Binding specificity and mRNA targets of a C. elegans PUF protein, FBF-1

Sequence-specific RNA-protein interactions underlie regulation of many mRNAs. Here we analyze the RNA sequence specificity of Caenorhabditis elegans FBF-1, a founding member of the PUF protein family. Like other PUF proteins, FBF-1 binds to the 3' UTR of target mRNAs and decreases expression of those target genes. Here, we show that FBF-1 and its close relative, FBF-2, bind with similar affinity to multiple RNA sites. We use mutagenesis and in vivo selection experiments to identify nucleotides that are essential for FBF-1 binding. The binding elements comprise a "core" central region and flanking sequences. The core region is similar but distinct from the binding sites of other PUF proteins. We combine the identification of binding elements with informatics to predict new FBF-1 binding sites in a C. elegans 3' UTR database. These data identify a set of new candidate mRNA targets of FBF-1 and FBF-2.     

LIP-1 phosphatase controls the extent of germline proliferation in Caenorhabditis elegans

Caenorhabditis elegans germline cells are maintained in an undifferentiated and mitotically dividing state by Notch signaling and the FBF (for fem-3 binding factor) RNA-binding protein. Here, we report that the LIP-1 phosphatase, a proposed homolog of mitogen-activated protein (MAP) kinase phosphatases, is required for the normal extent of germline proliferation, and that lip-1 controls germline proliferation by regulating MAP kinase activity. In wild-type germ lines, LIP-1 protein is present in the proximal third of the mitotic region, consistent with its effect on germline proliferation. We provide evidence that lip-1 expression in the germline mitotic region is controlled by a combination of GLP-1/Notch signaling and FBF repression. Unexpectedly, FBF controls the accumulation of lip-1 mRNA, and therefore is likely to control its stability or 3'-end formation. In a sensitized mutant background, LIP-1 can function as a pivotal regulator of the decision between proliferation and differentiation. The control of germline proliferation by LIP-1 has intriguing parallels with the control of stem cells and progenitor cells in vertebrates.     

Reduction of Derlin activity suppresses Notch-dependent tumours in the C. elegans germ line

Regulating the balance between self-renewal (proliferation) and differentiation is key to the long-term functioning of all stem cell pools. In the Caenorhabditis elegans germline, the primary signal controlling this balance is the conserved Notch signaling pathway. Gain-of-function mutations in the GLP-1/Notch receptor cause increased stem cell self-renewal, resulting in a tumour of proliferating germline stem cells. Notch gain-of-function mutations activate the receptor, even in the presence of little or no ligand, and have been associated with many human diseases, including cancers. We demonstrate that reduction in CUP-2 and DER-2 function, which are Derlin family proteins that function in endoplasmic reticulum-associated degradation (ERAD), suppresses the C. elegans germline over-proliferation phenotype associated with glp-1(gain-of-function) mutations. We further demonstrate that their reduction does not suppress other mutations that cause over-proliferation, suggesting that over-proliferation suppression due to loss of Derlin activity is specific to glp-1/Notch (gain-of-function) mutations. Reduction of CUP-2 Derlin activity reduces the expression of a read-out of GLP-1/Notch signaling, suggesting that the suppression of over-proliferation in Derlin loss-of-function mutants is due to a reduction in the activity of the mutated GLP-1/Notch(GF) receptor. Over-proliferation suppression in cup-2 mutants is only seen when the Unfolded Protein Response (UPR) is functioning properly, suggesting that the suppression, and reduction in GLP-1/Notch signaling levels, observed in Derlin mutants may be the result of activation of the UPR. Chemically inducing ER stress also suppress glp-1(gf) over-proliferation but not other mutations that cause over-proliferation. Therefore, ER stress and activation of the UPR may help correct for increased GLP-1/Notch signaling levels, and associated over-proliferation, in the C. elegans germline.     

Dose-dependent control of proliferation and sperm specification by FOG-1/CPEB

RNA-binding proteins control germline development in metazoans. This work focuses on control of the C. elegans germline by two RNA-binding proteins: FOG-1, a CPEB homolog; and FBF, a PUF family member. Previous studies have shown that FOG-1 specifies the sperm fate and that FBF promotes proliferation. Here, we report that FOG-1 also promotes proliferation. Whereas fbf-1 fbf-2 double mutants make approximately 120 germ cells, fog-1; fbf-1 fbf-2 triple mutants make only approximately 10 germ cells. The triple mutant germline divides normally until early L2, when germ cells prematurely enter meiosis and begin oogenesis. Importantly, fog-1/+; fbf-1 fbf-2 animals make more germ cells than fbf-1 fbf-2 double mutants, demonstrating that one dose of wild-type fog-1 promotes proliferation more effectively than two doses - at least in the absence of FBF. FOG-1 protein is barely detectable in proliferating germ cells, but abundant in germ cells destined for spermatogenesis. Based on fog-1 dose effects, together with the gradient of FOG-1 protein abundance, we suggest that low FOG-1 promotes proliferation and high FOG-1 specifies spermatogenesis. FBF binds specifically to regulatory elements in the fog-1 3'UTR, and FOG-1 increases in animals lacking FBF. Therefore, FBF represses fog-1 expression. We suggest that FBF promotes continued proliferation, at least in part, by maintaining FOG-1 at a low level appropriate for proliferation. The dose-dependent control of proliferation and cell fate by FOG-1 has striking parallels with Xenopus CPEB, suggesting a conserved mechanism in animal development.     

EGO-1, a putative RNA-directed RNA polymerase, promotes germline proliferation in parallel with GLP-1/notch signaling and regulates the spatial organization of nuclear pore complexes and germline P granules in Caenorhabditis elegans

Caenorhabditis elegans EGO-1, a putative cellular RNA-directed RNA polymerase, promotes several aspects of germline development, including proliferation, meiosis, and gametogenesis, and ensures a robust response to RNA interference. In C. elegans, GLP-1/Notch signaling from the somatic gonad maintains a population of proliferating germ cells, while entry of germ cells into meiosis is triggered by the GLD-1 and GLD-2 pathways. GLP-1 signaling prevents germ cells from entering meiosis by inhibiting GLD-1 and GLD-2 activity. We originally identified the ego-1 gene on the basis of a genetic interaction with glp-1. Here, we investigate the role of ego-1 in germline proliferation. Our data indicate that EGO-1 does not positively regulate GLP-1 protein levels or GLP-1 signaling activity. Moreover, GLP-1 signaling does not positively regulate EGO-1 activity. EGO-1 does not inhibit expression of GLD-1 protein in the distal germline. Instead, EGO-1 acts in parallel with GLP-1 signaling to influence the proliferation vs. meiosis fate choice. Moreover, EGO-1 and GLD-1 act in parallel to ensure germline health. Finally, the size and distribution of nuclear pore complexes and perinuclear P granules are altered in the absence of EGO-1, effects that disrupt germ cell biology per se and probably limit germline growth.     

Multi-pathway control of the proliferation versus meiotic development decision in the Caenorhabditis elegans germline

An important event in the development of the germline is the initiation of meiotic development. In Caenorhabditis elegans, the conserved GLP-1/Notch signaling pathway regulates the proliferative versus meiotic entry decision, at least in part, by spatially inhibiting genes in the gld-1 and gld-2 parallel pathways, which are proposed to either inhibit proliferation and/or promote meiotic development. Mutations that cause constitutive activation of the GLP-1 pathway, or inactivation of both the gld-1 and gld-2 parallel pathways, result in a tumorous germline in which all cells are thought to be proliferative. Here, to analyze proliferation and meiotic entry in wild-type and mutant tumorous germlines, we use anti-REC-8 and anti-HIM-3 specific antibodies as markers, which under our fixation conditions, stain proliferative and meiotic cells, respectively. Using these makers in wild-type animals, we find that the border of the switch from proliferation to meiotic entry is staggered in late-larval and adult germlines. In wild-type adults, the switch occurs between 19 and 26 cell diameters from the distal end, on average. Our analysis of mutants reveals that tumorous germlines that form when GLP-1 is constitutively active are completely proliferative, while tumors due to inactivation of the gld-1 and gld-2 pathways show evidence of meiotic entry. Genetic and time course studies suggest that a third pathway may exist, parallel to the GLD-1 and GLD-2 pathways, that promotes meiotic development.     

Eukaryotic translation initiation factor 5B activity regulates larval growth rate and germline development in Caenorhabditis elegans

In C. elegans, a population of proliferating germ cells is maintained via GLP-1/Notch signaling; in the absence of GLP-1 signaling, germ cells prematurely enter meiosis and differentiate. We previously identified ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we report that iffb-1 (initiation factor five B) is an ego gene. iffb-1 encodes the sole C. elegans isoform of eukaryotic translation initiation factor 5B, a protein essential for translation. We have used RNA interference and a deletion mutation to determine the developmental consequences of reduced iffb-1 activity. Our data indicate that maternal iffb-1 gene expression is sufficient for embryogenesis, and zygotic iffb-1 expression is required for development beyond late L1/early L2 stage. Partial reduction in iffb-1 expression delays larval development and can severely disrupt proliferation and differentiation of germ cells. We hypothesize that germline development is particularly sensitive to iffb-1 expression level.     

The establishment of Caenorhabditis elegans germline pattern is controlled by overlapping proximal and distal somatic gonad signals

We investigated the control of proliferation and differentiation in the larval Caenorhabditis elegans hermaphrodite germ line through analysis of glp-1 and lag-2 mutants, cell ablations, and ultrastructural data. After the first several rounds of germ cell division, GLP-1, a receptor of the LIN-12/Notch family, governs germline proliferation. We analyzed the proximal proliferation (Pro) phenotype in glp-1(ar202) and found that initial meiosis was delayed and spatially mispositioned. This is due, at least in part, to a heightened response of the mutant GLP-1 receptor to multiple sources of the somatic ligand LAG-2, including the proximal somatic gonad. We investigated whether proximal LAG-2 affects germline proliferation in the wild type. Our results indicate that (1) LAG-2 is necessary for GLP-1-mediated germline proliferation and prevention of early meiosis, and (2) several distinct anatomical sources of LAG-2 in the larval somatic gonad functionally overlap to promote proliferation and prevent early meiosis. Ultrastructural studies suggest that mitosis is not restricted to areas of direct DTC-germ line contact and that the germ line shares a common cytoplasm in larval stages. We propose that downregulation of the GLP-1 signaling pathway in the proximal germ line at the time of meiotic onset is under tight temporal and spatial control.     

Bipartite interaction sites differentially modulate RNA-binding affinity of a protein complex essential for germline stem cell self-renewal

In C. elegans, PUF proteins promote germline stem cell self-renewal. Their functions hinge on partnerships with two proteins that are redundantly required for stem cell maintenance. Here we focus on understanding how the essential partner protein, LST-1, modulates mRNA regulation by the PUF protein, FBF-2. LST-1 contains two nonidentical sites of interaction with FBF-2, LST-1 A and B. Our crystal structures of complexes of FBF-2, LST-1 A, and RNA visualize how FBF-2 associates with LST-1 A versus LST-1 B. One commonality is that FBF-2 contacts the conserved lysine and leucine side chains in the KxxL motifs in LST-1 A and B. A key difference is that FBF-2 forms unique contacts with regions N- and C-terminal to the KxxL motif. Consequently, LST-1 A does not modulate the RNA-binding affinity of FBF-2, whereas LST-1 B decreases RNA-binding affinity of FBF-2. The N-terminal region of LST-1 B, which binds near the 5′ end of RNA elements, is essential to modulate FBF-2 RNA-binding affinity, while the C-terminal residues of LST-1 B contribute strong binding affinity to FBF-2. We conclude that LST-1 has the potential to impact which mRNAs are regulated depending on the precise nature of engagement through its functionally distinct FBF binding sites.     

Caenorhabditis elegans germline patterning requires coordinated development of the somatic gonadal sheath and the germ line

Interactions between the somatic gonad and the germ line influence the amplification, maintenance, and differentiation of germ cells. In Caenorhabditis elegans, the distal tip cell/germline interaction promotes a mitotic fate and/or inhibits meiosis through GLP-1/Notch signaling. However, GLP-1-mediated signaling alone is not sufficient for a wild-type level of germline proliferation. Here, we provide evidence that specific cells of the somatic gonadal sheath lineage influence amplification, differentiation, and the potential for tumorigenesis of the germ line. First, an interaction between the distal-most pair of sheath cells and the proliferation zone of the germ line is required for larval germline amplification. Second, we show that insufficient larval germline amplification retards gonad elongation and thus delays meiotic entry. Third, a more severe delay in meiotic entry, as is exhibited in certain mutant backgrounds, inappropriately juxtaposes undifferentiated germ cells with cells of the proximal sheath lineage, leading to the formation of a proximal germline tumor derived from undifferentiated germ cells. Tumors derived from dedifferentiated germ cells, however, respond to the proximal interaction differently depending on the mutant background. Our study underscores the importance of strict developmental coordination between neighboring tissues. We discuss these results in the context of mechanisms that may underlie tumorigenesis.     

Wnt signaling is a key mediator of Cdx1 expression in vivo

In the mouse, Cdx1 is essential for normal anteroposterior vertebral patterning through regulation of a subset of Hox genes. Retinoic acid (RA) and certain Wnts have also been implicated in vertebral patterning, although the relationship between these signaling pathways and the regulation of mesodermal Hox gene expression is not fully understood. Prior work has shown that Cdx1 is a direct target of both Wnt and retinoid signaling pathways, and might therefore act to relay these signals to the Hox genes. Wnt and RA are believed to impact on Cdx1 through an atypical RA-response element (RARE) and Lef/Tcf-response elements (LRE), respectively, in the proximal promoter. To address the roles of these regulatory motifs and pathways, we derived mice mutated for the LRE or the LRE plus the RARE. In contrast to RARE-null mutants, which exhibit limited vertebral defects, LRE-null and LRE+RARE-null mutants exhibited vertebral malformations affecting the entire cervical region that closely phenocopied the malformations seen in Cdx1-null mutants. Mutation of the LRE also greatly reduced induction of Cdx1 by RA, demonstrating a requirement for Wnt signaling in the regulation of this gene by retinoids. LRE and LRE+RARE mutants also exhibited vertebral fusions, suggesting a defect in somitogenesis. As Wnt signaling is implicated in somitogenesis upstream of the Notch pathway, it is conceivable that Cdx1 might play a role in this process. However, none of the Notch pathway genes assessed was overtly affected.     

Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline

The C. elegans germline provides an excellent model for analyzing the regulation of stem cell activity and the decision to differentiate and undergo meiotic development. The distal end of the adult hermaphrodite germline contains the proliferative zone, which includes a population of mitotically cycling cells and cells in meiotic S phase, followed by entry into meiotic prophase. The proliferative fate is specified by somatic distal tip cell (DTC) niche-germline GLP-1 Notch signaling through repression of the redundant GLD-1 and GLD-2 pathways that promote entry into meiosis. Here, we describe characteristics of the proliferative zone, including cell cycle kinetics and population dynamics, as well as the role of specific cell cycle factors in both cell cycle progression and the decision between the proliferative and meiotic cell fate. Mitotic cell cycle progression occurs rapidly, continuously, with little or no time spent in G1, and with cyclin E (CYE-1) levels and activity high throughout the cell cycle. In addition to driving mitotic cell cycle progression, CYE-1 and CDK-2 also play an important role in proliferative fate specification. Genetic analysis indicates that CYE-1/CDK-2 promotes the proliferative fate downstream or in parallel to the GLD-1 and GLD-2 pathways, and is important under conditions of reduced GLP-1 signaling, possibly corresponding to mitotically cycling proliferative zone cells that are displaced from the DTC niche. Furthermore, we find that GLP-1 signaling regulates a third pathway, in addition to the GLD-1 and GLD-2 pathways and also independent of CYE-1/CDK-2, to promote the proliferative fate/inhibit meiotic entry.     

Caenorhabditis elegans DAZ-1 is expressed in proliferating germ cells and directs proper nuclear organization and cytoplasmic core formation during oogenesis

The deleted in azoospermia (DAZ) family genes encode potential RNA-binding proteins that are expressed exclusively in germ cells in a wide range of metazoans. We have previously shown that mutations in daz-1, the only DAZ family gene in Caenorhabditis elegans, cause pachytene stage arrest of female germ cells but do not affect spermatogenesis. In this study, we report that DAZ-1 protein is most abundantly expressed in proliferating female germ cells, in a manner independent of the GLP-1 signaling pathway. DAZ-1 is dispensable in males but it is expressed also in male mitotic germ cells. Detailed phenotypic analyses with fluorescence microscopy and transmission electron microscopy have revealed that loss of daz-1 function causes multiple abnormalities as early as the onset of meiotic prophase, which include aberrant chromatin structure, small nucleoli, absence of the cytoplasmic core, and precocious cellularization. Although the reduced size of nucleoli is indicative of a low translational activity in these cells, artificial repression of general translation in the germline does not phenocopy the daz-1 mutant. Thus, we propose that DAZ-1 in C. elegans plays essential roles in female premeiotic and early meiotic germ cells, probably via regulating the translational activity of specific target genes required for the progression of oogenesis.     

Caenorhabditis elegans atx-2 promotes germline proliferation and the oocyte fate

In the Caenorhabditis elegans germline, proliferation is induced by Notch-type signaling. Entry of germ cells into meiosis is triggered by activity of the GLD-1 and GLD-2 pathways, which function redundantly to promote meiosis and/or inhibit proliferation. Activation of the germline Notch-type receptor, GLP-1, ultimately inhibits the activities of the GLD-1 and GLD-2 pathways. We previously identified several ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we show that atx-2 is an ego gene. Our data suggest that ATX-2 is not a positive regulator of the GLP-1 signaling pathway and GLP-1 signaling is not the sole positive regulator of ATX-2 activity. Moreover, our data indicate that GLP-1 must have an additional function, which may be to repress activity of a third meiotic entry pathway that would work in parallel with the GLD-1 and GLD-2 pathways. In addition to its role in proliferation, ATX-2 acts downstream of FOG-2 to promote the female germline fate.     

Scratching the niche that controls Caenorhabditis elegans germline stem cells

The Caenorhabditis elegans gonad provides a well-defined model for a stem cell niche and its control of self-renewal and differentiation. The distal tip cell (DTC) forms a mesenchymal niche that controls germline stem cells (GSCs), both to generate the germline tissue during development and to maintain it during adulthood. The DTC uses GLP-1/Notch signaling to regulate GSCs; germ cells respond to Notch signaling with a network of RNA regulators to control the decision between self-renewal and entry into the meiotic cell cycle.