To determine the number of ovarioles in wild type and mutant ovaries, ovaries were stained with antibodies against Vasa and Engrailed, a transcription factor that is expressed only in niche cells of the germaria [52 (link)]. The number of niches was determined by counting Engrailed positive cells adjacent to Vasa positive cells.
Several nuclear parameters were assessed. To determine intensity of GFP-BAF, emerin, and lamin at the nuclear envelope or within the GSC nucleus, a line segment was drawn across each nucleus. The rim intensity was defined as the average grey value of the two intersection points between the line and the nuclear envelope. The interior intensity was defined as the mean grey value along the line that is inside of the nucleus. For these experiments, three replicates were performed, corresponding to three different slides made for each genotype. In each case, imaging parameters were kept the same among experiments and samples. The middle section of each GSC was chosen for quantification. Background was subtracted for these quantifications. To measure nuclear roundness, each nucleus was traced based on lamin staining in ImageJ and roundness was determined as 4*area/(π*major_axis^2).
Several mitotic parameters were assessed. Mitotic stages were determined based on staining patterns of α-tubulin, Cnn, and H3S10p using the following criteria (Fig. S2): 1) Prophase was defined as H3S10p staining that was restricted to the periphery and evidence of microtubules nucleation at the periphery of the nuclear envelope, 2) Prometaphase was defined as chromosomes marked by H3S10p that were partially condensed and microtubules that were beginning to connect chromosomes to the spindle poles, 3) Metaphase was defined as chromosomes marked with H3S10p that were fully condensed and aligned at the metaphase plates, and microtubules formed the characteristic fusiform metaphase spindle, 4) Anaphase was defined as chromosomes marked with H3S10p that were separated into two clusters that each had individual chromosome arms that were visible, and 5) Telophase was defined as H3S10p staining that was weaker and the presence of a central spindle at the mid-plane of the cell. To determine alignment of microtubules (MTs), images were visualized in ImageJ and misaligned MTs were defined as MTs that cross each other’s path at the metaphase plate. The percentage of MT coverage of chromosomes was quantified in maximum projection images of metaphase GSCs that were stained with antibodies against H3S10p and α-tubulin. The coverage was defined as a ratio of the chromosome length that was covered by MTs divided by the entire length of the chromosome mass. The intensity of CID and CENP-C in images of metaphase GSCs were quantified in Image J using the sum slices projection method. Only well-separated CID and CENP-C foci were included. For each centromere, background was subtracted with 50 pixels rolling ball radius. Total grey values were reported for both CID and CENP-C staining. Different genotypes were imaged using the same parameters and at the same time.
To quantify DCP-1 intensity in wing discs, discs were hand dissected from third instar larvae of the indicated genotypes and stained with antibodies against cleaved DCP-1 and DAPI. Samples were imaged using a confocal microscopy under the same setting at the same time. DCP-1 intensity was quantified from a summed z-projection with background subtracted using image J (50 pixel rolling ball radius). The total intensity was divided by the area of the wing disc to control for size differences. Resulting DCP-1 mean intensity was graphed. At least three replicates were performed for each genotype.
Several nuclear parameters were assessed. To determine intensity of GFP-BAF, emerin, and lamin at the nuclear envelope or within the GSC nucleus, a line segment was drawn across each nucleus. The rim intensity was defined as the average grey value of the two intersection points between the line and the nuclear envelope. The interior intensity was defined as the mean grey value along the line that is inside of the nucleus. For these experiments, three replicates were performed, corresponding to three different slides made for each genotype. In each case, imaging parameters were kept the same among experiments and samples. The middle section of each GSC was chosen for quantification. Background was subtracted for these quantifications. To measure nuclear roundness, each nucleus was traced based on lamin staining in ImageJ and roundness was determined as 4*area/(π*major_axis^2).
Several mitotic parameters were assessed. Mitotic stages were determined based on staining patterns of α-tubulin, Cnn, and H3S10p using the following criteria (Fig. S2): 1) Prophase was defined as H3S10p staining that was restricted to the periphery and evidence of microtubules nucleation at the periphery of the nuclear envelope, 2) Prometaphase was defined as chromosomes marked by H3S10p that were partially condensed and microtubules that were beginning to connect chromosomes to the spindle poles, 3) Metaphase was defined as chromosomes marked with H3S10p that were fully condensed and aligned at the metaphase plates, and microtubules formed the characteristic fusiform metaphase spindle, 4) Anaphase was defined as chromosomes marked with H3S10p that were separated into two clusters that each had individual chromosome arms that were visible, and 5) Telophase was defined as H3S10p staining that was weaker and the presence of a central spindle at the mid-plane of the cell. To determine alignment of microtubules (MTs), images were visualized in ImageJ and misaligned MTs were defined as MTs that cross each other’s path at the metaphase plate. The percentage of MT coverage of chromosomes was quantified in maximum projection images of metaphase GSCs that were stained with antibodies against H3S10p and α-tubulin. The coverage was defined as a ratio of the chromosome length that was covered by MTs divided by the entire length of the chromosome mass. The intensity of CID and CENP-C in images of metaphase GSCs were quantified in Image J using the sum slices projection method. Only well-separated CID and CENP-C foci were included. For each centromere, background was subtracted with 50 pixels rolling ball radius. Total grey values were reported for both CID and CENP-C staining. Different genotypes were imaged using the same parameters and at the same time.
To quantify DCP-1 intensity in wing discs, discs were hand dissected from third instar larvae of the indicated genotypes and stained with antibodies against cleaved DCP-1 and DAPI. Samples were imaged using a confocal microscopy under the same setting at the same time. DCP-1 intensity was quantified from a summed z-projection with background subtracted using image J (50 pixel rolling ball radius). The total intensity was divided by the area of the wing disc to control for size differences. Resulting DCP-1 mean intensity was graphed. At least three replicates were performed for each genotype.
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