A Gain-of-Function Suppressor Screen for Genes Involved in Dorsal–Ventral Boundary Formation in the Drosophila Wing

Abstract

The Drosophila wing primordium is subdivided into a dorsal (D) and a ventral (V) compartment by the activity of the LIM-homeodomain protein Apterous in D cells. Cell interactions between D and V cells induce the activation of Notch at the DV boundary. Notch is required for the maintenance of the compartment boundary and the growth of the wing primordium. Beadex, a gain-of-function allele of dLMO, results in increased levels of dLMO protein, which interferes with the activity of Apterous and results in defects in DV axis formation. We performed a gain-of-function enhancer-promoter (EP) screen to search for suppressors of Beadex when overexpressed in D cells. We identified 53 lines corresponding to 35 genes. Loci encoding for micro-RNAs and proteins involved in chromatin organization, transcriptional control, and vesicle trafficking were characterized in the context of dLMO activity and DV boundary formation. Our results indicate that a gain-of-function genetic screen in a sensitized background, as opposed to classical loss-of-function-based screenings, is a very efficient way to identify redundant genes involved in a developmental process. I N multicellular organisms, initially homogenous sheets of cells are often subdivided into adjacent cell populations by the activity of certain transcription factors (reviewed in Irvine and Rauskolb 2001). In many cases, cell interactions between these populations lead to the restricted expression of signaling molecules at their boundaries, which organize growth and/or the pattern of nearby cells. The stability of these boundaries frequently relies on the acquisition of differential cell affinities between adjacent populations. When these boundaries behave as lineage restriction boundaries, these populations are called compartments (García-Bellido et al. 1973). The Drosophila wing primordium, a monolayered epithelium that gives rise to the adult wing and part of the thorax, is subdivided into an anterior and a posterior compartment by the activity of the homeodomain transcription factors Engrailed and Invected in posterior cells (García-Bellido and Santamaria 1972;Lawrence and Morata 1976;Tabata et al. 1995;Zecca et al. 1995). During larval development, the wing primordium suffers a secondary compartment subdivision. The activity of the LIMhomeodomain transcription factor Apterous (Ap) is responsible for this later subdivision into a dorsal (D) and a ventral (V) compartment (Diaz-Benjumea and Cohen 1993).Ap has three functions in wing development. It is responsible for the establishment of the Notch-dependent signaling center, the generation of a lineage restriction at the DV boundary, and the acquisition of a dorsal identity during cell differentiation. Ap exerts these functions through three classes of target genes. The complementary expression of Serrate and Delta, two ligands of the receptor Notch, to D and V cells, respectively, initiates a cascade of short-range cell interactions that lead to the activation of Notch at the DV boundary ( Figure 1B The activity of ...

Dynamic isomiR regulation in Drosophila development

“…Indeed, miR-282, 26% of whose embryonic expression is driven by isomiR-As, has been genetically linked to dorso-ventral boundary establishment in Drosophila embryos (Bejarano et al 2008). Indeed, stabilization of miRNAs by adenylation has been previously shown in black cottonwood (Lu et al 2009) and mouse (Katoh et al 2009).…”

Polycomb preferentially targets stalled promoters of coding and noncoding transcripts

“…Although mir-282 and mir-275 lack thorough functional evidence, several EP insertions at the mir-282 locus were identified in screens for wing development and circadian rhythm (Molnar et al 2006;Bejarano et al 2008;Dubruille et al 2009) while the presumptive core promoter of pri-mir-275 maps exactly to the cuckold mutation (chr2L: 7,423,896-7,423,995) (Castrillon et al 1993), strongly suggesting that the observed behavioral defect for cuc 1 is caused by a deregulation of the pri-mir-275 transcript. In Drosophila, <50% of all miRNA loci overlap with protein-coding transcripts with the majority remaining as intergenic.…”

Viability, Longevity, and Egg Production of Drosophila melanogaster Are Regulated by the miR-282 microRNA

“…The latter notion is further supported by our findings that the mir-282 gene is active at the pupal stage and it is conserved in holometabolic insects, which undergo complete metamorphosis. Moreover, in genetic interaction screens, mir-282 overexpression was found to have effect on pupal wing development and nervous system function (Bejarano et al 2008;Cao et al 2008).…”