The Spalt Transcription Factors Generate the Transcriptional Landscape of the Drosophila melanogaster Wing Pouch Central Region.

The Drosophila genes spalt major (salm) and spalt-related (salr) encode Zn-finger transcription factors regulated by the Decapentaplegic (Dpp) signalling pathway in the wing imaginal disc. The function of these genes is required for cell survival and proliferation in the central region of the wing disc, and also for vein patterning in the lateral regions. The identification of direct Salm and Salr target genes, and the analysis of their functions, are critical steps towards understanding the genetic control of growth and patterning of the Drosophila wing imaginal disc by the Dpp pathway. To identify candidate Salm/Salr target genes, we have compared the expression profile of salm/salr knockdown wing discs with control discs in microarray experiments. We studied by in situ hybridization the expression pattern of the genes whose mRNA levels varied significantly, and uncovered a complex transcription landscape regulated by the Spalt proteins in the wing disc. Interestingly, candidate Salm/Salr targets include genes which expression is turned off and genes which expression is positively regulated by Salm/Salr. Furthermore, loss-of-function phenotypic analysis of these genes indicates, for a fraction of them, a requirement for wing growth and patterning. The identification and analysis of candidate Salm/Salr target genes opens a new avenue to reconstruct the genetic structure of the wing, linking the activity of the Dpp pathway to the development of this epithelial tissue.

Genetic annotation of gain-of-function screens using RNA interference and in situ hybridization of candidate genes in the Drosophila wing.

Gain-of-function screens in Drosophila are an effective method with which to identify genes that affect the development of particular structures or cell types. It has been found that a fraction of 2-10% of the genes tested, depending on the particularities of the screen, results in a discernible phenotype when overexpressed. However, it is not clear to what extent a gain-of-function phenotype generated by overexpression is informative about the normal function of the gene. Thus, very few reports attempt to correlate the loss- and overexpression phenotype for collections of genes identified in gain-of-function screens. In this work we use RNA interference and in situ hybridization to annotate a collection of 123 P-GS insertions that in combination with different Gal4 drivers affect the size and/or patterning of the wing. We identify the gene causing the overexpression phenotype by expressing, in a background of overexpression, RNA interference for the genes affected by each P-GS insertion. Then, we compare the loss and gain-of-function phenotypes obtained for each gene and relate them to its expression pattern in the wing disc. We find that 52% of genes identified by their overexpression phenotype are required during normal development. However, only in 9% of the cases analyzed was there some complementarity between the gain- and loss-of-function phenotype, suggesting that, in general, the overexpression phenotypes would not be indicative of the normal requirements of the gene.

A gain-of-function screen identifying genes required for growth and pattern formation of the Drosophila melanogaster wing.

The Drosophila melanogaster wing is a model system for analyzing the genetic control of organ size, shape, and pattern formation. The formation of the wing involves a variety of processes, such as cell growth, proliferation, pattern formation, and differentiation. These developmental processes are under genetic control, and many genes participating in specific aspects of wing development have already being characterized. In this work, we aim to identify novel genes regulating wing growth and patterning. To this end, we have carried out a gain-of-function screen generating novel P-UAS (upstream activating sequences) insertions allowing forced gene expression. We produced 3,340 novel P-UAS insertions and isolated 300 that cause a variety of wing phenotypes in combination with a Gal4 driver expressed exclusively in the central domain of the presumptive wing blade. The mapping of these P-UAS insertion sites allowed us to identify the gene that causes the gain-of-function phenotypes. We show that a fraction of these phenotypes are related to the induction of cell death in the domain of ectopic gene expression. Finally, we present a preliminary characterization of a gene identified in the screen, the function of which is required for the development of the L5 longitudinal vein.

Characterization of dSnoN and its relationship to Decapentaplegic signaling in Drosophila.

Vertebrate members of the ski/snoN family of proto-oncogenes antagonize TGFbeta and BMP signaling in a variety of experimental situations. This activity of Ski/SnoN proteins is related to their ability to interact with Smads, the proteins acting as key mediators of the transcriptional response to the TGFbeta superfamily members. However, despite extensive efforts to identify the physiological roles of the Ski/SnoN proteins, it is not yet clear whether they participate in regulating Activin and/or BMP signaling during normal development. It is therefore crucial to examine their roles in vivo mostly because of the large number of known Ski/SnoN-interacting proteins and the association between the up-regulation of these genes and cancer progression. Here we characterize the Drosophila homolog to vertebrate ski and snoN genes. The Drosophila dSnoN protein retains the ability of its vertebrate counterparts to antagonize BMP signaling in vivo and in cultured cells. dSnoN does not interfere with Mad phosphorylation but it interacts genetically with Mad, Medea and dSmad2. Mutations in either the Smad2-3 or Smad4 putative binding sites of dSnoN prevent the antagonism of dSnoN towards Dpp signaling, although homozygous flies for these mutations or for a genetic deficiency of the locus are viable and have wings of normal size and pattern.