(A-J) Expression of markers in testes from Hs-bam flies before (A,C,E,G,I) or after (B,D,F,H,J) induction of ectopic Bam. Hubs are outlined in all panels. (A-B) Zfh-1 marks CPC nuclei before heat-shock; these appear closer to the hub following heat-shock. (B, inset) Somatic (Vasa-negative) nuclei adjacent to the hub incorporate BrdU (green) in vitro after 5 heat-shocks. (C-D) Mature cyst cells normally express Eya; this marker remains off in somatic cells adjacent to the hub after induction of ectopic Bam. (E-F) The hub marker CB03470 is unchanged after heat-shocks, indicating that somatic cells adjacent to the hub do not acquire hub cell identity under these conditions. (G-H) CB03690 marks CPC and hub cell nuclei. CPC nuclei are closer to the hub after expression of ectopic Bam. (I-J) CC01872 is predominantly expressed throughout somatic cells, revealing thin cytoplasmic extensions on CPCs (arrow, inset) contacting the hub. After heat-shock, the distribution of CC01872 is altered, with GFP-positive somatic cells surrounding and broadly contacting the hub. Scale bars = 10μm.

(A,B,D,E) In Hs-bam testes, GMA driven marks cortical F-Actin in germ cells (green) 1B1 (red) marks fusomes; Armadillo (red) marks the hub; and DAPI (blue) marks nuclei (genotype: Hs-bam; nanos-Gal4-VP16/+; UAS-GMA/+) (A′,B′,D′,E′). GMA (green) channel. (A) Before expression of ectopic Bam, GSCs extend short Actin-rich projections (arrowhead) into the hub, while 4- and 8-cell spermatogonia lack protrusions (arrow), and gonialblasts display randomly oriented protrusions (open arrowhead). (B) After expression of ectopic Bam, spermatogonial cysts (arrow) often do not contact the hub. (C) The percentage of testes containing germ cells less than 3μm from the hub throughout the time course of ectopic Bam expression. (D) Testes appear phenotypically normal after 5 days of recovery; a rosette of polarized GSCs (arrowhead) flanked by CPCs surrounds the hub and spermatogonia (arrow) fill the testis. (E) Fine Actin-based protrusions on the surface of a 4-cell spermatogonial cyst (inset) at 1 day of recovery. (F) The percentage of testes containing 4-cell (blue) and 8-cell (purple) spermatogonial cysts with protrusions before, during and after recovery. Scale bars = 10μm.

(A-F) Hs-bam testes stained with Vasa (red, germ cells); Armadillo (green, hub, *); 1B1 (green, fusomes); and DAPI (blue, nuclei). (A) Without heat-shocks, testes are indistinguishable from wild-type and contain GSCs (arrowhead) and CPCs (arrow); spermatogonial cysts and spermatocytes are positioned progressively further from the hub. (B-F) Increasing heat-shocks depletes GSCs, but spermatogonial cysts and Vasa-negative cells (arrows) remain. (G) The average number of GSCs/testis (± standard deviation) falls with increasing ectopic Bam expression. (H) The average number of GSCs, gonialblasts and 2-cell cysts/testis (± standard deviation) inversely correlates with the number of heat-shocks. (I-J) Testes stained with TUNEL labeling (green, apoptotic cells); DAPI (blue, nuclei); Fasciclin III (red, hub, *). TUNEL-positive GSCs were not detected in wild-type or Hs-bam testes after 5 heat-shocks; apoptotic spermatogonial cysts were observed as expected. Scale bars = 10μm.

(A-C) Vasa (red) marks germ cells, Fasciclin III (green) marks the hub (asterisk), 1B1 (green) marks fusomes, and DAPI (blue) marks nuclei. (A) One day after withdrawal of ectopic Bam, niches are often filled with somatic cells but become indistinguishable from wild-type by (B) 4 and (C) 6 days of recovery. (D) The average number of GSCs/testis (± standard deviation) and (E) the percentage of testes containing GSCs rise during recovery. (F-G) Anillin (green) marks ring canals, Fasciclin III and 1B1 (red) mark the hub and fusomes, respectively, and DAPI (blue) marks nuclei in testes from Hs-bam flies. (F) Before heat-shock, stable ring canals are observed in spermatogonial cysts (arrow); smaller transient ring canal remnants are only apparent between GSCgonialblast pairs (open arrowhead). (G) After 5 heat-shocks and 2 days of recovery, ring canal remnants appear in cells near the hub (2 visible in this focal plane, arrowheads, insets); open ring canals remain in spermatogonial cysts displaced from the hub (arrow). Scale bars = 10μm.

(A-D) Examples of GSCs (arrowheads), spermatogonia (open arrowheads) and CPC nuclei (arrow) are indicated. (A) Testes from flies misexpressing moderate levels of UAS-SOCS36E maintain GSCs, as revealed by immunostaining for GFP (green, marks nuclei of cells misexpressing SOCS36E), Armadillo and 1B1 (blue, marks the hub and fusomes, respectively), and Vasa (red, marks germ cells). (B-D) Testes immunostained for GFP (SOCS36E misexpressing cells), 1B1, marks fusomes, and Armadillo, marks the hub (green); Vasa (red), marks germ cells; and DAPI (blue). (B) Before heat-shock, testes from flies containing Hs-bam and UAS-SOCS36E have wild-type morphology; GFP is undetectable as expected. Heat-shock-induced misexpression of (C) both SOCS36E (green nuclei) and Bam or (D) Bam alone yields morphologically indistinguishable testes: GSCs are lost but CPCs (arrows) and spermatogonia (open arrowheads) remain. (E) The average number of GSCs (± standard deviation) and (F) the percentage of testes containing GSCs from testes represented in panels B-D. GSCs in testes misexpressing SOCS36E and Bam (blue) are significantly reduced (asterisks) at 5 days of recovery compared to testes misexpressing Bam alone (purple). Scale bars = 10μm.