SYGL-1 and LST-1 link niche signaling to PUF RNA repression for stem cell maintenance in Caenorhabditis elegans

Abstract

Central questions in regenerative biology include how stem cells are maintained and how they transition from self-renewal to differentiation. Germline stem cells (GSCs) in Caeno-rhabditis elegans provide a tractable in vivo model to address these questions. In this system, Notch signaling and PUF RNA binding proteins, FBF-1 and FBF-2 (collectively FBF), maintain a pool of GSCs in a naïve state. An open question has been how Notch signaling modulates FBF activity to promote stem cell self-renewal. Here we report that two Notch targets, SYGL-1 and LST-1, link niche signaling to FBF. We find that SYGL-1 and LST-1 proteins are cytoplasmic and normally restricted to the GSC pool region. Increasing the distribution of SYGL-1 expands the pool correspondingly, and vast overexpression of either SYGL-1 or LST-1 generates a germline tumor. Thus, SYGL-1 and LST-1 are each sufficient to drive "stemness" and their spatial restriction prevents tumor formation. Importantly, SYGL-1 and LST-1 can only drive tumor formation when FBF is present. Moreover, both proteins interact physically with FBF, and both are required to repress a signature FBF mRNA target. Together, our results support a model in which SYGL-1 and LST-1 form a repressive complex with FBF that is crucial for stem cell maintenance. We further propose that progression from a naïve stem cell state to a state primed for differentiation relies on loss of SYGL-1 and LST-1, which in turn relieves FBF target RNAs from repression. Broadly, our results provide new insights into the link between niche signaling and a downstream RNA regulatory network and how this circuitry governs the balance between self-renewal and differentiation.

Fig 1. SYGL-1 and LST-1 proteins are spatially restricted to the GSC pool region.

(A) Schematic of adult distal gonad. The progenitor zone (PZ) includes a distal pool of germline stem cells (GSC) and a proximal pool of cells primed to differentiate [11]. The conventional metric for axis position is number of germ cell diameters from the distal end (gcd). Somatic niche for GSCs (gray); naïve stem cell state (yellow circles); early meiotic prophase (green crescents); primed transiting state (yellow to green gradient). Asterisk marks distal end. (B) Genetic pathway of GSC regulation. (C and D) Schematics of sygl-1 and lst-1 loci (top) and transgenes (bottom). Epitope tagged endogenous alleles are: sygl-1(q964)[3xMYC::sygl-1], sygl-1(q983)[3xOLLAS::sygl-1] and sygl-1(q1015)[sygl-1::1xV5]; lst-1(q1004)[lst-1::3xV5] and lst-1(q1008)[lst-1::3xOLLAS]. Colored boxes, sygl-1 or lst-1 exons; gray boxes, untranslated regions; orange boxes and triangles, epitopes. Bars below schematic, deletions; asterisk, nonsense mutation. See Methods for updated gene structures. (E-J) SYGL-1 and LST-1 proteins in dissected adult gonads. (E-H) Representative slice or (I-J) maximum intensity z-projections of distal gonad stained with α-FLAG (SYGL-1, magenta), α-HA (LST-1, yellow), α-GLD-1 (green), and DAPI (cyan). Dashed line, gonadal outline; asterisk, distal end. Scale bar is 20 μm in all images, except 5 μm in (E) and (G) insets. (E) sygl-1(q828); qSi49[P_sygl-1::3xFLAG::sygl-1::sygl-1 3’end]. (F) sygl-1(q828). (G) lst-1(ok814); qSi22[P_lst-1::lst-1::1xHA::lst-1 3’end]. (H) lst-1(ok814). See S1A–S1C Fig for whole gonad images. (K and L) Extent of SYGL-1 and LST-1 expression along the gonadal axis, estimated with functional epitope-tagged proteins. Expression is robust distally and graded proximally. Proximal boundaries were estimated by eye as the point at which staining becomes barely detected. nd, not determined. See S1D and S1E Fig for data supporting functionality of epitope-tagged proteins and see S2 Fig for characterization of sygl-1 or lst-1 mutants.

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[P_sygl-1::3xFLAG::sygl-1::sygl-1 3’end]. (D) sygl-1(q828); qSi150[P_sygl-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[P_sygl-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.

Fig 3. Ubiquitous germline expression of SYGL-1 or LST-1 drives tumor formation.

(A) Protocol to induce ubiquitous germline expression of SYGL-1 or LST-1. See text for explanation and S4A and S4B Fig for tumor penetrance over generations. (B-D) Schematics of transgenes. The mex-5 promoter and tbb-2 3’UTR were used to promote ubiquitous germline expression. (E-J) Young adult gonads stained with mitotic marker α-REC-8 (yellow), α-FLAG (SYGL-1 or LST-1, magenta), M-phase marker α-PH3 (white), and DAPI (cyan). Images are either single slice (E, G, I) or maximum intensity z-projections (F, H, J). Conventions as in Fig 1E–1J; scale bar is 20 μm. (E and F) Genotype for ubiquitous SYGL-1: sygl-1(tm5040); qSi235[P_mex-5::3xFLAG::sygl-1::tbb-2 3’end]. (G and H) Genotype for ubiquitous LST-1: lst-1(ok814); qSi267[P_mex-5::lst-1::3xFLAG::tbb-2 3’end]. (I and J) Genotype for ubiquitous GFP::H2B control, weSi2[P_mex-5::GFP::his-58::tbb-2 3’end] [91]. See S4C–S4K Fig for further characterization.

Fig 4. SYGL-1 and LST-1 tumor formation relies on FBF.

(A-I) Epistasis tests using sygl-1(ubiq) or lst-1(ubiq) transgenes. All images are dissected young adult gonads stained with sperm marker SP56 (red) and DAPI (cyan). (A-C) Epistasis with glp-1. (A) GSC defect in glp-1(q46) null: the few GSCs in L1 larvae differentiate as sperm [14]. (B and C) Germline tumor in sygl-1(ubiq); glp-1(q46) null and lst-1(ubiq); glp-1(q46) null. (D-F) Epistasis with lst-1 sygl-1. (D) GSC defect in lst-1(ok814) sygl-1(tm5040) double mutant is indistinguishable from that of glp-1 null [18]. (E and F) Germline tumor in lst-1(ok814) sygl-1(tm5040); sygl-1(ubiq) and in lst-1(ok814) sygl-1(tm5040); lst-1(ubiq). (G-I) Epistasis test with fbf-1 fbf-2. GSC defect in fbf-1(ok91) fbf-2(q704) double mutant: GSCs made in larvae but not maintained past late L4 when all differentiate as sperm at 15°C and 20°C [15]. At 25°C, a small number of GSCs is maintained in adults [40]. (H and I) GSC defect similar to that of fbf-1 fbf-2 double mutant in fbf-1(ok91) fbf-2(q704) sygl-1(ubiq) and fbf-1(ok91) fbf-2(q704) lst-1(ubiq). See S5 Fig for confirmation that SYGL-1 and LST-1 are expressed and functional in these strains, and for characterization of these strains at 25°C. Conventions as in Fig 1E–1J; scale bar is 20 μm. In all strains, sygl-1(ubiq) is qSi235[P_mex-5::3xFLAG::sygl-1::tbb-2 3’end] and lst-1(ubiq) is qSi267[P_mex-5:: lst-1::3xFLAG::tbb-2 3’end]. (J) Summary of epistasis results. (K) Revised genetic model for GSC regulation. See text for further explanation.

Fig 5. SYGL-1 and LST-1 interact physically with FBF.

(A) Yeast two hybrid assay. Full length SYGL-1 or LST-1 was fused to Gal4 activation domain (AD); PUF repeats of FBF-1(121–614) or FBF-2(121–632) were fused to LexA binding domain (BD). Interaction activates transcription of HIS3 gene. (B and C) Yeast growth assays tested interaction between SYGL-1 and FBF (B) or LST-1 and FBF (C). Yeast strains were monitored for growth on synthetic defined media (SD), either lacking histidine or with histidine as a control. A HIS3 competitive inhibitor (3-AT) improved stringency. (D) SYGL-1 and FBF-2 co-immunoprecipitation (IP). Western blots probed with α-FLAG to detect SYGL-1, α-V5 to detect FBF-2, and anti-α-tubulin as a loading control. 2% of input lysates and 20% of IP elutes were loaded. Exposure times of input and IP lanes are different, so band intensities are not comparable. RNA degradation by RNase A was confirmed. Genotypes for each lane: (1) sygl-1(tm5040); qSi235[P_mex-5::3xFLAG::sygl-1::tbb-2 3’end]; (2) sygl-1(tm5040); fbf-2(q931)[3xV5::fbf-2] qSi235[P_mex-5::3xFLAG::sygl-1::tbb-2 3’end]; (3) sygl-1(tm5040); qSi297[P_mex-5::3xMYC::sygl-1::tbb-2 3’end]; (4) sygl-1(tm5040); fbf-2(q932)[3xV5::fbf-2] qSi297[P_mex-5::3xMYC::sygl-1::tbb-2 3’end]. See S7 Fig for data supporting functionality of epitope-tagged FBF-2. (E) Quantitative PCR of two signature FBF target mRNAs and a control mRNA after α-FLAG IP, using either 3xFLAG::sygl-1(ubiq) for the experiment or 3xMYC::sygl-1(ubiq) as the control. Abundance of mRNAs in input (gray bars) and IPs (blue bars) was calculated with the ΔΔ C_T method, using rps-25 for normalization. Error bar indicates standard error. Asterisks indicate a statistically significant difference by 1-way ANOVA with Tukey HSD post hoc test. * p<0.05, ** p<0.01.

Fig 6. SYGL-1 and LST-1 repress gld-1 expression post-transcriptionally in GSC pool.

(A) Schematic of gld-1(q361), a missense allele with a null phenotype [45] that generates mRNA and protein normally [30]. The smFISH probe set spanned the locus. See text for details. (B-E) GLD-1(q361) protein in distal gonads, stained with α-GLD-1 (green) and DAPI (cyan). Genotypes are: (B) gld-2(q497) gld-1(q361); (C) sygl-1(q828) gld-2(q497) gld-1(q361); (D) lst-1(ok814) gld-2(q497) gld-1(q361); (E) lst-1(ok814) sygl-1(q828) gld-2(q497) gld-1(q361). (F) Quantitation of GLD-1(q361). α-GLD-1 intensities in 0–20 μm (1-~5 gcd) from the distal end were averaged and plotted. Box plot conventions as in Fig 2F, genotypes as in Fig 6B–6E. Asterisks indicate a statistically significant difference by 1-way Welch’s ANOVA with Games Howell post hoc test. ** p<0.001, * p< 0.05. Number of gonads examined: Control, n = 23; sygl-1, n = 26; lst-1, n = 24; lst-1 sygl-1, n = 38. (G-J) gld-1(q361) transcripts in distal gonads, probed using smFISH (white) and DAPI (cyan). Genotypes as in Fig 6B–6E. All gonads (100%) had mRNA distributions as shown: control, n = 32; sygl-1, n = 35; lst-1, n = 41; lst-1 sygl-1, n = 38. (K and L) Pink arrows, nascent transcripts in nucleus. Yellow arrowheads, mature mRNAs in cytoplasm. Top, gld-1 RNA; bottom, RNA merged with DAPI. (K) Magnifications from boxed areas in (G). In the presence of wild-type sygl-1 and lst-1, distal germ cells possess nuclear transcripts, but little cytoplasmic mRNA, whereas proximal germ cells have both. (L) Magnifications from boxed areas in (J). Without sygl-1 and lst-1, both distal and proximal germ cells contain nuclear and cytoplasmic gld-1 transcripts. See S8 Fig for confirmation of gld-1 probe specificity. All images are maximum intensity z-projections, except (K) and (L) show a single z-slice. Conventions as in Fig 1E–1J; scale bar is 20 μm in all images, except 2 μm in (K) and (L). n, number of gonadal arms.

Fig 7. Models for stem cell pool regulation.

(A) In each schematic, wild-type or manipulated extents of SYGL-1 (magenta) and LST-1 (orange) are shown above and GSC pool sizes are shown below. Wild type: GSC pool size corresponds to SYGL-1 rather than LST-1 extent; sygl-1 mutant: pool size smaller than wild type and likely determined by smaller LST-1 extent; lst-1 mutant: pool size not determined experimentally but likely similar to wild type, because progenitor zone is nearly the same size as normal; Extended SYGL-1 expression: moderate increase in SYGL-1 extent expands GSC pool (tbb-2 3’UTR transgene); Ubiquitous SYGL-1 expression: major expansion of SYGL-1 forms a massive tumor; Ubiquitous LST-1 expression: major expansion of LST-1 forms a massive tumor. (B) FBF forms a complex with SYGL-1 or LST-1 to repress differentiation RNAs. Red bars indicate repression; large pale blue circle represents an RNP granule. See text for explanation. (C) Loss of SYGL-1 and LST-1 triggers the switch from a naïve state to one primed-for-differentiation. See text for explanation.