Fig. 6.
Effect of LST-1 distribution on GSC pool size. (A) Schematic of emb-30 assay to estimate GSC pool size. This assay resolves two pools in the PZ, one inferred to be the GSC pool (Cinquin et al., 2010). Left: at permissive temperature, the emb-30(ts) PZ appears normal with M-phase germ cells (pink) scattered throughout and GLD-1 levels (green) gradually increasing as germ cells move proximally towards meiotic entry. Middle: after shifting emb-30(ts) from permissive to restrictive temperature, PZ germ cells stop dividing, stop moving proximally and arrest in either of two states. Distal PZ cells arrest in mitotic M-phase and express the PH3 M-phase marker, but not the GLD-1 differentiation marker; proximal PZ cells enter meiotic prophase and express abundant GLD-1 but no PH3. Right: inference of emb-30 results for wild-type PZ. (B) Quantitation of GSC pool sizes for CRISPR/Cas9-induced endogenous LST-1 variants (left) or LST-1 transgenes (right). After emb-30 temperature shift, we visualized the distal pool by morphology of DAPI-stained nuclei (see Materials and methods). Individual data points are plotted as pink or blue circles; boxes indicate 25-75% quantile; middle line indicates the median; whiskers indicate minimum and maximum values. P-values were calculated using a two-tailed t-test, assuming equal variance and comparing each LST-1(1-210) variant to its respective LST-1(wt) control. ***P<0.001. Endogenous variants were scored in a sygl-1(ø) background; genotypes and sample sizes: wtV5 is lst-1(q1004) sygl-1(q828); emb-30(tn377ts) (n=25) and 1-210V5 is lst-1(q1115) sygl-1(q828); emb-30(tn377ts) (n=30). Transgenic variants were scored in sygl-1(+) with genotypes and sample sizes as follows: wtHA is lst-1(ok814); qSi22; emb-30(tn377ts) (n=7) and 1-210HA is lst-1(ok814); qSi300; emb-30(tn377ts) (n=8). (C) Schematic of transgene driving ubiquitous germline expression of LST-1(1-210). (D) Representative z-projected confocal image of a germline tumor driven by a single-copy transgene as in C and lacking endogenous LST-1. Anti-PH3 antibodies (magenta); DAPI (cyan). (E-G) Schematics illustrating the effects of LST-1 spatial extent on GSC pool size. Top: lst-1 RNA (black) and LST-1 protein (magenta), with line thickness corresponding to quantity and line length to extent of expression. Bottom: effects on GSC pool size. Black arrow, switch from self-renewal to differentiation. (E) Wild type: lst-1 RNA and LST-1 protein are both restricted to distal germline and GSC pool size is similarly restricted. (F) LST-1(1-210): lst-1(1-210) RNA is slightly more abundant than in wild type, but still restricted to distal germline (from Fig. 4). By contrast, LST-1(1-210) protein is more abundant than wild type and expands proximally (from Fig. 3). The GSC pool expands correspondingly (from Fig. 5). (G) Ubiquitous LST-1(1-210): major expansion of LST-1 leads to germline tumor formation (from Fig. 5). The switch to differentiation fails to occur (red X).