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

Kershner, Aaron M.; Shin, Heaji; Hansen, Tyler J.; Kimble, Judith

doi:10.1073/pnas.1401861111

Cited by 102 publications

(266 citation statements)

References 46 publications

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“…RNAi feeding experiments were carried out following established protocols (Kershner et al 2014). For multiple gene knockdowns, HT115 bacteria containing lst-1 or sygl-1 RNAi vectors were grown separately in overnight cultures and then seeded to RNAi plates in equal volumes.…”

Section: Rnai Experimentsmentioning

confidence: 99%

The PUF binding landscape in metazoan germ cells

Prasad

Porter

Kroll-Conner

et al. 2016

RNA

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PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, including FBF, is a global view of their binding sites in their native context (i.e., their "binding landscape"). To understand the interactions underlying FBF function, we used iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to determine binding landscapes of C. elegans FBF-1 and FBF-2 in the germline tissue of intact animals. Multiple iCLIP peak-calling methods were compared to maximize identification of both established FBF binding sites and positive control target mRNAs in our iCLIP data. We discovered that FBF-1 and FBF-2 bind to RNAs through canonical as well as alternate motifs. We also analyzed crosslinking-induced mutations to map binding sites precisely and to identify key nucleotides that may be critical for FBF-RNA interactions. FBF-1 and FBF-2 can bind sites in the 5 ′ UTR, coding region, or 3 ′ UTR, but have a strong bias for the 3 ′ end of transcripts. FBF-1 and FBF-2 have strongly overlapping target profiles, including mRNAs and noncoding RNAs. From a statistically robust list of 1404 common FBF targets, 847 were previously unknown, 154 were related to cell cycle regulation, three were lincRNAs, and 335 were shared with the human PUF protein PUM2.

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Section: Rnai Experimentsmentioning

confidence: 99%

The PUF binding landscape in metazoan germ cells

Prasad

Porter

Kroll-Conner

et al. 2016

RNA

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“…We chose to further examine the bn18 mutant as it has the largest proliferative zone and therefore should be more comparable to wild type (Table 1). Currently, there is no direct readout for GLP-1 activity and reporters for GLP-1 transcriptional targets are just being developed (Kershner et al 2014). Instead, we used changes in GLD-1 levels as an indirect readout because GLP-1 signaling indirectly inhibits accumulation of GLD-1 protein in distal germ cells (Hansen et al 2004b); following shift of glp-1(bn18) to 25°, distal GLD-1 levels are elevated at the 2-hr time point (P. Fox and T. Schedl, unpublished results), indicating that GLP-1 activity has decreased significantly before the 2-hr point, which is propagated to alter GLD-1 protein levels through changes in translational regulation of the gld-1 mRNA (Lee and Schedl 2010).…”

Section: Cells Throughout the Proliferative Zone Rapidly And Synchronmentioning

confidence: 99%

Analysis of Germline Stem Cell Differentiation Following Loss of GLP-1 Notch Activity in Caenorhabditis elegans

Fox

Schedl

2015

Genetics

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Stem cells generate the differentiated progeny cells of adult tissues. Stem cells in the Caenorhabditis elegans hermaphrodite germline are maintained within a proliferative zone of 230 cells, 20 cell diameters in length, through GLP-1 Notch signaling. The distal tip cell caps the germline and supplies GLP-1-activating ligand, and the distal-most germ cells that occupy this niche are likely self-renewing stem cells with active GLP-1 signaling. As germ cells are displaced from the niche, GLP-1 activity likely decreases, yet mitotically cycling germ cells are found throughout the proliferative zone prior to overt meiotic differentiation. Following loss of GLP-1 activity, it remains unclear whether stem cells undergo transit-amplifying (TA) divisions or more directly enter meiosis. To distinguish between these possibilities we employed a temperature-sensitive (ts) glp-1 mutant to manipulate GLP-1 activity. We characterized proliferative zone dynamics in glp-1(ts) mutants at permissive temperature and then analyzed the kinetics of meiotic entry of proliferative zone cells after loss of GLP-1. We found that entry of proliferative zone cells into meiosis following loss of GLP-1 activity is largely synchronous and independent of their distal-proximal position. Furthermore, the majority of cells complete only a single mitotic division before entering meiosis, independent of their distal-proximal position. We conclude that germ cells do not undergo TA divisions following loss of GLP-1 activity. We present a model for the dynamics of the proliferative zone that utilizes cell cycle rate and proliferative zone size and output and incorporates the more direct meiotic differentiation of germ cells following loss of GLP-1 activity.

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“…SYTO-12 (Life Technologies) staining for apoptotic cells was performed as described (Gumienny et al 1999). Antibody staining of dissected gonads was carried out as described (Kershner et al 2014). Primary antibodies were used at the following dilutions: rabbit anti-GLP-1 (1:20) (Crittenden et al 1994), rabbit anti-REC-8 (1:500; SDIX), rabbit anti-HIM-3 (1:200) (Zetka et al 1999), mouse anti-DAO-5 (1:200; Developmental Studies Hybridoma Bank), and mouse anti-MPK-1(YT) (1:400; Sigma).…”

Section: Immunocytochemistrymentioning

confidence: 99%

Nucleotide levels regulate germline proliferation through modulating GLP-1/Notch signaling inC. elegans

et al. 2016

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Animals alter their reproductive programs to accommodate changes in nutrient availability, yet the connections between known nutrient-sensing systems and reproductive programs are underexplored, and whether there is a mechanism that senses nucleotide levels to coordinate germline proliferation is unknown. We established a model system in which nucleotide metabolism is perturbed in both the nematode Caenorhabditis elegans (cytidine deaminases) and its food (Escherichia coli); when fed food with a low uridine/thymidine (U/T) level, germline proliferation is arrested. We provide evidence that this impact of U/T level on the germline is critically mediated by GLP-1/Notch and MPK-1/MAPK, known to regulate germline mitotic proliferation. This germline defect is suppressed by hyperactivation of glp-1 or disruption of genes downstream from glp-1 to promote meiosis but not by activation of the IIS or TORC1 pathways. Moreover, GLP-1 expression is post-transcriptionally modulated by U/T levels. Our results reveal a previously unknown nucleotide-sensing mechanism for controlling reproductivity.

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

Cited by 102 publications

References 46 publications

The PUF binding landscape in metazoan germ cells

The PUF binding landscape in metazoan germ cells

Analysis of Germline Stem Cell Differentiation Following Loss of GLP-1 Notch Activity in Caenorhabditis elegans

Nucleotide levels regulate germline proliferation through modulating GLP-1/Notch signaling inC. elegans

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