Systematic interactome mapping and genetic perturbation analysis of a C. elegans TGF-beta signaling network

To initiate a system-level analysis of C. elegans DAF-7/TGF-beta signaling, we combined interactome mapping with single and double genetic perturbations. Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. Coaffinity purification (co-AP) assays in mammalian cells confirmed the overall quality of this network. Systematic perturbations of the network using RNAi, both in wild-type and daf-7/TGF-beta pathway mutant animals, identified nine DAF-7/TGF-beta signaling modifiers, seven of which are conserved in humans. We show that one of these has functional homology to human SNO/SKI oncoproteins and that mutations at the corresponding genetic locus daf-5 confer defects in DAF-7/TGF-beta signaling. Our results reveal substantial molecular complexity in DAF-7/TGF-beta signal transduction. Integrating interactome maps with systematic genetic perturbations may be useful for developing a systems biology approach to this and other signaling modules.     

A protein domain-based interactome network for C. elegans early embryogenesis

Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or "interactome" networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early-embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms.     

Chromatin regulation during C. elegans germline development

Recent studies in Caenorhabditis elegans implicate PcG- and NuRD-like chromatin regulators in the establishment and maintenance of germline-soma distinctions. Somatic cells appear to utilize NuRD-related nucleosome-remodeling factors to overwrite germline-specific chromatin states that are specified through PcG-like activities. The germline, in turn, may rely on an asymmetrically inherited inhibitor to prevent chromatin reorganization that would otherwise erase pluripotency.     

The oogenic germline starvation response in C. elegans

Many animals alter their reproductive strategies in response to environmental stress. Here we have investigated how L4 hermaphrodites of Caenorhabditis elegans respond to starvation. To induce starvation, we removed food at 2 h intervals from very early- to very late-stage L4 animals. The starved L4s molted into adulthood, initiated oogenesis, and began producing embryos; however, all three processes were severely delayed, and embryo viability was reduced. Most animals died via 'bagging,' because egg-laying was inhibited, and embryos hatched in utero, consuming their parent hermaphrodites from within. Some animals, however, avoided bagging and survived long term. Long-term survival did not rely on embryonic arrest but instead upon the failure of some animals to produce viable progeny during starvation. Regardless of the bagging fate, starved animals showed two major changes in germline morphology: All oogenic germlines were dramatically reduced in size, and these germlines formed only a single oocyte at a time, separated from the remainder of the germline by a tight constriction. Both changes in germline morphology were reversible: Upon re-feeding, the shrunken germlines regenerated, and multiple oocytes formed concurrently. The capacity for germline regeneration upon re-feeding was not limited to the small subset of animals that normally survive starvation: When bagging was prevented ectopically by par-2 RNAi, virtually all germlines still regenerated. In addition, germline shrinkage strongly correlated with oogenesis, suggesting that during starvation, germline shrinkage may provide material for oocyte production. Finally, germline shrinkage and regeneration did not depend upon crowding. Our study confirms previous findings that starvation uncouples germ cell proliferation from germline stem cell maintenance. Our study also suggests that when nutrients are limited, hermaphrodites scavenge material from their germlines to reproduce. We discuss our findings in light of the recently proposed state of dormancy, termed Adult Reproductive Diapause.     

Quantitative analysis of germline mitosis in adult C. elegans

Certain aspects of the distal gonad of C. elegans are comparable to niche/stem cell systems in other organisms. The distal tip cell (DTC) caps a blind-ended tube; only the distal germ cells maintain proliferation in response to signaling from the DTC via the GLP-1/Notch signaling pathway in the germ line. Fruitful comparison between this system and other stem cell systems is limited by a lack of basic information regarding germ cell division behavior in C. elegans. Here, we explore the spatial pattern of cell division frequency in the adult C. elegans germ line relative to distance from the distal tip. We mapped the positions of actively dividing germline nuclei in over 600 fixed gonad preparations including the wild type and a gain-of-function ligand-responsive GLP-1 receptor mutant with an extended mitotic zone. One particularly surprising observation from these data is that the frequency of cell divisions is lower in distal-most cells-cells that directly contact the distal tip cell body-relative to cells further proximal, a difference that persists in the gain-of-function GLP-1 mutant. These results suggest that cell division frequency in the distal-most cells may be suppressed or otherwise controlled in a complex manner. Further, our data suggest that the presence of an active cell division influences the probability of observing simultaneous cell divisions in the same gonad arm, and that simultaneous divisions tend to cluster spatially. We speculate that this system behaves similarly to niche/stem cell/transit amplifying cell systems in other organisms.     

PAN-1, a P-granule component important for C. elegans fertility, has dual roles in the germline and soma

In Caenorhabditis elegans, P granules are germline-specific, RNA-containing granules that segregate into the germline precursor cell during early embryogenesis. In this short report, PAN-1, which previously has been found by others in screens for genes causing larval molting defects, is identified here as a novel P-granule component and a binding partner of GLH-1 (Germline RNA Helicase-1), a constitutive, germline-specific, P-granule protein. The PAN-1 predicted protein contains multiple leucine-rich repeats (LRRs) and regions with similarities to F-box proteins. Antibodies raised against PAN-1 reveal it is present both in the soma and the germline. In the germline, PAN-1 uniquely localizes to P granules from the first larval stage onward and is unusual for a P-granule component in lacking recognizable RNA binding motifs. Homozygous pan-1(gk142) deletion worms arrest as larvae that are unable to molt and this phenotype is also seen with pan-1(RNAi) into wild type worms. pan-1(RNAi) into the somatic RNAi-defective strain rrf-1(pk1417) bypasses the larval arrest and allows an assessment of PAN-1 function in the germline. We find pan-1(RNAi) is variably effective in knocking down PAN-1 protein and results in adult progeny that display multiple germline defects. These phenocopies range from under-proliferation of the germline, as also seen with loss of GLH-1, to the induction of endomitotic replication in oocytes, both defects that result in sterility, to fertile animals with significantly reduced progeny numbers. Thus, while loss of PAN-1 in the soma inhibits molting, this report demonstrates that PAN-1 is also a P-granule component that is essential for fertility.     

The C. elegans glycopeptide hormone receptor ortholog, FSHR-1, regulates germline differentiation and survival

BACKGROUND: The mammalian glycopeptide hormone receptors (GPHRs) are key regulators of reproductive development, and their homologs are widely distributed throughout the animal kingdom. The C. elegans genome encodes a single GPHR family member, FSHR-1, which shares equal identity to the FSH, LH, and TSH receptors from mammals. RESULTS: Because loss of fshr-1 function does not produce a visible phenotype in C. elegans, we conducted a genome-wide RNAi-feeding screen to identify genes that perform functions that overlap with those of fshr-1. This approach led to the identification of the PUF family members fbf-1 and fbf-2 (the fbfs). Whereas a weak reduction in fbf activity caused little or no discernable effect in the wild-type, an equivalent loss in the fshr-1(0) mutant background resulted in a highly penetrant germline-masculinization phenotype. Furthermore, many fshr-1(0);fbf(RNAi) animals failed to maintain a germline stem cell niche. We also show that fshr-1 and the fbfs promote germline survival and prevent apoptosis with fog-1 and fog-3 and that simultaneous loss of fshr-1 and the fbfs can override the canonical requirement for fog-1 and fog-3 in the execution of the male-germline fate. Finally, we provide evidence that FSHR-1 controls germline processes nonautonomously via the soma and that FSHR-1 acts through a canonical signaling pathway involving Galpha(s) and adenyl cyclase. CONCLUSIONS: Our results indicate a conserved role for GPHR family receptors in controlling germline development and fertility. Our data suggest a model whereby FSHR-1 signaling acts in parallel to the known sex-determination pathway to control multiple aspects of germline development.     

SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development

SR-protein kinases (SRPKs) and their substrates, serine/arginine-rich pre-mRNA splicing factors, are key components of splicing machinery and are well conserved across phyla. Despite extensive biochemical investigation, the physiological functions of SRPKs remain unclear. In the present study, cDNAs for SPK-1, a C. elegans SRPK homologue, and CeSF2, an SPK-1 substrate, were cloned. SPK-1 binds directly to and phosphorylates the RS domain of CeSF2 in vitro. Both spk-1 and CeSF2 are predominantly expressed in germlines. RNA interference (RNAi) experiments revealed that spk-1 and CeSF2 play an essential role at the embryonic stage of C. elegans. Furthermore, RNAi studies demonstrated that spk-1 is required for germline development in C. elegans. We provide evidence that RNAi, achieved by the soaking of L1 larvae, is beneficial in the study of gene function in post-embryonic germline development.     

An extracellular matrix protein prevents cytokinesis failure and aneuploidy in the C. elegans germline

Interactions between extracellular matrix (ECM) proteins and their transmembrane receptors mediate cytoskeletal reorganization and corresponding changes in cell shape during cell migration, adhesion, differentiation and polarization. Cytokinesis is the final step in cell division as cells employ a contractile ring composed of actin and myosin to partition one cell into two. Cells undergo dramatic changes in cell shape during the division process, creating new membrane and forming an extracellular invagination called the cleavage furrow. However, existing models of cytokinesis include no role for the ECM. In a recent paper, we demonstrate that depletion of a large secreted protein, hemicentin, results in membrane destabilization, cleavage furrow retraction and cytokinesis failure in C. elegans germ cells and in preimplantation mouse embryos.Here, we demonstrate that cytokinesis failure produces tetraploid intermediate cells with multipolar spindles, providing a potential explanation for the large number of aneuploid progeny observed among C. elegans hemicentin mutant hermaphrodites.Key words: aneuploidy, cytokinesis, extracellular matrix, C. elegans, cleavage furrow, hemicentin, tetraploid intermediateThe karyotype of C. elegans has five autosomes and one or two X chromosomes in males and hermaphrodites, respectively. The majority of self-progeny produced by wild-type hermaphrodites are hermaphrodites (∼99.8%), while rare meiotic nondisjunction of the X chromosome produces nullo-X gametes and 0.2% males. Mutations in over 30 genes result in a 10–150-fold increase in the frequency of males among hermaphrodite self-progeny, due to increases in defects in X chromosome segregation.¹ The majority of these ‘him’ (high incidence of males) loci are genes that encode proteins associated with the intracellular machinery of meiotic chromosome segregation.^2,3 Unique among him genes, the him-4 locus encodes hemicentin, a large, highly conserved component of the extracellular matrix (ECM).⁴ In addition to defects in germline chromosome segregation, him-4 mutants have pleiotropic defects in somatic cell adhesion and migration.^1,4 The extracellular distribution of hemicentin at cell junctions that are defective in him-4 mutants dovetails with current models of cell adhesion and migration.⁵ However, it leaves unexplained several questions about how a secreted ECM component promotes correct chromosome segregation in the C. elegans germline.C. elegans hermaphrodite gonads are composed of two U-shaped tubes, and gametogenesis proceeds sequentially from the distal to the proximal end of each tube. Germ cells in C. elegans have incomplete cleavage furrows that connect them to a central cytoplasmic core, allowing distal cells to act as “nurses” while allowing more mature proximal oocytes to fill with bulk cytoplasm.^6–8 Several genetic and cytogenetic observations suggest a mitotic rather than a meiotic origin for germline chromosome segregation defects observed in the absence of hemicentin.⁴ For example, jackpots of male progeny from individual hermaphrodites and nullisomy of primary meiocytes in him-4 mutants suggest a defect in a mitotic germline stem cell rather than in a post-mitotic process. Our recent work describing hemicentin localization at the cleavage furrows of dividing cells in the early mouse embryo and C. elegans germline, in addition to membrane destabilization, cleavage furrow retraction and cytokinesis failure in the absence of hemicentin, suggests that hemicentin has an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows.⁹Aneuploid cells are frequently observed in, and may be associated with the generation of, human tumor cells. Recent work from several laboratories suggests that cytokinesis failure is one of several mechanisms whereby tumor cells generate tetraploid intermediates that result in the production of aneuploid daughter cells in subsequent cell divisions. One proposed mechanism for the generation of aneuploid daughter cells from a tetraploid intermediate is thought to involve multipolar mitotic spindles that result in asymmetric mitoses.^10–13To determine whether a similar mechanism might be responsible for the aneuploidy observed in the absence of hemicentin, him-4 (rh319) animals were examined for multipolar mitotic spindles. A significant fraction (14%) of mitotic germ cells have multipolar spindles that are not observed in a wild-type background (Fig. 1 and Fig. 1).Open in a separate windowFigure 1Multinucleate germ cells and multipolar germ cells observed in the mitotic zone of him-4 mutant hermaphrodites. (A) PH::RFP and histone::GFP in the mitotic region of wild-type (left) and him-4 (rh319) hermaphrodite gonads. Large numbers of multinucleate cells are observed among mitotic germ cells in mutant gonads (arrows). (B) PH::RFP and tubulin::GFP in the mitotic region of wild-type (left) and him-4 (rh319) hermaphrodite gonads. A significant fraction (14^–16.

Table 1

Severity and types of defective germ cells in him-4 gonads