PUF-8, a Pumilio Homolog, Inhibits the Proliferative Fate in the Caenorhabditis elegans Germline

Stem cell populations are maintained by keeping a balance between self-renewal (proliferation) and differentiation of dividing stem cells. Within the Caenorhabditis elegans germline, the key regulator maintaining this balance is the canonical Notch signaling pathway, with GLP-1/Notch activity promoting the proliferative fate. We identified the Pumilio homolog, PUF-8, as an inhibitor of the proliferative fate of stem cells in the C. elegans germline. puf-8(0) strongly enhances overproliferation of glp-1(gf) mutants and partially suppresses underproliferation of a weak glp-1(lf) mutant. The germline tumor that is formed in a puf-8(0); glp-1(gf) double mutant is due to a failure of germ cells to enter meiotic prophase. puf-8 likely inhibits the proliferative fate through negatively regulating GLP-1/Notch signaling or by functioning parallel to it.


puf-8
Pumilio PUF germline stem cells proliferative fate Notch signaling Successful reproduction in many organisms hinges on their ability to produce gametes throughout much of their adult lives. Prolonged gamete production relies on the maintenance of a population of undifferentiated germline stem cells (GSCs). A balance between proliferation (self-renewal) and differentiation (meiotic entry) allows GSCs to be maintained while still producing the required gametes. The C. elegans germline has emerged as a powerful model to study the control of this balance. Located at the distal end of each hermaphrodite gonad arm is a population of~200 to 250 mitotically dividing cells, with approximately 30% to 40% of these cells being in premeiotic Sphase (Fox et al. 2011;Killian and Hubbard 2005;Lamont et al. 2004). The proliferative fate of the stem cells is maintained in the distal gonad primarily through the activity of the conserved GLP-1/Notch signaling pathway (Figure 1) Schedl 2006, 2012;Kimble and Crittenden 2007). GLP-1/Notch signaling likely activates the transcription of genes required to promote the proliferative fate, which genes are yet to be fully described. GLP-1/Notch signaling levels are thought to decrease as cells move proximally, allowing the activities of at least two downstream genetic pathways to increase (Eckmann et al. 2004;Hansen et al. 2004b;Kadyk and Kimble 1998). These two path-ways, the gld-1 and gld-2 pathways, promote entry into meiosis and/or inhibit the proliferative fate (Figure 1), and function redundantly; when one pathway is inactivated, the other pathway is sufficient to promote entry into meiosis and/or inhibit the proliferative fate. However, if both pathways are inactivated, little or no entry into meiosis occurs, and a germline tumor of proliferative germ cells is formed (Eckmann et al. 2004;Hansen et al. 2004b;Kadyk and Kimble 1998). Inhibition of the gld-1 and gld-2 pathways in the distal end of the gonad is accomplished, at least in part, through the activities of fbf-1 and fbf-2, which encode homologs of Drosophila Pumilio. FBF inhibits these pathways, at least in part, through binding to the 39UTRs of gld-1 and gld-3, preventing their translation (Eckmann et al. 2004;Suh et al. 2009) Pumilio homologs, or PUF proteins (Pumilio and FBF), are a family of conserved proteins that bind RNA and affect the translation, stability, and/or localization of target mRNAs (Quenault et al. 2011;Wickens et al. 2002). C. elegans contains multiple PUF proteins, including FBF-1 and FBF-2, which participate in various aspects of germline development and maintenance. For example, in addition to their role in promoting the proliferative fate of GSCs, FBF-1 and FBF-2 promote the switch from spermatogenesis to oogenesis in germline sex determination by inhibiting the activity of fem-3 (Zhang et al. 1997). PUF-3/11 control the growth of oocytes, whereas another group of PUF proteins, PUF-5/6/7, control the formation of oocytes (Hubstenberger et al. 2012;Lublin and Evans 2007). PUF-8 has multiple functions in germline development, including inhibiting dedifferentiation of primary spermatocytes, functioning redundantly with FBF-1 to promote the sperm/oocyte switch, and functioning redundantly with the KH-domain containing protein MEX-3 to promote mitosis in the GSCs (Ariz et al. 2009;Bachorik and Kimble 2005;Mainpal et al. 2011;Subramaniam and Seydoux 2003). Here, we demonstrate that PUF-8 also functions within the C. elegans germline to inhibit the proliferative fate of GSCs.

RESULTS
teg-2(oz192) enhances glp-1(gf) mutations and suppresses a glp-1(lf) mutation To identify additional regulators functioning in the proliferative fate vs. meiotic entry decision, a mutant screen was conducted for mutations that enhance the overproliferative phenotype of the weak glp-1 gain-of-function allele, glp-1(oz112oz120gf), resulting in germline tumor formation (Wang et al. 2012). We have previously shown that two genes identified in this screen, teg-1 and teg-4 (tumorous enhancer of glp-1(oz112oz120)), encode likely splicing factors (Mantina et al. 2009;Wang et al. 2012). Here, we describe the characterization and cloning of another gene identified in this screen, teg-2. We demonstrate that teg-2 is allelic to puf-8, which encodes an RNA binding Pumilio homolog (Wickens et al. 2002); therefore, we will refer to teg-2 as puf-8 throughout this paper.
Although puf-8 single mutants show no germline overproliferation due to a defect in the proliferative fate vs. meiotic entry decision (Bachorik and Kimble 2005;Subramaniam and Seydoux 2003), puf-8(0) is a strong enhancer of glp-1(gf) in both males and hermaphrodites ( Figure 2; Table S1). puf-8(0); glp-1(gf) animals which is expressed on the somatic DTC at the distal end of the gonad, interacts with the GLP-1/Notch receptor that is expressed on the germ cells. This is thought to cause the intracellular portion of GLP-1, ICD, to translocate to the nucleus and interact with the LAG-1 transcription factor, turning on genes necessary for proliferation. GLP-1/Notch signaling inhibits the activities of two redundant pathways (GLD-1 and GLD-2 pathways), through the activity of two PUF proteins, FBF-1 and FBF-2. The GLD-1 and GLD-2 pathways promote meiotic entry and/or inhibit the proliferative fate. (B) Gene model of puf-8 illustrating the positions of the exons (boxes) and introns (connecting lines), as well as the 59 and 39 UTRs (black boxes) and the region encoding the eight PUF domain repeats (gray box). Also shown is the location of the puf-8 (oz192) lesion, which results in a premature stop codon in the second PUF repeat, and the deletion associated with the puf-8(q725) allele.
have completely tumorous germlines, with no evidence of any cells entering into meiotic prophase, even at the 15°permissive temperature. This enhancement is not specific to the glp-1 allele because puf-8(0) enhances multiple glp-1(gf) alleles (Table 1). In addition, puf-8(0) enhances when only one copy of glp-1(gf) is present (Table  1), further suggesting that puf-8(0) is a strong enhancer of glp-1(gf). Furthermore, loss of puf-8 activity partially suppresses the premature meiotic entry phenotype of the glp-1(bn18) partial loss-offunction allele ( Table 2). The interaction between puf-8(0) and both loss and gain-of-function alleles of glp-1 suggests that puf-8 functions as an inhibitor of the proliferative fate and/or promoter of the meiotic fate.
teg-2 and puf-8 are allelic We used SNP mapping to narrow the critical region containing teg-2 to a 98-kb region on chromosome II containing 18 genes. Sequencing of one of these genes, puf-8, revealed a G to T transversion at position 937 from the initiator ATG. This mutation is predicted to cause a premature amber stop codon in the second of the eight PUF repeats; therefore, the mutation likely results in a strong loss-of-function or null allele. To determine whether teg-2 and puf-8 are allelic, we performed a complementation test using the puf-8(q725) deletion allele (Bachorik and Kimble 2005) and found that puf-8(q725)/teg-2(oz192) rol-6(e187); glp-1(ar202gf) animals have a tumorous germline at 15°( n . 20). Therefore, teg-2 is allelic with puf-8, which encodes a homolog of Drosophila Pumilio ( Figure S1).
dissected gonads were all anti-REC-8(+) and anti-HIM-3(2) at both the late L2 and late L3 stages, n = 18 for each). Therefore, cells are unlikely to have entered into meiosis early in development and then dedifferentiated, resulting in a tumor in adult animals. We conclude that the tumor observed in puf-8(0); glp-1(gf) animals is not due to dedifferentiation of male germ cells, but rather due to a disruption of the proliferative fate vs. meiotic entry balance.
puf-8 functions upstream of, or parallel to, the glp-1/Notch signaling pathway To better understand how puf-8 may be regulating the proliferative fate vs. differentiation decision, we sought to determine where puf-8 may function in the genetic pathway regulating this decision by performing genetic epistasis experiments. Because puf-8 appears to inhibit the proliferative fate and/or promote meiotic entry, it is possible that puf-8 functions in either the gld-1 or gld-2 pathways, which also function to inhibit the proliferative fate and/or promote meiotic entry (Figure 1). Because the gld-1 and gld-2 pathways function redundantly, if puf-8 were to function in either of these pathways, puf-8 (0) would likely form a synthetic tumor when the function of the other pathway, not containing puf-8, was eliminated. We found that animals lacking puf-8 activity do not form synthetic tumors when the activities of any of the genes in the gld-1 or gld-2 pathways are also eliminated ( Table 3), suggesting that puf-8 likely does not function in either of these two pathways. It is possible that puf-8 may have a minor role in either the gld-1 or gld-2 pathways and that reducing its function does not reduce pathway function enough to form a synthetic tumor when the activity of the other pathway is also reduced. However, given that puf-8(0) appears to be a stronger enhancer of glp-1(gf) than any of the known components of the gld-1 and gld-2 pathways, we consider this unlikely. Therefore, puf-8 may function as a negative regulator of GLP-1/Notch signaling, or parallel to this signaling pathway.
To determine whether puf-8 is a general negative regulator of Notch signaling, we tested for an interaction with the Notch signaling pathway in other Notch-regulated cell fate decisions, including the anchor cell vs. ventral uterine precursor cell decision, and the redundant L1 arrest phenotype of lin-12(q269) glp-1(231) animals [LIN-12 is the other Notch receptor in C. elegans and functions redundantly with GLP-1 in certain developmental processes (Austin and Kimble 1989;Yochem and Greenwald 1989)]. We found no interaction between puf-8 and the Notch signaling pathway in either of these contexts (Table S2), suggesting that puf-8 is not a general negative regulator of Notch signaling but rather may function specifically in the germline to negatively regulate GLP-1/Notch signaling or function in parallel to the GLP-1/Notch signaling pathway to inhibit the proliferative fate.
PUF-8 is expressed throughout the entire proliferative zone and in early meiotic cells The PUF-8 protein was previously shown to be enriched in the distal end of the gonad (Ariz et al. 2009); however, it was unclear as to its spatial distribution pattern relative to where cells show signs of entering meiotic prophase. To determine whether its spatial distribution in the distal end could help explain how PUF-8 may be functioning in regulating the proliferative fate vs. meiotic entry decision, we measured the accumulation of PUF-8::GFP relative to the progression of cells from the proliferative fate to meiotic entry. We constructed an integrated single copy puf-8::gfp::tap transgenic line (ugSi1), which largely rescues multiple puf-8(0) phenotypes, and found that PUF-8::GFP is expressed throughout the distal end of the gonad, with slightly lower levels within the first third to half of the proliferative zone ( Figure 3). Protein levels gradually decrease proximally until eventually plateauing at~40 cell diameters, well beyond where all cells n 100 0 0 10 glp-1(oz264) f 100 0 0 9 puf-8(oz192); glp-1(ar202) 0 100 0 35 puf-8(q725); glp-1(ar202) 0 100 0 35 puf-8(oz192); glp-1(ar202)/+ g 0 7 4 2 6 2 7 puf-8(oz192); glp-1(oz264) 0 100 0 23 puf-8(q725); glp-1(oz264) 0 9 8 2 5 4 a All animals maintained at 15°. For each genotype, gonads were dissected and stained with DAPI, anti-REC-8 antibodies, and anti-HIM-3 antibodies. All animals were dissected at 1 d after the fourth larval stage. b A gonad arm was scored as wild-type if no overproliferation was observed. c A gonad arm with a complete tumor contained no anti-HIM-3(+) cells or fully differentiated cells. Rather, all cells were anti-REC-8(+). d A gonad arm with an incomplete tumor contained extensive over-proliferation throughout the gonad arm; however, small pockets of anti-HIM-3(+) cells, or sperm, were observed. e Number of dissected gonad arms analyzed. f The GLP-1(oz264) protein contains a G to E amino acid substitution at amino acid 528 of the GLP-1 protein (Kerins et al. 2010). g Actual genotype puf-8(oz192) unc-4(e120)/puf-8(oz192); glp-1(ar202)/+. n a Homozygous strains were used and maintained at 15°(permissive temperature) then shifted to the test restrictive temperature as L4 animals and allowed to self-fertilize. Progeny were scored 3 d later by Nomarski optics, after having grown to adulthood at the restrictive temperature. b Germline proliferation defective (Glp); animals lacked a distal proliferative zone resulting in only sperm being present in the gonad arm. c Number of gonad arms analyzed.
normally have entered into meiotic prophase (Crittenden et al. 1994;Hansen et al. 2004a). Within the proliferative zone,~30% to 40% of germ cells are in premeiotic S-phase (Fox et al. 2011). Therefore, assuming that these~30% to 40% premeiotic cells are located in the more proximal end of the proliferative zone, PUF-8 is at its maximum expression level in the approximate region of the proliferative zone where cells are likely entering pre-meiotic S-phase, consistent with a role in facilitating the switch from proliferation to meiosis.

DISCUSSION
A defining feature of stem cells is their ability to produce both selfrenewing and differentiating daughter cells. For GSCs, a disruption in the balance between self-renewal (proliferation) and differentiation (meiotic entry) can have detrimental effects on the reproductive fitness of the animal. Therefore, animals have evolved robust systems to regulate the balance between the proliferative fate and meiotic entry, including redundant pathways and modulating factors. Although the GLP-1/Notch signaling pathway is the major regulator of this balance in the C. elegans germline, other factors have been identified that modulate this pathway, work parallel to it, or are regulated by it (Hansen and Schedl 2013;Kimble and Crittenden 2007). We have identified the PUF protein, PUF-8, as an additional player in regulating this balance. PUF-8 inhibits the proliferative fate in the C. elegans germline, either through inhibition of the GLP-1/Notch signaling pathway, or functioning parallel to it (Figure 4). PUF proteins and the maintenance of stem cells Maintenance of stem cells by PUF proteins has been found in a number of other systems. In Drosophila, Pumilio is expressed in GSCs, and its absence causes female GSCs to prematurely differentiate as cystoblasts (Forbes and Lehmann 1998;Lin and Spradling 1997). In mouse hematopoietic stem cells, both Pum1 and Pum2 are highly transcribed (Spassov and Jurecic 2003). Mouse Pum2 is also highly expressed in male germ cells and, when mutated, pum2(0) animals have significantly smaller testes (Xu et al. 2007). In addition, in the C. elegans germline, the two closely related PUF proteins, FBF-1 and FBF-2, function downstream of GLP-1/Notch signaling to promote the proliferative fate in adult animals (Figure 1) (Crittenden et al. 2002). Therefore, Pumilio proteins appear to have conserved functions in regulating stem cell behavior. However, most PUF proteins that have been implicated in regulating GSC behavior appear to promote the proliferative fate. Here, we have demonstrated that PUF-8 also functions in the opposite direction, to inhibit the proliferative fate. We have demonstrated that puf-8 is unlikely to function in either the gld-12 and gld-22redundant pathways that function downstream of glp-1 signaling. Therefore, puf-8 is likely to function upstream of these pathways, possibly as a negative regulator of GLP-1/Notch signaling, or in a pathway that functions parallel to GLP-1/Notch signaling (Figure 4, B and C). None of the core components of glp-1 signaling contain a canonical PUF-8 binding site in their 39 UTRs (Stumpf et al. 2008), and none of these components were identified in a immunoprecipitation of potential PUF-8 mRNA targets (Mainpal et al. 2011); therefore, if puf-8 does inhibit glp-1 signaling, it likely does so indirectly, possibly by inhibiting the activity of a positive regulator.
Whether PUF-8 functions to inhibit the activity of the GLP-1/ Notch signaling pathway or functions in a parallel pathway, its expression throughout the proliferative zone, and well past the region where all cells have entered into meiotic prophase (Figure 3), suggests a model in which PUF-8 dampens the overall signal promoting the proliferative fate. In this model, in the very distal end of the gonad where the germ cells are close to the distal tip cell (DTC) and GLP-1/ Notch signaling levels are thought to be at their peak, this high level of signaling is able to overcome any inhibition of the proliferative fate provided by PUF-8. However, as cells move more proximally, the DTC is less able to promote high GLP-1/Notch signaling levels, and PUF-8 continues to inhibit the proliferative fate, either through contributing to the decrease in GLP-1/Notch signaling levels ( Figure 4B) or by inhibiting the proliferative fate independent of GLP-1/Notch signaling ( Figure 4C).
Although these models take into account the expression of PUF-8 throughout much of the distal end of the gonad, they also assume that PUF-8 is active in all areas where it is expressed. It remains possible that PUF-8 activity is controlled through a post-translational modification, or through the binding of a spatially regulated cofactor, which limits PUF-8's inhibition of the proliferative fate to cells in the region of the distal gonad where they enter meiotic prophase. Indeed, PUF proteins in other systems work cooperatively with other proteins to regulate target gene activity. For example, the Drosophila protein Nanos works with Pumilio to repress the translation of hunchback mRNA during embryonic patterning (Barker et al. 1992;Tautz 1988). In this system, Pumilio is expressed throughout the embryo (Macdonald 1992), but is only active in the posterior end where Nanos protein is localized (Murata and Wharton 1995). Therefore, it is possible that PUF-8 is not active throughout the entire mitotic zone but rather is limited to certain regions due to the activity of another protein. The nos-3 gene, which encodes a protein with similarity to Drosophila Nanos, n Table 3 puf-8 is unlikely to function in the gld-1 or gld-2 pathways
has previously been shown to inhibit the proliferative fate and/or promote meiotic entry (Hansen et al. 2004b); however, nos-3 appears to function in the gld-1 pathway, whereas puf-8 does not (Table 3). Therefore, if PUF-8 functions with another protein that spatially regulates its activity, this other protein likely is not NOS-3.
PUF-8 inhibits the proliferative fate at two stages in germ cell progression A portion of puf-8(0) single mutants were previously shown to form a germline tumor in the proximal end of their gonads (Subramaniam and Seydoux 2003). In animals that form a proximal tumor, GSCs enter the path to differentiation and enter into meiosis normally; however, some cells destined for the male fate fail to properly progress through meiotic prophase but rather dedifferentiate and reenter the mitotic cell cycle. Therefore, PUF-8 actively limits germ cell proliferation in the proximal gonad by preventing dedifferentiation of male germ cells undergoing spermatogenesis. We have demonstrated that puf-8 also inhibits the proliferative fate in GSCs, prior to them entering into meiotic prophase. This inhibition is distinct from the previously described dedifferentiation inhibition because it inhibits the proliferative fate of cells that have not yet entered into meiotic prophase, and is irrespective of the eventual sex of the cells. Therefore, puf-8 inhibits the proliferative fate in two distinct stages of germ cell development ( Figure 4A).
It is not unique for a single factor to inhibit the proliferative fate in more than one step in the formation of a gamete. For example, gld-1, which encodes a KH-domain containing translational inhibitor (Jones and Schedl 1995), also inhibits the proliferative fate in C. elegans in two stages of germ cell development. Like puf-8, gld-1 inhibits proliferation and/or promotes meiotic entry of GSCs (Francis et al. 1995b). In addition, like puf-8, gld-1 inhibits dedifferentiation of cells that have entered meiotic prophase; however, while puf-8 inhibits dedifferentiation of male germ cells (Subramaniam and Seydoux 2003), gld-1 inhibits dedifferentiation of female germ cells (Francis et al. 1995a). Some of the cells within the gld-1 single mutant tumor are positive for somatic cell markers, suggesting that they are differentiating, similar to cells in a mammalian teratoma (Biedermann et al. 2009;Ciosk et al. 2006). It is intriguing that two factors, which are both likely to regulate gene targets through mRNA translation and/or stability, inhibit the proliferative fate in, at least, two stages of germ cell development.
PUF-8 has other germline functions besides inhibition of the proliferative fate ( Figure 4A), including promoting stem cell maintenance (Ariz et al. 2009;Bachorik and Kimble 2005;Mainpal et al. 2011) and inhibiting the male sexual fate in hermaphrodites (Bachorik and Kimble 2005). The PUF-8 homolog in the closely related nematode, C. briggsae, also functions in germline sex determination, although to  (Ariz et al. 2009;Bachorik and Kimble 2005), inhibition of the proliferative fate in GSCs (this work), inhibition of the male sexual fate (Bachorik and Kimble 2005), and inhibition of dedifferentiation of male germ cells progressing through meiosis (Subramaniam and Seydoux 2003). (B and C) Models of PUF-8 function in regulating the balance between the proliferative fate and meiotic entry. Illustration of distal end of gonad showing the DTC (yellow), mitotic cells (green) and meiotic cells (red). (B) PUF-8 may inhibit the activity of GLP-1/Notch signaling. GLP-1/ Notch signaling levels are thought to be high at the very distal end, and lower as cells progress proximally. In this model, PUF-8 contributes to the lowering of GLP-1/Notch signaling levels, allowing cells to enter meiotic prophase. The inhibition of GLP-1/Notch signaling may not be direct, but rather PUF-8 may inhibit the activity of a positive regulator of GLP-1/Notch signaling. (C) PUF-8 may inhibit the proliferative fate in a pathway that is parallel to the GLP-1/Notch signaling pathway. In both models (B and C) PUF-8 is shown to be active throughout the distal end of the gonad; however, it is possible that another factor or post-translational modification causes PUF-8 not to be active in some regions where it is expressed. promote sperm production (Beadell et al. 2011). C. elegans PUF-8 has also been shown to have a somatic role in vulval development (Walser et al. 2006). Therefore, PUF-8 likely has many mRNA targets, many of which will be specific to only one of PUF-8's functions. A clear future direction to understanding how PUF-8 functions in regulating the proliferative fate vs. meiotic entry decision will be to identify the mRNA targets that are specifically involved in this decision.