Figure 1.
Psc and Su(z)2 are redundant and required in the CySC but not the GSC lineage.
(A–C): Analysis of testes with Psc and Su(z)2 double mutant clones generated in the CySC lineage (C587-GAL4;FRT42D, Df(2R)Su(z)21.b8/FRT42D, ubi-nGFP;UAS-FLP/tub-GAL80ts) and control (C587-GAL4;FRT42D/FRT42D, ubi-nGFP;UAS-FLP/tub-GAL80ts). (A–A') Marked nGFP-negative Psc and Su(z)2 double mutant cell aggregate (dashed line). (B–B') Control clones of normal cyst cells homozygous for FRT42D. (A, B) Phase; (A', B') nGFP. Testes examined 8 days after clone induction. (C): Percentage of testes with a visible abnormal cell aggregate at the tip of the testis scored at different time points after induction of Psc and Su(z)2 double mutant clones in the CySC lineage. (Red line) Testes with Psc and Su(z)2 double mutant clones. (Blue line) FRT42D controls. (D–E): Phase-contrast images of apical tip of testes with (D) simultaneous RNAi knockdown of Psc and Su(z)2 (C587-GAL4;UAS-Psc-RNAi/UAS-Su(z)2-RNAi;tub-GAL80ts) or (E) single RNAi knockdown of either Psc (C587-GAL4;UAS-Psc-RNAi/CyO;tub-GAL80ts) or Su(z)2 (C587-GAL4;UAS-Su(z)2-RNAi/CyO;tub-GAL80ts) alone in the CySC lineage (phenotypically indistinguishable). Testes 12 days after RNAi induction. Arrow marks spermatocytes. (F–H): Analysis of testes with Psc and Su(z)2 double mutant clones generated in the GSC lineage. (F–F') Psc and Su(z)2 double mutant clones homozygous mutant for Df(2R)Su(z)21.b8 deficiency. (G–G') Control germline clones homozygous for FRT42D. Bright field (F, G); nGFP (F', G'). Arrows mark germline clones. Testes 7 days after clone induction. (H): Percentage of testes with Psc and Su(z)2 double mutant (red line) or wild-type control (blue line) spermatocyte cysts scored at different time points after eclosion. Data reported as average +/− S.D. (I–J): Phase images of apical tip of testes with RNAi knockdown in the GSC lineage of (I) both Psc and Su(z)2 or (J) either Psc or Su(z)2 controls (phenotypically indistinguishable). Testes 12 days after RNAi induction. Spermatocytes marked by arrows. Scale bars: 50 μm.
Figure 2.
Cells in Psc and Su(z)2 double mutant aggregates are mitotically active.
(A–D) Phase-contrast and bright field images of apical region of testes with Psc and Su(z)2 double mutant clones generated by heat shock FLP and marked by the absence of nGFP (hs-FLP;FRT42D, Df(2R)Su(z)21.b8/FRT42D, ubi-nGFP) (A, A') Day 4, (B, B') day 8, (C, C') day 12, and (D, D') day 16 after clone induction. (Dashed line) Abnormal Psc and Su(z)2 mutant cell aggregates. (Arrows) Germline clones. Phase (A, B, C), nGFP (A', B', C', D'). Bright field (D). (E–E’’’): Confocal microscopy projection through a testis containing a Psc and Su(z)2 mutant aggregate 16 days after clone induction immunostained for (E–E’’’) phospho-Histone H3 (pH3) to mark mitotically active cells, (E’’) Vasa to mark germ cells, and (E’’’) DAPI to mark DNA. (E) merged image of E'-E’’’. Scale bars: 50 μm.
Figure 3.
Loss of Psc and Su(z)2 function in the CySC lineage impairs GSC maintenance non-cell autonomously.
(A): Maintenance of marked nGFP-negative late germline clones (spermatocyte and spermatid) in testes with (red line) Psc and Su(z)2 double mutant aggregates (hs-FLP;FRT42D, Df(2R)Su(z)21.b8/FRT42D, ubi-nGFP) or (blue line) FRT42D control (hs-FLP;FRT42D/FRT42D, ubi-nGFP) clones generated randomly in the GSC and CySC lineages by heat shock and scored at different times after clone induction. Data reported as average +/− S.D. (B–C): Schematic representation of testes containing a hub (blue) (B) enclosed by the mutant cell aggregate or (C) displaced to the side of a mutant cell aggregate and accessible to GSCs. (White) nGFP negative GSC clone. (Black) abnormal mutant cell aggregate. (D–E): Apical tip of a testis 8 days after clone induction showing hub enclosed by mutant cell aggregate (enclosed hub). Testis stained with antibodies against (D, D’’’) nGFP, arrow shows enclosed hub lacking GSCs; (D') Fas3 to mark the hub, (D’’, D’’’) Vasa to mark germ cells, and (D’’’) DAPI to mark DNA. (E) Close up of hub taken from merged images D–D’’ with Fas3 in blue. White arrow marks the hub; bracket shows presence of spermatocytes and lack of early germ cells. (F): Quantification of marked nGFP negative germline clones in testes with enclosed hub 15 days after clone induction. (G–H): Apical tip of a testis 8 days after clone induction showing a hub outside the mutant cell aggregate and associated with neighboring GSCs (accessible hub). (G, D’’’) Testis stained with antibodies against nGFP, (G', white arrow) Fas3 to mark the hub, (G’’, D’’’) Vasa to mark germ cells, and (G’’’) DAPI to mark DNA. (H) Merged close up of hub taken from images G–G’’ with Fas3 in blue. White arrow marks the hub, yellow arrow marks nGFP negative GSC, arrowheads mark germline clones. (I): Quantification of accessible hubs in testes with marked nGFP germline clones 15 days after clone induction. Scale bars 50 µm except in E and H where scale bar is 20 µm.
Figure 4.
Cells in mutant aggregates express some markers of somatic cells of the testis.
(A–I'): Immunostain of testes with abnormal Psc and Su(z)2 double mutant cell aggregates 8 days after clone induction (hs-FLP;FRT42D, Df(2R)Su(z)21.b8/FRT42D, ubi-nGFP). Testis immunostained with DAPI (blue), and the following antibodies: anti-GFP (green), (A–A’’) anti-Vasa, (B–B’’) anti-Zfh-1, (C–C’’) anti-Eya, (D–D') anti-Tj, (E–E’’) anti-Fas3, (F–F’’) anti-E-Cad, (G–G’’) anti-Arm, (H–H’’) anti-Cactus, and (I–I’’) anti-N-Cad. Stain of control testes with FRT42D clones shown in Fig. S4. Scale bars: 50 μm.
Figure 5.
Psc and Su(z)2 are required in the CySC lineage for repression of Abd-B.
(A–B’’’): Immunofluorescence images of testes from (A–A’’’) newly eclosed males with wild-type control or (B–B’’’) Psc and Su(z)2 double mutant clones generated in the CySC lineage (C587-GAL4;FRT42D, Df(2R)Su(z)21.b8/FRT42D, ubi-nGFP;UAS-FLP/tub-GAL80ts), immunostained with anti-ABD-B (red), anti-Vasa (A', A’’, yellow), anti-GFP (B', B’’, green), and DAPI (blue). Arrow marks nucleus of a sheath cell. (C–D’’’): Testes with simultaneous RNAi knockdown of both Psc and Su(z)2 in the CySC lineage (C587-GAL4;UAS-Psc-RNAi/UAS-Su(z)2-RNAi;tub-GAL80ts) immunostained with anti-ABD-B (red), anti-Vasa (C', C’’, yellow), anti-Zfh-1 (D', D’’’ green), anti-Tj (D’’, grey), and DAPI (C’’’, blue). Scale bars: 50 μm.
Figure 6.
Ectopic expression of Abdominal-B in the CySC lineage partially phenocopies loss of Psc and Su(z)2.
(A–B): Phase images of apical tip of testis 12 days after (A;C–F’’’) forced ectopic expression of Abd-B in the CySC lineage (C587-GAL4;UAS-Abd-B;tub-GAL80ts) and (B) control (UAS-Abd-B;tub-GAL80ts). Spermatocytes marked by bracket. (C–F’’’): Testes immunostained with (C, C') anti-ABDB, (C, C’’, D, D’’) anti-Zfh-1, (C, C’’’) anti-Tj, (D, D') anti-Eya, (D, D’’’, E, E’’’, F, F’’’) anti-Vasa, (D, D’’’, E, E’’’, F, F’’,) anti-Fas3, (E, E’’) anti-N-Cad, and (F, F') anti-Arm, antibodies. Scale bars: 50 µm.