The CRL2LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development

The molecular mechanisms that regulate cell cycle progression in a developmental context are poorly understood. Here, we show that the leucine-rich repeat protein LRR-1 promotes cell cycle progression during C. elegans development, both in the germ line and in the early embryo. Our results indicate that LRR-1 acts as a nuclear substrate-recognition subunit of a Cullin 2-RING E3 ligase complex (CRL2(LRR-1)), which ensures DNA replication integrity. LRR-1 contains a typical BC/Cul-2 box and binds CRL2 components in vitro and in vivo in a BC/Cul-2 box-dependent manner. Loss of lrr-1 function causes cell cycle arrest in the mitotic region of the germ line, resulting in sterility due to the depletion of germ cells. Inactivation of the DNA replication checkpoint signaling components ATL-1 and CHK-1 suppresses this cell cycle arrest and, remarkably, restores lrr-1 mutant fertility. Likewise, in the early embryo, loss of lrr-1 function induces CHK-1 phosphorylation and a severe cell cycle delay in P lineage division, causing embryonic lethality. Checkpoint activation is not constitutive in lrr-1 mutants but is induced by DNA damage, which may arise due to re-replication of some regions of the genome as evidenced by the accumulation of single-stranded DNA-replication protein A (ssDNA-RPA-1) nuclear foci and the increase in germ cell ploidy in lrr-1 and lrr-1; atl-1 double mutants, respectively. Collectively, these observations highlight a crucial function of the CRL2(LRR-1) complex in genome stability via maintenance of DNA replication integrity during C. elegans development.     

C. elegans RNA-binding proteins PUF-8 and MEX-3 function redundantly to promote germline stem cell mitosis

Maintenance of mitotically cycling germline stem cells (GSCs) is vital for continuous production of gametes. In worms and insects, signaling from surrounding somatic cells play an essential role in the maintenance of GSCs by preventing premature differentiation. In addition, germ cell proteins such as the Drosophila Pumilio and Caenorhabditis elegans FBF, both members of the PUF family translational regulators, contribute to GSC maintenance. FBF functions by suppressing GLD-1, which promotes meiotic entry. However, factors that directly promote GSC proliferation, rather than prevent differentiation, are not known. Here we show that PUF-8, another C. elegans member of the PUF family and MEX-3, a KH domain translational regulator, function redundantly to promote GSC mitosis. We find that PUF-8 protein is highly enriched in mitotic germ cells, which is similar to the expression pattern of MEX-3 described earlier. The puf-8(−) mex-3(−) double mutant gonads contain far fewer germ cells than both single mutants and wild-type. While these cells lack mitotic, meiotic and sperm markers, they retain the germ cell-specific P granules, and are capable of gametogenesis if GLP-1, which normally blocks meiotic entry, is removed. Significantly, we find that at least one of these two proteins is essential for germ cell proliferation even in meiotic entry-defective mutants, which otherwise produce germ cell tumors. We conclude PUF-8 and MEX-3 contribute to GSC maintenance by promoting mitotic proliferation rather than by blocking meiotic entry.     

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°.     

C. elegans CAND-1 regulates cullin neddylation, cell proliferation and morphogenesis in specific tissues

Cullin-RING ubiquitin ligases (CRLs) are critical regulators of multiple developmental and cellular processes in eukaryotes. CAND1 is a biochemical inhibitor of CRLs, yet has been shown to promote CRL activity in plant and mammalian cells. Here we analyze CAND1 function in the context of a developing metazoan organism. Caenorhabditis elegans CAND-1 is capable of binding to all of the cullins, and we show that it physically interacts with CUL-2 and CUL-4 in vivo. The covalent attachment of the ubiquitin-like protein Nedd8 is required for cullin activity in animals and plants. In cand-1 mutants, the levels of the neddylated isoforms of CUL-2 and CUL-4 are increased, indicating that CAND-1 is a negative regulator of cullin neddylation. cand-1 mutants are hypersensitive to the partial loss of cullin activity, suggesting that CAND-1 facilitates CRL functions. cand-1 mutants exhibit impenetrant phenotypes, including developmental arrest, morphological defects of the vulva and tail, and reduced fecundity. cand-1 mutants share with cul-1 and lin-23 mutants the phenotypes of supernumerary seam cell divisions, defective alae formation, and the accumulation of the SCF^LIN-23 target the glutamate receptor GLR-1. The observation that cand-1 mutants have phenotypes associated with the loss of the SCF^LIN-23 complex, but lack phenotypes associated with other specific CRL complexes, suggests that CAND-1 is differentially required for the activity of distinct CRL complexes.     

Dedifferentiation of primary spermatocytes into germ cell tumors in C. elegans lacking the pumilio-like protein PUF-8

PUF proteins are a conserved family of RNA binding proteins that regulate RNA stability and translation by binding to specific sequences in 3'-untranslated regions. Drosophila PUMILIO and C. elegans FBF are essential for self-renewal of germline stem cells, suggesting that a common function of PUF proteins may be to sustain mitotic proliferation of stem cells. Here, we show that PUF-8, the C. elegans PUF most related to PUMILIO, performs a different function in germ cells that have begun meiosis: in primary spermatocytes, puf-8 is required to maintain meiosis and prevent the return to mitosis. Primary spermatocytes lacking PUF-8 complete meiotic prophase but do not undergo normal meiotic divisions. Instead, they dedifferentiate back into mitotically cycling germ cells and form rapidly growing tumors. These findings reveal an unexpected ability for germ cells that have completed meiotic prophase to return to the mitotic cycle, and they support the view that PUF proteins regulate multiple transitions during germline development.     

ZTF-8 Interacts with the 9-1-1 Complex and Is Required for DNA Damage Response and Double-Strand Break Repair in the C. elegans Germline

Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs) can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR) and DSB repair (DSBR) within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to γ-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline.     

Automated and customizable quantitative image analysis of whole Caenorhabditis elegans germlines

Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-content image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: (1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and (2) assessment of these individual nuclei based on their position within the germline. We show the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double-strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields using the C. elegans germline as a model system.     

Gld-1, a Tumor Suppressor Gene Required for Oocyte Development in Caenorhabditis Elegans

We have characterized 31 mutations in the gld-1 (defective in germline development) gene of Caenorhabditis elegans. In gld-1(null) hermaphrodites, oogenesis is abolished and a germline tumor forms where oocyte development would normally occur. By contrast, gld-1(null) males are unaffected. The hermaphrodite germline tumor appears to derive from germ cells that enter the meiotic pathway normally but then exit pachytene and return to the mitotic cycle. Certain gld-1 partial loss-of-function mutations also abolish oogenesis, but germ cells arrest in pachytene rather than returning to mitosis. Our results indicate that gld-1 is a tumor suppressor gene required for oocyte development. The tumorous phenotype suggests that gld-1(+) may function to negatively regulate proliferation during meiotic prophase and/or act to direct progression through meiotic prophase. We also show that gld-1(+) has an additional nonessential role in germline sex determination: promotion of hermaphrodite spermatogenesis. This function of gld-1 is inferred from a haplo-insufficient phenotype and from the properties of gain-of-function gld-1 mutations that cause alterations in the sexual identity of germ cells.     

Mammalian E-type Cyclins Control Chromosome Pairing,Telomere Stability and CDK2 Localization in Male Meiosis

Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis.     

Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation

Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis.     

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.     

CRL2(LRR-1) targets a CDK inhibitor for cell cycle control in C. elegans and actin-based motility regulation in human cells

The Cip/Kip CDK inhibitor (CKI) p21(Cip1/WAF1) has?a critical role in the nucleus to limit cell proliferation by inhibiting CDK-cyclin complexes. In contrast, cytoplasmic p21 regulates cell survival and the actin cytoskeleton. These divergent functions for p21 in different cellular compartments suggest the necessity for complex regulation. In this study, we identify the CRL2(LRR-1) ubiquitin ligase as a conserved regulator of Cip/Kip CKIs that promotes the degradation of C. elegans CKI-1 and human p21. The nematode CRL2(LRR-1) complex negatively regulates nuclear CKI-1 levels to ensure G1-phase cell cycle progression in germ cells. In contrast, human CRL2(LRR1) targets cytoplasmic p21, acting as a critical regulator of cell motility that promotes a nonmotile stationary cell state by preventing p21 from inhibiting the Rho/ROCK/LIMK pathway. Inactivation of human CRL2(LRR1) leads to the activation of the actin-depolymerizing protein cofilin, dramatic reorganization of the actin cytoskeleton, and increased cell motility.     

A dynamic DUO of regulatory proteins coordinates gamete specification and germ cell mitosis in the angiosperm male germline

The production of two functional sperm cells within each male gametophyte is essential for double fertilization in flowering plants and involves a single mitotic division of the male germ cell and cell specification to produce functional gametes. Several proteins that are important regulators of male germ cell division have been identified as well as the R2R3 MYB protein DUO1 that has a dual role in cell division and cell specification. We recently identified a novel regulatory protein DUO3, that has overlapping roles with DUO1 in cell division and specification and shows similarity to GON4 related cell lineage regulators in animals. DUO3 also has important roles outside the germline and is required for embryo patterning and meristem function. We outline the regulatory roles of DUO3 in male germline development and its possible mechanisms of action as a lineage regulator in current models that link germ cell cycle control and gamete specification.Key words: DUO3, male germline development, cell cycle, cell specification, Arabidopsis, pollen, GON4-LThe two sperm cells required for double fertilization in flowering plants are produced after an asymmetric division of the haploid microspore produces a large vegetative cell and a smaller germ cell, thereby establishing the male germline (reviewed in ref. 1; Fig. 1A). The germ cell is engulfed within the vegetative cell cytoplasm where it divides to produce the two sperm cells. The germ cell also goes through a process of specification, with ∼6,000 genes expressed in sperm cells,² many of which show specific or enhanced expression in the male germline and/or are essential for fertilization.^2–4 Since 2005 a number of proteins with important regulatory roles in either germ cell division^5–9 or both germ cell division and specification^10–12 have been described, enabling the formulation of basic models for the regulation of male germline development.^12,13 In our recent publication¹⁴ we identify a novel regulatory protein, DUO POLLEN3 (DUO3) that has essential roles in germ cell division and specification, as well as vital sporophytic functions. Here we present the role of DUO3 in an emerging model for the regulation of male germline development in Arabidopsis (Fig. 1B) and briefly discuss the wider role and possible mechanism of DUO3 function.Open in a separate windowFigure 1Overview of male gametophyte development in arabidopsis (a) and model of germ cell cycle progression and specification in the male germline (B). (A) Schematic of the distinct morphological stages of male gametophyte development in arabidopsis. Diploid pollen mother cells undergo meiotic division to produce a tetrad of haploid microspores. the released microspores undergo a highly asymmetric division to produce a bicellular pollen grain with a small germ cell engulfed within the cytoplasm of a large vegetative cell. Whilst the vegetative cell exits the cell cycle, the germ cell undergoes a further mitotic division to produce twin sperm cells. (B) a schematic model integrating the control of cell proliferation and sperm cell specification in male germline development of arabidopsis. after microspore division, the cell cycle inhibitors KrP6 and KrP7 are present in the newly formed germ cell. transient expression of FBL17 leads to the degradation of these KRPs, allowing CDKA/CYCD to phosphorylate RBR, thereby relieving RBR-mediated repression of the E2F/DP pathway and progression of the germ cell through S-phase. Gamete specification begins shortly after germ cell division, where the co-expression of DUO1 and DUO3 in the germ cell leads to the activation of common and distinct germline differentiation genes. Once S-phase is complete, the DuO1-dependent activation of the G₂/m phase regulator CYCB1;1 promotes germ cell cycle progression and entry into mitosis. In parallel, DUO3 also controls G₂/m transition, by an unknown mechanism that acts independently of cYcB1;1 expression. DUO1 and DUO3 therefore integrate germline differentiation with cell cycle progression. Ultimately, the cooperation of these parallel pathways results in a pair of fully differentiated sperm cells equipped with a complement of essential germline factors such as GcS1 that are required for successful gamete fusion.     

The Utilization during Mitotic Cell Division of Loci Controlling Meiotic Recombination and Disjunction in DROSOPHILA MELANOGASTER

To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by UV and X rays.—Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells.—The chromosome instability produced by mei-41 alleles is the consequence of chromosome breakage, that of mei-9 alleles is primarily due to chromosome breakage and, to a lesser extent, to an elevated frequency of mitotic recombination, whereas no predominant mechanism responsible for the instability caused by c(3)G alleles is discernible. Since these three loci are defective in their responses to mutagen damage, their effects on chromosome stability in nonmutagenized cells are interpreted as resulting from an inability to repair spontaneous lesions. Both mei-W68 and mei-S282 increase mitotic recombination (and in mei-W68, to a lesser extent, chromosome loss) in the abdomen but not the wing. In the abdomen, the primary effect on chromosome stability occurs during the larval period when the abdominal histoblasts are in a nondividing (G2) state.—Mitotic recombination is at or above control levels in the presence of each of the recombination-defective meiotic mutants examined, suggesting that meiotic and mitotic recombination are under separate genetic control in Drosophila.—Of the six mutants examined that are defective in processes required for regular meiotic chromosome segregation, four (l(1)TW-6^cs, ca^nd, mei-S332, ord) affect mitotic chromosome behavior. At semi-restrictive temperatures, the cold sensitive lethal l(1)TW-6^cs causes very frequent somatic spots, a substantial proportion of which are attributable to nondisjunction or loss. Thus, this locus specifies a function essential for chromosome segregation at mitosis as well as at the first meiotic division in females. The patterns of mitotic effects caused by ca^nd, mei-S332, and ord suggest that they may be leaky alleles at essential loci that specify functions common to meiosis and mitosis. Mutants at the two remaining loci (nod, pal) do not affect mitotic chromosome stability.     

Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule-severing activity during both meiosis and mitosis

The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1's meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.     

Microtubule severing by the katanin complex is activated by PPFR-1–dependent MEI-1 dephosphorylation

Katanin is an evolutionarily conserved microtubule (MT)-severing complex implicated in multiple aspects of MT dynamics. In Caenorhabditis elegans, the katanin homologue MEI-1 is required for meiosis, but must be inactivated before mitosis. Here we show that PPFR-1, a regulatory subunit of a trimeric protein phosphatase 4 complex, enhanced katanin MT-severing activity during C. elegans meiosis. Loss of ppfr-1, similarly to the inactivation of MT severing, caused a specific defect in meiosis II spindle disassembly. We show that a fraction of PPFR-1 was degraded after meiosis, contributing to katanin inactivation. PPFR-1 interacted with MEL-26, the substrate recognition subunit of the CUL-3 RING E3 ligase (CRL3^MEL-26), which also targeted MEI-1 for post-meiotic degradation. Reversible protein phosphorylation of MEI-1 may ensure temporal activation of the katanin complex during meiosis, whereas CRL3^MEL-26-mediated degradation of both MEI-1 and its activator PPFR-1 ensure efficient katanin inactivation in the transition to mitosis.     

Differential conservation and divergence of fertility genes boule and dazl in the rainbow trout

Background

The genes boule and dazl are members of the DAZ (Deleted in Azoospermia) family encoding RNA binding proteins essential for germ cell development. Although dazl exhibits bisexual expression in mitotic and meiotic germ cells in diverse animals, boule shows unisexual meiotic expression in invertebrates and mammals but a bisexual mitotic and meiotic expression in medaka. How boule and dazl have evolved different expression patterns in diverse organisms has remained unknown.

Methodology and Principal Findings

Here we chose the fish rainbow trout (Oncorhynchus mykiss) as a second lower vertebrate model to investigate the expression of boule and dazl. By molecular cloning and sequence comparison, we identified cDNAs encoding the trout Boule and Dazl proteins, which have a conserved RNA-recognition motif and a maximal similarity to their homologs. By RT-PCR analysis, adult RNA expression of trout boule and dazl is restricted to the gonads of both sexes. By chromogenic and two-color fluorescence in situ hybridization, we revealed bisexual and germline-specific expression of boule and dazl. We found that dazl displays conserved expression throughout gametogenesis and concentrates in the Balbinani''s body of early oocytes and the chromatoid body of sperm. Surprisingly, boule exhibits mitotic and meiotic expression in the male but meiosis-specific expression in the female.

Conclusions

Our data underscores differential conservation and divergence of DAZ family genes during vertebrate evolution. We propose a model in which the diversity of boule expression in sex and stage specificity might have resulted from selective loss or gain of its expression in one sex and mitotic germ cells.     

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.     

Synaptonemal Complex Components Promote Centromere Pairing in Pre-meiotic Germ Cells

Mitosis and meiosis are two distinct cell division programs. During mitosis, sister chromatids separate, whereas during the first meiotic division, homologous chromosomes pair and then segregate from each other. In most organisms, germ cells do both programs sequentially, as they first amplify through mitosis, before switching to meiosis to produce haploid gametes. Here, we show that autosomal chromosomes are unpaired at their centromeres in Drosophila germline stem cells, and become paired during the following four mitosis of the differentiating daughter cell. Surprisingly, we further demonstrate that components of the central region of the synaptonemal complex are already expressed in the mitotic region of the ovaries, localize close to centromeres, and promote de novo association of centromeres. Our results thus show that meiotic proteins and meiotic organization of centromeres, which are key features to ensure reductional segregation, are laid out in amplifying germ cells, before meiosis has started.