GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult C. elegans Germ Line

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.     

Protein kinase CK2 both promotes robust proliferation and inhibits the proliferative fate in the C. elegans germ line

Stem cells are capable of both self-renewal (proliferation) and differentiation. Determining the regulatory mechanisms controlling the balance between stem cell proliferation and differentiation is not only an important biological question, but also holds the key for using stem cells as therapeutic agents. The Caenorhabditis elegans germ line has emerged as a valuable model to study the molecular mechanisms controlling stem cell behavior. In this study, we describe a large-scale RNAi screen that identified kin-10, which encodes the β subunit of protein kinase CK2, as a novel factor regulating stem cell proliferation in the C. elegans germ line. While a loss of kin-10 in an otherwise wild-type background results in a decrease in the number of proliferative cells, loss of kin-10 in sensitized genetic backgrounds results in a germline tumor. Therefore, kin-10 is not only necessary for robust proliferation, it also inhibits the proliferative fate. We found that kin-10’s regulatory role in inhibiting the proliferative fate is carried out through the CK2 holoenzyme, rather than through a holoenzyme-independent function, and that it functions downstream of GLP-1/Notch signaling. We propose that a loss of kin-10 leads to a defect in CK2 phosphorylation of its downstream targets, resulting in abnormal activity of target protein(s) that are involved in the proliferative fate vs. differentiation decision. This eventually causes a shift towards the proliferative fate in the stem cell fate decision.     

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.     

A role for Caenorhabditis elegans chromatin-associated protein HIM-17 in the proliferation vs. meiotic entry decision

Chromatin-associated protein HIM-17 was previously shown to function in the chromosomal events of meiotic prophase. Here we report an additional role for HIM-17 in regulating the balance between germ cell proliferation and meiotic development. A cryptic function for HIM-17 in promoting meiotic entry and/or inhibiting proliferation was revealed by defects in germline organization in him-17 mutants grown at high temperature (25°) and by a synthetic tumorous germline phenotype in glp-1(ar202); him-17 mutants at 15°.     

JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage

Adult stem cells are the most primitive cells of a lineage and are distinguished by the properties of self-renewal and multipotency. Coordinated control of stem cell proliferation and multilineage differentiation is essential to ensure a steady output of differentiated daughter cells necessary to maintain tissue homeostasis. However, little is known about the signals that coordinate stem cell proliferation and daughter cell differentiation. Here we investigate the role of the conserved JAK/STAT signaling pathway in the Drosophila intestinal stem cell (ISC) lineage. We show first, that JAK/STAT signaling is normally active in both ISCs and their newly formed daughters, but not in terminally differentiated enteroendocrine (ee) cells or enterocyte (EC) cells. Second, analysis of ISC lineages shows that JAK/STAT signaling is necessary but not sufficient for daughter cell differentiation, indicating that competence to undergo multilineage differentiation depends upon JAK/STAT. Finally, our analysis reveals JAK/STAT signaling to be a potent regulator of ISC proliferation, but not ISC self-renewal. On the basis of these findings, we suggest a model in which JAK/STAT signaling coordinates the processes of stem cell proliferation with the competence of daughter cells to undergo multilineage differentiation, ensuring a robust cellular output in the lineage.     

Characterization of a germ-line proliferation mutation in C. elegans.

The C. elegans germ line is generated by extensive proliferation of the two germ-line progenitor cells present in newly hatched larvae. We describe genetic and phenotypic characterization of glp-4, a locus whose product is required for normal proliferation of the germ line. glp-4(bn2ts) mutant worms raised at the restrictive temperature contain approximately 12 germ nuclei, in contrast to the 700-1000 present in wild-type adults. The few germ cells present in sterile glp-4 adults appear to be arrested at prophase of the mitotic cell cycle. This cell-cycle disruption prevents the germ cells from entering meiosis and differentiating into gametes. Shifting sterile glp-4 worms to the permissive temperature enables their germ cells to undergo extensive proliferation and form gametes, demonstrating that the bn2-induced cell-cycle arrest is reversible and that proliferation and differentiation of germ cells can be uncoupled from development of the somatic gonad. The glp-4(bn2ts) mutation can be used to generate large populations of worms that are severely depleted in germ cells, facilitating determination of whether any gene of interest is expressed in the germ line or soma or both.     

Enhancers of Glp-1, a Gene Required for Cell-Signaling in Caenorhabditis Elegans, Define a Set of Genes Required for Germline Development

The distal tip cell (DTC) regulates the proliferation or differentiation choice in the Caenorhabditis elegans germline by an inductive mechanism. Cell signaling requires a putative receptor in the germline, encoded by the glp-1 gene, and a putative signal from the DTC, encoded by the lag-2 gene. Both glp-1 and lag-2 belong to multigene gene families whose members are essential for cell signaling during development of various tissues in insects and vertebrates as well as C. elegans. Relatively little is known about how these pathways regulate cell fate choice. To identify additional genes involved in the glp-1 signaling pathway, we carried out screens for genetic enhancers of glp-1. We recovered mutations in five new genes, named ego (enhancer of glp-1), and two previously identified genes, lag-1 and glp-4, that strongly enhance a weak glp-1 loss-of-function phenotype in the germline. Ego mutations cause multiple phenotypes consistent with the idea that gene activity is required for more than one aspect of germline and, in some cases, somatic development. Based on genetic experiments, glp-1 appears to act upstream of ego-1 and ego-3. We discuss the possible functional relationships among these genes in light of their phenotypes and interactions with glp-1.     

POS-1 and GLD-1 repress glp-1 translation through a conserved binding-site cluster

RNA-binding proteins (RBPs) coordinate cell fate specification and differentiation in a variety of systems. RNA regulation is critical during oocyte development and early embryogenesis, in which RBPs control expression from maternal mRNAs encoding key cell fate determinants. The Caenorhabditis elegans Notch homologue glp-1 coordinates germline progenitor cell proliferation and anterior fate specification in embryos. A network of sequence-specific RBPs is required to pattern GLP-1 translation. Here, we map the cis-regulatory elements that guide glp-1 regulation by the CCCH-type tandem zinc finger protein POS-1 and the STAR-domain protein GLD-1. Our results demonstrate that both proteins recognize the glp-1 3′ untranslated region (UTR) through adjacent, overlapping binding sites and that POS-1 binding excludes GLD-1 binding. Both factors are required to repress glp-1 translation in the embryo, suggesting that they function in parallel regulatory pathways. It is intriguing that two equivalent POS-1–binding sites are present in the glp-1 3′ UTR, but only one, which overlaps with a translational derepression element, is functional in vivo. We propose that POS-1 regulates glp-1 mRNA translation by blocking access of other RBPs to a key regulatory sequence.     

Sex-lethal Facilitates the Transition From Germline Stem Cell to Committed Daughter Cell in the Drosophila Ovary

In Drosophila, the female-specific SEX-LETHAL (SXL) protein is required for oogenesis, but how Sxl interfaces with the genetic circuitry controlling oogenesis remains unknown. Here we use an allele of sans fille (snf) that specifically eliminates SXL protein in germ cells to carry out a detailed genetic and cell biological analysis of the resulting ovarian tumor phenotype. We find that tumor growth requires both Cyclin B and zero population growth, demonstrating that these mutant cells retain at least some of the essential growth-control mechanisms used by wild-type germ cells. Using a series of molecular markers, we establish that while the tumor often contains at least one apparently bona fide germline stem cell, the majority of cells exhibit an intermediate fate between a stem cell and its daughter cell fated to differentiate. In addition, snf tumors misexpress a select group of testis-enriched markers, which, remarkably, are also misexpressed in ovarian tumors that arise from the loss of bag of marbles (bam). Results of genetic epistasis experiments further reveal that bam's differentiation-promoting function depends on Sxl. Together these data demonstrate a novel role for Sxl in the lineage progression from stem cell to committed daughter cell and suggest a model in which Sxl partners with bam to facilitate this transition.     

TDIF Peptide Signaling Regulates Vascular Stem Cell Proliferation via the WOX4 Homeobox Gene in Arabidopsis

The indeterminate nature of plant growth and development depends on the stem cell system found in meristems. The Arabidopsis thaliana vascular meristem includes procambium and cambium. In these tissues, cell–cell signaling, mediated by a ligand-receptor pair made of the TDIF (for tracheary element differentiation inhibitory factor) peptide and the TDR/PXY (for TDIF RECEPTOR/ PHLOEM INTERCALATED WITH XYLEM) membrane protein kinase, promotes proliferation of procambial cells and suppresses their xylem differentiation. Here, we report that a WUSCHEL-related HOMEOBOX gene, WOX4, is a key target of the TDIF signaling pathway. WOX4 is expressed preferentially in the procambium and cambium, and its expression level was upregulated upon application of TDIF in a TDR-dependent manner. Genetic analyses showed that WOX4 is required for promoting the proliferation of procambial/cambial stem cells but not for repressing their commitment to xylem differentiation in response to the TDIF signal. Thus, at least two intracellular signaling pathways that diverge after TDIF recognition by TDR might regulate independently the behavior of vascular stem cells. Detailed observations in loss-of-function mutants revealed that TDIF-TDR-WOX4 signaling plays a crucial role in the maintenance of the vascular meristem organization during secondary growth.     

Evidence for Chromatin-Remodeling Complex PBAP-Controlled Maintenance of the Drosophila Ovarian Germline Stem Cells

In the Drosophila oogenesis, germline stem cells (GSCs) continuously self-renew and differentiate into daughter cells for consecutive germline lineage commitment. This developmental process has become an in vivo working platform for studying adult stem cell fate regulation. An increasing number of studies have shown that while concerted actions of extrinsic signals from the niche and intrinsic regulatory machineries control GSC self-renewal and germline differentiation, epigenetic regulation is implicated in the process. Here, we report that Brahma (Brm), the ATPase subunit of the Drosophila SWI/SNF chromatin-remodeling complexes, is required for maintaining GSC fate. Removal or knockdown of Brm function in either germline or niche cells causes a GSC loss, but does not disrupt normal germline differentiation within the germarium evidenced at the molecular and morphological levels. There are two Drosophila SWI/SNF complexes: the Brm-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex. More genetic studies reveal that mutations in polybromo/bap180, rather than gene encoding Osa, the BAP complex-specific subunit, elicit a defect in GSC maintenance reminiscent of the brm mutant phenotype. Further genetic interaction test suggests a functional association between brm and polybromo in controlling GSC self-renewal. Taken together, studies in this paper provide the first demonstration that Brm in the form of the PBAP complex functions in the GSC fate regulation.     

Wnt control of stem cells and differentiation in the intestinal epithelium

The intestinal epithelium represents a very attractive experimental model for the study of integrated key cellular processes such as proliferation and differentiation. The tissue is subjected to a rapid and perpetual self-renewal along the crypt-villus axis. Renewal requires division of multipotent stem cells, still to be morphologically identified and isolated, followed by transit amplification, and differentiation of daughter cells into specialized absorptive and secretory cells. Our understanding of the crucial role played by the Wnt/beta-catenin signaling pathway in controlling the fine balance between cell proliferation and differentiation in the gut has been significantly enhanced in recent years. Mutations in some of its components irreversibly lead to carcinogenesis in humans and in mice. Here, we discuss recent advances related to the Wnt/beta-catenin signaling pathway in regulating intestinal stem cells, homeostasis, and cancer. We emphasize how Wnt signaling is able to maintain a stem cell/progenitor phenotype in normal intestinal crypts, and to impose a very similar phenotype onto colorectal adenomas.     

EGFR and Notch signaling respectively regulate proliferative activity and multiple cell lineage differentiation of Drosophila gastric stem cells

Quiescent, multipotent gastric stem cells (GSSCs) in the copper cell region of adult Drosophila midgut can produce all epithelial cell lineages found in the region, including acid-secreting copper cells, interstitial cells and enteroendocrine cells, but mechanisms controlling their quiescence and the ternary lineage differentiation are unknown. By using cell ablation or damage-induced regeneration assays combined with cell lineage tracing and genetic analysis, here we demonstrate that Delta (Dl)-expressing cells in the copper cell region are the authentic GSSCs that can self-renew and continuously regenerate the gastric epithelium after a sustained damage. Lineage tracing analysis reveals that the committed GSSC daughter with activated Notch will invariably differentiate into either a copper cell or an interstitial cell, but not the enteroendocrine cell lineage, and loss-of-function and gain-of-function studies revealed that Notch signaling is both necessary and sufficient for copper cell/interstitial cell differentiation. We also demonstrate that elevated epidermal growth factor receptor (EGFR) signaling, which is achieved by the activation of ligand Vein from the surrounding muscle cells and ligand Spitz from progenitor cells, mediates the regenerative proliferation of GSSCs following damage. Taken together, we demonstrate that Dl is a specific marker for Drosophila GSSCs, whose cell cycle status is dependent on the levels of EGFR signaling activity, and the Notch signaling has a central role in controlling cell lineage differentiation from GSSCs by separating copper/interstitial cell lineage from enteroendocrine cell lineage.     

Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in Drosophila

In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of mei-P26 3′UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Our genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage.     

Localization and function of Bam protein require the benign gonial cell neoplasm gene product.

Division of a female Drosophila stem cell produces a daughter stem cell and a cystoblast. The cystoblast produces a syncytial cluster of 16 cells by precisely four mitotic divisions and incomplete cytokinesis. Mutations in genes required for cystoblast differentiation, such as bag-of-marbles, block syncytial cluster formation and produce a distinctive "tumorous" or hyperplastic germ cell phenotype. In this paper, we compare the oogenic phenotype of benign gonial cell neoplasm mutations to that of mutations in bam. The data indicate that, like bam, bgcn is required for cystoblast development and that germ cells lacking bgcn become trapped in a stem cell-like state. One indication that germ cells lacking bgcn cannot form cystoblasts is that bgcn stem cells resist genetic ablation by Bam misexpression. Misexpression of Bam eliminates wild-type stem cells, apparently by inducing them to divide as cystoblasts. bgcn stem cells remain active when Bam is misexpressed, probably because they cannot adopt the cystoblast fate. Bgcn activity is not required for Bam protein expression but is essential for the localization of Bam protein to the fusome. Together, the results suggest that Bam and Bgcn cooperatively regulate cystoblast differentiation by controlling localization of Bam protein to the fusome.     

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans.