a, Schematic diagram of the apical tip of the testis. Hub cells (red) contact SSCs (light grey) and GSCs (green). SSCs produce cyst cells (dark grey) that envelop developing germline cells (green). b-f, Immunofluorescence images of the apical tip of the adult testis from flies heat shocked to generate labelled dividing cells expressing nuclear β-galactosidase (β-gal). Testes were stained with antibodies to DE-cadherin or Fasciclin III (FasIII), β-gal, and Traffic Jam (TJ) or 4,6-diamidino-2-phenylindole (DAPI). b, One day after heat shock, three β-gal⁺ (arrowheads, green; second panel) and TJ⁺ somatic cells (red; fourth panel) are near the FasIII⁺ (blue; third panel) hub (asterisk). c, Five days after heat shock two β-gal⁺ SSCs (arrowheads, green; right panel) contact hub cells (outline/DE-cadherin, red). A β-gal⁺ SSC daughter cell (arrow) is also present. d, Lower magnification view of c. β-Gal⁺ somatic cells (arrows, green) are present along the length of the testis. e, Five days after heat shock, the hub (outline, DE-cadherin, red) contains two β-gal⁺ cells (arrowheads, green; right panel). f, Fifteen days after heat shock, two β-gal⁺ hub cells (green, arrowheads; second panel) co-stain with FasIII (blue, outline; third panel) and TJ (red; fourth panel). Scale bars: b, d, 20 μm; c, e, f, 10 μm.

a, b, d, Immunofluorescence images of testes from wild-type (OregonR) (a, b) or updGAL4-UAS-gfp (d) adult flies fed BrdU. a, Left panel: BrdU⁺ cells (green) surround the FasIII⁺ hub (red, outline). Two BrdU⁺ GSCs (asterisks) and a BrdU⁺ somatic cell (arrow) are adjacent to the apical hub (outline) after a 1-day chase. Right panel: no BrdU⁺ hub cells are detected. b, A BrdU⁺ hub cell (arrow) is present after a 3-day chase (left panel). FasIII (middle panel); BrdU (right panel). c, Hub cells stained with DE-cadherin (red; middle panel) and GFP (green; right panel) in updGAL4-UAS-gfp testis. d, A BrdU⁺ hub cell (arrow) is detected after a 7-day chase. The middle panel shows the GFP channel; the right panel shows the BrdU channel. Scale bars: 10 μm.

a-d, Confocal images of updGAL4-UAS-gfp testes. a, b, Testes stained for Vasa (blue; middle panels), phospho-histone-H3 (pHH3, red; right panels) and GFP (green). Phospho-histone-H3⁺ SSCs (arrowheads) are observed near the hub. c, Testes stained for TJ (blue; middle panel), phospho-histone-H3 (green; right panel) and GFP (green; right panel). The hub is outlined. A mitotic CPC (arrow) surrounds a mitotic GSC (arrowhead). d, Testes stained with TJ (blue; middle panel), phosphohistone-H3 (red; right panel) and GFP (green). A mitotic CPC (arrows) one-cell distance from the hub (outline) is shown. e, Graph depicting marked somatic cell type frequency from all testes assayed at various time points. f, Graph depicting marked somatic cell type frequency from testes containing marked somatic cells at various time points. g-i, A testis 10 days after heat shock stained for β-gal⁺ (green; bottom panels), DE-cadherin (red) and DAPI (blue). A β-gal⁺ hub cell (g, indented arrowhead), β-gal⁺ SSC (h, i, arrowhead) and β-gal⁺ CPC (i, arrow) are shown. Z-section images (1 μm) are denoted by depth in the lower left corner. Scale bars: 10 μm.

a, Ten days after heat shock a series of GFP⁺ late cyst cells (green, arrows) and a single labelled hub cell are present (arrowhead in b). GFP (green), DE-cadherin (red) and DAPI (blue) are shown. b, Higher magnification (×63) of the hub in a (outline, DE-cadherin, red) with a GFP⁺ hub cell (arrowhead, right panel). c, Five days after heat shock GFP⁺ germline and somatic cells are maintained near the DE-cadherin⁺ hub (red). Late GFP⁺ germline (arrowhead) and somatic cell (arrow) clones are present basally. d, Five days after heat shock GFP⁺ shg^IG29 mutant late somatic cells are present (arrow), but no GFP⁺ clones are present near the DE-cadherin⁺ hub (red) at the apical tip. e, f, Fifteen days after heat shock GFP⁺ hub cells are present from wild-type (e) and shg^IG29 (f) somatic clones (arrowheads). GFP⁺ shg^IG29 hub cells display loss of DE-cadherin (red) at the hub interface (inset, DE-cadherin channel). g, h, Testes from 1-day-old c587GAL4 (g) and c587GAL4/UAS-shgRNAi (h) males stained with DE-cadherin (red) and TJ (green). i, j, Testes from 15-day-old c587GAL4 (i) and c587GAL4/UAS-shgRNAi (j) males. Note the loss of DE-cadherin (red) and TJ (green) stain in j. k, l, Fifteen days after heat shock distorted GFP⁺ hub cells are present from esg^G66 (k) and esg^L2 (l) mutant somatic clones (compare to e). The hub is marked with DE-cadherin (red). m, n, esg^shof/CyO; osk/osk (m) and esg^shof/esg^shof; osk/osk (n) testes stained for FasIII (red, insets) and TJ (green). Scale bars: a, c, d, 20 μm; e-n, 10 μm.