Fig 1.
POS-1 is a germline RNA binding protein required for posterior cell fate specification in the early embryo [9].
A. Diagram of a C. elegans hermaphrodite gonad [1]. B. The pattern of POS-1 expression in early embryos is shown in green. In older embryos POS-1 is eventually restricted to the P-lineage [9]. C. In the genetic background used in this study (DG4222), the endogenous pos-1 locus, which contains two CCCH-type zinc finger domains (labeled in pink), has an N-terminal gfp::tev::3xflag tag [26]. D. The ΔUTR allele replaces 241 of the 314 nucleotides of the pos-1 3′UTR with a 23 nucleotide jump board sequence [27]. GLD-1, FBF-1, and POS-1 binding motifs are shown. E. Chromatogram of the mutant confirming the deletion and jump board insertion. A portion of each flanking homology arm are shown on either side of the incorporated sequence.
Table 1.
Genotype.
Fig 2.
The pos-1 3′UTR contributes to reproductive robustness and germline formation.
A. Violin plots of total brood for WT UTR (gray) and ∆UTR (orange) strains. The red lines indicate the median. Each point represents the total number of embryos produced by a single hermaphrodite. Asterisks indicate statistical significance in a one-way ANOVA with Bonferroni correction for multiple hypothesis testing. B. Box-and-whisker plot representing the hatch rate (viable/total brood) of embryos from panel A. Each dot represents the hatch rate of embryos from a single hermaphrodite. Red points indicate far outliers in a Tukey analysis (far outliers are at least 3X larger or smaller than the inner quartile range). C. Box-and-whisker plot depicting the sterility rate (sterile/sterile + fertile) of viable worms from panel B once they reach adulthood. Each dot represents the ratio of sterile F1 hermaphrodites to total viable hermaphrodites. Orange points indicate outliers in a Tukey analysis (1.5X larger or smaller than the inner quartile range). Far outliers and statistical significance are represented as in panel A. D. 20X DIC and GFP images of GFP-WT UTR and GFP-∆UTR mutants at 20°C. The yellow asterisk indicates the vulva, and the dotted white line represents each gonad arm, if present. Scale bars: 30 μm. E. Violin plot of total brood comparing between GFP-WT UTR (gray) and the GFP-∆UTR (green) at 20°C and 25°C. Median bars, statistical significance, and individual points are represented as in panel A. F. Box-and-whisker plot depicting the hatch rate of embryos from panel E. Each dot represents the hatch rate of embryos produced from a single worm as in panel B. G. Box-and-whisker plot depicting the sterility rate of viable progeny from panel F at adulthood. The representation is as described for panel C.
Fig 3.
Germline phenotypes in the pos-1 ∆3′UTR mutant.
A. DIC image of a GFP-∆UTR mutant with a polynucleated oocyte (cyan arrows–each points to a different nucleus within the same oocyte). B. Stacked bar graph showing the fraction of worms with polynucleated oocytes (cyan) or normal oocytes (gray) in germline images. Asterisks indicate statistical significance in a Pearson’s chi square test with Bonferroni correction for multiple hypothesis testing. C. DIC image of a GFP-∆UTR oblong oocyte phenotype. The length of most proximal oblong oocyte is marked in red. D. Stacked bar graph showing fraction of worms with oblong oocytes (red) or normal oocytes (gray) in germline images. Statistical significance is denoted as in B. E. DIC image of a representative GFP-∆UTR mutant with protruding vulva phenotype (pvul, yellow arrow). F. DIC image of a GFP-∆UTR mutant with a multiple vulva (muv) phenotype. Each abnormal vulva location is marked with a green arrow. The scale bars represent 30 μm in all images.
Fig 4.
POS-1 protein expression is affected in the germline and embryos of 3′∆UTR mutants.
A. DIC and GFP images of GFP-WT UTR and GFP-∆UTR germlines (white outline) revealing POS-1 protein overexpression differences between genotype and condition. The scale bars represent 30 μm. B. Plot of germline fluorescence intensity as a function of distance from the distal end, with average GFP::POS-1 fluorescence averaged across twenty equal width bins representing the full length of the germline. The points represent the average fluorescence intensity from multiple images within a given genotype and condition; the error bars represent the standard error of the mean. Statistical significance is shown with asterisks as labeled in the key. P values were calculated with a one-way ANOVA with Bonferroni correction for multiple hypothesis testing. C. DIC and GFP image of GFP-WT UTR and GFP-∆UTR embryo at the two-cell stage. The posterior pole is oriented to the right. Scale bars: 10 μm. D. Average GFP::POS-1 fluorescence intensity across the entire embryo is plotted as a function of time from embryogenesis movies collected in five-minute intervals over 90 minutes of early embryonic development. Statistical significance is represented as in panel B.
Fig 5.
Germline progenitor specification is aberrant in pos-1 Δ3′UTR mutants.
A. Brood size of mCherry::pgl-1 marked GFP-WT UTR and GFP-ΔUTR strains. The representations are as in Fig 2A. B. Hatch rate of the embryos from panel A. The representation is as in Fig 2B. C. Sterility rate of the viable progeny from panel B. The graph is represented as in Fig 2C. D. DIC, GFP, and mCherry images of embryos from GFP-WT UTR or GFP-ΔUTR animals tagged with mCherry::PGL-1 showing an example of increased germline progenitor cells in embryos in the 3′UTR deletion mutant. Scale Bars: 20 μm. E. Fraction of animals with less than (blue), equal to (gray), or more than (red) the expected number of germline progenitor cells in embryo images binned by developmental stage. Statistically significant differences from a Pearson’s Chi Square test with Bonferroni correction are marked with asterisks.
Fig 6.
Germline proliferation is impacted in pos-1 3′UTR mutants.
A. DIC and mCherry images of mCherry::PGL-1 marked GFP-WT UTR or GFP-ΔUTR mutants as a function of stage from L1 larva to adults. Scale bars: 20 μm. B. Violin plots of germline length as a function of developmental stage. Each point is the length of a single gonad arm as defined by the zone of mCherry::PGL-1 expression. Gray represents GFP-WT UTR animals, orange denotes GFP-ΔUTR animals. The red bar indicates the median. The asterisks indicate statistical significance in a one-way ANOVA with Bonferroni correction for multiple hypothesis testing.
Fig 7.
RNA sequencing reveals transcriptome-wide changes in pos-1 3′UTR mutants.
A. Volcano and MA plots of differentially expressed genes from GFP-tagged pos-1 WT and 3′UTR mutants grown at 20ºC. Downregulated genes that meet filtering criteria are shown in red, upregulated genes are shown in blue. Genes that are unchanged or do not meet the filtering criteria are shown in gray. The pos-1, glp-1, and neg-1 genes are labeled in both plots. B. Volcano and MA plots of the differentially regulated germline genes. The representation is the same as in panel A. C. Differentially expressed gene sets from WormCat analyses of both up and downregulated genes. The WormCat categories are organized by function. The colors denote statistical significance in WormCat 2.0, and the size of the circle represents the number of genes in the category impacted.