Germline Stem Cell Differentiation Entails Regional Control of Cell Fate Regulator GLD-1 in Caenorhabditis elegans

Abstract

Germline stem cell differentiation in Caenorhabditis elegans is controlled by glp-1 Notch signaling. Cell fate regulator GLD-1 is sufficient to induce meiotic entry and expressed at a high level during meiotic prophase, inhibiting mitotic gene activity. glp-1 signaling and other regulators control GLD-1 levels post-transcriptionally (low in stem cells, high in meiotic prophase), but many aspects of GLD-1 regulation are uncharacterized, including the link between glp-1-mediated transcriptional control and post-transcriptional GLD-1 regulation. We established a sensitive assay to quantify GLD-1 levels across an 35-cell diameter field, where distal germline stem cells differentiate proximally into meiotic prophase cells in the adult C. elegans hermaphrodite, and applied the approach to mutants in known or proposed GLD-1 regulators. In wild-type GLD-1 levels elevated 20-fold in a sigmoidal pattern. We found that two direct transcriptional targets of glp-1 signaling, lst-1 and sygl-1, were individually required for repression of GLD-1. We determined that lst-1 and sygl-1 act in the same genetic pathway as known GLD-1 translational repressor fbf-1, while lst-1 also acts in parallel to fbf-1, linking glp-1-mediated transcriptional control and post-transcriptional GLD-1 repression. Additionally, we estimated the position in wild-type gonads where germ cells irreversibly commit to meiotic development based on GLD-1 levels in worms where glp-1 activity was manipulated to cause an irreversible fate switch. Analysis of known repressors and activators, as well as modeling the sigmoidal accumulation pattern, indicated that regulation of GLD-1 levels is largely regional, which we integrated with the current view of germline stem cell differentiation.

Small RNA in situ hybridization in Caenorhabditis elegans, combined with RNA-seq, identifies germline-enriched microRNAs

“…This could relate to findings in (Akay et al, 2013) that both miR35 and let7 families genetically interact with GLD-1, a well-studied RNA binding protein. While Brenner and Schedl (2016) present convincing evidence that GLD-1 is not a target of the miR35s , we suggest GLD-1 may indirectly regulate these two miRNA families and that when one miRNA family is missing, the other increases in expression. In Fig.…”

“…As described above, deletion of all miR35 family members results in embryonic lethality; however adding back maternal expression of the miR35–41 gene cluster or of each individual miR35 member, results in normal embryonic development (Alvarez-Saavedra and Horvitz, 2010), implying redundancy of the miR35 s and a vital role for this family in embryonic development. In addition, McJunkin and Ambros (2014) have demonstrated that maternal, along with zygotic, miR35s act early in development to insure maximal fertility of the adult progeny, while Brenner and Schedl (2016) quantified the marked reduction in proliferation in the C. elegans germline with loss of all miR35 family members. In the miR51 family, miR-56 is the only member we identified as germline-enriched.…”

Keeping stem cells under control: New insights into the mechanisms that limit niche‐stem cell signaling within the reproductive system

“…The distal tip cell expresses the Notch ligand LAG‐2 while the germ line expresses the receptor GLP‐1. Notch pathway activation within germ cells induces the transcription of a number of target genes whose products act in concert with additional factors to repress germ cell entry into meiosis (Brenner and Schedl, ). Thus, by using the Notch pathway, the C. elegans distal tip cells directly and precisely regulate the size of the germ line stem cell population (Byrd and Kimble, ; Kimble, ).…”

GLP-1 Notch - LAG-1 CSL control of the germline stem cell fate is mediated by transcriptional targetslst-1andsygl-1

“…were exported, stitched in pairs, using Bio-Formats and Stitching plugin in Fiji(Preibisch 857 et al 2009;Linkert et al 2010;Schindelin et al 2012). In Fiji, DAPI images were used to 858 draw a line, starting at the distal end to the desired cd(Brenner and Schedl 2016).859 860 FBF-2 protein was quantified 35 cd from the distal end of the germlines, whereas 861 quantification of LAG-1, SYGL-1 and LST-1 was done using 25 cd. A width of 75 pixel 862 was used to collect protein levels for each line.…”