Limits...
Coordinate regulation of stem cell competition by Slit-Robo and JAK-STAT signaling in the Drosophila testis.

Stine RR, Greenspan LJ, Ramachandran KV, Matunis EL - PLoS Genet. (2014)

Bottom Line: CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy.Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels.Our work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). Here, we show a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. Our work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche.

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Robo2 and Abl alter cell-cell adhesion to control CySC maintenance.(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 Abl4 CySCs (arrowheads) are present at high numbers in each testis, while (B) the number of Abl4 CySCs expressing ECad RNAi (arrowhead) remains low. At 10 days ACI, (C) marked Abl4 CySCs (arrowheads) are present in high numbers per testis, while (D) the number of Abl4 CySCs expressing β-cat RNAi (arrowhead) remains low. At 2 days ACI, (E) robo2  GSCs (asterisk), but not CySCs are present while (F) robo2  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.
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pgen-1004713-g004: Robo2 and Abl alter cell-cell adhesion to control CySC maintenance.(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 Abl4 CySCs (arrowheads) are present at high numbers in each testis, while (B) the number of Abl4 CySCs expressing ECad RNAi (arrowhead) remains low. At 10 days ACI, (C) marked Abl4 CySCs (arrowheads) are present in high numbers per testis, while (D) the number of Abl4 CySCs expressing β-cat RNAi (arrowhead) remains low. At 2 days ACI, (E) robo2 GSCs (asterisk), but not CySCs are present while (F) robo2 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.

Mentions: As mentioned above, testes with Abl mutant CySC clones retain a full complement of GSCs, while testes with Socs36E mutant CySCs do not [18], indicating that Abl and Socs36E may control stem cell competition in different ways. Abl is known to affect adhesion levels in multiple tissues [26], [48], [52], [53], usually by destabilizing adherens junctions. Therefore, we focused on cadherins, rather than integrins as a potential mechanism for adhesion-based stem cell competition in Abl mutant CySCs. We first hypothesized that ECad mediated adhesion is normally attenuated by Abl kinase in CySCs. If this is true, reducing ECad in CySCs lacking Abl kinase activity should eliminate their competitive advantage. We induced control (Abl4) and experimental (Abl4 with ECad RNAi) CySC clones at similar frequencies, but by 8 days ACI, the latter clones did not outcompete their neighbors. Instead, they were present in similar numbers as wild type CySCs (Figure 4A, 4B, Table 4). This indicates that Abl mutant CySCs require ECad to outcompete their neighbors for occupancy in the niche. While Abl and ECad could be working in parallel pathways, we suspect that Abl normally destabilizes ECad adherens junctions to mediate CySC competition. In this case, knocking down ECad would cause Abl- CySCs to lose their competitive advantage compared to neighboring wild type cells. Because ECad RNAi is not completely efficient at knocking down ECad levels (predicted efficiency, 48.7–50.3%, [54]), the residual ECad would still be stabilized in Abl mutant CySCs, leading to an intermediate phenotype where cells are better maintained than in clones expressing ECad RNAi alone. This is what we observe and our data suggests that Abl-mediated competition relies on increased ECad-mediated adhesion to the hub. Consistent with this idea, ectopic expression of ECad within individual CySCs was sufficient to cause them to outcompete neighboring wild type CySCs but not GSCs from the niche (Figure S6C, Table 2). Taken together, these data suggest that Abl kinase activity destabilizes ECad-mediated adherens junction complexes in CySCs to attenuate CySC-hub cell adhesion. However, as we have not shown a direct interaction between Abl and ECad in the testis niche, it is possible that these molecules act in parallel.


Coordinate regulation of stem cell competition by Slit-Robo and JAK-STAT signaling in the Drosophila testis.

Stine RR, Greenspan LJ, Ramachandran KV, Matunis EL - PLoS Genet. (2014)

Robo2 and Abl alter cell-cell adhesion to control CySC maintenance.(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 Abl4 CySCs (arrowheads) are present at high numbers in each testis, while (B) the number of Abl4 CySCs expressing ECad RNAi (arrowhead) remains low. At 10 days ACI, (C) marked Abl4 CySCs (arrowheads) are present in high numbers per testis, while (D) the number of Abl4 CySCs expressing β-cat RNAi (arrowhead) remains low. At 2 days ACI, (E) robo2  GSCs (asterisk), but not CySCs are present while (F) robo2  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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4222695&req=5

pgen-1004713-g004: Robo2 and Abl alter cell-cell adhesion to control CySC maintenance.(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 Abl4 CySCs (arrowheads) are present at high numbers in each testis, while (B) the number of Abl4 CySCs expressing ECad RNAi (arrowhead) remains low. At 10 days ACI, (C) marked Abl4 CySCs (arrowheads) are present in high numbers per testis, while (D) the number of Abl4 CySCs expressing β-cat RNAi (arrowhead) remains low. At 2 days ACI, (E) robo2 GSCs (asterisk), but not CySCs are present while (F) robo2 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.
Mentions: As mentioned above, testes with Abl mutant CySC clones retain a full complement of GSCs, while testes with Socs36E mutant CySCs do not [18], indicating that Abl and Socs36E may control stem cell competition in different ways. Abl is known to affect adhesion levels in multiple tissues [26], [48], [52], [53], usually by destabilizing adherens junctions. Therefore, we focused on cadherins, rather than integrins as a potential mechanism for adhesion-based stem cell competition in Abl mutant CySCs. We first hypothesized that ECad mediated adhesion is normally attenuated by Abl kinase in CySCs. If this is true, reducing ECad in CySCs lacking Abl kinase activity should eliminate their competitive advantage. We induced control (Abl4) and experimental (Abl4 with ECad RNAi) CySC clones at similar frequencies, but by 8 days ACI, the latter clones did not outcompete their neighbors. Instead, they were present in similar numbers as wild type CySCs (Figure 4A, 4B, Table 4). This indicates that Abl mutant CySCs require ECad to outcompete their neighbors for occupancy in the niche. While Abl and ECad could be working in parallel pathways, we suspect that Abl normally destabilizes ECad adherens junctions to mediate CySC competition. In this case, knocking down ECad would cause Abl- CySCs to lose their competitive advantage compared to neighboring wild type cells. Because ECad RNAi is not completely efficient at knocking down ECad levels (predicted efficiency, 48.7–50.3%, [54]), the residual ECad would still be stabilized in Abl mutant CySCs, leading to an intermediate phenotype where cells are better maintained than in clones expressing ECad RNAi alone. This is what we observe and our data suggests that Abl-mediated competition relies on increased ECad-mediated adhesion to the hub. Consistent with this idea, ectopic expression of ECad within individual CySCs was sufficient to cause them to outcompete neighboring wild type CySCs but not GSCs from the niche (Figure S6C, Table 2). Taken together, these data suggest that Abl kinase activity destabilizes ECad-mediated adherens junction complexes in CySCs to attenuate CySC-hub cell adhesion. However, as we have not shown a direct interaction between Abl and ECad in the testis niche, it is possible that these molecules act in parallel.

Bottom Line: CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy.Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels.Our work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Stem cells in tissues reside in and receive signals from local microenvironments called niches. Understanding how multiple signals within niches integrate to control stem cell function is challenging. The Drosophila testis stem cell niche consists of somatic hub cells that maintain both germline stem cells and somatic cyst stem cells (CySCs). Here, we show a role for the axon guidance pathway Slit-Roundabout (Robo) in the testis niche. The ligand Slit is expressed specifically in hub cells while its receptor, Roundabout 2 (Robo2), is required in CySCs in order for them to compete for occupancy in the niche. CySCs also require the Slit-Robo effector Abelson tyrosine kinase (Abl) to prevent over-adhesion of CySCs to the niche, and CySCs mutant for Abl outcompete wild type CySCs for niche occupancy. Both Robo2 and Abl phenotypes can be rescued through modulation of adherens junction components, suggesting that the two work together to balance CySC adhesion levels. Interestingly, expression of Robo2 requires JAK-STAT signaling, an important maintenance pathway for both germline and cyst stem cells in the testis. Our work indicates that Slit-Robo signaling affects stem cell function downstream of the JAK-STAT pathway by controlling the ability of stem cells to compete for occupancy in their niche.

Show MeSH
Related in: MedlinePlus