Fig. 5

Apontic functions as a feedback inhibitor of STAT in the testes CySCs. a-c Testes stained with antibodies recognizing STAT (magenta) and Tj (white, somatic cells including the hub: blue outline) and counterstained with DAPI (blue, nuclei). Arrowheads indicate CySCs (first tier of Tj+ cells around the hub). Insets display STAT expression alone. a Control testis shows wild-type STAT expression: most detectable STAT is found in the GSCs around the hub (arrows), but it decreases in gonialblasts and CySCs (arrowheads). b More STAT is detectable when apt is reduced in somatic cells via c587-Gal4. c STAT expression is reduced in the CySCs when apt is over-expressed via c587-Gal4. d Nuclear STAT (nSTAT) levels were quantified in CySCs and normalized to DAPI intensity. Tj staining was utilized to outline nuclei of CySCs for measurement (see Methods). c587-Gal4; aptRNAi and c587-Gal4;; UAS-apt were normalized to the RNAi-alone control to obtain a relative expression level. Somatic reduction of apt significantly increases nSTAT levels in CySCs, while heightened levels of apt significantly reduces nSTAT in CySCs. e-h Optical sections projected into 2D of testes stained with Zfh-1 (magenta), Eya (white), and NCad (white) antibodies. Insets display Zfh-1 expression. Hub, indicated by NCad expression, is outlined in blue. A single copy mutation in Stat92E (g) or reduction of Stat92E via c587-Gal4 (h) in testes with reduced apt in CySCs and early cyst cells suppresses the expansion of the Zfh-1+ population observed when apt alone is reduced (f) and is similar to the control (e). i Quantification of the Zfh-1+ population of indicated genotypes. Genetic reduction of Stat92E function (globally or in CySCs and early cyst cells) when apt is reduced via c587-Gal4 results in a wild-type quantity of Zfh-1+ cells. j Proportion of Zfh-1+ cells that did not co-express the differentiation marker Eya in testes from the genotypes in (i). One copy of a Stat92E mutation in an apt deficient background restores the Zfh-1+/Eya- percentage to wild type. For all images scale bar = 20μm. Two-tailed t-tests were utilized to assess significance in (d) and (i), while a two-tailed Fisher’s exact test was used in (j), where *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, and n.s. denotes not significant. All statistics were performed between the experimental genotype and aptRNAi/+ (d, j) or c587>mGFP (i) controls unless otherwise indicated by a black bar. “n” indicates number of testes scored