Fig. 5: Pax acts downstream of Wts to regulate the proliferation of ISC.
a Total RNA for real-time PCR was collected from whole midguts of the indicated genotypes: MyoIAts>Ctrl, MyoIAts>Pax RNAi, MyoIAts>Yki RNAi and MyoIAts>Pax RNAi;Yki RNAi (n = 3 for each group). b–e’ Adult midguts containing GFP-positive MARCM clones of control (b–b’), wtsx1 (c–c’), HA-Pax (d–d’) and wtsx1 in the presence of Pax (e–e’). Midguts were dissected 6 days after clone induction and immunostained with DAPI (nuclei, blue). Clones are marked by GFP. f Quantification of the cell number per clone in adult midguts from the indicated genotypes in (b–e’) at 2, 4, and 6 days after clone induction (n = 41, 72, 63, 67, 48, 92, 42, 80, 45, 74, 65, 29). g–n’ Adult midguts of bantam-lacZ;Esgts>Ctrl (g–g’), bantam-lacZ;Esgts>Pax RNAi (h–h’), Diap1-lacZ;Esgts>Ctrl (i–i’), Diap1-lacZ;Esgts>Pax RNAi (j–j’), bantam-lacZ;MyoIAts>Ctrl (k–k’), bantam-lacZ;MyoIAts>Pax RNAi (l–l’), Diap1-lacZ;MyoIAts>Ctrl (m–m’) and Diap1-lacZ;MyoIAts>Pax RNAi (n–n’) were immunostained with β-gal (red) and DAPI (nuclei, blue). Note, Bantam is specifically expressed in precursor cells and EEs, and Diap1 is upregulated in both precursors (GFP- lacZ+, white arrows) and ECs (GFP+ lacZ+, orange arrows) when Pax is knocked down in ECs. o–p Relative mRNA levels of Hippo signaling targets Diap1 and ex were analyzed by real-time PCR. Total RNA was collected from whole midguts of the indicated genotypes: Esgts>Ctrl, Esgts>Pax RNAi, MyoIAts>Ctrl, and MyoIAts>Pax RNAi (n = 3 for each group). The relative mRNA level was normalized with that of Esg-GFP in (o). Three independent experiments were performed, and the error bars are mean ± SEM. P values of significance (indicated with asterisks, NS no significance ≥0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001) were calculated by two-tailed Student’s t-test (o, p) and one-way ANOVA with Tukey’s test (a, f). Scale bars: 30 µm. Confocal images were taken from the boundary region between R4c and R5a of the posterior midgut.
