Fig. 2

PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. a A diagram of a portion of the genome of the GZ264 strain that contains the GFP::PCN-1 transgene (isIs17) inserted at an undefined genomic location. The pie-1 promoter drives transcription in the germline and is fused to the coding regions of GFP (red) and PCN-1(blue). Boxes indicate exons with light shading for the 3’ UTR, straight lines indicate promoters, and peaked lines indicate introns. The strain contains an intact pcn-1 locus on chromosome IV. Scale bar is 100 bp. b-e Confocal microscope images of a hermaphrodite gonad at the adult stage. The dotted white line outlines the gonad, and the dashed white line marks the end of the progenitor zone. Note that not all nuclei are in the focal plane. The asterisk marks the position of the distal tip cell. The inset shows an enlargement of the region outlined by a white rectangle - scale bars are in panel E. Main scale bar is 10um, inset scale bar is 1um. Single arrowheads (in inset) and double arrowheads (outside of inset) mark nuclei that were negative for EdU staining (in gap phase) and positive for GFP::PCN-1 accumulation. White or red mark GFP::PCN-1 accumulation visualized by antibody staining (b, d, e). White or green mark EdU staining visualized by click chemistry (c, d, e). Yellow indicates overlap (d). Blue marks DAPI staining for DNA (e). GFP::PCN-1 accumulated in all progenitor zone nuclei in an adult hermaphrodite, whereas only about half the nuclei were positive for EdU staining. f A diagram of the pcn-1(am315) genomic locus. The endogenous pcn-1 promoter drives transcription and is fused to the coding regions of 3xFLAG epitope tag (red) inserted in-frame immediately after the ATG start codon to create an N-terminally tagged fusion protein expressed from the endogenous locus. g-j Confocal microscope images of a pcn-1(am315)/+ hermaphrodite gonad at the late L4 stage. The dotted white line outlines the gonad, and the dashed white line marks the end of the progenitor zone. Note that not all nuclei are in the focal plane. The asterisk marks the position of the distal tip cell. The inset shows an enlargement of the region outlined by a white rectangle – scale bars are in panel J. Main scale bar is 10um, inset scale bar is 1um. Single and double arrowheads mark nuclei that were negative for EdU staining (in gap phase) and positive for FLAG::PCN-1 accumulation. The arrow points to an anaphase nucleus. Main scale bar is 10um, inset scale bar is 1um. White or red mark FLAG::PCN-1 visualized by antibody staining (g, i, j); it accumulated in all progenitor zone nuclei. White or green mark EdU staining visualized by click chemistry (h, i, j). Yellow indicates overlap (i). Blue marks DAPI staining for DNA (j). Longer exposures of panels B and G show that all nuclei are PCN-1 positive. Longer exposures of panels D and I show that a 30 min EdU pulse, which marks S-phase nuclei, stains about 50% of progenitor zone nuclei. Antibody staining for WAPL-1 was used to define progenitor zone cells. k Western blot probed with anti-FLAG antibody. Lane 1: Markers, with sizes indicated in kDa. Lanes 2, 3, and 4: 1.5ul, 7.5ul, and 15ul protein lysate from pcn-1(am315flag)/nT1g animals, respectively. Lane 5: 7.5ul protein lysate from wild-type animals. The Western blot shows an ~ 35 kDa band specific to the lanes containing pcn-1(am315flag) (arrowhead on right), which corresponds well with the expected 31.75 kDa size. l, m Quantification of fluorescence intensity of EdU (l) and FLAG::PCN-1 (m). Background values were obtained from image regions without tissue. Nuclei were selected in the DAPI channel and assigned to phases as follows: nuclei without WAPL-1 signal were assigned to meiosis (n = 73), nuclei with both WAPL-1 and EdU signal were assigned to S-phase (n = 104), and nuclei with WAPL-1 but without EdU signal were assigned to gap phases (n = 36). ANOVA with Tukey’s Honest Significant Difference post-hoc was used to compare the mean fluorescence intensity of nuclei; NS indicates P > 0.01, * P < 0.01, ** P < .001, *** P < .0001. The distributions of gap and S-phase EdU intensity were mutually exclusive; the FLAG::PCN-1 distributions of gap and S-phase display 74% overlap