Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans

Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from... Background: The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E. Results: To study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of- function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development. Conclusions: In the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor cells is distinct from somatic cells, as it does not heavily rely on cyclic accumulation of classic cell cycle proteins. Keywords: PCNA, S-phase, C. elegans, Germline, Cell cycle, pcn-1 * Correspondence: kornfeld@wustl.edu; ts@wustl.edu Kerry Kornfeld and Tim Schedl contributed equally to this work. Department of Developmental Biology, Washington University in St. Louis, 660 S. Euclid Ave, St. Louis, MO 63108, USA Department of Genetics, Washington University in St. Louis, 660 S. Euclid Ave, St. Louis, MO 63108, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 2 of 9 Background short (e.g. 30 min) exposure, which might reflect defects in Regulation of cell proliferation is key to a wide range of ingestion and/or transport of EdU ([11], our unpublished health concerns, from infertility to wound healing and can- observations). To clarify the relationships between PCN-1 cer.LikeGoldilocks, everytissueinevery living organism accumulation and nucleotide analog incorporation as markers must find the level of cell division that is “just right” in of S-phase, we developed methods to combine these order to survive, develop, and pass on genetic information. two approaches. The cell cycle coordinates duplication and segregation of To visualize PCN-1 in the adult germline, we used both genetic and cellular material to two daughter cells. CRISPR/Cas9 genome editing to modify the native pcn-1 Cell cycle regulators change abundance or localization at locus to encode a 3xFLAG epitope at the N-terminus of different stages of DNA replication and cell division. For PCN-1. Surprisingly, FLAG::PCN-1 accumulated in all example, cyclin E accumulates during the G1-S-phase tran- nuclei in the germline progenitor zone. By contrast, a sition. This pattern is generally conserved between multiple short pulse of EdU revealed that only about half of these eukaryotic kingdoms of life. However, the adult C. elegans nuclei were in S-phase. These results suggest that the germline accumulates cyclin E in all cell cycle phases, sug- accumulation and localization of PCN-1 is regulated dif- gesting that this tissue may utilize distinctive mechanisms ferently in the C. elegans germline, where it is present in of cell cycle control [1]. all progenitor zone nuclei, compared to the embryo, The distal C. elegans hermaphrodite germline contains where it is restricted to nuclei in S-phase. Furthermore, the only stem cells in the adult C. elegans (Fig. 1a). The we demonstrated that pcn-1 is an essential gene in C. somatic distal tip cell (DTC) surrounds the syncytial elegans necessary for both adult fertility and embryonic distal-most nuclei and provides the niche to maintain development. These results extend the understanding of these stem cells in their proliferative fate. The ~ 20 the accumulation and function of PCN-1 in C. elegans, cell-diameter long distal region, which includes the mi- an important model system for studies of germline de- totically cycling germline stem and progenitor cells and velopment and cell cycle regulation. meiotic S-phase cells but not cells in meiotic prophase, is called the progenitor zone [2–5]. As germ cells move Methods away from the DTC, the cells finish the mitotic cell C. elegans strains were maintained at 20 °C on 6 cm cycle, enter the meiotic cell cycle, undergo meiotic dishes of nematode growth media (NGM) seeded with S-phase, and enter prophase I of meiosis [3]. OP50 E. coli [12]. The wild-type strain was Bristol N2. The proliferating cell nuclear antigen (PCNA or The GZ264 strain contains the GFP::PCN-1 transgene PCN-1 in C. elegans) is an essential processivity factor (isIs17) inserted at an undefined location in the genome, for DNA polymerase δ (Reviewed in [6]). As its name and it is wild-type at the pcn-1 locus [9]. To engineer a suggests, PCNA was identified by its nuclear accumula- FLAG epitope tag into the endogenous pcn-1 locus, we tion in proliferating cells [7]. In mammals, antibody rec- used the co-CRISPR approach [13]. The injection mix ognition of PCNA is an often-used technique to identify contained Cas9-expressing pDD162 (gift from Mike cells in S-phase [8]. Brauchle et al. [9] established Nonet) at 50 ng/μl, dpy-10 guide plasmid (pMN3153) at PCN-1 as a useful in vivo marker of S-phase in C. ele- 20 ng/μl, dpy-10(cn64) ssDNA repair template AFZF827 gans early embryos, a stage when the cell cycle involves at 500 nM, pcn-1 guide plasmids (pZK21 and pZK23) at only negligible gap phases. In transgenic worms that 40 ng/μl for each plasmid, and a ssDNA repair template express a green fluorescent protein GFP::PCN-1 fusion (ZK071 containing the 3xflag sequence) at 600 nM. We protein under the control of the pie-1 promoter, injected the gonads of P0 wild-type hermaphrodites, GFP::PCN-1 localizes to the nucleus during S-phase, selected F1 progeny that displayed the Rol phenotype, resulting in a bright fluorescent signal. At nuclear enve- and used PCR to select animals with the desired geno- lope breakdown (beginning of mitosis), GFP::PCN-1 type. Two independently derived strains contained the localizes to the cytoplasm, resulting in a diffuse, low same insertion of the 22 amino acid coding region for level signal. Similarly, GFP::PCNA protein injected into the 3xFLAG epitope after the initial methionine codon the gonad serves as a marker of S-phase in both C. ele- of pcn-1. We named these alleles pcn-1(am315) and gans pronuclei and early embryonic divisions [10]. pcn-1(am316) (strains WU1764 and WU1765, respect- For studies of cell cycle dynamics in the C. elegans ively). We used a GFP-tagged balancer chromosome to adult germline, labeling with nucleotide analogs such as maintain the strain pcn-1(am315)/nT1g due to the ob- 5-ethynyl-2′-deoxyuridine (EdU) or 5-bromo-2′-deoxyuridine served maternal-effect lethal phenotype. (BrdU) has been the gold standard to identify S-phase [1, 2]. To confirm expression of the full-length FLAG::PCN-1 However, these chemicals must enter C. elegans by feeding or product, we performed a Western blot. In brief, animals soaking, which limits the utility of this approach. For example, were washed from one 12 cm NGM dish and rinsed 4 some older adult animals fail to label with EdU following a times with M9 buffer. Protease inhibitor (Thermo Scientific Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 3 of 9 Fig. 1 Diagram of distal C. elegans germline and experimental workflow. a The syncytial distal progenitor zone (highlighted in red based on WAPL-1 antibody staining) contains mitotically cycling stem and progenitor cells and cells in meiotic S-phase. The distal tip cell (DTC) provides GLP-1 signal (Notch ligand) to maintain the stem cell fate of these cells. As cells migrate away from the DTC, they exit the progenitor zone and enter meiotic prophase. b Workflow used to assay the relationship between PCN-1 accumulation and S-phase (EdU labeling) in the C. elegans germline Pierce Protease Inhibitor Mini Tablets Cat# 88665) was used at 2× listed concentration in the second two washes. An equal volume of 2× lysis-and-loading buffer (100 mM TrisHCl pH 6.8, 200 mM DTT, 4% SDS, 0.2% Bromophe- nol Blue, 20% Glycerol) was added, and the samples were frozen at − 80 °C overnight. The samples were microwaved, boiled, sonicated in an ice bath, and boiled again. Samples (1.5, 7.5, or 15 uL) and a size reference (7uL, AccuRuler Prestained Protein Ladder LambdaBio Cat# G02101) were loaded into a pre-cast gel (BioRad Mini-PROTEAN TGX 4–20% polyacrylamide gel Cat#456–1093) in 1X Tris/ Gly- cine/ SDS buffer and run at 180 V for ~ 1 h in an ice bath. Samples were transferred to activated membrane (Millipore Immobilon-P) in 1X Tris/ Glycine/ 20%Methanol buffer at 350 mA for ~ 1 h in an ice bath. The membrane was incu- bated in blocking buffer (5% non-fat milk in 1X PBS + 0.5% Tween-20) for ~ 1 h at room temperature with agitation. Primary mouse-anti-FLAG antibody (Sigma M2 F-1804) was diluted 1:10,000 in blocking buffer and applied over- night at 4 °C with agitation. The membrane was rinsed 3× with PBSTw. Secondary anti-mouse HRP antibody (Cell Signaling) was diluted 1:2000 in blocking buffer and applied for ~ 1 h at room temperature with agitation. The mem- brane was rinsed 3× with PBSTw, incubated with substrate (ThermoScientific SuperSignal West Femto Maximum Sensitivity Substrate) for ~ 1 min, and imaged on a Chemi- Doc imaging system. Protein size was estimated from con- ceptually translated DNA sequence using the Science Gateway Protein Molecular Weight Calculator (http:// www.sciencegateway.org/tools/proteinmw.htm). To visualize both FLAG::PCN-1 accumulation and cells in S-phase, we cultured fourth larval stage (L4) and young adult progeny of heterozygous pcn-1(am315/+) parent animals on EdU-labeled E. coli MG1693 for 30 min and promptly dissected [1]. Animals were fixed with 3% paraformaldehyde for 10 min and post-fixed in 100% methanol at − 20 °C overnight. Primary mouse-anti-FLAG antibody (Sigma M2 F-1804) was di- luted 1:1000 and rabbit-anti-WAPL antibody (Novus 49300002) was diluted 1:20000 in 30% goat serum and applied overnight. Secondary goat-anti-mouse-Alexa-647 antibody and secondary goat-anti-rabbit-Alexa-594 anti- body (Life Technologies) were diluted 1:400 in 30% goat serum and applied for 4 h. The Click-iT EdU Fluor 488 Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 4 of 9 reaction was performed according to manufacturer instruc- (egg-to-larva and egg-to-adult), the identical crosses tions (ThermoFisher Cat#C10350). Animals were mounted were performed, and F1 animals were allowed to lay in Vectashield + DAPI on agarose pads and imaged using a eggs for several hours. These eggs were transferred to a 63×/1.4NA Plan Apo oil immersion objective on a Zeiss fresh dish and counted 24 h later, the unhatched eggs Observer Z1 inverted microscope equipped with a were counted to estimate the number of larvae, and the Perkin-Elmer Ultraview Vox spinning disc confocal system adults were counted 48 h later. using Volocity software. Exposure times were 150 ms for EdU, 50 ms for FLAG::PCN-1, and 500 ms for DAPI. Im- Results ages were processed with Fiji [14] and Adobe Illustrator. PCN-1 accumulated during all cell cycle phases in the GFP::PCN-1 was visualized as described above, except pri- germline progenitor zone mary rabbit-anti-GFP (gift from Swathi Arur) and secondary PCN-1 accumulation has been monitored using an inte- goat-anti-rabbit-Alexa-594 were used (Fig. 1b). grated transgene that encodes a GFP::PCN-1 fusion pro- To quantify fluorescence intensity, we used the region tein (Fig. 2a). In C. elegans early embryos, PCN-1 of interest ROI and Measure tools in Fiji. In brief, a cell accumulates during S-phase but not during M phase, indi- was selected in a single plane based on DAPI channel cating that it is regulated during the cell cycle [9, 10]. and assigned to S-phase or gap phase based on the Thus, we anticipated that PCN-1 would display a similar presence or absence of EdU. A single ROI was drawn to accumulation pattern in the larval and adult germline. To surround the DAPI signal, and the mean intensity of test this prediction, we monitored S-phase nuclei using a DAPI, EdU, and PCN-1 was obtained for the ROI. Back- short, 30 min pulse of EdU and we monitored PCN-1 ground intensities of all three channels were very low expression with an anti-GFP antibody to detect the and comparable, and therefore no background subtrac- GFP::PCN-1 fusion protein (Fig. 1b). The nucleotide ana- tion was performed. log EdU is used as a selective marker of DNA synthesis as Statistical analyses were performed in R [15] using R it is incorporated into DNA during S-phase. In the germ- studio [16] and ggplot2 [17]. Data were compared using line, S-phase occurs only in progenitor zone cells, which an ANOVA with Tukey’s Honest Significant Difference include the stem cells (Fig. 1a). Consistent with previous post-hoc. All error bars shown in figures represent the publications, approximately 50% of progenitor zone nuclei mean +/− standard deviation. were EdU positive, indicating that these cells were in We determined the genotype and phenotype of animals S-phase (Fig. 2c)[1, 2, 18]. By contrast, all progenitor zone descended from P0 heterozygous pcn-1(am315)/+ or nuclei were GFP::PCN-1 positive, irrespective of their cell pcn-1(am316)/+ hermaphrodites. F1 animals were cul- cycle stage (Fig. 2b). Thus, about half the progenitor zone tured individually for 3 to 4 days from egg. Each animal nuclei were positive for both EdU and GFP::PCN-1, indi- was observed for viability and fertility. Animals which de- cating that cells in S-phase display nuclear accumulation posited no eggs on the substrate were dissected to allow of PCN-1 (Fig. 2d). However, about half the progenitor unlaid F2 eggs to develop. One to 2 days later, F2 progeny zone nuclei were negative for EdU and positive for were examined for viability. Genotype was established by GFP::PCN-1, indicating nuclei that were not in S-phase, performing PCR analysis on individual F1 animals. Her- and presumably were in G2, M, or the short G1 phase, maphrodites were defined as “fertile” if they deposited one also accumulated PCN-1 (Fig. 2d, e). It is notable that the or more eggs on the agar surface that hatched and devel- GFP fluorescence signal in the germline displayed ex- oped into larvae. Animals were defined as “sterile” if they tremely low intensity, and we could only detect the deposited no eggs on the agar surface, or “dead eggs” if all GFP::PCN-1 fusion protein with long exposure times for deposited eggs failed to hatch. GFP fluorescence in live animals or after staining with Heterozygous animals were fertile and did not display anti-GFP antibodies. obvious defects when cultured in standard laboratory con- Two features of the transgenic strain raise the possibil- ditions. The GFP-tagged balancer chromosome nT1g was ity that the accumulation pattern of the GFP::PCN-1 fu- used to track the pcn-1 genotype in alargernumberof sion protein may not accurately reflect the accumulation animals. For brood size assays, P0 pcn-1(am315)/nT1g pattern of endogenous PCN-1; (1) fusion to the large males were mated to fog-2(q71) females. The non-green F1 GFP protein may affect the accumulation pattern or (2) progeny of this cross, pcn-1(am315/+); fog-2(q71/+) were the transgene uses the heterologous pie-1 promoter and is synchronized at the mid-L4 stage, isolated to individual integrated at a random site in the genome, not the en- NGM dishes, and moved to fresh dishes every 24 h. Parent dogenous locus (Fig. 2a). To address these concerns, we animals which fled the dish or died of matricidal hatch- used the CRISPR/Cas9 technique to edit the C. elegans ing were excluded. The F2 progeny were counted ~ genome. We chose the small FLAG epitope tag, reasoning 2 days later until the cessation of reproduction. that it is less likely to influence protein behavior than fog-2(q71/+) served as the control. For survival assays GFP. However, we cannot exclude the possibility that the Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 5 of 9 Fig. 2 (See legend on next page.) Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 6 of 9 (See figure on previous page.) 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 FLAG tagged protein does not behave identically to the Several features of the expression pattern were not- endogenous protein. We inserted coding sequence for able. In metaphase and anaphase nuclei (identified by three copies of the FLAG epitope tag at the N-terminus of DAPI morphology), FLAG::PCN-1 signal appeared PCN-1 into the native pcn-1 locus (Fig. 2f). Two inde- somewhat diffuse and to surround the chromatin (Fig. pendently derived strains with the identical insertion were 2g). By contrast, FLAG::PCN-1 signal in G1, S, and G2 generated, and we named these alleles pcn-1(am315) and nuclei overlapped with both chromatin and the nucle- pcn-1(am316). The presence of a full-length FLAG::PCN-1 olus. Similar to the results described above with fusion protein was confirmed by performing an anti-FLAG GFP::PCN-1, we detected multiple examples of nuclei in Western blot (Fig. 2k). gap phase which displayed no EdU incorporation but To monitor the expression pattern, we dissected and did display nuclear FLAG::PCN-1 accumulation (Fig. immunostained animals using an antibody directed 2g–j). The same accumulation pattern was observed against the FLAG epitope. The antibody staining was per- with both pcn-1(am315) and pcn-1(am316) strains. formed on the progeny of pcn-1(am315/+) animals, and PCNA has been reported to show a punctate nuclear thus included animals of three genotypes: pcn-1(am315/ pattern in S-phase of mammalian cells [19, 20]. We did am315), pcn-1(am315/+) and pcn-1(+/+). While the not observe punctate FLAG::PCN-1 staining; it is pos- genotype of each individual was not determined using sible that harsh fixation conditions contribute to the lack molecular approaches, we observed that the majority of of punctate staining. animals showed the same pattern of FLAG::PCN-1 in To explore the relationship between PCN-1 expression the distal germline. Specifically, FLAG::PCN-1 was and S-phase in detail, we quantified the intensity of detected in all cells in the progenitor zone (Fig. 2g). We FLAG::PCN-1 and EdU signal (following a 30 min label- interpreted these animals as a mixture of pcn-1(am315/ ing) in individual distal germline nuclei (Fig. 2l–m). Mei- am315) and pcn-1(am315/+). Thus, it is likely that otic prophase cells that are not replicating DNA were pcn-1(am315/am315) and pcn-1(am315/+) animals used to establish the level of background noise. For EdU, have a similar pattern of expression. A minority of the current gold-standard for S-phase labeling in C. ele- animals displayed no staining. We interpreted these gans, progenitor zone nuclei either displayed very low animals as pcn-1(+/+). intensity signal, similar to background, or substantial Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 7 of 9 signal; we assigned these nuclei to gap phase or S-phase, strain displayed abnormal, tortuous intestinal morph- respectively. By contrast, all of the progenitor zone nuclei ology after only 2–3 days of adulthood and a higher rate displayed FLAG::PCN-1 staining that was higher than of vulval extrusion. The self-fertile brood size was not background, indicating that all of these cells accumulate different from wild type, but the lifespan was slightly re- the protein. Thus, the presence of FLAG::PCN-1 cannot duced (GZ264 16.6 ± 5.0 n = 60; N2 20.5 ± 5.1 n = 120, be used to reliably determine if a cell is in S-phase or gap Log-Rank Test P = .0000019). These phenotypes could phase. Cells in S-phase had a higher average level of be caused by expression of the GFP::PCN-1 fusion pro- FLAG::PCN-1 staining than cells in gap phase, but the dis- tein, a deleterious effect of insertion of the transgene tributions were highly overlapping: 71% of the true into the genome at a site of functional significance, or S-phase nuclei and 81% of the true gap phase nuclei (74% additional background effects. of total nuclei) were in the ambiguous range of To determine how the insertion of the sequence en- FLAG::PCN-1 intensity (Fig. 2l–m). Thus, FLAG::PCN-1 coding the FLAG epitope at the pcn-1 locus affected appears to be continually present in nuclei, although it ac- gene function, we analyzed heterozygous and homozy- cumulates to higher levels during S phase. gous mutant animals. To rigorously quantify the pene- In the C. elegans early embryo, GFP::PCN-1 shows nu- trance of phenotypes, we determined the genotype and clear accumulation only during S-phase [9, 10]). A limita- phenotype of 31 animals descended from pcn-1(am315)/ tion of our use of FLAG::PCN-1 is that visualization cannot + hermaphrodites (Table 1a). Genotype was established be performed in live embryos. Thus, our results do not es- by performing PCR analysis on individual animals. Het- tablish the expression pattern of FLAG::PCN-1 in early em- erozygous animals were fertile and did not display obvious bryos. By contrast to embryos, our results indicate that in defects when cultured in standard laboratory conditions. the L4 and adult germline progenitor zone, PCN-1 accu- Animals homozygous for either pcn-1(am315) or mulates throughout the cell cycle and its presence cannot pcn-1(am316) that descended from heterozygous P0 be used as a reliable marker of S-phase (Fig. 3). It is likely hermaphrodites developed to adulthood and displayed that germline PCN-1 is regulated by mechanisms other sterility or maternal-effect embryonic lethality (Table 1a). than degradation/translation and nuclear localization. These homozygous mutant animals produced few F2 homozygous embryos. Some F1 homozygous mutant her- pcn-1 was necessary for robust fertility and embryonic maphrodites did not lay any embryos and displayed F2 development embryos retained inside the uterus. Other F1 homozygous The GZ264 strain that contains the GFP::PCN-1 trans- mutant hermaphrodites deposited a few embryos on the gene (isIs17) and is wild-type at the pcn-1 locus (Fig. 2a) substrate, but these embryos failed to develop. This result is viable and fertile. Compared to wild-type animals, this was confirmed in an independent experiment (without PCR genotyping) examining 60 F1 progeny of heterozygous P0 animals; 15 of these animals (25%) produced no viable ab progeny. These results indicate that both pcn-1(am315) and pcn-1(am316) are loss-of-function alleles, and pcn-1 func- tion is necessary to promote hermaphrodite fertility. In addition, pcn-1 appears to have a function early in develop- ment that can be maternally rescued, since pcn-1 homozy- gous mutants derived from heterozygous hermaphrodites develop to adulthood, whereas pcn-1 homozygous mutants derived from pcn-1 homozygous hermaphrodites displayed embryonic lethality. Fig. 3 The adult germline and embryo show distinct patterns of To determine if the pcn-1 mutation causes a dominant PCN-1 nuclear accumulation. The inner circle represents the stages effect, we analyzed survival and fertility in heterozygous of the cell cycle; DNA replication occurs in S-phase (green), mitosis animals under standard laboratory conditions. Progeny of occurs in M phase (pink), and these are separated by G2 and a very short G1 phase (blue) [1]. Germline stem cells progress to meiotic pcn-1(am315/+) heterozygous mutants displayed the same S-phase and meiosis (gray). The outer circle represents nuclear rates of egg hatching (98%) and survival to adulthood accumulation of PCN-1 (red). Germline stem cells in C. elegans (96%) as progeny of pcn-1(+/+) animals (Table 1b). The display a more typical mitotic cell cycle with gap phases and also self-fertile brood size of pcn-1(am315/+) hermaphrodites display constant (non-cycling) accumulation of several proteins, was not significantly reduced compared to pcn-1(+/+) her- including PCN-1 and CYE-1, which have been reported to display cell cycle regulated accumulation in other tissues and organisms (a) maphrodites (Table 1b). These results indicate that the [1, 22]. By contrast, early embryos in C. elegans display a simplified FLAG::PCN-1 protein does not interfere with the en- cell cycle (S,M), and PCN-1, like several other cell cycle proteins, dogenous protein function and pcn-1(lf) mutations are re- show nuclear accumulation only during S-phase (b)[9, 10] cessive for the fertility and survival phenotypes. Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 8 of 9 Table 1 Genetic analysis of pcn-1 fertility. A) Thirty-one self-progeny of heterozygous pcn-1(am315flag)/+ hermaphrodites were analyzed by PCR of single animals to determine genotype after egg-laying was complete. Full genotypes: pcn-1(+/+), pcn-1(am315/ +), and pcn-1(am315/am315). Hermaphrodites were scored as “fertile” if they deposited one or more eggs on the agar surface that hatched and developed into larvae. Animals were scored as “sterile” if they deposited no eggs on the agar surface, or if all deposited eggs failed to hatch. B) The percent of eggs that generate larvae and adults was determined for pcn-1(+/+); fog-2(q71/+) and pcn- 1(am315flag/+); fog-2(q71/+) hermaphrodites, as described in the methods. n refers to total number of eggs analyzed. A chi-square test was used to compare the larval and adult survival. The total self-fertile brood size was determined as described in the methods. ANOVA was used to compare the brood size. Mean ± s.d. n refers to total number of parental worms analyzed. NS indicates P > 0.01, * P < 0.01, ** P < .001, *** P < .0001 A Genotype pcn-1 (+/+) pcn-1 (flag/+) pcn-1 (flag/flag) Fertile 7 17 0 Sterile 0 0 7 B Parental Genotype pcn-1 (+/+) pcn-1 (flag/+) Larval survival (%) 98.1 (n = 376) 98.1 (n = 324) NS Adult survival (%) 96.0 (n = 376) 95.7 (n = 324) NS Total Brood Size 305.4 ± 35.5 (n = 15) 332.5 ± 35.7 (n = 21) NS Discussion in all nuclei in the progenitor zone. Significantly, nuclei We set out to engineer an endogenous non-feeding marker that were negative for EdU staining, and thus not in for S-phase in the C. elegans germline. We chose the gene S-phase, displayed nuclear PCN-1 accumulation (Fig. 3). PCN-1 for this purpose, as it has been used as an S-phase Similar results were previously observed for cyclin E accu- marker in mammals and in the C. elegans embryo. Both the mulation (CYE-1 in C. elegans)[1]. These observations GFP::PCN-1 transgene and our CRISPR-engineered reinforce that mitotic cell cycle regulation in the germline FLAG::PCN-1 allele showed that PCN-1 localizes to the nu- stem and progenitor cells is distinct from somatic cells [1]. cleus in all cells in the progenitor zone, regardless of The C. elegans proliferating cell nuclear antigen homolog whether the cells are in S-phase or gap phase. Therefore, pcn-1 was necessary for robust fertility and embryonic PCN-1 was not a useful marker of S-phase under our condi- development. tions. The localization of PCN-1 to all nuclei in the progeni- tor zone was similar to the pattern of cyclin E1 (CYE-1) in Abbreviations the C. elegans germline [1]. While this result means PCN-1 C. elegans: Caenorhabditis elegans; GFP: Green fluorescent protein; PCNA: Proliferating cell nuclear antigen; PFA: Paraformaldehyde is not an S-phase marker, it reveals that the C. elegans germ- line likely regulates S-phase in an unusual way. No previous publications have described the pcn-1 Acknowledgements We are grateful to Swathi Arur for rabbit-anti-GFP antibodies, the E. coli stock loss-of-function phenotype. The C. elegans database center for MG1693, Wormbase, and the Caenorhabditis Genetics Center (WormBase WS257) lists a deletion allele, pcn-1(tm3241) which is funded by the National Institutes of Health Office of Research Infra- that is predicted to be a loss-of-function mutation. A structure Programs (P40OD010440) for strains, Mike Nonet and Scott Dour for advice, reagents, and microscope use for CRISPR/Cas9 genome engineer- strain that contains this allele displays a lethal or sterile ing, Zach Pincus for injection microscope use, Ariz Mohammad for the bal- phenotype, consistent with our findings; however, the anced strain, Andrea Scharf and James Tan for assistance with Western blot pcn-1(tm3241) strain has not been backcrossed, and these preparation, the Kornfeld and Schedl labs for feedback on this manuscript, and the anonymous reviewers for suggestions that greatly improved the phenotypes might reflect background mutations [21]. We manuscript. found pcn-1(am315) animals showed a maternal-effect embryonic lethal phenotype. While it is unclear whether Funding am315 is a hypomorph or null allele, our results show that This work was supported in part by NIH grant R01 AG02656106A1 to KK, NIH pcn-1 is necessary for fertility. grant R01 GM100756 to TS, and a NSF predoctoral fellowship DGE-1143954 and DGE-1745038 to ZK. Neither the NIH nor the NSF had any role in the de- sign of the study, collection, analysis, and interpretation of data, nor in writ- Conclusions ing the manuscript. We established the nuclear accumulation pattern of PCN-1 in the larval and adult germline by analyzing Availability of data and materials FLAG::PCN-1 encoded by the native locus and All data generated or analyzed during this study are included in this published GFP::PCN-1 encoded by an integrated transgene. Both fu- article. The genetic reagents used and generated during the current study are sion proteins displayed similar patterns and accumulated available from the corresponding authors on reasonable request. Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 9 of 9 Authors’ contributions C. elegans germ line. Dev Biol. 2007;308:206–21. https://doi.org/10.1016/j. ZK, KK, and TS designed the study. ZK performed experiments and wrote the ydbio.2007.05.019. manuscript. ZK, KK, and TS revised the manuscript. All authors read and 19. Hahn AT, Jones JT, Meyer T. Quantitative analysis of cell cycle phase approved the final manuscript. durations and PC12 differentiation using fluorescent biosensors. Cell Cycle. 2009;8:1044–52. https://doi.org/10.4161/cc.8.7.8042. 20. Leonhardt H, Rahn H-P, Weinzierl P, Sporbert A, Cremer T, Zink D, et al. Ethics approval and consent to participate Dynamics of DNA replication factories in living cells. J Cell Biol. 2000;149: Not applicable 271–80. http://jcb.rupress.org/content/149/2/271. Accessed 25 July 2017 21. Consortium, T. C. E. D. M, Barstead R, Moulder G, Cobb B, Frazee S, Competing interests Henthorn D, Holmes J, Jerebie D, Landsdale M, Osborn J, Pritchett C, The authors declare that they have no competing interests. Robertson J, Rummage J, Stokes E, Vishwanathan M, Mitani S, Gengyo-Ando K, Funatsu OR. Large-scale screening for targeted knockouts in the Caenorhabditis elegans genome. G3. 2012;2:1415–25. https://doi.org/10. Publisher’sNote 1534/g3.112.003830. Springer Nature remains neutral with regard to jurisdictional claims in published 22. Leonardi E, Girlando S, Serio G, Mauri FA, Perrone G, Scampini S, et al. PCNA maps and institutional affiliations. and Ki67 expression in breast carcinoma: correlations with clinical and biological variables. J Clin Pathol. 1992;45:416–9. https://www.ncbi.nlm.nih.gov/ Received: 12 February 2018 Accepted: 15 May 2018 pmc/articles/PMC495304/pdf/jclinpath00419-0052.pdf.Accessed 19May 2017 References 1. Fox PM, Vought VE, Hanazawa M, Lee M-H, Maine EM, Schedl T. Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline. Development. 2011;138:2223–34. https://doi.org/10.1242/ dev.059535. 2. Crittenden SL, Leonhard KA, Byrd DT, Kimble J. Cellular analyses of the mitotic region in the Caenorhabditis elegans adult germ line. Mol Biol Cell. 2006;17:3051–61. https://doi.org/10.1091/mbc.E06-03-0170. 3. Fox PM, Schedl T. Analysis of germline stem cell differentiation following loss of GLP-1 notch activity in Caenorhabditis elegans. Genetics. 2015;201 September:167–84. https://doi.org/10.1534/genetics.115.178061. 4. Hansen D, Schedl T. Stem cell proliferation versus meiotic fate decision in Caenorhabditis elegans. Adv Exp Med Biol. 2013;757:71–99. 5. Pazdernik N, Schedl T. Introduction to germ cell development in caenorhabditis elegans. Adv Exp Med Biol. 2013;757:1–16. 6. Moldovan GL, Pfander B, Jentsch S. PCNA, the maestro of the replication fork. Cell. 2007;129:665–79. 7. Miyachi K, Fritzler MJ, Tan EM. Autoantibody to a nuclear antigen in proliferating cells. J Immunol. 1978;121:2228–34. http://www.jimmunol.org/ content/121/6/2228. Accessed 25 Jan 2017 8. Galand P, Degraef C. Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues. Cell Prolif. 1989;22:383–92. https:// doi.org/10.1111/j.1365-2184.1989.tb00223.x. 9. Brauchle M, Baumer K, Gönczy P. Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos. Curr Biol. 2003;13:819–27. https://doi.org/10.1016/S0960- 9822(03)00295-1. 10. Kisielewska J, Lu P, Whitaker M. GFP-PCNA as an S-phase marker in embryos during the first and subsequent cell cycles. Biol Cell. 2005;97:221–9. https:// doi.org/10.1042/BC20040093. 11. Cinquin A, Chiang M, Paz A, Hallman S, Yuan O, Vysniauskaite I, et al. Intermittent stem cell cycling balances self-renewal and senescence of the C. elegans germ line. PLoS Genet. 2016;12:e1005985. https://doi.org/10. 1371/journal.pgen.1005985. 12. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. https://doi.org/10.1002/cbic.200300625. 13. Arribere JA, Bell RT, Fu BXH, Artiles KL, Hartman PS, Fire AZ. Efficient marker- free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics. 2014;198:837–46. https://doi.org/10.1534/ genetics.114.169730. 14. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. https://doi.org/10.1038/nmeth.2019. 15. R Core Team. R: a language and environment for statistical computing. 2013. http://www.r-project.org/. 16. RStudio Team. RStudio: integrated development environment for R. 2015. http://www.rstudio.com/. 17. Wickham H. ggplot2: elegant graphics for data analysis. New York: Springer- Verlag; 2009. http://ggplot2.org. 18. Jaramillo-Lambert A, Ellefson M, Villeneuve AM, Engebrecht J. Differential timing of S phases, X chromosome replication, and meiotic prophase in the http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Developmental Biology Springer Journals

Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans

BMC Developmental Biology , Volume 18 (1) – May 30, 2018
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Abstract

Background: The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E. Results: To study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of- function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development. Conclusions: In the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor cells is distinct from somatic cells, as it does not heavily rely on cyclic accumulation of classic cell cycle proteins. Keywords: PCNA, S-phase, C. elegans, Germline, Cell cycle, pcn-1 * Correspondence: kornfeld@wustl.edu; ts@wustl.edu Kerry Kornfeld and Tim Schedl contributed equally to this work. Department of Developmental Biology, Washington University in St. Louis, 660 S. Euclid Ave, St. Louis, MO 63108, USA Department of Genetics, Washington University in St. Louis, 660 S. Euclid Ave, St. Louis, MO 63108, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 2 of 9 Background short (e.g. 30 min) exposure, which might reflect defects in Regulation of cell proliferation is key to a wide range of ingestion and/or transport of EdU ([11], our unpublished health concerns, from infertility to wound healing and can- observations). To clarify the relationships between PCN-1 cer.LikeGoldilocks, everytissueinevery living organism accumulation and nucleotide analog incorporation as markers must find the level of cell division that is “just right” in of S-phase, we developed methods to combine these order to survive, develop, and pass on genetic information. two approaches. The cell cycle coordinates duplication and segregation of To visualize PCN-1 in the adult germline, we used both genetic and cellular material to two daughter cells. CRISPR/Cas9 genome editing to modify the native pcn-1 Cell cycle regulators change abundance or localization at locus to encode a 3xFLAG epitope at the N-terminus of different stages of DNA replication and cell division. For PCN-1. Surprisingly, FLAG::PCN-1 accumulated in all example, cyclin E accumulates during the G1-S-phase tran- nuclei in the germline progenitor zone. By contrast, a sition. This pattern is generally conserved between multiple short pulse of EdU revealed that only about half of these eukaryotic kingdoms of life. However, the adult C. elegans nuclei were in S-phase. These results suggest that the germline accumulates cyclin E in all cell cycle phases, sug- accumulation and localization of PCN-1 is regulated dif- gesting that this tissue may utilize distinctive mechanisms ferently in the C. elegans germline, where it is present in of cell cycle control [1]. all progenitor zone nuclei, compared to the embryo, The distal C. elegans hermaphrodite germline contains where it is restricted to nuclei in S-phase. Furthermore, the only stem cells in the adult C. elegans (Fig. 1a). The we demonstrated that pcn-1 is an essential gene in C. somatic distal tip cell (DTC) surrounds the syncytial elegans necessary for both adult fertility and embryonic distal-most nuclei and provides the niche to maintain development. These results extend the understanding of these stem cells in their proliferative fate. The ~ 20 the accumulation and function of PCN-1 in C. elegans, cell-diameter long distal region, which includes the mi- an important model system for studies of germline de- totically cycling germline stem and progenitor cells and velopment and cell cycle regulation. meiotic S-phase cells but not cells in meiotic prophase, is called the progenitor zone [2–5]. As germ cells move Methods away from the DTC, the cells finish the mitotic cell C. elegans strains were maintained at 20 °C on 6 cm cycle, enter the meiotic cell cycle, undergo meiotic dishes of nematode growth media (NGM) seeded with S-phase, and enter prophase I of meiosis [3]. OP50 E. coli [12]. The wild-type strain was Bristol N2. The proliferating cell nuclear antigen (PCNA or The GZ264 strain contains the GFP::PCN-1 transgene PCN-1 in C. elegans) is an essential processivity factor (isIs17) inserted at an undefined location in the genome, for DNA polymerase δ (Reviewed in [6]). As its name and it is wild-type at the pcn-1 locus [9]. To engineer a suggests, PCNA was identified by its nuclear accumula- FLAG epitope tag into the endogenous pcn-1 locus, we tion in proliferating cells [7]. In mammals, antibody rec- used the co-CRISPR approach [13]. The injection mix ognition of PCNA is an often-used technique to identify contained Cas9-expressing pDD162 (gift from Mike cells in S-phase [8]. Brauchle et al. [9] established Nonet) at 50 ng/μl, dpy-10 guide plasmid (pMN3153) at PCN-1 as a useful in vivo marker of S-phase in C. ele- 20 ng/μl, dpy-10(cn64) ssDNA repair template AFZF827 gans early embryos, a stage when the cell cycle involves at 500 nM, pcn-1 guide plasmids (pZK21 and pZK23) at only negligible gap phases. In transgenic worms that 40 ng/μl for each plasmid, and a ssDNA repair template express a green fluorescent protein GFP::PCN-1 fusion (ZK071 containing the 3xflag sequence) at 600 nM. We protein under the control of the pie-1 promoter, injected the gonads of P0 wild-type hermaphrodites, GFP::PCN-1 localizes to the nucleus during S-phase, selected F1 progeny that displayed the Rol phenotype, resulting in a bright fluorescent signal. At nuclear enve- and used PCR to select animals with the desired geno- lope breakdown (beginning of mitosis), GFP::PCN-1 type. Two independently derived strains contained the localizes to the cytoplasm, resulting in a diffuse, low same insertion of the 22 amino acid coding region for level signal. Similarly, GFP::PCNA protein injected into the 3xFLAG epitope after the initial methionine codon the gonad serves as a marker of S-phase in both C. ele- of pcn-1. We named these alleles pcn-1(am315) and gans pronuclei and early embryonic divisions [10]. pcn-1(am316) (strains WU1764 and WU1765, respect- For studies of cell cycle dynamics in the C. elegans ively). We used a GFP-tagged balancer chromosome to adult germline, labeling with nucleotide analogs such as maintain the strain pcn-1(am315)/nT1g due to the ob- 5-ethynyl-2′-deoxyuridine (EdU) or 5-bromo-2′-deoxyuridine served maternal-effect lethal phenotype. (BrdU) has been the gold standard to identify S-phase [1, 2]. To confirm expression of the full-length FLAG::PCN-1 However, these chemicals must enter C. elegans by feeding or product, we performed a Western blot. In brief, animals soaking, which limits the utility of this approach. For example, were washed from one 12 cm NGM dish and rinsed 4 some older adult animals fail to label with EdU following a times with M9 buffer. Protease inhibitor (Thermo Scientific Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 3 of 9 Fig. 1 Diagram of distal C. elegans germline and experimental workflow. a The syncytial distal progenitor zone (highlighted in red based on WAPL-1 antibody staining) contains mitotically cycling stem and progenitor cells and cells in meiotic S-phase. The distal tip cell (DTC) provides GLP-1 signal (Notch ligand) to maintain the stem cell fate of these cells. As cells migrate away from the DTC, they exit the progenitor zone and enter meiotic prophase. b Workflow used to assay the relationship between PCN-1 accumulation and S-phase (EdU labeling) in the C. elegans germline Pierce Protease Inhibitor Mini Tablets Cat# 88665) was used at 2× listed concentration in the second two washes. An equal volume of 2× lysis-and-loading buffer (100 mM TrisHCl pH 6.8, 200 mM DTT, 4% SDS, 0.2% Bromophe- nol Blue, 20% Glycerol) was added, and the samples were frozen at − 80 °C overnight. The samples were microwaved, boiled, sonicated in an ice bath, and boiled again. Samples (1.5, 7.5, or 15 uL) and a size reference (7uL, AccuRuler Prestained Protein Ladder LambdaBio Cat# G02101) were loaded into a pre-cast gel (BioRad Mini-PROTEAN TGX 4–20% polyacrylamide gel Cat#456–1093) in 1X Tris/ Gly- cine/ SDS buffer and run at 180 V for ~ 1 h in an ice bath. Samples were transferred to activated membrane (Millipore Immobilon-P) in 1X Tris/ Glycine/ 20%Methanol buffer at 350 mA for ~ 1 h in an ice bath. The membrane was incu- bated in blocking buffer (5% non-fat milk in 1X PBS + 0.5% Tween-20) for ~ 1 h at room temperature with agitation. Primary mouse-anti-FLAG antibody (Sigma M2 F-1804) was diluted 1:10,000 in blocking buffer and applied over- night at 4 °C with agitation. The membrane was rinsed 3× with PBSTw. Secondary anti-mouse HRP antibody (Cell Signaling) was diluted 1:2000 in blocking buffer and applied for ~ 1 h at room temperature with agitation. The mem- brane was rinsed 3× with PBSTw, incubated with substrate (ThermoScientific SuperSignal West Femto Maximum Sensitivity Substrate) for ~ 1 min, and imaged on a Chemi- Doc imaging system. Protein size was estimated from con- ceptually translated DNA sequence using the Science Gateway Protein Molecular Weight Calculator (http:// www.sciencegateway.org/tools/proteinmw.htm). To visualize both FLAG::PCN-1 accumulation and cells in S-phase, we cultured fourth larval stage (L4) and young adult progeny of heterozygous pcn-1(am315/+) parent animals on EdU-labeled E. coli MG1693 for 30 min and promptly dissected [1]. Animals were fixed with 3% paraformaldehyde for 10 min and post-fixed in 100% methanol at − 20 °C overnight. Primary mouse-anti-FLAG antibody (Sigma M2 F-1804) was di- luted 1:1000 and rabbit-anti-WAPL antibody (Novus 49300002) was diluted 1:20000 in 30% goat serum and applied overnight. Secondary goat-anti-mouse-Alexa-647 antibody and secondary goat-anti-rabbit-Alexa-594 anti- body (Life Technologies) were diluted 1:400 in 30% goat serum and applied for 4 h. The Click-iT EdU Fluor 488 Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 4 of 9 reaction was performed according to manufacturer instruc- (egg-to-larva and egg-to-adult), the identical crosses tions (ThermoFisher Cat#C10350). Animals were mounted were performed, and F1 animals were allowed to lay in Vectashield + DAPI on agarose pads and imaged using a eggs for several hours. These eggs were transferred to a 63×/1.4NA Plan Apo oil immersion objective on a Zeiss fresh dish and counted 24 h later, the unhatched eggs Observer Z1 inverted microscope equipped with a were counted to estimate the number of larvae, and the Perkin-Elmer Ultraview Vox spinning disc confocal system adults were counted 48 h later. using Volocity software. Exposure times were 150 ms for EdU, 50 ms for FLAG::PCN-1, and 500 ms for DAPI. Im- Results ages were processed with Fiji [14] and Adobe Illustrator. PCN-1 accumulated during all cell cycle phases in the GFP::PCN-1 was visualized as described above, except pri- germline progenitor zone mary rabbit-anti-GFP (gift from Swathi Arur) and secondary PCN-1 accumulation has been monitored using an inte- goat-anti-rabbit-Alexa-594 were used (Fig. 1b). grated transgene that encodes a GFP::PCN-1 fusion pro- To quantify fluorescence intensity, we used the region tein (Fig. 2a). In C. elegans early embryos, PCN-1 of interest ROI and Measure tools in Fiji. In brief, a cell accumulates during S-phase but not during M phase, indi- was selected in a single plane based on DAPI channel cating that it is regulated during the cell cycle [9, 10]. and assigned to S-phase or gap phase based on the Thus, we anticipated that PCN-1 would display a similar presence or absence of EdU. A single ROI was drawn to accumulation pattern in the larval and adult germline. To surround the DAPI signal, and the mean intensity of test this prediction, we monitored S-phase nuclei using a DAPI, EdU, and PCN-1 was obtained for the ROI. Back- short, 30 min pulse of EdU and we monitored PCN-1 ground intensities of all three channels were very low expression with an anti-GFP antibody to detect the and comparable, and therefore no background subtrac- GFP::PCN-1 fusion protein (Fig. 1b). The nucleotide ana- tion was performed. log EdU is used as a selective marker of DNA synthesis as Statistical analyses were performed in R [15] using R it is incorporated into DNA during S-phase. In the germ- studio [16] and ggplot2 [17]. Data were compared using line, S-phase occurs only in progenitor zone cells, which an ANOVA with Tukey’s Honest Significant Difference include the stem cells (Fig. 1a). Consistent with previous post-hoc. All error bars shown in figures represent the publications, approximately 50% of progenitor zone nuclei mean +/− standard deviation. were EdU positive, indicating that these cells were in We determined the genotype and phenotype of animals S-phase (Fig. 2c)[1, 2, 18]. By contrast, all progenitor zone descended from P0 heterozygous pcn-1(am315)/+ or nuclei were GFP::PCN-1 positive, irrespective of their cell pcn-1(am316)/+ hermaphrodites. F1 animals were cul- cycle stage (Fig. 2b). Thus, about half the progenitor zone tured individually for 3 to 4 days from egg. Each animal nuclei were positive for both EdU and GFP::PCN-1, indi- was observed for viability and fertility. Animals which de- cating that cells in S-phase display nuclear accumulation posited no eggs on the substrate were dissected to allow of PCN-1 (Fig. 2d). However, about half the progenitor unlaid F2 eggs to develop. One to 2 days later, F2 progeny zone nuclei were negative for EdU and positive for were examined for viability. Genotype was established by GFP::PCN-1, indicating nuclei that were not in S-phase, performing PCR analysis on individual F1 animals. Her- and presumably were in G2, M, or the short G1 phase, maphrodites were defined as “fertile” if they deposited one also accumulated PCN-1 (Fig. 2d, e). It is notable that the or more eggs on the agar surface that hatched and devel- GFP fluorescence signal in the germline displayed ex- oped into larvae. Animals were defined as “sterile” if they tremely low intensity, and we could only detect the deposited no eggs on the agar surface, or “dead eggs” if all GFP::PCN-1 fusion protein with long exposure times for deposited eggs failed to hatch. GFP fluorescence in live animals or after staining with Heterozygous animals were fertile and did not display anti-GFP antibodies. obvious defects when cultured in standard laboratory con- Two features of the transgenic strain raise the possibil- ditions. The GFP-tagged balancer chromosome nT1g was ity that the accumulation pattern of the GFP::PCN-1 fu- used to track the pcn-1 genotype in alargernumberof sion protein may not accurately reflect the accumulation animals. For brood size assays, P0 pcn-1(am315)/nT1g pattern of endogenous PCN-1; (1) fusion to the large males were mated to fog-2(q71) females. The non-green F1 GFP protein may affect the accumulation pattern or (2) progeny of this cross, pcn-1(am315/+); fog-2(q71/+) were the transgene uses the heterologous pie-1 promoter and is synchronized at the mid-L4 stage, isolated to individual integrated at a random site in the genome, not the en- NGM dishes, and moved to fresh dishes every 24 h. Parent dogenous locus (Fig. 2a). To address these concerns, we animals which fled the dish or died of matricidal hatch- used the CRISPR/Cas9 technique to edit the C. elegans ing were excluded. The F2 progeny were counted ~ genome. We chose the small FLAG epitope tag, reasoning 2 days later until the cessation of reproduction. that it is less likely to influence protein behavior than fog-2(q71/+) served as the control. For survival assays GFP. However, we cannot exclude the possibility that the Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 5 of 9 Fig. 2 (See legend on next page.) Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 6 of 9 (See figure on previous page.) 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 FLAG tagged protein does not behave identically to the Several features of the expression pattern were not- endogenous protein. We inserted coding sequence for able. In metaphase and anaphase nuclei (identified by three copies of the FLAG epitope tag at the N-terminus of DAPI morphology), FLAG::PCN-1 signal appeared PCN-1 into the native pcn-1 locus (Fig. 2f). Two inde- somewhat diffuse and to surround the chromatin (Fig. pendently derived strains with the identical insertion were 2g). By contrast, FLAG::PCN-1 signal in G1, S, and G2 generated, and we named these alleles pcn-1(am315) and nuclei overlapped with both chromatin and the nucle- pcn-1(am316). The presence of a full-length FLAG::PCN-1 olus. Similar to the results described above with fusion protein was confirmed by performing an anti-FLAG GFP::PCN-1, we detected multiple examples of nuclei in Western blot (Fig. 2k). gap phase which displayed no EdU incorporation but To monitor the expression pattern, we dissected and did display nuclear FLAG::PCN-1 accumulation (Fig. immunostained animals using an antibody directed 2g–j). The same accumulation pattern was observed against the FLAG epitope. The antibody staining was per- with both pcn-1(am315) and pcn-1(am316) strains. formed on the progeny of pcn-1(am315/+) animals, and PCNA has been reported to show a punctate nuclear thus included animals of three genotypes: pcn-1(am315/ pattern in S-phase of mammalian cells [19, 20]. We did am315), pcn-1(am315/+) and pcn-1(+/+). While the not observe punctate FLAG::PCN-1 staining; it is pos- genotype of each individual was not determined using sible that harsh fixation conditions contribute to the lack molecular approaches, we observed that the majority of of punctate staining. animals showed the same pattern of FLAG::PCN-1 in To explore the relationship between PCN-1 expression the distal germline. Specifically, FLAG::PCN-1 was and S-phase in detail, we quantified the intensity of detected in all cells in the progenitor zone (Fig. 2g). We FLAG::PCN-1 and EdU signal (following a 30 min label- interpreted these animals as a mixture of pcn-1(am315/ ing) in individual distal germline nuclei (Fig. 2l–m). Mei- am315) and pcn-1(am315/+). Thus, it is likely that otic prophase cells that are not replicating DNA were pcn-1(am315/am315) and pcn-1(am315/+) animals used to establish the level of background noise. For EdU, have a similar pattern of expression. A minority of the current gold-standard for S-phase labeling in C. ele- animals displayed no staining. We interpreted these gans, progenitor zone nuclei either displayed very low animals as pcn-1(+/+). intensity signal, similar to background, or substantial Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 7 of 9 signal; we assigned these nuclei to gap phase or S-phase, strain displayed abnormal, tortuous intestinal morph- respectively. By contrast, all of the progenitor zone nuclei ology after only 2–3 days of adulthood and a higher rate displayed FLAG::PCN-1 staining that was higher than of vulval extrusion. The self-fertile brood size was not background, indicating that all of these cells accumulate different from wild type, but the lifespan was slightly re- the protein. Thus, the presence of FLAG::PCN-1 cannot duced (GZ264 16.6 ± 5.0 n = 60; N2 20.5 ± 5.1 n = 120, be used to reliably determine if a cell is in S-phase or gap Log-Rank Test P = .0000019). These phenotypes could phase. Cells in S-phase had a higher average level of be caused by expression of the GFP::PCN-1 fusion pro- FLAG::PCN-1 staining than cells in gap phase, but the dis- tein, a deleterious effect of insertion of the transgene tributions were highly overlapping: 71% of the true into the genome at a site of functional significance, or S-phase nuclei and 81% of the true gap phase nuclei (74% additional background effects. of total nuclei) were in the ambiguous range of To determine how the insertion of the sequence en- FLAG::PCN-1 intensity (Fig. 2l–m). Thus, FLAG::PCN-1 coding the FLAG epitope at the pcn-1 locus affected appears to be continually present in nuclei, although it ac- gene function, we analyzed heterozygous and homozy- cumulates to higher levels during S phase. gous mutant animals. To rigorously quantify the pene- In the C. elegans early embryo, GFP::PCN-1 shows nu- trance of phenotypes, we determined the genotype and clear accumulation only during S-phase [9, 10]). A limita- phenotype of 31 animals descended from pcn-1(am315)/ tion of our use of FLAG::PCN-1 is that visualization cannot + hermaphrodites (Table 1a). Genotype was established be performed in live embryos. Thus, our results do not es- by performing PCR analysis on individual animals. Het- tablish the expression pattern of FLAG::PCN-1 in early em- erozygous animals were fertile and did not display obvious bryos. By contrast to embryos, our results indicate that in defects when cultured in standard laboratory conditions. the L4 and adult germline progenitor zone, PCN-1 accu- Animals homozygous for either pcn-1(am315) or mulates throughout the cell cycle and its presence cannot pcn-1(am316) that descended from heterozygous P0 be used as a reliable marker of S-phase (Fig. 3). It is likely hermaphrodites developed to adulthood and displayed that germline PCN-1 is regulated by mechanisms other sterility or maternal-effect embryonic lethality (Table 1a). than degradation/translation and nuclear localization. These homozygous mutant animals produced few F2 homozygous embryos. Some F1 homozygous mutant her- pcn-1 was necessary for robust fertility and embryonic maphrodites did not lay any embryos and displayed F2 development embryos retained inside the uterus. Other F1 homozygous The GZ264 strain that contains the GFP::PCN-1 trans- mutant hermaphrodites deposited a few embryos on the gene (isIs17) and is wild-type at the pcn-1 locus (Fig. 2a) substrate, but these embryos failed to develop. This result is viable and fertile. Compared to wild-type animals, this was confirmed in an independent experiment (without PCR genotyping) examining 60 F1 progeny of heterozygous P0 animals; 15 of these animals (25%) produced no viable ab progeny. These results indicate that both pcn-1(am315) and pcn-1(am316) are loss-of-function alleles, and pcn-1 func- tion is necessary to promote hermaphrodite fertility. In addition, pcn-1 appears to have a function early in develop- ment that can be maternally rescued, since pcn-1 homozy- gous mutants derived from heterozygous hermaphrodites develop to adulthood, whereas pcn-1 homozygous mutants derived from pcn-1 homozygous hermaphrodites displayed embryonic lethality. Fig. 3 The adult germline and embryo show distinct patterns of To determine if the pcn-1 mutation causes a dominant PCN-1 nuclear accumulation. The inner circle represents the stages effect, we analyzed survival and fertility in heterozygous of the cell cycle; DNA replication occurs in S-phase (green), mitosis animals under standard laboratory conditions. Progeny of occurs in M phase (pink), and these are separated by G2 and a very short G1 phase (blue) [1]. Germline stem cells progress to meiotic pcn-1(am315/+) heterozygous mutants displayed the same S-phase and meiosis (gray). The outer circle represents nuclear rates of egg hatching (98%) and survival to adulthood accumulation of PCN-1 (red). Germline stem cells in C. elegans (96%) as progeny of pcn-1(+/+) animals (Table 1b). The display a more typical mitotic cell cycle with gap phases and also self-fertile brood size of pcn-1(am315/+) hermaphrodites display constant (non-cycling) accumulation of several proteins, was not significantly reduced compared to pcn-1(+/+) her- including PCN-1 and CYE-1, which have been reported to display cell cycle regulated accumulation in other tissues and organisms (a) maphrodites (Table 1b). These results indicate that the [1, 22]. By contrast, early embryos in C. elegans display a simplified FLAG::PCN-1 protein does not interfere with the en- cell cycle (S,M), and PCN-1, like several other cell cycle proteins, dogenous protein function and pcn-1(lf) mutations are re- show nuclear accumulation only during S-phase (b)[9, 10] cessive for the fertility and survival phenotypes. Kocsisova et al. BMC Developmental Biology (2018) 18:12 Page 8 of 9 Table 1 Genetic analysis of pcn-1 fertility. A) Thirty-one self-progeny of heterozygous pcn-1(am315flag)/+ hermaphrodites were analyzed by PCR of single animals to determine genotype after egg-laying was complete. Full genotypes: pcn-1(+/+), pcn-1(am315/ +), and pcn-1(am315/am315). Hermaphrodites were scored as “fertile” if they deposited one or more eggs on the agar surface that hatched and developed into larvae. Animals were scored as “sterile” if they deposited no eggs on the agar surface, or if all deposited eggs failed to hatch. B) The percent of eggs that generate larvae and adults was determined for pcn-1(+/+); fog-2(q71/+) and pcn- 1(am315flag/+); fog-2(q71/+) hermaphrodites, as described in the methods. n refers to total number of eggs analyzed. A chi-square test was used to compare the larval and adult survival. The total self-fertile brood size was determined as described in the methods. ANOVA was used to compare the brood size. Mean ± s.d. n refers to total number of parental worms analyzed. NS indicates P > 0.01, * P < 0.01, ** P < .001, *** P < .0001 A Genotype pcn-1 (+/+) pcn-1 (flag/+) pcn-1 (flag/flag) Fertile 7 17 0 Sterile 0 0 7 B Parental Genotype pcn-1 (+/+) pcn-1 (flag/+) Larval survival (%) 98.1 (n = 376) 98.1 (n = 324) NS Adult survival (%) 96.0 (n = 376) 95.7 (n = 324) NS Total Brood Size 305.4 ± 35.5 (n = 15) 332.5 ± 35.7 (n = 21) NS Discussion in all nuclei in the progenitor zone. Significantly, nuclei We set out to engineer an endogenous non-feeding marker that were negative for EdU staining, and thus not in for S-phase in the C. elegans germline. We chose the gene S-phase, displayed nuclear PCN-1 accumulation (Fig. 3). PCN-1 for this purpose, as it has been used as an S-phase Similar results were previously observed for cyclin E accu- marker in mammals and in the C. elegans embryo. Both the mulation (CYE-1 in C. elegans)[1]. These observations GFP::PCN-1 transgene and our CRISPR-engineered reinforce that mitotic cell cycle regulation in the germline FLAG::PCN-1 allele showed that PCN-1 localizes to the nu- stem and progenitor cells is distinct from somatic cells [1]. cleus in all cells in the progenitor zone, regardless of The C. elegans proliferating cell nuclear antigen homolog whether the cells are in S-phase or gap phase. Therefore, pcn-1 was necessary for robust fertility and embryonic PCN-1 was not a useful marker of S-phase under our condi- development. tions. The localization of PCN-1 to all nuclei in the progeni- tor zone was similar to the pattern of cyclin E1 (CYE-1) in Abbreviations the C. elegans germline [1]. While this result means PCN-1 C. elegans: Caenorhabditis elegans; GFP: Green fluorescent protein; PCNA: Proliferating cell nuclear antigen; PFA: Paraformaldehyde is not an S-phase marker, it reveals that the C. elegans germ- line likely regulates S-phase in an unusual way. No previous publications have described the pcn-1 Acknowledgements We are grateful to Swathi Arur for rabbit-anti-GFP antibodies, the E. coli stock loss-of-function phenotype. The C. elegans database center for MG1693, Wormbase, and the Caenorhabditis Genetics Center (WormBase WS257) lists a deletion allele, pcn-1(tm3241) which is funded by the National Institutes of Health Office of Research Infra- that is predicted to be a loss-of-function mutation. A structure Programs (P40OD010440) for strains, Mike Nonet and Scott Dour for advice, reagents, and microscope use for CRISPR/Cas9 genome engineer- strain that contains this allele displays a lethal or sterile ing, Zach Pincus for injection microscope use, Ariz Mohammad for the bal- phenotype, consistent with our findings; however, the anced strain, Andrea Scharf and James Tan for assistance with Western blot pcn-1(tm3241) strain has not been backcrossed, and these preparation, the Kornfeld and Schedl labs for feedback on this manuscript, and the anonymous reviewers for suggestions that greatly improved the phenotypes might reflect background mutations [21]. We manuscript. found pcn-1(am315) animals showed a maternal-effect embryonic lethal phenotype. While it is unclear whether Funding am315 is a hypomorph or null allele, our results show that This work was supported in part by NIH grant R01 AG02656106A1 to KK, NIH pcn-1 is necessary for fertility. grant R01 GM100756 to TS, and a NSF predoctoral fellowship DGE-1143954 and DGE-1745038 to ZK. Neither the NIH nor the NSF had any role in the de- sign of the study, collection, analysis, and interpretation of data, nor in writ- Conclusions ing the manuscript. We established the nuclear accumulation pattern of PCN-1 in the larval and adult germline by analyzing Availability of data and materials FLAG::PCN-1 encoded by the native locus and All data generated or analyzed during this study are included in this published GFP::PCN-1 encoded by an integrated transgene. Both fu- article. 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BMC Developmental BiologySpringer Journals

Published: May 30, 2018

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