Sorokin, Elena P. et al. (2011) International Worm Meeting "Chemically reprogramming the adult sperm/oocyte fate decision."

Morgan, Clinton T., Sorokin, Elena P., Kimble, Judith

[International Worm Meeting, 2011]

The molecular basis of germ cell specification as sperm or oocyte remains poorly understood. In most animals (nematodes, fruitflies and mice), somatic signaling controls germline sexual fate, but the regulators in germ cells that direct the sperm or oocyte fate are best understood in C. elegans. Based on that knowledge, we recently devised a method to chemically reprogram germline sex: U0126, a MEK kinase inhibitor, transforms a puf-8; lip-1 masculinized germline to produce functional oocytes instead of sperm (Morgan et al., 2010). Here we report the use of this chemical method to analyze two critical aspects of germ cell fate reprogramming. First, we mapped the reprogramming to cells at the boundary of the mitotic and transition zone, where virtually all germ cells have entered the meiotic cell cycle. Consistent with this map, active MAP kinase and the terminal sperm fate regulator FOG-1 rapidly decrease in the transition zone after U0126 treatment. Therefore, the U0126-mediated effect on the sperm/oocyte decision occurs as germ cells enter meiotic prophase. Second, we used RNA-seq to profile changes in mRNA expression after chemical reprogramming. Specifically we compared RNAs from U0126-treated puf-8; lip-1 vs U0126-treated N2s and from U0126-treated vs DMSO vehicle-treated puf-8; lip-1 over an 18-hour time course. Preliminary results reveal a highly-enriched binding motif in the promoters of mRNAs differentially expressed upon reprogramming. Moreover, RNAi against the likely transcription factor affects germline sex. This approach therefore promises to extend our molecular understanding of germ cell fate determination to a transcriptional level. We conclude that chemical reprogramming provides an invaluable tool for analyzing the sperm/oocyte decision. Morgan, C. T., Lee, M. H., Kimble, J., 2010. Chemical reprogramming of Caenorhabditis elegans germ cell fate. Nat Chem Biol 6, 102-4.

Sorensen, Erika B. et al. (2015) International Worm Meeting "ChIP-Seq analysis of GLP-1/Notch signaling target genes in germline stem cells."

Sorensen, Erika B., Kimble, Judith, Groth, Amy C., Sorokin, Elena P.

[International Worm Meeting, 2015]

The C. elegans mesenchymal niche for germline stem cells (GSCs) employs GLP-1/Notch signaling to maintain a pool of ~50 GSCs (1,2). Loss-of-function Notch mutants cannot maintain GSCs (3) and gain-of-function mutants cause germline tumors (4). All metazoan Notch signaling relies on cleavage of the Notch receptor to generate the Notch intracellular domain (NICD), which moves into the nucleus and forms a ternary complex with DNA-binding protein LAG-1 and transcriptional co-activator LAG-3/SEL-8. To date, identification of direct transcriptional targets of GLP-1/Notch signaling in the germline has relied on indirect methods and revealed four possible genes: fbf-2 (5), lip-1 (6), lst-1 and sygl-1 (7). We set out to test the validity of these proposed targets and to identify others by chromatin immunoprecipitation (ChIP). To this end, we generated Mos-mediated transgenic lines expressing epitope-tagged versions of the GLP-1 receptor and LAG-1 DNA-binding protein. The glp-1 transgene includes the glp-1 genomic locus plus a C-terminal 6xmyc6HIS tag; it rescues glp-1 null homozygotes to fertility and enables visualization of both transmembrane and intranuclear GLP-1 NICD. The lag-1 transgene is driven by the germline-specific mex-5 promoter and contains an N-terminal 6xFLAG tag; it rescues homozygotes for the unusual germline-specific lag-1(q426) allele from 75% sterility/25% embryonic lethality to 100% fertility and viability. GLP-1::6xmyc6HIS is expressed in the distal germline and 6XFLAG::LAG-1 is expressed in the germline. ChIP-Seq with these epitope-tagged Notch signaling components enriches for the sygl-1 and lst-1 promoters, confirming their identification as direct targets, but not for fbf-1 or lip-1, casting doubt on those genes as direct Notch targets. In addition, our ChIP-Seq data identifies other likely targets currently being assayed for biological effects.References: (1) Angelo and Van Gilst (2009) Science 326(5955): 954; (2) Cinquin et al. (2010) PNAS 107(5): 2048; (3) Austin and Kimble (1987) Cell 51: 589; (4) Berry et al. (1997) Development 124: 925; (5) Lamont et al. (2004) Dev Cell 7(5): 697; (6) Lee et al. (2006) EMBO J 25(1): 88; (7) Kershner et al. (2014) PNAS 111(10): 3739.

Sorensen, Erika et al. (2019) International Worm Meeting "MicroRNA depletion results in Notch loss of function phenotypes and loss of stem cell totipotency."

Ballard, Joseph, Kimble, Judith, Jones, Hunter, Sorensen, Erika, Sorokin, Elena, Geier, Ben Geier, Groth, Amy Groth, Doenier, Emma

[International Worm Meeting, 2019]

Notch signaling regulates stem cells and differentiation during normal animal development and when dysregulated can lead to cancer. In the model organism C. elegans, Notch signaling functions to maintain germline stem cells (GSCs) in a totipotent state (capable of differentiating into all cell types) by promoting the expression of target genes that function in GSC maintenance. Recently, microRNAs (miRNAs) mir-61 and mir-250, termed collectively mir61-250, were identified as Notch target genes in GSCs. miRNAs are non-coding RNAs that act post-transcriptionally to limit the expression of other genes. To ask if mir61-250 affects GSC maintenance, we performed assays using a CRISPR-Cas9 generated mutant, which lacks the mir61-250 promoter. Unfortunately, no GSC defect was observed. This lack of defect is consistent with previous reports which show that single-gene miRNA deletions fail to produce strong mutant phenotypes unless placed in a sensitized background. Progressing forward, our lab has placed mir61-250 mutant animals in sensitized backgrounds and assayed them for defects in animal development, GSCs, and fertility. We find that mir61-250 mutants exhibit developmental deformities in their egg laying apparatus and have fewer GSCs. In addition, we find that loss of these miRNAs promotes reprograming from a germ cell fate to a somatic fate.

Sorensen, Erika B. et al. (2017) International Worm Meeting "ChIP-Seq reveals miRNA61-250 and deps-1 as Notch target genes in germline stem cells."

Kimble, Judith, Noble, Dan C., Porter, Doug F., Sorensen, Erika B., Sorokin, Elena P., Doenier, Emma, Lee, ChangHwan, Groth, Amy C.

[International Worm Meeting, 2017]

Notch signaling activates transcription of target genes and thereby regulates stem cells and differentiation broadly during animal development. The nematode C. elegans employs Notch signaling to maintain germline stem cells (GSCs) (1). Previous work identified two Notch targets with a candidate gene approach (2). Here we used chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) to identify GSC Notch targets on a genomic scale. ChIPs of the LAG-1/CSL DNA binding protein identified 384 significant peaks, of which 8 were also identified in parallel ChIPs of the GLP-1/Notch receptor. Two of these 8 common peaks belonged to the targets already known. In addition, we found peaks in the promoters of RNA regulators. One peak was in the promoter of mir-61 and mir-250, termed collectively mir61-250 (3). In situ hybridization demonstrates that these miRNAs are transcribed in GSCs and that their transcription is Notch-dependent. To ask if mir61-250 affects GSC maintenance, we used CRISPR-Cas9 (4,5) to delete the mir61-250 promoter. The resultant mir61-250 promoter deletion abolishes transcription of the miRNAs and therefore provides a loss-of-function mutant. Yet no GSC defect was seen. One explanation is that mir61-250 is functionally redundant with other genes. We are currently determining if miRNA overexpression or the presence of a miRNA sponge affects GSCs (6). Another ChIP peak was in the promoter of deps-1, a protein localized to the P-granule (7). Single molecule fluorescence in situ hybridization (smFISH) demonstrates that deps-1 expression in GSCs is Notch-dependent. We are exploring how these newly identified Notch target genes function as RNA regulators to control GSC maintenance. (1) Kimble and Crittenden. (2007) Annu Rev Cell Dev Biol, 23: 405; (2) Kershner et al. (2014) PNAS,111(10): 3739; (3) Martinez et al. (2008) Genome Res 18: 2005; (4) Arribere et al. (2014) Genetics 198(3): 837; (5) Paix et al. (2014) Genetics 198(4): 1347; (6) Ebert and Sharp (2010) RNA, 16: 2043; (7) Spike et al. (2007) Dev, 135: 983.