heptanoicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lipídeos/análise , Lovastatina/farmacologia , Ácido Mevalônico/farmacologia , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Mutação , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Ligação Proteica/efeitos dos fármacos , Prenilação de Proteína/efeitos dos fármacos , Pirróis/farmacologia , Sinvastatina/farmacologia , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Hydroxymethylglutaryl coenzyme A reductase (HMGCoAR) is required for isoprenoid and cholesterol biosynthesis. In Drosophila, reduced HMGCoAR activity results in germ cell migration defects. We show that pharmacological HMGCoAR inhibition alters zebrafish development and germ cell migration. Embryos treated with atorvastatin (Lipitor) exhibited germ cell migration defects and mild morphologic abnormalities. The effects induced by atorvastatin were completely rescued by prior injection of mevalonate, the product of HMGCoAR activity, or the prenylation precursors farnesol and geranylgeraniol. In contrast, squalene, a cholesterol intermediate further down the pathway, failed to rescue statin-induced defects. Moreover, pharmacologic inhibition of geranylgeranyl transferase 1 (GGT1) protein prenylation activity also resulted in abnormal germ cell migration. Thus, our pharmacological inhibition-and-rescue approach provided detailed information about the elements of isoprenoid biosynthesis that contribute to germ cell migration. Together with data from Drosophila (Santos and Lehmann, this issue), our results highlight a conserved role for protein geranylgeranylation in this context.
