Figure 3. N/Src synergy induces both MMP1 and apoptosis.
(A–L) Immunofluorescence for MMP1 (A–F) and cleaved caspase 3 (cl-casp3, G–L) in wing discs expressing UAS constructs under vgGal4. Together, Nact and Src42ACA cause robust activation of both MMP1 (A) and cl-casp3 (G), which is strongly reduced by BskDN (E, K). The combination of Nact and Mef2 results in an increase in MMP1 (F) but little effect on cc3 (L). (M) qPCR for egr and wgn in wing discs overexpressing genes as indicated under the vgGal4 driver reveals that both transcripts are strongly downregulated when Nact and Src42ACA are coexpressed. Scale bar: 100 μm.
Figure 3—figure supplement 1. Gal4/UAS titration does not affect the N/Src phenotype.
(A, C) One copy each of UAS-Nact, UAS-Src42ACA, and UAS-GFP (three UAS transgenes total) were driven with vgGal4 in the wing disc, and compared to (B, D) wing discs expressing only UAS-Nact and UAS-Src42ACA (two UAS transgenes total) with vgGal4. Discs were stained for MMP1 (A, B) or cleaved caspase 3 (C, D). Scale bar: 100 μM.
Figure 3—figure supplement 2. A heterozygous null mutation of Notch can rescue lethality and phenotype of Src alone.
N55e11/FM7C;UAS-Src64B virgins were crossed to vgGal4 males at 18°C (A, B) and the resultant female progeny were scored. N55e11/+;vgGal4/UAS-Src64B flies were more viable (B, n = 126 over four independent experiments) than their FM7C/+;vgGal4/UAS-Src64B siblings (A, n = 16), and show a rescued phenotype similar to that of N55e11/+;vgGal4/+ controls (C). FM7C/+;vgGal4/UAS-Src64B (A) wings were indistinguishable from vgGal4/UAS-Src64B (D) wings. (E–H) Immunostaining for MMP1 (E, G) or cleaved caspase 3 (F, H) in wing discs with genotypes (D, E) FM7iGFP/+;vgGal4/UAS-Src64B or (F, G) N55e11/+;vgGal4/UAS-Src64B. Scale bar: 100 μm. (I–K) d10338 (Exelixis Src42A allele) lethality and phenotype at 25°C can be partially rescued by Notch RNAi. vgGal4/d10338 flies (I) are largely pupal lethal (n = 3 viable adults compared to 62 vgGal4/CyO-Tb siblings from the same cross) and the few escapers have no wings. In contrast, vgGal4/d10338, UAS-NRNAi flies (J) have narrow, short, and shriveled wings and much lower lethality (n = 67, compared to 108 vgGal4/CyO siblings from the same cross.) The wing phenotype appears to be a more severe version of the phenotype of vgGal4/UAS-NRNAi flies (K).