A functional genomic screen for cardiogenic genes using RNA interference in developing Drosophila embryos

Abstract

Identifying genetic components is an essential step toward understanding complex developmental processes. The primitive heart of the fruit fly, the dorsal vessel, which is a hemolymph-pumping organ, has provided a unique model system to identify cardiogenic genes and to further our understanding of the molecular mechanisms of cardiogenesis. Using RNA interference in developing Drosophila embryos, we performed a genomewide search for cardiogenic genes. Through analyses of the >5,800 genes that cover Ϸ40% of all predicted Drosophila genes, we identified a variety of genes encoding transcription factors and cell signaling proteins required for different steps during heart development. Analysis of mutant heart phenotypes and identified genes suggests that the Drosophila heart tube is segmentally patterned, like axial patterning, but assembled with regional modules. One of the identified genes, simjang, was further characterized. In the simjang mutant embryo, we found that within each segment a subset of cardial cells is missing. Interestingly, the simjang gene encodes a protein that is a component of the chromatin remodeling complex recruited by methyl-CpG-DNA binding proteins, suggesting that epigenetic information is crucial for specifying cardiac precursors. Together, these studies not only identify key regulators but also reveal mechanisms underlying heart development. O ver the last decade significant progress has been made in our understanding of the molecular details of cardiac morphogenesis. These advances can be attributed to the fact that many embryonic events involved in cardiogenesis are remarkably conserved among distinct species (1-3). In the Drosophila embryo, bilaterally symmetric groups of cardiac precursor cells in the dorsal-most mesoderm, specified by positional information provided by intrinsic cell-autonomous factors and extrinsic signals, meet in the dorsal midline to form a linear heart tube (3). Unlike the vertebrate linear heart tube, which proceeds further to looping and multichamber formation, the Drosophila heart remains a contractile linear tube at maturity. Two major classes of cell observed within the Drosophila cardiogenic repertoire are cardial cells (cardioblasts), which form the lumen of the linear heart tube, and pericardial cells, which flank and are associated with cardial cells (4). Cardial cells eventually form the cardiac muscles and are the contractile cells of the heart, whereas pericardial cells do not express muscle proteins but may function in hemolymph filtration. The linear heart tube shows clear structural differences along its length after its formation in late embryogenesis. The posterior three segments constitute the heart, a contractile tube with a wide bore involved in the forward pumping of hemolymph. Three pairs of specialized inflow valves (ostia) are present in the heart to facilitate the lateral entry of hemolymph (5, 6). Anterior to the heart is a narrower section, termed the aorta, that encompasses four segments. Despite its apparently simple structure an...

Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors

“…Thus, the ability of Lb to act either as repressor or as activator suggests a context-dependent interaction with cofactors. Of note, several miroarray identified Lb targets have also been found in the RNAi-based screen for genes involved in heart morphogenesis (see Supplementary Table S5; Kim et al 2004).…”

Development of RNAi Libraries for Target Validation and Therapeutics

“…The population will be analyzed 24,48,96,120,168 and 240 hours after transfection to determine the percentage of cells and the levels of expression in the population. It is predicted that at early times, there will be a large distribution of levels of expressed GFP within the population since non-integrated plasmids will be expressing GFP and that at later times a more homogenous population will be obtained consisting primarily of single integrants at the Flp site.…”

“…An alternative approach to that taken in generating the random siRNA library (14), would be to restriction digest cDNA to generate a library of long dsRNAs. Long dsRNA libraries have been used to analyze gene function in Drosophila (23,24) but potential off-target effects and induction of the stress response are issues for mammalian gene silencing. Thus, the focus of this study was to analyze the efficiency, selectivity and toxicity of long dsRNAs in mammalian cells and to determine if long dsRNAs lead to the desired biological response in these cells, as a first step to generate long dsRNA libraries.…”

Premios nobel en Fisiología o Medicina y Química, año 2006. Una nueva dimensión del ARN en la regulación de la expresión genética y como herramienta experimental y terapéutica

“…En poco tiempo ya se han generado nuevas investigaciones en busca del conocimiento de la fisiopatología molecular de enfermedades con alteraciones en la regulación de la expresión de genes y su intrincada relación con esos procesos celulares y como una aproximación para conocer el posible papel del ARNi en los mecanismos patógenos de varias enfermedades comunes, crónicas e infecciosas que incluyen enfermedades virales, influenza, hepatitis, cáncer, malformaciones congénitas, hipercolesterolemia, algunas enfermedades cardiacas, neurodegenerativas y cerebrovasculares, entre otras (36)(37)(38)(39)(40)(41)(42)(43). Por ejemplo, en el cáncer, las células escapan de los mecanismos normales que regulan la expresión de genes específicos cuyos productos controlan la proliferación, la diferenciación y la muerte celular.…”

“…Las alteraciones de la expresión de genes que codifican proteínas específicas y que no codifican proteínas como los miARN, han sido reportadas en cáncer lo cual sugiere que en la regulación de la expresión genética, el ARNi podría contribuir en el proceso de su desarrollo (39,40). En otras entidades como en los trastornos inflamatorios, cardiovasculares y neurodegenerativos, el uso de ds-ARN cortos sintetizados químicamente y a través del desarrollo de sistemas de expresión que producen de manera estable siARN han permitido confirmar que estas patologías también afrontan los mecanismos de regulación del ARNi, alteraciones y cambios en expresión de genes específicos (41)(42)(43)(44).…”