Additional file 5: Figure S5. of Apontic regulates somatic stem cell numbers in Drosophila testes

Apt limits the GSC population at the hub interface and E-cadherin expression. A-B) Single optical sections of testes stained with antibodies that recognize Vasa (magenta), Tj (white), and Fas3 (white, to label the hub). The hub is outlined in blue. Scale bars = 10 μm. Testes from homozygous apt KG05830 males exhibit a significant increase in GSCs (magenta) contacting the hub (B), relative to wild type (A). C) Number of GSCs at the hub interface for the indicated genotypes. D-E) Images of testes stained with antibodies specific for E-cadherin (magenta and insets), Vasa (white), and DAPI (blue). Scale bars = 20 μm. An increase or mislocalization of E-cadherin expression is observed in the cells surrounding the hub, including the Vasa + GSCs (arrows) in a testis from an apt KG05830 homozygous male (E), compared to a w 1118 testis (D, arrows), where it is barely detected outside the hub. Images were taken under the same conditions. F-G) Single optical sections of testes stained for Apt (magenta and insets), Tj (white), and DAPI (blue). Arrows indicate GSCs; arrowheads show CySCs. Scale bars = 10 μm. F) apt KG05830 /+ heterozygotes show no significant reduction of Apt protein in CySCs and a mild reduction in GSCs. G) In homozygous mutant males, Apt expression is reduced in CySCs (first tier of Apt+/Tj + cells proximal to the hub: arrowheads) but is not detected in the germline (arrows, the presence of a cell is indicated by DAPI). H) Quantification of the relative expression levels of Apt protein in the stem cell populations adjacent to the hub for the indicated genotypes. "n" is the number of testes examined with the number of cells in parentheses. Statistical significance was tested via two-tailed t-tests, where *p < 0.05, ***p < 0.005, ****p < 0.0001, and n.s. = not significant. Experimental genotypes were tested against Canton S, unless indicated by a bar. (TIF 14942 kb

Additional file 4: Figure S4. of Apontic regulates somatic stem cell numbers in Drosophila testes

Somatic reduction of apt heightens STAT expression in the CySCs. A-B) Testes stained with antibodies recognizing STAT (magenta), Tj (white, somatic cells), and Fas3 (white, hub: blue outline) and counterstained with DAPI (blue, nuclei). Arrowheads indicate CySCs (first tier of Tj + cells around the hub). Scale bars = 20 μm. Insets display STAT expression, alone. A) Control testis shows wild - type STAT expression: most detectable STAT is found in the GSCs around the hub (labeled arrows), but it decreases in gonialblasts and CySCs (arrowheads). A Tj + cell distal from the hub shows undetectable levels of nSTAT (unlabeled arrow). B) More STAT is detectable when apt is reduced in somatic cells via Tj-Gal4. Tj + cells several cell diameters away from the hub displayed high levels of nSTAT (asterisks). C) 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). Tj-Gal4;aptRNAi was normalized to the Tj-Gal4 or aptRNAi-alone controls to obtain a relative expression level. Somatic reduction of apt significantly increases nSTAT levels in CySCs. Two-tailed t-tests were used to test for significance, as indicated. “n” provides the total number of testes examined for each genotype, while the number of individual cells analyzed is given in parentheses. (TIF 8047 kb