Genome-wide phenotypic RNAi screen in theDrosophilawing: global parameters

Abstract

We have screened a collection of UAS-RNAi lines targeting 10920 Drosophila protein-coding genes for phenotypes in the adult wing. We identified 3653 genes (33%) whose knock-down causes either larval/pupal lethality or a mutant phenotype affecting the formation of a normal wing. The most frequent phenotypes consist in changes in wing size, vein differentiation and patterning, defects in the wing margin and in the apposition of the dorsal and ventral wing surfaces. We also defined 16 functional categories encompassing the most relevant aspect of each protein function, and assigned each Drosophila gene to one of these functional groups. This allowed us to identify which mutant phenotypes are enriched within each functional group. Finally, we used previously published gene expression datasets to determine which genes are or are not expressed in the wing disc. Integrating expression, phenotypic and molecular information offers considerable precision to identify the relevant genes affecting wing formation and the biological processes regulated by them.

Genome-wide phenotypic RNAi screen in theDrosophilawing: phenotypic description of functional classes

“…We arranged the wing phenotypes of the Gal4/UAS-RNAi combinations presented in the preceding manuscript ( López-Varea et al 2021 ) into sets, each including the genes belonging to each molecular group. Each mutant wing was described using a set of terms that summarize its main phenotypic characteristics.…”

“…A list with all wing pictures included in this manuscript is presented in Supplementary Table S3. Pictures of wings included in López-Varea et al (2021) and Ostalé et al (2021) are also included in Supplementary Table S3. All wing pictures we have were submitted to the Figshare repository: https://doi.org/10.6084/m9.figshare.16624645.v1 ; https://doi.org/10.6084/m9.figshare.16624630.v1 ; https://doi.org/10.6084/m9.figshare.16624603.v1 ; and https://doi.org/10.6084/m9.figshare.16624591.v1…”

“…In this manuscript, we described in more detail the following molecular classes defined in López-Varea et al (2021 ): Ribosomal biology (RIB), Cell adhesion genes (CA), Protein biology (PRO), RNA biology (RNA), DNA biology (DNA), Cell signaling (SIG), Cellular metabolism (MET), Cuticle proteins (CUT), Coding genes without conserved sequence domains (CG), and Coding genes with conserved sequence domains CGh. The classes Immune responses (IMM) and Cell death (CD) are only included in Supplementary Table S1 and were not further analyzed because these genes rarely affect wing formation.…”

“…For the description of mutant phenotypes, we develop a simplified terminology reflecting the morphological changes observed in each mutant wing such as its size [S and S(L)]), wing size and patterning of veins (S-P), differentiation of veins (V− and V+), defects in the wing margin (WM), defects in dorsoventral wing surfaces adhesion (WA), defects in wing pigmentation (WP), changes in trichome size, number, polarity, or spacing (cell differentiation; CD), changes in overall wing shape (WS), and other wing defects including incomplete unfolding of the wing surfaces (WF), appearance of necrotic patches and wing cuticle with abnormal appearance or lack of rigidity (wing differentiation; WD). All cases in which the wing fails to form were described as “nW” (no wing), phenotypes affecting the bristles of the wing margin as “Q−” and phenotypes of ectopic bristle formation as “Q+.” A list of all abbreviations can be found in Figure 1 , Supplementary Table S2, and in the accompanying manuscript ( López-Varea et al 2021 ). Some mutant phenotypes are more informative than others with regard to the function of the corresponding gene during wing development.…”

A promoter-proximal silencer modifies the activity of a shared enhancer to mediate divergent expression of nub and pdm2 paralogs in wing development

“…Classical regulatory alleles of nub cause severe growth and tissue organization defects of the adult wing (Cifuentes & García-Bellido, 1997;Ng et al, 1995). Surprisingly, however, when Nubbin protein is specifically depleted during wing development, either through RNAi knockdown (López-Varea et al, 2021) or somatic CRISPR targeting of the gene locus (Figure 1C), the wings appear normal. To examine if the lack of wing phenotype could be due to a redundant function of the paralogous pdm2 gene, we repeated the somatic CRISPR loss-offunction experiments in the background of the pdm2 E46 allele, which produces a truncated protein as a result of a premature termination codon prior to the homeodomain (Yeo et al, 1995).…”