Discovery of two GLP-1/Notch target genes that account for the role of GLP-1/Notch signaling in stem cell maintenance

Abstract

A stem cell's immediate microenvironment creates an essential "niche" to maintain stem cell self-renewal. Many niches and their intercellular signaling pathways are known, but for the most part, the key downstream targets of niche signaling remain elusive. Here, we report the discovery of two GLP-1/Notch target genes, lst-1 (lateral signaling target) and sygl-1 (synthetic Glp), that function redundantly to maintain germ-line stem cells (GSCs) in the nematode Caenorhabditis elegans. Whereas lst-1 and sygl-1 single mutants appear normal, lst-1 sygl-1 double mutants are phenotypically indistinguishable from glp-1/Notch mutants. Multiple lines of evidence demonstrate that GLP-1/Notch signaling activates lst-1 and sygl-1 expression in GSCs within the niche. Therefore, these two genes fully account for the role of GLP-1/Notch signaling in GSC maintenance. Importantly, lst-1 and sygl-1 are not required for GLP-1/Notch signaling per se. We conclude that lst-1 and sygl-1 forge a critical link between Notch signaling and GSC maintenance.

Fig. 1.

Identification of lst-1 and sygl-1 as candidate GSC regulators. (A) DTC (red) uses GLP-1/Notch signaling to maintain GSCs within the niche. Asterisk marks distal end. (B) Fifteen genes (asterisk) are shared between lists of putative Notch and FBF targets. Double RNAi of two common genes, lst-1 and sygl-1, caused a Glp phenotype. Also see Tables S1 and S2. (C) lst-1 and sygl-1 genes. Yellow, exons; red lines, LAG-1 binding sites (LBS); black bar, deletions; purple line, probe for in situ hybridizations (exons only). (D) LST-1 and SYGL-1 proteins and their predicted motifs.

Fig. 2.

lst-1 and sygl-1 function redundantly to promote GSC self-renewal in larvae and adults. (A–D) DIC- and DAPI-stained images of gonads dissected from L4 hermaphrodites. Asterisk marks distal end; dotted line outlines germ line plus somatic gonadal cells; arrows mark mature sperm. (A–C) Wild type (100% non-Glp; n > 100), lst-1(ok814) (100% non-Glp; n = 146), and sygl-1(tm5040) (100% non-Glp; n = 159) all produce normal germ lines. (D) lst-1 sygl-1 double mutants produce Glp germ lines with only a few differentiated sperm (100% Glp; n = 76). (E) Gonad from lst-1 sygl-1 L4 with a somatic GFP marker (green), a sperm marker (red) and DNA staining (blue). All nonsperm cells expressed somatic GFP. Each gonadal arm contained 14 ± 3 sperm (n = 9) on average (from three to four premeiotic germ cells). (F) Total premeiotic germ cells (GC#) in entire L3 gonad, scored with PGL-1 germ cell marker. For wild-type and each single mutant, n = 5; for lst-1 sygl-1 double mutant, n = 9. (G–H) Representative confocal images of wild-type late L4 larval germ lines treated with RNAi for 48 h; DTC expresses GFP (green). Same conventions as in A–D; mitotic zone scored by presence of mitotic marker REC-8 (yellow) and absence of crescent-shaped DAPI staining typical of early meiotic prophase nuclei (white arrowheads). Anti–DAO-5 (red, nucleolar marker) counter stain facilitates scoring of DAPI crescents. (G) Germ line treated with empty RNAi vector possesses a mitotic zone. (H) Germ line treated with lst-1 sygl-1 double RNAi lacks a mitotic zone and hence lacks GSCs.

Fig. 3.

lst-1 and sygl-1 are targets of GLP-1/Notch activation. (A and B) lst-1 and sygl-1 mRNA expression in wild-type young adult gonads. (C and D) Expression of lst-1 and sygl-1 in GSCs within the niche requires GLP-1/Notch. Shown are L4 gonads. GLP-1(+): lst-1, 97% positive (n = 37); sygl-1, 97% positive (n = 36). GLP-1(-): lst-1, 0% positive (n = 28); sygl-1, 0% positive (n = 33). (E) Wild-type sygl-1 promoter drives reporter expression in germ cells within the niche (100% GFP-positive; n = 45), whereas 4× LBS mut sygl-1 promoter does not (0% distal positive; n = 46). Filled white triangles mark nuclei positive for H2B::GFP; empty white triangles mark nuclei negative for H2B::GFP. Because reporters were occasionally silenced, only germ lines expressing GFP proximally were scored (SI Materials and Methods). (A–E) Asterisk marks distal end.

Fig. 4.

lst-1 and sygl-1 do not affect GLP-1/Notch signaling. (A) GLP-1/Notch reporter assay exploits sygl-1(tm5040) deletion mutant (see Results). Conventions same as Fig. 1C. (B and C) L4 hermaphrodite gonads were probed for GLP-1/Notch reporter by in situ hybridization. Shown are representative images of distal gonads. Both mutants have tumorous germ lines that appear the same. Asterisk marks distal end. (B) The gld-2 gld-1 lst-1 sygl-1 quadruple mutant stains positively for the GLP-1/Notch reporter (solid bracket) (91% positive; n = 35). (C) The gld-2 gld-1 lst-1 sygl-1; glp-1 quintuple mutant does not stain positively for the GLP-1/Notch reporter (dashed bracket) (0% positive; n = 27). (D) Model for lst-1 and sygl-1 function in the GSC self-renewal pathway.