Ecad overexpression or Abl knockdown partially rescues loss of Robo2 mutant CySC clones.

a<p>Testes with CySC clones = testes with GFP⁺, Zfh-1⁺ cells/total testes scored (percentage).</p>b<p>Testes with >50% marked CySCs = testis where GFP⁺, Zfh-1⁺ cells>GFP⁻, Zfh-1⁺ cells/total testes scores (percentage).</p>c+e<p>Wild type clones expressing Ecad or Abl-RNAi = wild type MARCM Frt 40A flies driving (c) Ecad or (e) Abl-RNAi specifically in induced clones.</p>d+f<p>Robo2¹ clones expressing Ecad or Abl-RNAi = Robo2¹ MARCM flies driving (c) Ecad or (e) Abl-RNAi specifically in induced clones.</p>g<p>P value compared to Robo2¹ clones.</p>h<p>P value compared to wild type clones.</p><p>ND = Not Determined, ACI = After Clone Induction,</p><p>* = P value<.05,</p><p>** = P value<.01.</p><p>Ecad overexpression or Abl knockdown partially rescues loss of Robo2 mutant CySC clones.</p

The phosphorylation state of β-catenin affects CySC maintenance in the <i>Drosophila</i> testis.

a<p>Testes with CySC clones = testes with GFP⁺, Zfh-1⁺ cells/total testes scored (percentage).</p>b<p>P value vs Robo2 RNAi with Arm overexpression.</p><p>** = P value<.01.</p><p>*** = P value<.001.</p><p>The phosphorylation state of β-catenin affects CySC maintenance in the <i>Drosophila</i> testis.</p

Components of the Slit-Robo pathway are expressed in the <i>Drosophila</i> testis stem cell niche.

<p>(A) The <i>Drosophila</i> testis apex. Germline stem cells (GSCs) and somatic cyst stem cells (CySCs) contact the hub. GSCs divide asymmetrically to give differentiating daughters that undergo four rounds of mitosis, producing interconnected spermatogonia that will eventually form sperm. CySCs divide to give non-mitotic somatic cyst cells that encase and support differentiating spermatogonia. (B) Confocal image of a testis apex containing a GFP enhancer trap that reports the expression of <i>robo2</i>. GFP (green) is expressed in the hub (outlined), CySCs and early cyst cell daughters (arrows), but not in GSCs (arrowhead) or their progeny. Inset is GFP channel alone. (C and E) Confocal sections of wild type testes with Vasa staining the germline (red). (C) Robo2 protein (green) is enriched at the cell surface around hub cells (arrowhead) and CySCs (arrow). (D) Robo2 alone (green channel). (E) Slit protein (green) is enriched in hub cells (outlined). (F) Slit alone (green channel). DNA stained with DAPI (blue), scale bars = 10 µm.</p

The Robo2 receptor is autonomously required for CySC maintenance in the testis.

<p>(A–B) Confocal sections of testes with Zfh-1 staining CySCs and early cyst cell daughters (red). Negatively marked mosaic clones are identified by absence of GFP (green). (A) At 2 days ACI, wild type CySC clones (outlined, yellow) and GSC clones (outlined, white) are both present in the testis while (B) <i>robo2¹</i> mutant CySCs are absent at 2 days ACI and only GSC clones (outlined, white) are present. For comparison, examples of GFP⁺ GSCs (arrowheads) and CySCs (arrows) are indicated. A′ and B′ show GFP panels alone. (C–D) Confocal sections of testes with Traffic jam staining the hub, CySCs and cyst cells (red). Positively marked mosaic clones are identified by presence of GFP (green). (C) At 2 days ACI, marked wild type CySCs (arrows) are present close to the hub while differentiating cyst cells are present further from the hub. (D) <i>robo2¹</i> mutant CySCs are absent near the hub but <i>robo2¹</i> null differentiating cysts cells (arrowheads) are present far from the hub (yellow asterisk, below plane of focus). C′ and D′ show Traffic Jam staining alone. (E–F) Confocal sections of testes with Eya staining differentiating late cyst cells (red). Positively marked mosaic clones are identified by presence of GFP (green). (E) Eya⁺ wild type cyst cell clones (arrowheads) and (F) <i>robo2¹</i> cyst cell clones (arrowheads) are present outside the niche at 2 days ACI. Early cyst cell clones (both wild type and <i>robo2¹</i>) do not express Eya (arrows) E′ and F′ show Eya staining alone. (G–H) Confocal sections of testes with Zfh-1 staining CySCs and early cyst cell daughters (red). Positively marked mosaic clones are identified by presence of GFP (green). (G) At 2 days ACI, overexpression of ECad rescues <i>robo2</i> null clones, which are capable of dividing (arrowhead indicates mitotic GFP⁺ CySC, DNA condensed, and nuclear GFP and Zfh-1 proteins appear cytoplasmic). (H) <i>Robo2</i> null CySCs rescued with ECad (arrowhead) are maintained at 5 days ACI and produce progeny (arrows). G′ and H′ show Zfh-1 staining alone. Hubs outlined in white, DNA stained with DAPI (blue), scale bars = 10 µm.</p

<i>Robo2</i> transcription levels in the testis depend on JAK-STAT signaling.

<p>(A–D) Whole testis in situ hybridization of <i>robo2</i> mRNA. Hubs are marked with an asterisk. In (A) temperature sensitive Stat92E flies (<i>Stat92E^ts</i>) and (C) <i>hs-Upd</i> flies at 18°C, <i>robo2</i> is expressed in the hub and surrounding early CySCs and daughters, consistent with the wild type <i>robo2</i> mRNA expression pattern (not shown). (B) In <i>Stat92E^ts</i> flies shifted to the restrictive temperature (29°C) for 24 hours, <i>robo2</i> expression is abolished. (D) In <i>hs-Upd</i> flies heat shocked for 45 minutes at 37°C, <i>robo2</i> expression expands throughout the testis apex. (E–F) Confocal sections of testes containing the <i>Robo2-GFP</i> enhancer trap with Zfh-1 staining CySCs and early cyst cell daughters (red) and GFP reporting <i>robo2</i> expression (green). Fewer GFP⁺ cyst lineage cells are found in (E) <i>Robo2-GFP</i> testes than in (F) <i>Robo2-GFP</i> testes overexpressing Upd. Hubs outlined in white, DAPI stains DNA (blue), scale bars = 10 µm.</p

Abl kinase activity is required to prevent CySC overcompetition.

<p>(A) Confocal section of testis expressing an Abl∶GFP fusion protein. Abl is expressed in both germline and somatic lineages and is enriched at cell surfaces. The position of the hub is marked with an asterisk. (B) Structure of the Abl kinase protein with two <i>Abl</i> alleles described. (C–H) Confocal sections of testes with Zfh-1 staining CySCs and early cyst cell daughters (red). Positively marked mosaic clones are identified by presence of GFP (green). At 2 days ACI, (C) wild type CySC clones (arrowhead) are detected in similar numbers to (D) <i>Abl⁴</i> mutant CySC clones (arrowhead). At 8 days ACI, (E) the number of marked wild type CySCs per testis remains low while (F) the number of marked <i>Abl⁴</i> mutant CySCs per testis increases and few unmarked CySCs are detected. (G) At 8 days ACI, the number of marked <i>Abl⁴</i> mutant CySCs per testis does not increase compared to wild type when Abl is resupplied while (H) Abl^KinaseDead expression fails to rescue the increase in marked <i>Abl⁴</i> mutant CySCs per testis. Hubs outlined in white, DNA stained with DAPI (Blue), scale bars = 10 µm.</p

β-Cat knockdown prevents Abl⁴ mutant CySC clones from outcompeting their neighbors and taking over the niche.

a<p>Testes with CySC clones = testes with GFP⁺, Zfh-1⁺ cells/total testes scored (percentage).</p>b<p>Testes with >50% marked CySCs = testis where GFP⁺, Zfh-1⁺ cells>GFP⁻, Zfh-1⁺ cells/total testes scores (percentage).</p>c<p>Wild type clones expressing β-Cat RNAi = Wild type Frt 40A MARCM flies driving β-Cat RNAi specifically in induced clones.</p>d<p>Abl⁴ clones expressing β-Cat RNAi = Abl⁴ MARCM flies driving β-Cat RNAi specifically in induced clones.</p>e<p>P value vs Abl⁴ clones.</p><p>ND = Not Determined, ACI = After Clone Induction,</p><p>* = P value<.05,</p><p>** = P value<.01.</p><p>β-Cat knockdown prevents Abl⁴ mutant CySC clones from outcompeting their neighbors and taking over the niche.</p

Abl kinase activity is required to attenuate CySC competition in the <i>Drosophila</i> testis.

a<p>Testes with CySC clones = testes with GFP⁺, Zfh-1⁺ cells/total testes scored (percentage).</p>b<p>Testes with >50% marked CySCs = testis where GFP⁺, Zfh-1⁺ cells>GFP⁻, Zfh-1⁺ cells/total testes scores (percentage).</p>c<p>Abl⁴ MARCM clones expressing Abl = Abl⁴ MARCM flies driving UAS-Abl specifically in induced clones.</p>d<p>Abl⁴ MARCM clones expressing Abl^KinaseDead = Abl⁴ MARCM flies driving UAS-Abl^KinaseDead specifically in induced clones.</p><p>ND = Not Determined, ACI = After Clone Induction,</p><p>* = p value<.05 vs Wild type clones,</p><p>*** = p value<.001 vs Wild type clones.</p><p>Abl kinase activity is required to attenuate CySC competition in the <i>Drosophila</i> testis.</p

Robo2 and Abl alter cell-cell adhesion to control CySC maintenance.

<p>(A–F) Confocal sections of testes with Zfh-1 staining CySCs and early cyst cell daughters (red). Positively marked mosaic clones are identified by presence of GFP (green). At 8 days ACI (A) control <i>Abl⁴</i> CySCs (arrowheads) are present at high numbers in each testis, while (B) the number of <i>Abl⁴</i> CySCs expressing ECad RNAi (arrowhead) remains low. At 10 days ACI, (C) marked <i>Abl⁴</i> CySCs (arrowheads) are present in high numbers per testis, while (D) the number of <i>Abl⁴</i> CySCs expressing β-cat RNAi (arrowhead) remains low. At 2 days ACI, (E) <i>robo2</i> null GSCs (asterisk), but not CySCs are present while (F) <i>robo2</i> null CySC expressing Abl RNAi (arrowhead) are present in the niche and produce cyst cell daughter cells (arrows). Hubs outlined in white, DNA stained with DAPI (blue), scale bars = 10 µm.</p