Crittenden, Sarah L. et al. (2017) International Worm Meeting "Visualizing nuclear GLP-1/Notch NICD."

Battula, Sindhu, Lynch, Tina R., Kimble, Judith, Sorensen, Erika, Lee, ChangHwan, Seidel, Hannah S., Lei, Cecilia, Crittenden, Sarah L.

[International Worm Meeting, 2017]

Notch is a conserved regulator of stem cells and differentiation. Upon activation by ligand, the Notch receptor is cleaved, releasing its intracellular domain (NICD) for assembly of a nuclear complex and transcriptional activation of downstream targets. Our work focuses on Notch signaling in the C. elegans germline stem cell niche, where well-defined signaling and receiving cells facilitate quantitative analyses. We recently reported that the transcriptional response to GLP-1/Notch activation occurs in a steep gradient across the germline stem cell pool (Lee et al., 2016). An outstanding question is whether this transcriptional gradient reflects a corresponding gradient in GLP-1/Notch activation. We are visualizing nuclear GLP-1/Notch NICD as a readout of GLP-1/Notch activation. Our reagents include MOS-SCI transgenes encoding a tagged GLP-1 receptor that rescues a glp-1 null allele as well as CRISPR-inserted tags at the endogenous locus. To date, we have detected signal with Myc, V5, OLLAS and Halo tags. The signal is low, however, and we are working on methods to bring the signal into a quantifiable range without changing receptor abundance. Our progress will be reported at the meeting.

Crittenden, Sarah et al. (2011) International Worm Meeting "The sexually dimorphic germline stem cell niche."

Knobel, Karla, Mohanti, Ipsita, Byrd, Dana, Kimble, Judith, Crittenden, Sarah

[International Worm Meeting, 2011]

Germline stem cells (GSCs) maintain the adult germline tissue in both sexes. Distal tip cells (DTCs) contribute to the niche for GSCs in both sexes, but the DTCs are sexually dimorphic with respect to cell number (1 per gonadal arm in hermaphrodites, 2 in males) and expression of the Notch ligand, LAG-2 (high in hermaphrodites, low in males (Chesney et al., 2009)). Moreover germ cells within the niche are sexually dimorphic: the spermatogenic male germ cells divide two times faster than oogenic hermaphrodite germ cells throughout the mitotic zone (D. Morgan et al., 2010). The male GSC niche therefore provides a distinct but related mode of GSC control. In adult hermaphrodite germlines, which are oogenic, the distal tip cell (DTC) and Notch signaling maintain an immature, stem-cell-like state in germ cells that reside within 6-8 cell diameters from the distal end (Cinquin et al., 2010). We have now identified a similar distal pool of stem-cell-like germ cells in males that reside within 11-13 cell diameters of the distal end. The distal pool, which extends further from the distal end in males, also contains more germ cells (hermaphrodite: 45-70 gc, male: 60-91 gc). Therefore, the distal pool of males has distinct properties and may have distinct modes of regulation. Characteristics of DTC cellular architecture correlate with the distal pool. The hermaphrodite DTC cell body forms a cap covering the first 4 rows of germ cells; this cap extends both short intercalating processes to embrace germ cells within the first 9 rows from the distal end as well as longer processes along the surface nearly to the point of meiotic entry. We have found that male DTCs also have a cap and short intercalating processes as well as longer superficial processes. Our model is that the cap defines the region of highest Notch signaling and that intercalating processes may also anchor the GSCs distally. Knowledge of the Notch ligands expressed by the DTC in each sex is essential for understanding the niche. We are currently analyzing the battery of DSL ligands for both expression and function in the DTCs of both sexes.

Crittenden, Sarah L. et al. (2015) International Worm Meeting "Sexually dimorphic GSCs and development of tools to manipulate them."

Kimble, Judith, Crittenden, Sarah L., Lee, ChangHwan, Ryan, Anne R., Taylor, Brandon, Mohanty, Ipsita

[International Worm Meeting, 2015]

In C. elegans, germline stem cells (GSCs) are maintained by interaction with their niche (distal tip cell (DTC)) in both sexes and the primary GSC regulators are the same in both sexes (Notch signaling, LST-1, SYGL-1 and FBF). Yet the niches are sexually dimorphic with respect to cell number and Notch ligand expression (1), and germ cells within the niche are sexually dimorphic with respect to cell cycle rate (2). Previous work delineated a likely hermaphrodite GSC pool extending 6-8 cell diameters from the distal end and containing 45-70 germ cells (3). We now delineate a likely male GSC pool and find that it extends further (11-13 cell diameters from the distal end) and contains more germ cells (60-90). Notch targets, lst-1 and sygl-1, are restricted to a germline region corresponding roughly to the GSC pool in hermaphrodites (4; Kimble lab, unpublished) and preliminary results indicate that sygl-1 expression expands in males, as might be expected for its larger GSC pool. We conclude that the DTC niches and GSC pools are sexually dimorphic and likely have distinct modes of regulation in the two sexes.We are interested in whether the DTC also regulates germline sex determination. To explore this question, we are using the tissue specific, inducible Q system to express the secreted masculinizing ligand, HER-1, in the DTC and other discrete tissues to assay effects on the sperm/oocyte decision. We have also generated lines expressing Cre in the germline to track GSCs in both male and female germlines. Progress will be reported.(1) Chesney et al., Developmental Biology 331 (2009) 14-25.(2) Morgan et al., Developmental Biology 346 (2010) 204-214.(3) Cinquin et al., PNAS 107 (2010) 2048-2053.(4) Kershner et al., PNAS 111 (2014) 3739-3744.

Crittenden, Sarah L. et al. (2013) International Worm Meeting "Progress on developing the Q system to study GSCs and their control."

Crittenden, Sarah L., Kimble, Judith, Mohanty, Ipsita

[International Worm Meeting, 2013]

The Q system (Potter et al., 2010) permits inducible gene expression in C. elegans (Wei et al., 2012) and is similar in principal to the widely used GAL4-UAS system. The QF transcriptional activator directs transcription via an upstream activating sequence (QUAS). But in addition, another protein, QS, can inhibit QF activation and QS repression can be relieved by a small molecule, quinic acid or QA. QA is non-toxic and can be fed to worms (Wei et al., 2012). This inducible system has the potential to control target genes in both space and time. A chemically inducible method to control gene expression in specific cells at specific times would be tremendously useful for analyses of germline stem cells (GSCs) and their niche, the somatic distal tip cell (DTC). To this end, we are generating mosSCI insertion transgenes that rely on DTC-specific and GSC-specific regulatory sequences to drive QF expression. We are also generating a QUAS driven nuclear GFP (fused to H2B). Once we have the QF/QUAS pair working well for spatial regulation, we will add the QS/QA pair for temporal regulation. Preliminary experiments are promising and results will be shared at the meeting.

Crittenden, Sarah et al. (2021) International Worm Meeting "FBF binding elements in the gld-1 3'UTR and their role in germline regulation"

Woodworth, Jennifer, Kimble, Judith, Kroll-Conner, Peggy, Wickens, Marv, Crittenden, Sarah

[International Worm Meeting, 2021]

PUF RNA binding proteins (for Pumilio and FBF) are key regulators of self-renewal in the C. elegans germline. Two nearly identical PUF proteins, FBF-1 and FBF-2 (collectively FBF), have long been known as self-renewal regulators of late larval and adult germline stem cells (GSCs) (1). Genomic studies using iCLIP identified a battery of FBF target RNAs as well as FBF binding elements (FBEs) within them (2). That knowledge of in vivo FBEs provides a powerful inroad for studying the direct effects of FBF binding on regulation of individual target RNAs and germ cell fate. A major target of FBF regulation is gld-1, which promotes meiotic progression and oogenesis (3). Strong evidence has accumulated for FBF repression of gld-1 in GSCs (1), but FBF had also been proposed to activate gld-1 to promote meiotic entry. (1,4). We have now begun to focus on FBEs in the gld-1 3'UTR to assess their molecular and biological roles. To this end, we used CRISPR/Cas9 gene editing to mutate the two canonical gld-1 FBEs, FBEa and FBEb, in the 3'UTR of endogenous gld-1. Phenotypically, FBEa and FBEb single mutants both maintain GSCs and are fertile; an FBEa FBEb double mutant was just recovered and is being characterized. Using GLD-1 immunostaining, we find that the FBEa single mutant makes higher than normal levels of GLD-1 protein in GSCs, while the FBEb single mutant makes wild-type levels in GSCs. We are currently examining gld-1 mRNA levels in these single mutants using smFISH, and soon will start analyzing the double mutant. 1 Crittenden et al.(2002).Nature, 417,660. 2 Porter, Prasad et al.(2019).G3,9,153. 3 Francis et al (1995) Genetics 139, 579 4 Suh et al.(2009).Genetics,181,1249.

Sarah Crittenden et al. (1999) International C. elegans Meeting "FBF, NANOS-3 and the regulation of germline fates"

Sarah Crittenden, Shanping Wang, Andrei Petcherski, Beilin Zhang, Maria Gallegos, Brian Kraemer, Gary Moulder, Judith Kimble, Robert Barstead, Marvin Wickens

[International C. elegans Meeting, 1999]

Germline fate decisions include mitosis or meiosis, survival or death, and spermatogenesis or oogenesis. FBF-1 and FBF-2 (FBF), nearly identical proteins in the Puf family (for Pumilio and FBF), regulate the hermaphrodite sperm-oocyte switch by repressing the fem-3 3'UTR (1). We now find that FBF also regulates germline proliferation. In fbf(dsRNAi) and fbf-1 fbf-2 double mutants, all germline cells enter meiosis and differentiate as sperm. Genetic experiments place fbf upstream of the gld-1 gld-2 control over meiosis. In gld-1 gld-2 double mutants, all germline cells remain mitotic (2); the same is true of fbf-1 fbf-2; gld-1 gld-2 quadruple mutants. Experiments to determine the regulatory relationships between fbf and glp-1 are in progress. In addition, we have found that fog-1 , which originally was identified by its role in sperm specification (3), also plays a role in germline proliferation. Therefore, FBF and FOG-1 provide key links between controls of proliferation and sex determination. Using two-hybrid screens with FBF as bait, we identified NOS-3. The C. elegans genome harbors three nanos homologs. NOS-3 interacts specifically with both FBF-1 and FBF-2, but NOS-1 or NOS-2 do not interact with either. To explore the functions of the three NOS proteins, we used RNAi and isolation of a nos-3 deletion mutant to show that the three nos genes influence the sperm/oocyte switch, and also all three are required for germline survival. The nos effect on germline survival does not require ced-3 , and therefore is unlikely to trigger the programmed cell death pathway. In contrast to the nos genes, fbf is not required for germline survival. Therefore, FBF and NOS act together for the sperm/oocyte switch, but have distinct roles in germline proliferation (FBF) and survival (NOS). We propose that distinct partners target FBF and other Puf proteins to different mRNAs, which in turn leads to diverse biological outcomes. 1. Zhang and Gallegos et al. (1997) Nature 390, 477-484. 2. Kadyk and Kimble (1998) Development 125, 1803-1813. 3. Barton and Kimble (1990) Genetics 125, 29-39.

Sarah L Crittenden et al. (2005) International Worm Meeting "C. elegans germline mitotic region possesses germline stem cells"

Myon Hee Lee, Dana T Byrd, Sarah L Crittenden, Judith Kimble, Kim A Leonhard

[International Worm Meeting, 2005]

The adult C. elegans germ line includes mitotically-dividing cells in the distal gonad and meiotic cells more proximally. A single somatic cell, the distal tip cell (DTC), provides a <sym08>stem cell niche<sym09> that promotes germline mitoses by GLP-1/Notch signaling. We will present experiments that address the self-renewal and division pattern of germline stem cells and that further define the DTC niche. To assay self-renewal, we have done BrdU pulse-chase experiments in germline cells in the mitotic region. Multiple regimens all gave the same result. A long pulse of BrdU (~24 hours) effectively labeled all germline cells in the mitotic region, but after a chase of 14 hours, virtually no label was detected in the mitotic region. Instead, the labeled cells were observed in the meiotic region of the gonad. We conclude that the labeled mitotically-dividing cells were able to generate a new population of unlabelled mitotically-dividing cells as well as differentiating gametes the two hallmarks of stem cells. We observed no asymmetric or oriented cell divisions. Our current model is that distal cells within the mitotic region reside in the DTC niche and remain undifferentiated, while more proximal cells begin the transition into differentiation.

Sarah L Crittenden et al. (2004) East Coast Worm Meeting "Characterization of the DTC niche and germline stem cells in C. elegans"

Dana Byrd, Sarah L Crittenden, Judith Kimble, Kim Leonhard

[East Coast Worm Meeting, 2004]

The mitotic region of the C. elegans germ line includes stem cells, as defined by its ability to maintain a proliferating population of germ cells as well as to generate gametes. [Note that all 'cells' in the germ line are part of a syncytium; we call them 'cells' for simplicity; all are partially enclosed by membranes and appear to behave as individuals within the mitotic region.] In young wild-type adults, the mitotic region is composed of about 225 germ cells that extend 18-20 germ cell diameters along the distal-proximal axis. The somatic distal tip cell (DTC) promotes proliferation by GLP-1/Notch signaling and provides the 'stem cell niche'. To ask whether contact between the DTC and the germ cells defines the extent of the mitotic region, we examined the DTC and its processes using a lag-2::GFP reporter. In animals of different ages and in mutants with longer and shorter mitotic regions we found that DTC process length does not correlate with the length of the mitotic region. These findings extend the work of Hall et al. (1999) and confirm the idea that the length of DTC processes does not define the extent of the mitotic region. In an attempt to identify classical germline stem cells, we are characterizing cell cycles and orientation of mitotic spindles within the germline mitotic region. Preliminary results using BrdU, Cy3-dUTP and anti-PH3 reveal no differences along the distal-proximal axis with respect to frequency of mitosis or time from BrdU incorporation to M phase. In addition, the orientation of mitotic spindles appears random throughout the region. We suggest that stem cells in the C. elegans germ line may be controlled at a population level rather than by asymmetric divisions. Although we see no difference in cell cycle timing or orientation of cell divisions along the distal-proximal axis, germ cells in the mitotic region are not uniform by other criteria. First, cells in the adult mitotic region do not respond uniformly to removal of GLP-1 signaling (shift of glp-1(ts) from permissive to restrictive temperature). Instead, entry into meiosis occurs in a wave that begins with the most proximal cells and progresses to the most distal cells. Second, cells within the mitotic region do not express regulators of the mitosis/meiosis decision uniformly (e.g., see Hansen et al., 2004). Our current model is that the mitotic region of the germ line includes distal cells that reside within the DTC niche and remain undifferentiated, while more proximal cells that have left the niche begin the transition into differentiation. Before the more proximal cells can leave the mitotic cell cycle and enter meiosis, they must achieve a critical level of regulatory activity that drives them forward into the meiotic cell cycle. Hall et al. (1999) Developmental Biology 212, 101-123 Hansen et al. (2004), Developmental Biology 268, 342-357

Ali Khan L et al. (1997) International C. elegans Meeting "MATERNAL GENE EMB-8 IS NECESSARY FOR ASYMMETRIC DISTRIBUTION OF DEVELOPMENTAL POTENTIALS IN EARLY C. ELEGANS EMBRYOS"

Siddiqui SS, Ali Khan L, Ali MY, Miwa J, Kikuno R, Kohara Y, Siddiqui ZK

[International C. elegans Meeting, 1997]

A number of observations suggest that emb-8 gene plays critical roles in C. elegans development. At non-permissive temperature, emb-8 embryos divides symmetrically; in 50% the first division produces two equal sized cells, and in the second cleavage both of these cells divide symmetrically again to produce four equal sized cells. In some embryos, these four cells fuse togather and produce two asymmetric cells. emb-8 variably mislocalizes P-granules, they are uniformly distributed in 1-cell embryos, are localized on both anterior AB, and posterior P1 cells of the 2-cell embryos, and are found in all four blastomeres of the 4-cell embryos. Crittenden et al., (1996) also observed mislocalization of the P-granules and Glp-1 in emb-8 embryos. emb-8 also appears to control the orientation of early mitotic spindles, similar to the par-3 muatnts (K. Kemphues and others). However complementation tests show that emb-8 and par-3 are different genes. We thank Sarah Crittenden, Judith Kimble and Ken Kemphues for sharing unpublished results.

Dyan Vogel et al. (2006) Development & Evolution Meeting "Cell Cycle Analysis of the Germline Mitotic Region in Adult Males"

Dyan Vogel, Judith Kimble, Sarah Crittenden

[Development & Evolution Meeting, 2006]

Organization of the germ line is similar in both adult hermaphrodites and males. In both sexes, cells divide mitotically at the distal end, in a region dubbed the &quot;mitotic region&quot;, and they progress through meiotic prophase more proximally. In adult hermaphrodites, all germ cells in the mitotic region are actively cycling with a cell cycle time of ~16-24 hours (Crittenden et al., submitted). After a 15-minute pulse of BrdU, only germ cells in the mitotic region were labeled. After a similarly short BrdU pulse followed by consecutively longer chase periods, all labeled cells moved proximally and entered meiosis. Importantly, no label-retaining germ cells were found in the mitotic region. Furthermore, the most distal mitotic germ cells had a lower mitotic index than more proximal mitotic region cells (Maciejowski et al, 2006, Crittenden et al., submitted). We have begun similar experiments in the adult male germline. Results will be presented at the meeting.