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

<div><p>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 <i>Caeno-rhabditis elegans</i> provide a tractable <i>in vivo</i> 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.</p></div

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

<p>(A-I) Epistasis tests using <i>sygl-1(ubiq)</i> or <i>lst-1(ubiq)</i> transgenes. All images are dissected young adult gonads stained with sperm marker SP56 (red) and DAPI (cyan). (A-C) Epistasis with <i>glp-1</i>. (A) GSC defect in <i>glp-1(q46)</i> null: the few GSCs in L1 larvae differentiate as sperm [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.ref014" target="_blank">14</a>]. (B and C) Germline tumor in <i>sygl-1(ubiq); glp-1(q46)</i> null and <i>lst-1(ubiq); glp-1(q46)</i> null. (D-F) Epistasis with <i>lst-1 sygl-1</i>. (D) GSC defect in <i>lst-1(ok814) sygl-1(tm5040)</i> double mutant is indistinguishable from that of <i>glp-1</i> null [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.ref018" target="_blank">18</a>]. (E and F) Germline tumor in <i>lst-1(ok814) sygl-1(tm5040); sygl-1(ubiq)</i> and in <i>lst-1(ok814) sygl-1(tm5040); lst-1(ubiq)</i>. (G-I) Epistasis test with <i>fbf-1 fbf-2</i>. GSC defect in <i>fbf-1(ok91) fbf-2(q704)</i> double mutant: GSCs made in larvae but not maintained past late L4 when all differentiate as sperm at 15°C and 20°C [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.ref015" target="_blank">15</a>]. At 25°C, a small number of GSCs is maintained in adults [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.ref040" target="_blank">40</a>]. (H and I) GSC defect similar to that of <i>fbf-1 fbf-2</i> double mutant in <i>fbf-1(ok91) fbf-2(q704) sygl-1(ubiq)</i> and <i>fbf-1(ok91) fbf-2(q704) lst-1(ubiq)</i>. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.s005" target="_blank">S5 Fig</a></b> 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 <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.g001" target="_blank">Fig 1E–1J</a></b>; scale bar is 20 μm. In all strains, <i>sygl-1(ubiq)</i> is <i>qSi235[P</i>_<i>mex-5</i>::<i>3xFLAG</i>::<i>sygl-1</i>::<i>tbb-2 3’end]</i> and <i>lst-1(ubiq)</i> is <i>qSi267[P</i>_<i>mex-5</i>:: <i>lst-1</i>::<i>3xFLAG</i>::<i>tbb-2 3’end]</i>. (J) Summary of epistasis results. (K) Revised genetic model for GSC regulation. See text for further explanation.</p

Extent of SYGL-1 expression domain correlates with size of GSC pool.

<p>(A) Schematics of transgenes. Conventions as in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.g001" target="_blank">Fig 1C</a></b>. Left, <i>sygl-1</i> 3’UTR transgene. Right, <i>tbb-2</i> 3’UTR transgene replaces <i>sygl-1</i> 3’UTR with <i>tbb-2</i> (β-tubulin) 3’UTR. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.s003" target="_blank">S3 Fig</a></b> for data supporting functionality of <i>tbb-2</i> 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 <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.g001" target="_blank">Fig 1E–1J</a></b>; scale bar is 20 μm. (B) <i>sygl-1(q828)</i>. (C) <i>sygl-1(q828); qSi49[P</i>_{<i>sygl-1</i>}::<i>3xFLAG</i>::<i>sygl-1</i>::<i>sygl-1 3’end]</i>. (D) <i>sygl-1(q828); qSi150[P</i>_{<i>sygl-1</i>}::<i>3xFLAG</i>::<i>sygl-1</i>::<i>tbb-2 3’end]</i>. (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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#sec014" target="_blank">Methods</a>). 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 <i>post hoc</i> test. **p<0.001, n.s. = non-significant. (G) <i>emb-30</i> assay to measure GSC pool size. An <i>emb-30</i> temperature-sensitive mutant stops germ cell movement by cell cycle arrest [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.ref029" target="_blank">29</a>]. 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 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.ref011" target="_blank">11</a>]. (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 <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.g001" target="_blank">Fig 1E–1J</a></b>; scale bar is 20 μm. (H) Control: <i>emb-30(tn377ts)</i>. (I) <i>sygl-1(tm5040); emb-30(tn377ts)</i>. (J) <i>sygl-1(tm5040); qSi150[P</i>_{<i>sygl-1</i>}::<i>3xFLAG</i>::<i>sygl-1</i>::<i>tbb-2 3’end]; emb-30(tn377ts)</i>. (K) GSC pool size estimates. Box plot conventions as in <b>Fig 2F</b>. 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 <i>post hoc</i> test. ** p<0.001. Genotypes as in <b>Fig 2H-2J</b>.</p

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

<p>(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 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.ref011" target="_blank">11</a>]. 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 <i>sygl-1 and lst-1</i> loci (top) and transgenes (bottom). Epitope tagged endogenous alleles are: <i>sygl-1(q964)[3xMYC</i>::<i>sygl-1]</i>, <i>sygl-1(q983)[3xOLLAS</i>::<i>sygl-1]</i> and <i>sygl-1(q1015)[sygl-1</i>::<i>1xV5]</i>; <i>lst-1(q1004)[lst-1</i>::<i>3xV5]</i> and <i>lst-1(q1008)[lst-1</i>::<i>3xOLLAS]</i>. Colored boxes, <i>sygl-1</i> or <i>lst-1</i> exons; gray boxes, untranslated regions; orange boxes and triangles, epitopes. Bars below schematic, deletions; asterisk, nonsense mutation. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#sec014" target="_blank">Methods</a> 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) <i>sygl-1(q828); qSi49[P</i>_{<i>sygl-1</i>}::<i>3xFLAG</i>::<i>sygl-1</i>::<i>sygl-1 3’end]</i>. (F) <i>sygl-1(q828)</i>. (G) <i>lst-1(ok814); qSi22[P</i>_<i>lst-1</i>::<i>lst-1</i>::<i>1xHA</i>::<i>lst-1 3’end]</i>. (H) <i>lst-1(ok814)</i>. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.s001" target="_blank">S1A–S1C Fig</a></b> 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 <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.s001" target="_blank">S1D and S1E Fig</a></b> for data supporting functionality of epitope-tagged proteins and see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.s002" target="_blank">S2 Fig</a></b> for characterization of <i>sygl-1</i> or <i>lst-1</i> mutants.</p

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

<p>(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 <i>HIS3</i> 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 <i>HIS3</i> 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) <i>sygl-1(tm5040); qSi235[P</i>_<i>mex-5</i>::<i>3xFLAG</i>::<i>sygl-1</i>::<i>tbb-2 3’end];</i> (2) <i>sygl-1(tm5040); fbf-2(q931)[3xV5</i>::<i>fbf-2] qSi235[P</i>_<i>mex-5</i>::<i>3xFLAG</i>::<i>sygl-1</i>::<i>tbb-2 3’end];</i> (3) <i>sygl-1(tm5040); qSi297[P</i>_<i>mex-5</i>::<i>3xMYC</i>::<i>sygl-1</i>::<i>tbb-2 3’end];</i> (4) <i>sygl-1(tm5040); fbf-2(q932)[3xV5</i>::<i>fbf-2] qSi297[P</i>_<i>mex-5</i>::<i>3xMYC</i>::<i>sygl-1</i>::<i>tbb-2 3’end]</i>. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007121#pgen.1007121.s007" target="_blank">S7 Fig</a></b> 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 <i>3xFLAG</i>::<i>sygl-1(ubiq)</i> for the experiment or <i>3xMYC</i>::<i>sygl-1</i>(<i>ubiq</i>) as the control. Abundance of mRNAs in input (gray bars) and IPs (blue bars) was calculated with the ΔΔ C_T method, using <i>rps-25</i> for normalization. Error bar indicates standard error. Asterisks indicate a statistically significant difference by 1-way ANOVA with Tukey HSD <i>post hoc</i> test. * p<0.05, ** p<0.01.</p

Models for stem cell pool regulation.

<p>(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; <i>sygl-1</i> mutant: pool size smaller than wild type and likely determined by smaller LST-1 extent; <i>lst-1</i> 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 (<i>tbb-2</i> 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.</p

FBF Binds Specifically to FBEs in <i>mpk-1</i> 3′UTR

<div><p>(A) Two predicted FBF binding elements in <i>mpk-1</i> 3′UTR.</p><p>(B) Schematic of yeast three-hybrid assay. Briefly, a hybrid RNA carrying the query sequence can bridge the LexA-MS2 and GAL4AD-FBF hybrid proteins if FBF binds, but it cannot bridge them if FBF fails to bind.</p><p>(C) Nucleotide sequences of predicted FBEs, aligned in register with their conserved UGURHHAU motifs (bold in gray boxes). Each wild-type sequence is followed by its mutant (*), in which UGU is replaced by aca (mutated nucleotides are lowercase). Controls included the <i>fem</i>-3 FBE in the <i>fem-3</i> 3′UTR, previously called the PME [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-b012" target="_blank">12</a>], which served as a positive control for FBF binding, and the <i>hb (hunchback)</i> NRE, which served as a negative control for FBF binding and a positive control for PUF-8 binding [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-b055" target="_blank">55</a>].</p><p>(D) Three-hybrid interactions assayed by β-galactosidase activity. Nomenclature and conventions are the same as in (C). Standard deviation bars were calculated from three independent experiments.</p><p>(E) Purified FBF-2 binds <i>mpk-1</i> FBEa and <i>mpk-1</i> FBEb in gel mobility assays, but not to mutants (*) with an altered consensus as detailed in (C). Apparent affinities of MPK-1 FBEa and FBEb are 93 nM and 320 nM, respectively.</p><p>(F) Coimmunoprecipitation of <i>mpk-1</i> mRNA with an epitope-tagged FBF. <i>eft-3</i> served as a negative control, and <i>gld-1</i> served as a positive control [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-b015" target="_blank">15</a>].</p><p>(G) Sequence alignment of <i>mpk-1</i> FBEs from C. elegans, <i>C. briggsae,</i> and C. remanei.</p></div

<i>mpk-1</i> Expression in the C. elegans Germline

<div><p>(A) Schematics of <i>mpk-1a</i> and <i>mpk-1b</i> mRNAs. Box, exon; connecting line, intron; ATG, initiation codon; TAG, termination codon. Below schematics: thick bars, extent of probes used for in situ hybridization; arrows, primer pairs used for RT-PCR.</p><p>(B) Semiquantitative RT-PCR of RNA prepared from adult hermaphrodites that either had an essentially normal germline [<i>glp-1(q224)</i> grown at 15 °C], or had virtually no germline [<i>glp-1(q224)</i> grown at 25 °C] (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#s4" target="_blank">Materials and Methods</a>). <i>unc-54</i> was used as a control.</p><p>(C) Western blot. MPK-1A protein is ≈45 kDa, MPK-1B is ≈55 kDa, and α-TUB is α-tubulin. Proteins were extracted from adult hermaphrodites that were either wild-type (wt), <i>glp-1(q224)</i> grown at 25 °C (GL−), or <i>mpk-1(ga117)</i> putative null homozygotes <i>[mpk-1(0)]</i>.</p><p>(D–F) In situ analysis of dissected adult hermaphrodite germlines. (D) Total <i>mpk-1</i> RNA was assessed using the <i>mpk-1ab</i> antisense probe shown in (A). (E) <i>mpk-1b</i> RNA was assessed using an isoform-specific antisense probe shown in (A). (F) Negative control, using an <i>mpk-1b</i>−specific sense probe.</p><p>(G–L) Immunocytochemistry of dissected adult hermaphrodite germlines. All were stained using both MPK-1 antibodies (G, I, K) and DAPI (H, J, L). Distal end, arrowhead; dotted lines, boundaries between regions of germline maturation [MR (mitotic region), TZ (transition zone), PR (pachytene region), OO (oocytes), SP (sperm)]; PEX (pachytene exit defect). (G, H) Same wild-type germline. (I, J) Same <i>mpk-1(0)</i> germline. (K, L) Same <i>mpk-1b(RNAi)</i> germline.</p></div

Regulation of MAPK Activity by PUF and MKPs

<div><p>(A) Conserved positive and negative regulators of MAPK expression and activity. See text for further explanation.</p><p>(B) MAPK regulation in the C. elegans germline. The distal end of the germline is controlled by Notch signaling from the distal tip cell (DTC), which provides the stem cell niche [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-b011" target="_blank">11</a>]. FBF/PUF RNA-binding proteins are present in the distalmost germ cells, which include stem cells. FBF maintains germ cells in a naïve and undifferentiated state, in part by repression of <i>mpk-1</i> expression (present work). In addition, FBF represses <i>lip-1/MKP</i> mRNA in the stem cell region [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030233#pgen-0030233-b023" target="_blank">23</a>]; more proximally, where FBF abundance decreases, LIP-1/MKP inhibits MPK-1/MAPK activity; yet more proximally, LET-60/RAS activates MPK-1/MAPK to promote oocyte differentiation and apoptosis. OO, oocyte.</p></div

Conservation of PUF Binding to Regulatory Elements in Human Erk2 and p38α 3′UTRs

<div><p>(A) Putative PUM2 binding elements (NREs) in Erk2 and p38α 3′UTRs; filled triangles, elements that bound in vitro; empty triangles, elements that did not bind in vitro.</p><p>(B) Nucleotide sequence of predicted NREs. Sequences are aligned in register with their conserved UGUANAU motif (bold in gray boxes). Mutated nucleotides are lowercase.</p><p>(C) Three-hybrid interactions assayed by β-galactosidase activity. Standard deviation bars were calculated from three independent experiments.</p><p>(D) Purified PUM2 binds Erk2 NRE as well as p38α NREa and NREb in gel mobility assays, but does not bind mutants (*) with an altered consensus as detailed in (B).</p><p>(E) Sequence alignment of Erk2 NREs from human and mouse.</p></div