Generation of the Peg-Gal4 line (see also Fig. S9)

The peg-Gal4 line was generated by CRISPR-mediated homology-directed genome editing using the strategy reported by Gratz et. al³⁵, co-injecting the following plasmids in a Vasa-cas9 Drosophila line: a pFCD4 vector carrying tandem RNA guides introduced with the primers Tan-CG17278_3′guide_Rv and Tan-CG17278_5′guide_Fw (see Table ^S4 for primer sequences), following the protocol described in³⁵; and the pTV[cherry]³⁶ vector carrying 1 kb homology arms targeting the 5′ and 3′ regions of peg (see Fig. ^S9) designed to remove the whole peg locus while maintaining its presumptive regulatory regions. Successful peg^pTV CRISPR-mediated homologous recombinants were screened for red eyes (w + marker within pTV[Cherry]) and sequenced. These flies expressed mCherry in some tissues but not in the imaginal discs, so we surmised that a large peg intronic region removed in the CRISPR-mediated homologous recombinants might carry a regulatory element required for peg expression in the imaginal discs. We therefore re-introduced the missing intronic region in our peg^pTV lines. For this, we first carried out a Cre-Lox recombination “flip-out” by crossing the peg^pTV the with a y w; snaSco/CyO, P{Crew}DH1 line obtained from the Kyoto stock centre, to remove most of the pTV[cherry] sequence, and leaving only an ATTp site, for site-directed transgenesis. Successful flip-out events were screened by loss of the w marker. We then cloned the missing intronic sequence into the RIV^Gal4 vector³⁶, amplified using the primers intron_fragment1_fw and intron_fragment1_rv (see Table ^S4 for primer sequences), and EcoRI restriction. The intron-carrying RIV^Gal4 vector was then used for site-specific transgenesis in our Cre-Lox recombined peg^pTV line, by co-injection with the act-phiC31-integrase vector, obtained from the DGRC (barcode 1368). Successful transformants were screened for recovery of the w + marker. These flies drive the expression of genes under the control of UAS in the wing imaginal discs with the same pattern of expression as peg; this is also a peg null allele since it lacks the peg ORF, this line is therefore called peg^Gal4. Plasmid injections were made in BestGene, or at the Drosophila transgenesis facility at the Madrid Centre of Molecular Biology.