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 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 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.

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.

Hunter CP et al. (1991) International C. elegans Meeting "NON-AUTONOMY OF HER-1 FUNCTION IMPLICATES CELL INTERACTIONS IN C. ELEGANS SEX DETERMINATION."

Hunter CP, Wood WB

[International C. elegans Meeting, 1991]

Activity of the her-l gene is required for normal male development in C. elegans. Loss-of-function her-l mutations cause feminization of XO animals (normally male) to produce fertile hermaphrodites, while gain-of-function her-l mutations cause masculinization of XX animals ( normally hermaphrodite) to produce pseudo-males. Genetic analysis indicated that her-l functions early in the hierarchy of regulatory interactions among the major sex determination genes (1). We have analyzed her-l genetic mosaics to determine which cells must express her-l for wild-type male development. If her-l functions cell- autonomously, then in XO animals her-l (+) cells will follow male fates, and her-1(-) cells will follow hermaphrodite fates. If her-l or a her-l regulated activity functions non-autonomously, then the her-l genotype of a cell may not determine its sexual phenotype. The phenotypes of XO her-l genetic mosaics are variable, but clearly show that both her-1(-) and her-l (+) cells can follow either hermaphrodite or male fates. We conclude that her-l is neither necessary nor sufficient to cell-autonomously determine male sexual phenotypes and, therefore, that her-l or a her-l regulated activity must be able to function non-autonomously. The observed patterns of non-autonomous effects suggest that many or all cells express and respond to her-l activity. These findings implicate cell interactions in C. elegans sex determination. Earlier genetic analysis indicated that her-l might directly control tra-2 activity (1). Molecular characterization of the gene products of her-l and tra-2 (see abstracts by M. Perry et al. and P. Kuwabara et al) is consistent with a model in which a secreted ligand encoded by her-l interacts with a cell-surface receptor encoded by tra-2. We will discuss the possible role of such an interaction in coordinating the sex determination decision.

Ellis RE et al. (1991) International C. elegans Meeting "PROGRESS IN CLONING fog- 1"

Kimble JE, Ellis RE

[International C. elegans Meeting, 1991]

The fog-l gene plays an important role in the decision of germ cells to differentiate as either spermatocytes or oocytes (Doniach 1986, Barton and Kimble 1990). In fog-l mutants, all germ cells develop into oocytes. This is true for both males and hermaphrodites. However, these fog-l mutations do not appear to affect other aspects of sexual development. Furthermore, the fog-l mutant phenotype is not suppressed by mutations in any of the other genes known to control sex- determination. Thus fog-l might function to initiate spermatogenesis in response to other genes that regulate general sexdetermination. There are 42 fog-l mutations. These mutations arise at a frequency typical for knockout mutations in other C. elegans genes, and four of these fog-l alleles were isolated from a screen capable of recovering deletions. Thus it is likely that these mutations cause a loss of fog- l function. In order to study the mechanisms by which fog-l functions, and to determine how fog-l expression is regulated by the genes that control sex-determination, we are now cloning this gene. The fog-l gene is located on the left arm of LGI, between sup-ll and unc-ll, a region spanned by a large contig of about one megabase in size. By mapping DNA polymorphisms with respect to fog-l, we have narrowed down the region in which fog-l must be located, and are now injecting YAC DNA that covers this region in an attempt to rescue fog-l mutants.

Byrd, Dana T. et al. (2013) International Worm Meeting "Distal tip cell processes provide extensive contact between the germline stem cell pool and its cellular niche."

Byrd, Dana T., Schmitt, Katharyn, Kimble, Judith, Knobel, Karla, Crittenden, Sarah L.

[International Worm Meeting, 2013]

The germline stem cell niche in C. elegans includes the distal tip cell (DTC). The DTC has a complex cellular architecture; there is a cap that covers germ cells with a sheet of membrane, and long external processes that extend along the outside of the germline syncytium, contacting a subset of germ cells. The role of this architecture in germline stem cell biology, stem cell maintenance and the pattern of mitosis and differentiation in the germ line remains an open question. We have identified an additional region of DTC to germline contact: a plexus of membranous processes that surround and intercalate between adjacent germ cells in the distal germ line. We find that the end of the plexus region corresponds with the start of expression of a meiosis-promoting protein marker, GLD-1::GFP. Thus, germ cells within the cap and plexus regions have a high level of niche contact and low levels of a meiosis-promoting factor. One model is that increased niche contact leads to increased Notch signaling in this region and is important for maintaining the "stem cell-like" state of germ cells in the distal germ line. In addition, we find that formation and maintenance of the DTC processes depends on continued germ cell divisions. Thus signals from mitotic germ cells appear to influence niche architecture. This work lays a foundation for studying the complex cellular architecture of a niche, its role in regulating the self-renewal versus differentiation decision of stem cells and two-way communication between niche and stem cells.

Lackner MR et al. (1995) International C. elegans Meeting "The role of mpk-l in vulval development"

Kim SK, Lackner MR

[International C. elegans Meeting, 1995]

The mpk-l gene encodes a MAP kinase protein that is approximately 75% identical to vertebrate MAP kinases. Previously, we have shown that mpk-l plays an important role in ras-mediated induction of vulval cell fates, as reduction-offunction mutations in mpk-l completely suppress excess vulval differentiation caused by a gain-of-function mutation in let-60 ras. We have now characterized a strong reduction-offunction allele of mpk-l that results in temperature-sensitive vulvaless phenotype (84% Vul at 25 degrees Celsius) and a completely penetrant sterile phenotype at all temperatures. Epistasis analyses suggest that mpk-l acts downstream of lin-15 and let-60 ras, but upstream of the putative transcription factors lin-l and lin-31. To determine whether mpk-l is absolutely essential for vulval induction, or whether some induction may occur in the absence of mpk-l activity, we are in the process of isolating a null allele of mpk-l. We used mutant activated forms of mpk-l and Drosophila MEK to address the question of whether activation of these two kinases is sufficient to promote vulval fates in the absence of upstream signaling. Coexpression of both activated mpk-l and activated D-MEK causes a strong Muv phenotype (~90% Muv), whereas coexpression of wild-type mpk-l and wild-type D-MEK results in a weak Muv phenotype (~10% Muv). The strong Muv phenotype is unaffected by ablation of the anchor cell or a reduction-offunction mutation in lin-45 raf, consistent with activation of mpk-l and MEK being at least partially independent of upstream signaling. Our future plans are to identify targets of mpk-l by isolating suppressors of this strong Muv phenotype.