The fruit fly Drosophila melanogaster contains a primitive linear heart tube, providing a unique model system to study the heart development. Although during last decade studies of cardiac morphogenesis using this model animal identified important genes for cardiac development, many cardiogenic genes remain to be identified in order to gain insight into the genetic control mechanism of cardiac morphogenesis. Using information obtained Drosophila genome sequencing and and RNAi technology as a new strategy for blocking gene function we have initiated genome-wide screening of regulatory genes that are involved in Drosophila heart development. The term RNA interference (RNAi) describes the use of double-stranded RNA (dsRNA) to target specific mRNAs for degradation, thereby silencing their expression. For screening we have injected dsRNA into embryos that harbor beta-galactosidase transgene which is mainly expressed in cells of cardiogenic lineage. By monitoring change in expression pattern of beta-galactosidase transgene and morphology of the heart in the injected embryos we can easily identify potential cardiogenic genes. We have prepared dsRNAs by in vitro transcription using template DNAs from unigene set (6,000 genes). In addition, in order to test function of genes that are not included in a unigene set we have synthesized short dsRNAs (21-mer) and tested its RNAi activity in the injected embryos. We have found that the 21-mer dsRNA shows very effective and specific RNAi activity. When we have injected beta-galactosidase transgene dsRNA (21-mer) into the embryos, it silences beta-galactosidase gene expression without affecting normal embryonic development. Also, when we have injected the tinman dsRNA (21-mer), it produces embryos lacking tissues deribed from the dorsal mesoderm which mimics the loss-of-function phenotype of the endogenous tinman mutant embryo. Using this scheme we are in progress of identifying potential cardiogenic genes including both novel genes suchs as dhipk2 and known genes of which the cardiogenic function was not described previously. Because the genetic control mechanism of the embryonic cardiac development of the Drosophila is well conserved in mammallian heart development, identifying cardiogenic regulatory genes in Drosophila should help us understand the genetic control mechanism of the mammalian cardiac morphogenesis.
| Kim, Yong-Ou; Park, Sang-Joon; Balaban, Robert S et al. (2004) A functional genomic screen for cardiogenic genes using RNA interference in developing Drosophila embryos. Proc Natl Acad Sci U S A 101:159-64 |
