Extended Data Figure 4. Tissue-specific allele switching to determine the anatomical origin of organismal developmental delay in NRT-Wingless animals.

a, Cumulative pattern of vestigial-Gal4 activity in various organs precursors. Expression of vestigial-gal4 at any stage or place leads to excision of the stop cassette in Actin-FRT.stop.FRT-Lac-Z thus marking permanently the corresponding cells. As expected, nearly the whole wing and haltere discs were labelled at the end of larval development. In wing imaginal discs, only a few cells were β–Galactosidase-negative that did not overlap with the domain of Wingless expression (anti-Wg, red). In the eye antennal disc, the patterns of Wingless (white arrowhead) and β–Galactosidase expression are also nonoverlapping. Therefore, in combination with UAS-Flp, vestigial-gal4 is expected to excise an FRT cassette throughout the domain of wingless expression. Examination of the brain and CNS shows that vestigial-Gal4 is unexpectedly active in these tissues. b, In larvae of genotype vestigial-Gal4, UAS-Flp, wg[FRT.Wg.FRT NRT-Wg]/Cyo, most of the wingless-expressing cells in leg, haltere and wing imaginal discs, but not in the brain and CNS, were converted to expressing NRT wingless (anti-HA; green). c, Developmental timing in wg[KO; WT^Body; NRT-Wg^Discs] (vestigial-Gal4, UAS-Flp, wg[FRT.Wg.FRT NRT-Wg]) and control (vestigial-Gal4, UAS-Flp, wg[FRT.Wg.FRT NRT-Wg]/GlaBC) larvae (80 animals, 4 experiments). The two data sets cannot be statistically distinguished (p>0.05). d, Adult wing size for three genotypes: 1) In black, wg[KO; WT^Body; NRT-Wg^Discs/GlaBc obtained from selfed vestigial-Gal4, UAS-Flp, wg[FRT.Wg.FRT NRT-Wg]/GlaBc) (n=16), 2) in purple, wg[KO; WT^Body; NRT-Wg^Discs], obtained from homozygous vestigial-Gal4, UAS-Flp, wg[FRT.Wg.FRT NRT-Wg]) (n=15), and 3) in grey, wg[KO; NRT-Wg^Body; WT^Discs], obtained from homozygous vestigial-Gal4, UAS-Flp, wg[FRT. NRT-Wg.FRT Wg]) (n=13). e-g, Extent of distalless expression in wg[KO; WT^Body; NRT-Wg^Discs] heterozygotes (over Cyo; e) and homozygotes (f). All the discs were obtained from immobile larvae at the time of anterior spiracle eversion, an event that marks the onset of pupariation. The extent of the distalless domain was estimated from the surface area of a polygon drawn around the zone of immunoreactivity, as shown. The results, plotted in panel g, show a mild reduction in wg[KO; WT^Body; NRT-Wg^Discs] discs (n=13) compared to controls (n=20; *** = p<0.001; n.s. = not significantly different). Error bars represent standard deviation. Statistical significance was assessed by Student’s t-test.