Project description:The discovery of direct downstream targets of transcription factors (TFs) is necessary for understanding the genetic mechanisms underlying complex, highly regulated processes such as development. In this report, we have used a combinatorial strategy to conduct a genome-wide search for novel direct targets of Eyeless (Ey), a key transcription factor controlling early eye development in Drosophila. Like many other TFs, little is known for Ey direct downstream targets. To date, only one gene, sine oculis (so), has been identified as Ey direct targets in Drosophila. Therefore, it is crucial to identify additional targets in order to gain a better understanding of ey function. To overcome the lack of high quality consensus binding site sequences, phylogenetic shadowing of Ey binding sites in so was used to construct a position weight matrix (PWM) of the Ey protein. This PWM was then used for in silico prediction of potential binding sites in the Drosophila melanogaster genome. To reduce the false positive rate, conservation of these potential binding sites was assessed by comparing the genomic sequences from seven Drosophila species. In parallel, microarray analysis of wild-type versus ectopic ey-expressing tissue, followed by microarray-based epistasis experiments in an atonal (ato) mutant background, identified 188 genes induced by ey. Intersection of in silico predicted conserved Ey binding sites with the candidate gene list produced through expression profiling yields a list of 20 putative ey-induced, eye-enriched, ato-independent, direct targets of Ey, including so. The accuracy of this list of genes was confirmed using both in vitro and in vivo methods. Initial analysis reveals three genes, eyes absent, shifted, and Optix, as novel direct targets of Ey. These results suggest that the integrated strategy of computational biology, genomics, and genetics is a powerful approach that can be applied to systematically identify direct downstream targets for any transcription factor genome-wide.

Project description:The discovery of direct downstream targets of transcription factors (TFs) is necessary for understanding the genetic mechanisms underlying complex, highly regulated processes such as development. In this report, we have used a combinatorial strategy to conduct a genome-wide search for novel direct targets of Eyeless (Ey), a key transcription factor controlling early eye development in Drosophila. Like many other TFs, little is known for Ey direct downstream targets. To date, only one gene, sine oculis (so), has been identified as Ey direct targets in Drosophila. Therefore, it is crucial to identify additional targets in order to gain a better understanding of ey function. To overcome the lack of high quality consensus binding site sequences, phylogenetic shadowing of Ey binding sites in so was used to construct a position weight matrix (PWM) of the Ey protein. This PWM was then used for in silico prediction of potential binding sites in the Drosophila melanogaster genome. To reduce the false positive rate, conservation of these potential binding sites was assessed by comparing the genomic sequences from seven Drosophila species. In parallel, microarray analysis of wild-type versus ectopic ey-expressing tissue, followed by microarray-based epistasis experiments in an atonal (ato) mutant background, identified 188 genes induced by ey. Intersection of in silico predicted conserved Ey binding sites with the candidate gene list produced through expression profiling yields a list of 20 putative ey-induced, eye-enriched, ato-independent, direct targets of Ey, including so. The accuracy of this list of genes was confirmed using both in vitro and in vivo methods. Initial analysis reveals three genes, eyes absent, shifted, and Optix, as novel direct targets of Ey. These results suggest that the integrated strategy of computational biology, genomics, and genetics is a powerful approach that can be applied to systematically identify direct downstream targets for any transcription factor genome-wide. Keywords: genetic modification, cell type comparison

Project description:MicroRNA expression in the mouse eye.MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina . This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures. microRNA profiling of ocular tissues from mouse. In particular we analysed the cornea, lens, Retina Pigment Epithelium (RPE) and retina and compared them against RNA extracted from the entire eye. The purpose of this experiment was to understand which microRNAs are present nd/or show differential expression in the various structures of the eye (cornea, lens, RPE, retina). The samples numbered 1 & 2 (i.e. CORNEA1, CORNEA2 etc ) are biological replicates, prepared from tissues dissecyed from different groups of wild-type animals. RNA extracted from the entire eye (EYE) served as the unique reference sample. For each tissue to be analysed we performed the following hybridizations: - 2 slides for lens (LENS1, LENS2) vs entire eye (EYE) - 2 slides for RPE (RPE1, RPE2) vs entire eye (EYE) - 2 slides for retina (RETINA1, RETINA2) vs entire eye (EYE) - 2 slides for cornea (CORNEA1, CORNEA2) vs entire eye (EYE) - 1 slide for entire eye (EYE) vs entire eye (EYE)

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Integrative Genomic Analysis Reveals Novel Regulatory Mechanisms of eyeless During Drosophila Eye Development

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