The Jak-STAT Target Chinmo Prevents Sex Transformation of Adult Stem Cells in the Drosophila Testis Niche

Local signals maintain adult stem cells in many tissues. Whether the sexual identity of adult stem cells must also be maintained was not known. In the adult Drosophila testis niche, local Jak-STAT signaling promotes somatic cyst stem cell (CySC) renewal through several effectors, including the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo). Here, we find that Chinmo also prevents feminization of CySCs. Chinmo promotes expression of the canonical male sex determination factor DoublesexM (Dsx(M)) within CySCs and their progeny, and ectopic expression of DsxM in the CySC lineage partially rescues the chinmo sex transformation phenotype, placing Chinmo upstream of Dsx(M). The Dsx homolog DMRT1 prevents the male-to-female conversion of differentiated somatic cells in the adult mammalian testis, but its regulation is not well understood. Our work indicates that sex maintenance occurs in adult somatic stem cells and that this highly conserved process is governed by effectors of niche signals

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

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

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

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