Es: UAShdcRNAi2 (Autophagy milder phenotype) targets all three isoforms while UAShdcRNAi1 (stronger phenotype) and UAShdcRNAi3 (milder phenotype) are the same construct but inserted in diferent chromosomes and only target two of the 3 isoforms. Note that hub cell loss could not be tested using hdc mutants, as all alleles tested (hdc50, hdcFus-6, hdc43, hdcB4-3-20 and hdcKG09851) exhibited embryonic/larval lethality. For hdc over-expression two lines were used: UAS-hdcGSD404 (gift from D. Van Meyel), and UAS-hdcRA/C. To Epigenetic Reader Domain generate transgenicflies carrying UAS-hdcRA/C, the full length hdc sequence, corresponding to isoforms A/C, was amplified via PCR and cloned into the pUAST-attB vector to allow targeted integration into the att2 site on the third chromosome. Embryo injections were performed by Genetic Services Inc (Sudbury, MA).Candidate RNAi ScreenMale flies carrying RNAi transgenes targeting candidate genes were crossed to virgin females carrying the updGal4 driver. Crosses were set at 25uC and male flies were dissected upon eclosion (1d) or collected and maintained at 25uC before dissection at 10 days (10d). Suppression of expression of the UAS-rpr, UAS-rpr+UAS-hid, UAS-hdcRNAi1 and G-TRACE constructs during development was achieved using a temperature sensitive allele of Gal80 (Gal80ts). Flies were raised at 18uC and shifted to 29uC upon eclosion (day 1) to activate transgene expression for 10 days, or as otherwise noted. Expression of UAS-hdcRNAi2 and UAS-hdcRNAi3 was 18204824 suppressedHeadcase Regulates Maintenance of the Testis NicheFigure 7. Reduced headcase levels in hub cells results in loss of hub cells due to apoptosis. As hub cells are lost, stem cell niche architecture changes. However, severely compromised hubs composed of only 1 or 2 cells remain functional and capable of maintaining active stem cells around them. doi:10.1371/journal.pone.0068026.gduring development by raising flies at 18uC (ie., Gal80ts was not used).Statistical AnalysisFor the purpose of comparing hub cell averages between multiple genotypes/time points found on Figure 1 and Figure 2 the Kruskal allis one-way analysis of variance test was used, followed by a Dunn’s multiple comparison test. For Figure 4, a Welch’s t-test was used to determine the statistical significance of the difference in hub cell averages between pairs of columns (hdcRNAi1 vs hdcRNAi1+p35) (hdcRNAi3 vs hdcRNAi3+p35). For both comparisons, p-values were represented in graphs using the following asterisk code: * P20.05, **P20.01, ***P20.001. Statistical significance of the frequency of apoptotic hub cells was tested with the Fisher’s exact test, two tailed. A Chi-square test was used to analyze the G-trace data and the frequency of testes with hubrestricted vs non-restricted GFP expression.AntibodiesTestes were stained with: rabbit anti-Vasa (1:2,000) (gift from P. Lasko); mouse anti-Hdc(1:5) (gift from R. White); guinea pig antiZfh-1 (1:3,000) (gift from C. Doe); rabbit anti-Stat92E (1:800) (gift from D. Montell); mouse anti-Fasciclin III (7G10) (1:10); rat antiDE-cadherin (1:20), rat anti-DN-cadherin (1:20) and mouse antiarmadillo (1:15) (Developmental Studies Hybridoma Bank (developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences); rabbit anti-GFP(1:5000) (Molecular Probes). Secondary antibodies were diluted 1:500 (Molecular Probes).Apoptosis Assay Immunofluorescence and MicroscopyPhase co.Es: UAShdcRNAi2 (milder phenotype) targets all three isoforms while UAShdcRNAi1 (stronger phenotype) and UAShdcRNAi3 (milder phenotype) are the same construct but inserted in diferent chromosomes and only target two of the 3 isoforms. Note that hub cell loss could not be tested using hdc mutants, as all alleles tested (hdc50, hdcFus-6, hdc43, hdcB4-3-20 and hdcKG09851) exhibited embryonic/larval lethality. For hdc over-expression two lines were used: UAS-hdcGSD404 (gift from D. Van Meyel), and UAS-hdcRA/C. To generate transgenicflies carrying UAS-hdcRA/C, the full length hdc sequence, corresponding to isoforms A/C, was amplified via PCR and cloned into the pUAST-attB vector to allow targeted integration into the att2 site on the third chromosome. Embryo injections were performed by Genetic Services Inc (Sudbury, MA).Candidate RNAi ScreenMale flies carrying RNAi transgenes targeting candidate genes were crossed to virgin females carrying the updGal4 driver. Crosses were set at 25uC and male flies were dissected upon eclosion (1d) or collected and maintained at 25uC before dissection at 10 days (10d). Suppression of expression of the UAS-rpr, UAS-rpr+UAS-hid, UAS-hdcRNAi1 and G-TRACE constructs during development was achieved using a temperature sensitive allele of Gal80 (Gal80ts). Flies were raised at 18uC and shifted to 29uC upon eclosion (day 1) to activate transgene expression for 10 days, or as otherwise noted. Expression of UAS-hdcRNAi2 and UAS-hdcRNAi3 was 18204824 suppressedHeadcase Regulates Maintenance of the Testis NicheFigure 7. Reduced headcase levels in hub cells results in loss of hub cells due to apoptosis. As hub cells are lost, stem cell niche architecture changes. However, severely compromised hubs composed of only 1 or 2 cells remain functional and capable of maintaining active stem cells around them. doi:10.1371/journal.pone.0068026.gduring development by raising flies at 18uC (ie., Gal80ts was not used).Statistical AnalysisFor the purpose of comparing hub cell averages between multiple genotypes/time points found on Figure 1 and Figure 2 the Kruskal allis one-way analysis of variance test was used, followed by a Dunn’s multiple comparison test. For Figure 4, a Welch’s t-test was used to determine the statistical significance of the difference in hub cell averages between pairs of columns (hdcRNAi1 vs hdcRNAi1+p35) (hdcRNAi3 vs hdcRNAi3+p35). For both comparisons, p-values were represented in graphs using the following asterisk code: * P20.05, **P20.01, ***P20.001. Statistical significance of the frequency of apoptotic hub cells was tested with the Fisher’s exact test, two tailed. A Chi-square test was used to analyze the G-trace data and the frequency of testes with hubrestricted vs non-restricted GFP expression.AntibodiesTestes were stained with: rabbit anti-Vasa (1:2,000) (gift from P. Lasko); mouse anti-Hdc(1:5) (gift from R. White); guinea pig antiZfh-1 (1:3,000) (gift from C. Doe); rabbit anti-Stat92E (1:800) (gift from D. Montell); mouse anti-Fasciclin III (7G10) (1:10); rat antiDE-cadherin (1:20), rat anti-DN-cadherin (1:20) and mouse antiarmadillo (1:15) (Developmental Studies Hybridoma Bank (developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences); rabbit anti-GFP(1:5000) (Molecular Probes). Secondary antibodies were diluted 1:500 (Molecular Probes).Apoptosis Assay Immunofluorescence and MicroscopyPhase co.
