The JAK/STAT pathway is required for vein expression in the visceral muscles upon infection with Ecc15. (a) Immunostaining against lacZ and GFP of guts derived from vein-lacZ; howGal4TS UAS-GFP flies reveals than vein (nuclear signal) is induced upon infection in the circular visceral muscles. (b) RNAi silencing of vein in visceral muscles and reduction of Keren and Spitz in precursor cells blocked infection-induced proliferation (P < 0.05). Conversely, ectopic expression of the three EGFs (UAS-sKeren, UAS-sSpitz and UAS-vein1.2) was sufficient to trigger proliferation (P < 0.05). Mean values from five experiments (N = 10-20 guts each) ± SE are shown. ANOVA F = 62.96. df = 26. P < 0.0001. (c) Infection induced the JAK/STAT reporter gene, STAT-GFP, in visceral muscles in addition to ISCs. Representative images of UC and Ecc15-infected guts are shown. Guts of STAT-GFP flies were stained with DAPI and examined by fluorescence microscopy at ×20 original magnification. The microscopic focus was set to the external layer of the gut (top). Transverse sections show that expression of STAT-GFP is localized to the circular visceral muscle and progenitor cells (bottom). (d) The JAK/STAT pathway is required in both ISCs and visceral muscles to promote ISC proliferation. RNAi directed against STAT92E or expression of a dominant-negative form of Domeless in ISCs (esgGal4TS) or muscles (howGal4TS), but not enterocytes (Myo1AGal4TS) strongly reduced the number of mitotic ISCs in the guts of flies infected with Ecc15 (t = 16 hours; P < 0.05). Mean values from five experiments (N = 10-20 guts each) ± SE are shown. ANOVA F = 50.74. df = 15. P < 0.0001. (e) The induction of vein upon Ecc15 infection was reduced in flies with reduced JAK/STAT signaling in visceral muscles (P < 0.05). Tissue-specific silencing of upd3 in enterocytes also reduced vein expression (P < 0.05). RT-qPCR analysis of gut extracts from UC and Ecc15-infected flies (t = 8 hours). Levels of vein expression were normalized to RpL32. Mean values from four experiments (N = 20 guts each) ± SE are shown. ANOVA F = 14.83. df = 7. P < 0.0001.