Highlights d Germline MPK-1B is dispensable for proliferation of germline stem cells d Germline MPK-1B is essential for gametogenesis and fertility d Somatic MPK-1A is required for a high rate of GSC proliferation d Somatic MPK-1A promotes germline proliferation from the gut or somatic gonad

GLP-1/Notch signaling and a downstream RNA regulatory network maintain germline stem cells (GSCs) in Caenorhabditis elegans. In mutants lacking the GLP-1 receptor, all GSCs enter the meiotic cell cycle precociously and differentiate into sperm. This dramatic GSC 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 GSCs. 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 nine glp-1 alleles, two lst-1 alleles, and one allele of pole-1, which encodes the catalytic subunit of DNA polymerase ε. Three glp-1 alleles reside in Ankyrin (ANK) 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 one locus that interacts genetically with sygl-1 and generated useful mutations for further studies of GSC regulation.

Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) is a major positive regulator of cell proliferation that is often upregulated in cancer. Yet few studies have addressed ERK/MAPK regulation of proliferation within a complete organism. The C. elegans ERK/MAPK ortholog MPK-1 is best known for its control of somatic organogenesis and germline differentiation, but it also stimulates germline stem cell proliferation. Here we identify tissue-specific MPK-1 isoforms and characterize their distinct roles in germline function. The germline-specific MPK-1B isoform promotes germline differentiation, but has no apparent role in germline stem cell proliferation. By contrast, the soma-specific MPK-1A isoform promotes germline proliferation non-autonomously. Indeed, MPK-1A functions in the intestine or somatic gonad to promote germline proliferation, independently of its other known roles. We propose that a non-autonomous role of ERK/MAPK in stem cell proliferation may be conserved across species and other tissue types, with major clinical implications for cancer and other diseases.

The expression of genes encoding powerful developmental regulators is exquisitely controlled, often at multiple levels. Here, we investigate developmental expression of three conserved genes, Caenorhabditis elegans mpk-1, lag-1, and lag-3/sel-8, which encode homologs of ERK/MAPK and core components of the Notch-dependent transcription complex, respectively. We use single-molecule FISH (smFISH) and MATLAB to visualize and quantify nuclear nascent transcripts and cytoplasmic mRNAs as a function of position along the germline developmental axis. Using differentially labeled probes, one spanning an exceptionally long first intron and the other spanning exons, we identify two classes of active transcription sites (ATS). The iATS class, for “incomplete” ATS, harbors only partial nascent transcripts; the cATS class, for “complete” ATS, harbors full-length nascent transcripts. Remarkably, the frequencies of iATS and cATS are patterned along the germline axis. For example, most mpk-1 ATS are iATS in hermaphrodite germline stem cells, but most are cATS in differentiating stem cell daughters. Thus, mpk-1 ATS class frequencies switch in a graded manner as stem cell daughters begin differentiation. Importantly, the patterns of ATS class frequency are gene-, stage-, and sex-specific, and cATS frequency strongly correlates with transcriptional output. Although the molecular mechanism underlying ATS classes is not understood, their primary difference is the extent of transcriptional progression. To generate only partial nascent transcripts in iATS, progression must be slowed, paused, or aborted midway through the gene. We propose that regulation of ATS class can be a critical mode of developmental gene regulation.

6Genes encoding powerful developmental regulators are exquisitely controlled, often at multiple levels. 7Here, we use single molecule FISH (smFISH) to investigate nuclear active transcription sites (ATS) and 8 cytoplasmic mRNAs of three key regulatory genes along the C. elegans germline developmental axis. 9The genes encode ERK/MAP kinase and core components of the Notch-dependent transcription 10 complex. Using differentially-labeled probes spanning either a long first intron or downstream exons, we 11identify two ATS classes that differ in transcriptional progression: iATS harbor partial nascent transcripts 12 while cATS harbor full-length nascent transcripts. Remarkably, the frequencies of iATS and cATS are 13 patterned along the germline axis in a gene-, stage-and sex-specific manner. Moreover, regions with 14 more frequent iATS make fewer full-length nascent transcripts and mRNAs, whereas those with more 15 frequent cATS produce more of them. We propose that the regulated balance of these two ATS classes 16 has a major impact on transcriptional output during development.

Germline stem cells (GSCs) in C. elegans are maintained by GLP-1/Notch signaling from the niche and by a downstream RNA regulatory network. Loss of the GLP-1 receptor causes GSCs to precociously undergo meiotic differentiation, the Glp phenotype, due to a failure to self-renew. lst-1 and sygl-1 are functionally redundant direct targets of GLP-1 signaling whose gene products work with PUF RNA binding proteins to promote GSC self-renewal. Whereas single loss-of-function mutants are fertile, lst-1 sygl-1 double mutants are sterile and Glp. We set out to identify genes that function redundantly with either lst-1 or sygl-1 to maintain GSCs. To this end, we conducted forward genetic screens for Glp mutants in genetic backgrounds lacking functional copies of either lst-1 or sygl-1. The screens generated nine glp-1 alleles, two lst-1 alleles, and one allele of pole-1, which encodes the catalytic subunit of DNA polymerase ϵ. Three glp-1 alleles reside in Ankyrin (ANK) repeats not previously mutated. pole-1 single mutants have a low penetrance Glp that is enhanced by loss of either lst-1 or sygl-1. Thus, the screen uncovered one locus that interacts genetically with both lst-1 and sygl-1 and generated useful mutations for further studies of GSC regulation.

One distinguishing feature of eukaryotic genomes is the presence of introns within nascent transcripts. Genomic analyses of distantly related species, spanning yeast to humans, reveal conservation of intron size and position, suggesting an intron can be advantageous. Many studies have examined the relationship between short introns and gene expression; however, the effect of long introns and gene expression is not well understood. Critical developmental regulators often have an unusually long intron that is conserved in size and position and typically one of the first introns. We hypothesize that long first introns of developmental regulators affect gene expression. To investigate the relationship between a long first intron and gene expression, we have focused on the Caenorhabditis elegans gene encoding ERK/MAP kinase (mpk‐1). Like other metazoan ERK/MAP kinases, mpk‐1 harbors a long first intron in one of its isoforms. The mpk‐1b nascent transcripts include a small first exon and an 8.2 kb first intron, while mpk‐1a lacks that first exon and intron but shares other exons and introns with mpk‐1b. To visualize RNAs, we designed single molecule in situ hybridization (smFISH) probes targeting the 8.2 kb intron to visualize nascent transcripts as well as other probes targeting the shared exons to detect the mRNAs. We use confocal microscopy and MATLAB image processing to assess mpk‐1 transcription from germline stem cell self‐renewal to meiotic pachytene of differentiating cells. In parallel, we are using CRISPR to delete regions of the long mpk‐1b intron to assess its functional importance. Our plan is to compare mpk‐1 nascent and mature transcripts during germline development, both in wild type and in in mutants that lack part of most of that large intron. Our analysis will tell us if the mpk‐1b 8.2 intron is critical for the mpk‐1 regulatory program during germ cell development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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