Control of cell cycle timing during C. elegans embryogenesis

As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first 8 out of 10 rounds of embryonic cell division in Caenorhabditis elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P₃ (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.     

A novel function for the Sm proteins in germ granule localization during C. elegans embryogenesis

General mRNA processing factors are traditionally thought to function only in the control of global gene expression. Here we show that the Sm proteins, core components of the splicesome, also regulate germ granules during early C. elegans development. Germ granules are large cytoplasmic particles that localize to germ cells and their precursors during embryogenesis of diverse organisms. In C. elegans, germ granules, called P granules, are segregated to the germline precursor cells during embryogenesis by asymmetric cell division, and they remain in germ cells at all stages of development. We found that at least some Sm proteins are components of P granules. Moreover, disruption of Sm activity caused defects in P granule localization to the germ cell precursors during early embryogenesis. In contrast, loss of other splicing factor activities had no effect on germ granule control in the embryo. These observations suggest that the Sm proteins control germ granule integrity and localization in the early C. elegans embryo and that this role is independent of pre-mRNA splicing. Thus, a highly conserved splicing factor may have been adapted to control both snRNP biogenesis and the localization of components important for germ cell function.     

The many facets of SC function during C. elegans meiosis

Sexually reproducing organisms rely on meiosis for the formation of haploid gametes. This is achieved through two consecutive rounds of cell division (meiosis I and II) after one round of DNA replication. During the meiotic divisions, chromosomes face several challenges to ultimately ensure proper chromosome segregation. Unique events unfold during meiosis I to overcome these challenges. Homologous chromosomes pair, synapse, and recombine. A remarkable feature throughout this process is the formation of an evolutionarily conserved tripartite proteinaceous structure known as the synaptonemal complex (SC). It is comprised of two lateral elements, assembled along each axis of a pair of homologous chromosomes, and a central region consisting of transverse filaments bridging the gap between lateral elements. While the presence of the SC during meiosis has been appreciated now for 50 years (Moses, Biophys Biochem Cytol 2:215–218, 1956; Fawcett, J Biophys Biochem Cytol 2:403–406, 1956), its role(s) remain a matter of intense investigation. This review concentrates on studies performed in Caenorhabditis elegans, a powerful system for investigating meiosis. Studies in this organism are contributing to the unraveling of the various processes leading to the formation of the SC and the various facets of the functions it exerts throughout meiosis.The synaptonemal complex-50 years     

Network news: functional modules revealed during early embryogenesis in C. elegans

The functional module is fast becoming the operational unit of the postgenomics era. A new report in Nature by Gunsalus and colleagues describes, using a multiply supported network, functional modules within early C. elegans embryos and identifies several new components of known molecular machines (Gunsalus et al., 2005).     

Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTN gene produces 1-3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis.     

CSR-1 RNAi pathway positively regulates histone expression in C. elegans

Endogenous small interfering RNAs (endo-siRNAs) have been discovered in many organisms, including mammals. In C. elegans, depletion of germline-enriched endo-siRNAs found in complex with the CSR-1 Argonaute protein causes sterility and defects in chromosome segregation in early embryos. We discovered that knockdown of either csr-1, the RNA-dependent RNA polymerase (RdRP) ego-1, or the dicer-related helicase drh-3, leads to defects in histone mRNA processing, resulting in severe depletion of core histone proteins. The maturation of replication-dependent histone mRNAs, unlike that of other mRNAs, requires processing of their 3′UTRs through an endonucleolytic cleavage guided by the U7 snRNA, which is lacking in C. elegans. We found that CSR-1-bound antisense endo-siRNAs match histone mRNAs and mRNA precursors. Consistently, we demonstrate that CSR-1 directly binds to histone mRNA in an ego-1-dependent manner using biotinylated 2′-O-methyl RNA oligonucleotides. Moreover, we demonstrate that increasing the dosage of histone genes rescues the lethality associated with depletion of CSR-1 and EGO-1. These results support a positive and direct effect of RNAi on histone gene expression.     

Whole-genome sequencing and variant discovery in C. elegans

Massively parallel sequencing instruments enable rapid and inexpensive DNA sequence data production. Because these instruments are new, their data require characterization with respect to accuracy and utility. To address this, we sequenced a Caernohabditis elegans N2 Bristol strain isolate using the Solexa Sequence Analyzer, and compared the reads to the reference genome to characterize the data and to evaluate coverage and representation. Massively parallel sequencing facilitates strain-to-reference comparison for genome-wide sequence variant discovery. Owing to the short-read-length sequences produced, we developed a revised approach to determine the regions of the genome to which short reads could be uniquely mapped. We then aligned Solexa reads from C. elegans strain CB4858 to the reference, and screened for single-nucleotide polymorphisms (SNPs) and small indels. This study demonstrates the utility of massively parallel short read sequencing for whole genome resequencing and for accurate discovery of genome-wide polymorphisms.     

Systematic analysis of pleiotropy in C. elegans early embryogenesis

Pleiotropy refers to the phenomenon in which a single gene controls several distinct, and seemingly unrelated, phenotypic effects. We use C. elegans early embryogenesis as a model to conduct systematic studies of pleiotropy. We analyze high-throughput RNA interference (RNAi) data from C. elegans and identify "phenotypic signatures", which are sets of cellular defects indicative of certain biological functions. By matching phenotypic profiles to our identified signatures, we assign genes with complex phenotypic profiles to multiple functional classes. Overall, we observe that pleiotropy occurs extensively among genes involved in early embryogenesis, and a small proportion of these genes are highly pleiotropic. We hypothesize that genes involved in early embryogenesis are organized into partially overlapping functional modules, and that pleiotropic genes represent "connectors" between these modules. In support of this hypothesis, we find that highly pleiotropic genes tend to reside in central positions in protein-protein interaction networks, suggesting that pleiotropic genes act as connecting points between different protein complexes or pathways.     

Autophagy and apoptosis are redundantly required for C. elegans embryogenesis

Apoptosis, the main form of regulated (or programmed) cell death, allows the organism to tightly control cell numbers and tissue size, and to protect itself from potentially damaging cells. This type of cellular self-killing has long been assumed to be essential for early development. In the nematode Caenorhabditis elegans, however, the core apoptotic cell death pathway appears to be dispensable for embryogenesis when most developmental cell deaths take place: mutant nematodes defective for apoptosis develop into adulthood, with superficially normal morphology and behavior. Accumulating evidence indicates a similar situation in mammalian systems as well. For example, apoptosis-deficient mice can grow as healthy, fertile adults. These observations raise the possibility that alternative cell death mechanisms may compensate for the lack of apoptotic machinery in developing embryos. Interestingly, C. elegans embryogenesis can also occur without autophagy, an alternative form of cellular self-destruction (also called autophagic cell death). In an upcoming paper we report that simultaneous inactivation of the autophagic and apoptotic gene cascades in C. elegans arrests development at early stages, and the affected embryos exhibit severe morphological defects. Double-mutant nematode embryos deficient in both autophagy and apoptosis are unable to undergo body elongation or to arrange several tissues correctly. This novel function of autophagy genes in morphogenesis indicates a more fundamental role for cellular self-digestion in tissue patterning than previously thought.     

Degrade to create: developmental requirements for ubiquitin-mediated proteolysis during early C. elegans embryogenesis

The ubiquitin protein conjugation system tags proteins with the small polypeptide ubiquitin. Most poly-ubiquitinated proteins are recognized and degraded by the proteasome, a large multi-subunit protease. Ubiquitin-dependent protein degradation is used as a regulatory tool for many essential processes, the best studied of which is eukaryotic cell cycle progression. More recently, genetic studies in C. elegans have identified multiple roles for the ubiquitin system in early development, where ubiquitin-dependent protein degradation governs such diverse events as passage through meiosis, cytoskeletal regulation and cell fate determination.     

HMG-D and histone H1 interplay during chromatin assembly and early embryogenesis

HMG-D is an abundant chromosomal protein associated with condensed chromatin during the first nuclear cleavage cycles of the developing Drosophila embryo. We previously suggested that HMG-D might substitute for the linker histone H1 in the preblastoderm embryo and that this substitution might result in the characteristic less compacted chromatin. We have now studied the association of HMG-D with chromatin using a cell-free system for chromatin reconstitution derived from Drosophila embryos. Association of HMG-D with chromatin, like that of histone H1, increases the nucleosome spacing indicative of binding to the linker DNA between nucleosomes. HMG-D interacts with DNA during the early phases of nucleosome assembly but is gradually displaced as chromatin matures. By contrast, purified chromatin can be loaded with stoichiometric amounts of HMG-D, and this can be displaced upon addition of histone H1. A direct physical interaction between HMG-D and histone H1 was observed in a Far Western analysis. The competitive nature of this interaction is reminiscent of the apparent replacement of HMG-D by H1 during mid-blastula transition. These data are consistent with the hypothesis that HMG-D functions as a specialized linker protein prior to appearance of histone H1.     

The WAVE/SCAR complex promotes polarized cell movements and actin enrichment in epithelia during C. elegans embryogenesis

The WAVE/SCAR complex promotes actin nucleation through the Arp2/3 complex, in response to Rac signaling. We show that loss of WVE-1/GEX-1, the only C. elegans WAVE/SCAR homolog, by genetic mutation or by RNAi, has the same phenotype as loss of GEX-2/Sra1/p140/PIR121, GEX-3/NAP1/HEM2/KETTE, or ABI-1/ABI, the three other components of the C. elegans WAVE/SCAR complex. We find that the entire WAVE/SCAR complex promotes actin-dependent events at different times and in different tissues during development. During C. elegans embryogenesis loss of CED-10/Rac1, WAVE/SCAR complex components, or Arp2/3 blocks epidermal cell migrations despite correct epidermal cell differentiation. 4D movies show that this failure occurs due to decreased membrane dynamics in specific epidermal cells. Unlike myoblasts in Drosophila, epidermal cell fusions in C. elegans can occur in the absence of WAVE/SCAR or Arp2/3. Instead we find that subcellular enrichment of F-actin in epithelial tissues requires the Rac-WAVE/SCAR-Arp2/3 pathway. Intriguingly, we find that at the same stage of development both F-actin and WAVE/SCAR proteins are enriched apically in one epithelial tissue and basolaterally in another. We propose that temporally and spatially regulated actin nucleation by the Rac-WAVE/SCAR-Arp2/3 pathway is required for epithelial cell organization and movements during morphogenesis.