Egree the overall size of the hub can influence stem cell number, how stem cells respond to damage to the niche, and how a functional niche is maintained over time. Therefore, we designed an RNAi-based screen 1317923 to begin to address such questions.Results and Discussion headcase Function is Required for Maintenance of the He cell population spreads across the substrate can be calculated. A apical HubTo identify factors involved in regulating hub size and maintenance, we sought to reduce the expression of candidate genes specifically in hub cells. We employed the bipartite GAL4-UAS system, in combination with RNAi, to reduce candidate gene expression in hub cells using the updGAL4 `driver’ line. When expression of RNAi constructs was lethal or lead to developmental defects, a temperature sensitive allele ofHeadcase Regulates Maintenance of the Testis NicheFigure 1. hdc function is required to maintain hub cells in the Drosophila testis. (A) Schematic of the male germline stem cell niche. (B) Hub cell quantification at 1d(blue) and 10d(red) in controls and upon reduction of hdc. N = 30 for each genotype/time point. Mean and SD are shown; ***P,0.001 (Kruskal allis one-way analysis of variance). (C) Immunofluorescence image of wild-type (wt) testis. FasIII (hub, red), Vasa (germ line, green) (C’) Phase-contrast image of a wt testis. Asterisk denotes apical tip; transit-amplifying spermatogonia (black bar); spermatocytes (arrows). (D and D’) Reduction of hdc in hub cells leads to loss of hub cells and niche degeneration. Note absence of FasIII+ hub cells (red) and presence of large spermatocytes or mature sperm (D’) at the apical tip. (E and E’) Testis from wt larval (L3) male gonad showing Hdc expression in all cells at the apical tip. (F and F’) RNAi-mediated knock-down of hdc in hub cells results in loss of Hdc protein. Similar results were obtained for all RNAi lines tested. Scale bars, 20 mm. See also Table 1 and Figure 2. doi:10.1371/journal.pone.0068026.gGal80 (Gal80ts) was used to suppress Gal4 activity, and thus RNAi expression, until eclosion (hatching). Subsequently, the number of hub cells and stem cells was quantified in 1-day old (1d) and 10-day old (10d) adults. Hub cell number varies with genetic background, even within controls; therefore, the Title Loaded From File average number of hub cells was measured in a variety of genetic backgrounds at both time points. All genotypes tested had an average hub cell number ranging between 9 and 11 (Fig. 1B; Table 1), which is consistent with previously published data for wild-type testes [7] [23]. Importantly, theseresults verified that hub cell number does not change significantly within 10 days post-eclosion [23]. While knock-down of the majority of the candidates had no effect, reduction of the headcase (hdc) gene led to a dramatic loss of hub cells (Fig. 1B ; Table 1). In contrast, no evident phenotype was observed when hdc was over-expressed in the hub (Table 1). Staining with a Hdc antibody revealed cytoplasmic expression in all cells throughout the tip of wild-type testes and demonstrated efficient knock-down of Hdc expression in hub cells upon RNAimediated depletion (Fig. 1E,F). Loss of hub cells upon hdcHeadcase Regulates Maintenance of the Testis NicheTable 1. Results of candidate gene screen for regulators of hub size.Genotypehub cell number 1d+/2 std dev 10d 10.94+/22.45 (N = 31); 0 10.93+/22.07 (N = 30); 0 10.19+/22.83 (N = 16); 0 11.07+/22.40 (N = 29); 0 ND 4.20+/21.44 (N = 25); 0 5.17+/22.27 (N = 35); 0 1.6+/21.36 (N = 26); 35 8.34+/22.74 (N = 26); 0 9.04+.Egree the overall size of the hub can influence stem cell number, how stem cells respond to damage to the niche, and how a functional niche is maintained over time. Therefore, we designed an RNAi-based screen 1317923 to begin to address such questions.Results and Discussion headcase Function is Required for Maintenance of the Apical HubTo identify factors involved in regulating hub size and maintenance, we sought to reduce the expression of candidate genes specifically in hub cells. We employed the bipartite GAL4-UAS system, in combination with RNAi, to reduce candidate gene expression in hub cells using the updGAL4 `driver’ line. When expression of RNAi constructs was lethal or lead to developmental defects, a temperature sensitive allele ofHeadcase Regulates Maintenance of the Testis NicheFigure 1. hdc function is required to maintain hub cells in the Drosophila testis. (A) Schematic of the male germline stem cell niche. (B) Hub cell quantification at 1d(blue) and 10d(red) in controls and upon reduction of hdc. N = 30 for each genotype/time point. Mean and SD are shown; ***P,0.001 (Kruskal allis one-way analysis of variance). (C) Immunofluorescence image of wild-type (wt) testis. FasIII (hub, red), Vasa (germ line, green) (C’) Phase-contrast image of a wt testis. Asterisk denotes apical tip; transit-amplifying spermatogonia (black bar); spermatocytes (arrows). (D and D’) Reduction of hdc in hub cells leads to loss of hub cells and niche degeneration. Note absence of FasIII+ hub cells (red) and presence of large spermatocytes or mature sperm (D’) at the apical tip. (E and E’) Testis from wt larval (L3) male gonad showing Hdc expression in all cells at the apical tip. (F and F’) RNAi-mediated knock-down of hdc in hub cells results in loss of Hdc protein. Similar results were obtained for all RNAi lines tested. Scale bars, 20 mm. See also Table 1 and Figure 2. doi:10.1371/journal.pone.0068026.gGal80 (Gal80ts) was used to suppress Gal4 activity, and thus RNAi expression, until eclosion (hatching). Subsequently, the number of hub cells and stem cells was quantified in 1-day old (1d) and 10-day old (10d) adults. Hub cell number varies with genetic background, even within controls; therefore, the average number of hub cells was measured in a variety of genetic backgrounds at both time points. All genotypes tested had an average hub cell number ranging between 9 and 11 (Fig. 1B; Table 1), which is consistent with previously published data for wild-type testes [7] [23]. Importantly, theseresults verified that hub cell number does not change significantly within 10 days post-eclosion [23]. While knock-down of the majority of the candidates had no effect, reduction of the headcase (hdc) gene led to a dramatic loss of hub cells (Fig. 1B ; Table 1). In contrast, no evident phenotype was observed when hdc was over-expressed in the hub (Table 1). Staining with a Hdc antibody revealed cytoplasmic expression in all cells throughout the tip of wild-type testes and demonstrated efficient knock-down of Hdc expression in hub cells upon RNAimediated depletion (Fig. 1E,F). Loss of hub cells upon hdcHeadcase Regulates Maintenance of the Testis NicheTable 1. Results of candidate gene screen for regulators of hub size.Genotypehub cell number 1d+/2 std dev 10d 10.94+/22.45 (N = 31); 0 10.93+/22.07 (N = 30); 0 10.19+/22.83 (N = 16); 0 11.07+/22.40 (N = 29); 0 ND 4.20+/21.44 (N = 25); 0 5.17+/22.27 (N = 35); 0 1.6+/21.36 (N = 26); 35 8.34+/22.74 (N = 26); 0 9.04+.
