A sensitized genetic screen to identify regulators of Caenorhabditis elegans germline stem cells

Abstract GLP-1/Notch signaling and a downstream RNA regulatory network maintain germline stem cells in Caenorhabditis elegans. In mutants lacking the GLP-1 receptor, all germline stem cells enter the meiotic cell cycle precociously and differentiate into sperm. This dramatic germline stem cell defect is called the “Glp” phenotype. The lst-1 and sygl-1 genes are direct targets of Notch transcriptional activation and functionally redundant. Whereas single lst-1 and sygl-1 mutants are fertile, lst-1 sygl-1 double mutants are sterile with a Glp phenotype. We set out to identify genes that function redundantly with either lst-1 or sygl-1 to maintain germline stem cells. To this end, we conducted forward genetic screens for mutants with a Glp phenotype in genetic backgrounds lacking functional copies of either lst-1 or sygl-1. The screens generated 9 glp-1 alleles, 2 lst-1 alleles, and 1 allele of pole-1, which encodes the catalytic subunit of DNA polymerase ε. Three glp-1 alleles reside in Ankyrin repeats not previously mutated. pole-1 single mutants have a low penetrance Glp phenotype that is enhanced by loss of sygl-1. Thus, the screen uncovered 1 locus that interacts genetically with sygl-1 and generated useful mutations for further studies of germline stem cell regulation.


Introduction
Stem cells maintain a robust balance between self-renewal and differentiation to ensure tissue homeostasis despite physiological and environmental challenges. Failure to maintain that balance can lead to tissue dysfunction, disease, and death (Simons and Clevers 2011). Therefore, understanding the molecular circuitry governing stem cell regulation is critical. Yet biologically robust regulatory circuits are notoriously difficult to disentangle.
The C. elegans germline is a powerful system for the study of stem cell regulation (Hubbard and Schedl 2019). The adult hermaphrodite germline is contained in 2 U-shaped gonadal arms and produces oocytes; sperm are made during larval development and stored for later fertilization (Fig. 1a, top). Germline stem cells (GSCs) are maintained at the distal end of each gonadal arm by a single-celled somatic niche, while GSC daughters differentiate as they move proximally away from the niche and ultimately undergo oogenesis (Fig. 1a, middle) (Hubbard and Greenstein 2000).
The PUF hub is characterized by pervasive genetic redundancy. For example, mutants lacking 3 PUF homologs are able to sustain some GSC self-renewing divisions, but animals lacking all 4 homologs phenocopy glp-1 null mutants (Haupt et al. 2020). Moreover, single mutants lacking lst-1 or sygl-1 are fertile and similar to the wildtype, while lst-1 sygl-1 double mutants phenocopy glp-1 null mutants (Fig. 1c) (Kershner et al. 2014). The highly redundant nature of the PUF hub has hampered the identification of its component parts. Indeed, LST-1 and SYGL-1 were not identified using standard forward genetic approaches, but instead were discovered using a candidate gene approach (Kershner et al. 2014), leaving open the possibility that additional components remain unidentified. For example, the LST-1 or SYGL-1 proteins might work with other unknown redundant factors. Here, we describe the results of mutagenesis screens designed to identify regulators that function redundantly with lst-1 or sygl-1.

Strain maintenance
Unless noted otherwise, strains were maintained as previously described (Brenner 1974), at a temperature of 15 C. Balancers used to maintain recovered alleles were hT2[qIs48] (Siegfried and Kimble 2002) and hIn1 [unc-54(h1040)] (Zetka and Rose 1992). Table 1 lists the strains used and their genotypes.

Screen design and phenotype scoring
We screened for lst-1 or sygl-1 enhancers using a modified ethyl methanesulfonate (EMS) protocol (Brenner 1974). Fourth larval stage (L4) hermaphrodites were soaked in 25 mM EMS (Sigma: M0880) for 4 h at room temperature, washed with M9, and placed on plates. F1 progeny were singled onto individual Petri dishes and allowed to self at 15 C. F2 adult progeny were scored for sterility by dissecting scope, and then L4 larvae were scored for a Glp phenotype using a Zeiss Axioskop compound scope equipped with DIC Nomarski optics, as described (Kershner et al. 2014). Each screen was done in 2 ways-first with single mutants lst-1(ok814) and sygl-1(tm5040) (Fig. 1d, regimen 1) and then with each of the same mutants carrying a transgenic copy of wildtype glp-1 (Sorensen et al. 2020) in addition to an endogenous copy of wildtype glp-1 (Fig. 1d, regimen 2).

Allele identification
Following isolation of a mutant with a Glp phenotype, the starting lst-1 or sygl-1 allele was crossed away to test whether the Glp defect depended on loss of lst-1 or sygl-1. Mutations were then mapped to a chromosome and tested for their ability to complement alleles of likely candidate genes. Mutants that were fertile as single mutants and mapped to chromosome I were tested for complementation with lst-1(ok814) I. Briefly, the double mutant (e.g. mut-x sygl-1) was balanced over the green balancer hT2[qIs48], crossed to lst-1(ok814) sygl-1(tm5040)/hT2[qIs48] males, and nongreen L4 male progeny (e.g. mut-x sygl-1/lst-1 sygl-1) were scored for the Glp defect. Mutants that were sterile as single mutants and mapped to chromosome III were tested for complementation with the null allele glp-1(q175) III. Briefly, unc-32 glp-1(q175)/hT2[qIs48] males were mated to each suspected glp-1 allele and nongreen male progeny scored for the Glp defect. If an allele failed to complement either lst-1 or glp-1, then Sanger sequencing was used to identify the molecular lesion. The glp-1(q823) allele was sequenced 2,382 bp upstream of the 5 0 UTR and 927 bp downstream of the 3 0 UTR in addition to the exons and introns, but no lesion was found.
Whole-genome sequencing was used to identify the likely lesion in q831, which was sterile as a single mutant and mapped to the right arm of chromosome I. Briefly, we picked 570 adult homozygotes, isolated DNA with Puregene Core Kit A (Qiagen ID: Fig. 1. Genetic screens for synthetic Glp mutants. a) Top, adult hermaphrodite has 2-U-shaped gonadal arms (GSCs, yellow; blue, sperm; pink, oocytes). Sperm made during larval development are stored in spermatheca. Middle, wildtype germline with a GSC pool (yellow) distally and oocytes (pink) proximally. Bottom, Glp adult germline with only a few mature sperm (blue). b) Molecular regulation of GSC self-renewal. GLP-1/Notch signaling activates transcription of lst-1 and sygl-1, which are components of the PUF regulatory hub, along with fbf-1, fbf-2, puf-3, and puf-11 (Haupt et al. 2020). c) Adult germ cell (GC) numbers and phenotypes of specified genotypes. d) Strategies to identify genes that have a synthetic Glp phenotype with lst-1 or sygl-1. Regimen 1 mutagenizes lst-1(lf) or sygl-1(lf) homozygotes and scores for Glp sterility in the F 2 . Regimen 2 mutagenizes lst-1(lf) or sygl-1(lf) homozygotes that also carry a wildtype glp-1 transgene, glp-1(tg), to avoid isolation of glp-1 mutations. 158667) following the manufacturer's directions and submitted the DNA (100 ng) to the Wisconsin Biotechnology Core for sequencing using an Illumina MiSeq. The genome sequence was uploaded to a Galaxy server and analyzed by CloudMap, as previously described (Minevich et al. 2012). A premature stop codon occurred in 1 gene, F33H2.5, which resides on the right arm of chromosome I. q831 failed to complement F33H2.5 (gk49) (C. elegans Deletion Mutant Consortium 2012), and the premature stop codon was confirmed by Sanger sequencing of DNA from q831 homozygotes.
Assay for temperature sensitivity of glp-1 and pole-1 alleles Balanced strains carrying glp-1 or pole-1 alleles were maintained at 15 C, 20 C, or 25 C for at least 1 generation before homozygous glp-1 or pole-1 L4 progeny were scored for a Glp phenotype.

Allele
LG

Results and discussion
Screens for Glp mutants in lst-1 and sygl-1 single mutant backgrounds To identify new GSC regulators and perhaps new components of the PUF hub, we conducted genetic screens for mutations that cause a Glp phenotype in a lst-1(lf) or sygl-1(lf) single mutant background (Fig. 1d). Our initial screens simply mutagenized lst-1(lf) and sygl-1(lf) single mutants and scored their F2 progeny for the Glp phenotype (Fig. 1d, regimen 1). We screened 8,749 haploid genomes after mutagenesis of lst-1(lf) and 5,504 haploid genomes after mutagenesis of sygl-1(lf) ( Table 2). This first set of screens recovered 10 mutants. However, outcrossing revealed that all 10 mutants generated animals with a Glp phenotype after lst-1(lf) or sygl-1(lf) was removed. Nine mutations, alleles q817-q825, caused a fully penetrant Glp phenotype and mapped to chromosome III (Table 3). Because the glp-1 locus is large (7.4 kb) and located on chromosome III, these 9 mutations were likely glp-1 alleles. Indeed, all 9 failed to complement glp-1(null) ( Table 3). The 10th allele q831 caused a low penetrance Glp phenotype and was mapped to the right arm of chromosome I, at some distance from both sygl-1 and lst-1 loci. Therefore, this mutation must be a lesion in some other gene; its identity is described below. The initial screens were heavily biased for the recovery of glp-1 alleles. To limit the isolation of more glp-1 alleles, we introduced a transgenic copy of wildtype glp-1 into the lst-1(lf) and sygl-1(lf) single mutants (Fig. 1d, regimen 2; Table 2). The glp-1 transgene, qSi44 or glp-1(tg), is a single copy insertion of wildtype glp-1 on chromosome II that rescues a glp-1 null mutant (Sorensen et al. 2020). Using the same EMS mutagenesis procedure as before, we screened 7,922 lst-1(lf); glp-1(tg) haploid genomes and 3,868 sygl-1(lf); glp-1(tg) haploid genomes. No mutants with a Glp phenotype were isolated from lst-1(lf); glp-1(tg) but 2 were recovered from sygl-1(lf); glp-1(tg) ( Table 2). These mutations were subsequently determined to be alleles of lst-1 (see below). Table 3 summarizes the genetic characterization of alleles recovered from the screen, and Table 4 summarizes their molecular lesions. Our failure to recover sygl-1 alleles in the lst-1(lf) background shows that our screens were not performed to saturation. However, we note that the sygl-1 locus is relatively small (621 bp coding region) and therefore likely a poor mutagenesis target.

Gene(allele)
Type of mutation Nucleotide change Codon change Amino acid change Unknown Not found n/a n/a glp-1(q824) Substitution AC ! CA in intron 4 b n/a n/a glp-1(q825) Splice site G ! A n/a n/a lst-1(q826) n/a, not applicable. a See Methods for more details. b 184 bp from 5 0 splice site. (n > 50) and had the Glp phenotype (n ¼ 10). These lst-1 alleles will prove useful in future studies focused on lst-1 function.

Characterization of glp-1 alleles
We identified the molecular lesions in the glp-1 alleles with Sanger sequencing: q818, q821, and q822 were nonsense mutants; q817, q819, and q820 were missense mutants and q825 altered a 5 0 splice site ( Fig. 2b; Table 4). The q824 allele had a 2 bp change (AC ! CA) in intron 4 that did not affect the 5 0 or 3 0 splice sites or the branch point (Fig. 2b). We failed to determine the lesion in 1 allele, q823, despite sequencing all exons and introns plus 2,382 bp upstream of the transcription start site and 927 bp  Table 4 for molecular changes. b) The glp-1 locus generates 1 RNA isoform and 1 protein product. Regions within exons are colored according to protein domains: yellow, EGFlike (EGFL) repeats; green, lin-12/Notch Repeats (LNR); red, transmembrane domain ; dark blue, RAM domain; light blue, ANK repeats. Mutations in the ANK repeats that are shown below include those from this work (red) and those published previously (Austin and Kimble 1987;Kodoyianni et al. 1992;Berry et al. 1997;Dalfo et al. 2010;Thompson et al. 2013). Not shown are ANK repeat mutations isolated as intragenic suppressors of glp-1(q231) and glp-1(q224) (Lissemore et al. 1993). Ms, missense. c) Key features of glp-1 mutations in ANK repeats. See Table 4 for molecular changes in other glp-1 alleles and Table 5 for temperature sensitivity data. downstream of the 3 0 UTR. Nonetheless, the remaining 8 alleles were all previously unreported glp-1 lesions. The 3 glp-1 missense alleles-q817, q819, and q820-all carry amino acid changes in the ANK repeats ( Fig. 2b and c). ANK repeats are conserved across eukaryotes with roles in protein interaction, cell signaling, and disease (Roehl et al. 1996;Mosavi et al. 2004). Many previously identified glp-1 alleles also have changes in this region. Mutations in ANK repeats 1, 2, 4, and 5 all cause a temperature sensitive Glp phenotype (Kodoyianni et al. 1992;Berry et al. 1997;Nadarajan et al. 2009;Dalfo et al. 2010). Our 3 newly identified missense alleles occur in different repeats, ANK 3 (q819 and q820) and ANK 6 (q817) and they are not temperature sensitive (Table 5). All 3 affect conserved residues (Fig. 3). We conclude that the newly identified ANK missense mutations affect residues essential for GLP-1 function. These alleles should prove useful for investigating ANK repeats and their role in Notch signaling.

Characterization of pole-1(q831)
One mutant allele isolated in the sygl-1(lf) background, q831, mapped to the right arm chromosome I. Whole-genome sequencing revealed a nonsense mutation R1899Stop in F33H2.5 (Table 4), which encodes a C. elegans ortholog of the catalytic subunit of DNA polymerase e (Fig. 4a). We confirmed q831 as an allele of F33H2.5 by Sanger sequencing, and by its failure to complement gk49, a deletion allele in F33H2.5 that had been generated by the C. elegans Knockout Consortium (C. elegans Deletion Mutant Consortium 2012). F33H2.5 has been named pole-1 for its DNA polymerase e orthology.
The pole-1(q831) mutation was isolated because sygl-1(lf) pole-1(q831) double mutants had a Glp phenotype. During outcrossing, we found that pole-1(q831) single mutants were 100% sterile ( Fig. 4d-f). To ask if pole-1 sterility was due to a Glp defect, we examined L4 larvae under DIC/Normaski and also stained dissected gonads with a sperm-specific antibody (SP56) (Ward et al. 1986) and DAPI (Fig. 4b-f) (see Methods). Wildtype L4 gonads contain several hundred germ cells, with undifferentiated cells at the distal end and differentiated sperm at the proximal end (Fig. 4b). glp-1(null) L4 gonads, in contrast, contain only a few germ cells, all of which have differentiated into SP56-positive sperm extending to the distal end (Fig. 4c). Similar to glp-1(null) gonads, the pole-1(q831) gonads were physically smaller than wildtype; however, only 30% had differentiated sperm extending to the distal end and thus were Glp (Fig. 4d and f). The other 70% did not have Fig. 3. Amino acid alignment for ANK repeats glp-1 orthologs and in the paralog lin-12. Alleles from Fig. 2c are marked. Blue bar, mutation causes sterility at 25 C but not at 15 C; red bar, mutation causes sterility at 15 C, 20 C, and 25 C. The residue affected in gk872502 is marked by a gray bar, because it has not been tested for temperature sensitivity. ANK repeat location within each paralog is shown beside amino acids. See legend for conservation key.
sperm extending to the distal end and were designated nonGlp steriles ( Fig. 4e and f). We also observed a low penetrance Glp phenotype in the deletion strain pole-1(gk49) (Fig. 4a and f). Because the Glp phenotype was not fully penetrant at 20 C, we examined pole-1(q831) animals raised at 15 C and 25 C. Indeed, the Glp penetrance increased with the temperature-indicating that the Glp defect is temperature sensitive (Fig. 4f). In addition to germline defects, pole-1 mutants had a range of other defects, consistent with a broad role in development. For example, pole-1 mutants had vulval defects (Fig. 4f) and were uncoordinated. DNA polymerase e pole-1 was not previously been recognized critical for GSC maintenance, though other components of the DNA  (Pospiech and Syvä oja 2003). Dissected mid-L4 gonads stained with SP56 antibodies for sperm (red) and with DAPI for DNA (blue) (see Methods). Dotted line outlines each gonad; asterisk marks the distal end. Scale, 50 mm. b) Wildtype. c) glp-1 Glp germline. d) Glp pole-1(q831) germline. e) NonGlp pole-1(q831) germline. f) Low penetrance pole-1 Glp phenotype is enhanced by loss of sygl-1, not lst-1. Germline "normal" refers to an adult germline similar to wildtype in size and organization; "Glp" refers to a smaller than normal germline with sperm to distal end; "nonGlp" refers to a smaller than normal germline without sperm at the distal end. Vulva: "normal" refers a vulva similar to a wildtype morphology; "Vul" denotes Vulvaless; "Muv" denotes Multivulva. Temp refers to temperature at which animals were raised (see Methods). n, number of germlines or vulvas scored. replication machinery have been implicated in germ cell proliferation (Yoon et al. 2018).

Conclusions and future directions
The goal of the mutant screens in lst-1 and sygl-1 mutant backgrounds was to identify new regulators of GSC self-renewal. In particular, we sought to test the idea that the LST-1 and SYGL-1 proteins might work with other factors that were similarly redundant. The screens identified 9 alleles of glp-1, 2 alleles of lst-1, and 1 allele of pole-1-the C. elegans ortholog of DNA polymerase e. Although the screens were not saturated, identification of pole-1 with a low penetrance Glp phenotype demonstrates that additional genes likely await discovery. Any additional screens in lst-1 or sygl-1 null backgrounds should focus on the modified design with transgenic glp-1 to avoid isolation of more glp-1 alleles. Alternatively, overexpression of either lst-1 or sygl-1 causes a germline tumor (Shin et al. 2017) and so one might seek suppressors of those tumors or enhancers of the low penetrance pole-1 Glp phenotype.

Data availability
Strains are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.