SYGL-1 and LST-1 link niche signaling to PUF RNA repression for stem cell maintenance in Caenorhabditis elegans
Fig 2
Extent of SYGL-1 expression domain correlates with size of GSC pool.
(A) Schematics of transgenes. Conventions as in Fig 1C. Left, sygl-1 3’UTR transgene. Right, tbb-2 3’UTR transgene replaces sygl-1 3’UTR with tbb-2 (β-tubulin) 3’UTR. See S3 Fig for data supporting functionality of tbb-2 3’UTR transgene. (B-D) Extents of SYGL-1 protein in dissected adult gonads stained with α-FLAG (SYGL-1, magenta) and DAPI (cyan). Conventions as in Fig 1E–1J; scale bar is 20 μm. (B) sygl-1(q828). (C) sygl-1(q828); qSi49[Psygl-1::3xFLAG::sygl-1::sygl-1 3’end]. (D) sygl-1(q828); qSi150[Psygl-1::3xFLAG::sygl-1::tbb-2 3’end]. (E) Quantitation of SYGL-1 abundance, based on intensity of α-FLAG staining. Average intensity values were plotted against distance in microns along the gonadal axis (x-axis, top), which were converted to the conventional metric of germ cell diameters from distal end (x-axis, bottom) (see Methods). Lines, average intensity in arbitrary units (A.U.); shaded areas, standard deviation; n, number of gonadal arms. (F) Progenitor zone sizes. Averages and standard deviations for each genotype are as follows: (1) 231 ± 33 (n = 12); (2) 119 ± 17 (n = 22); (3) 117 ± 16 (n = 20); (4) 229 ± 16 (n = 15); (5) 234 ± 23 (n = 12); (6) 298 ± 34 (n = 13); (7) 292 ± 25 (n = 12). Bottom and top boundaries of each box, first and third quartiles; middle lines, median; red dots, mean; whiskers, minimum and maximum values. Asterisks indicate a statistically significant difference by Welch’s ANOVA with Games-Howell post hoc test. **p<0.001, n.s. = non-significant. (G) emb-30 assay to measure GSC pool size. An emb-30 temperature-sensitive mutant stops germ cell movement by cell cycle arrest [29]. At permissive temperature (15°C), the distal gonad appears normal, with scattered PH3-positive M-phase cells and graded GLD-1, a differentiation marker. A shift to restrictive temperature (25°C) reveals a distal pool of naïve stem-like germ cells arrested in M-phase and a proximal pool of germ cells primed to differentiate and hence expressing GLD-1 [11]. (H-J) GSC pool size correlates with SYGL-1 expression. Maximum intensity z-projected images of dissected gonads stained with α-PH3 (magenta), α-GLD-1 (green) and DAPI (cyan). Conventions as in Fig 1E–1J; scale bar is 20 μm. (H) Control: emb-30(tn377ts). (I) sygl-1(tm5040); emb-30(tn377ts). (J) sygl-1(tm5040); qSi150[Psygl-1::3xFLAG::sygl-1::tbb-2 3’end]; emb-30(tn377ts). (K) GSC pool size estimates. Box plot conventions as in Fig 2F. Averages and standard deviations for each genotype are as follows: (1) 35 ± 7; (2) 21 ± 7; (3) 43 ± 11; n>28 gonadal arm per genotype. Asterisks indicate a statistically significant difference by 1-way ANOVA with Tukey HSD post hoc test. ** p<0.001. Genotypes as in Fig 2H-2J.