Eleanor M. Maine

EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans

Abstract Background: Cell-fate determination requires that cells choose between alternative developmental pathways. For example, germ cells in the nematode worm Caenorhabditis elegans choose between mitotic and meiotic division, and between oogenesis and spermatogenesis. Germ-line mitosis depends on a somatic signal that is mediated by a Notch-type signaling pathway. The ego-1 gene was originally identified on the basis of genetic interactions with the receptor in this pathway and was also shown to be required for oogenesis. Here, we provide more insight into the role of ego-1 in germ-line development. Results: We have determined the ego-1 gene structure and the molecular basis of ego-1 alleles. Putative ego-1 null mutants had multiple, previously unreported defects in germ-line development. The ego-1 transcript was found predominantly in the germ line. The predicted EGO-1 protein was found to be related to the tomato RNA-directed RNA polymerase (RdRP) and to Neurospora crassa QDE-1, two proteins implicated in post-transcriptional gene silencing (PTGS). For a number of germ-line-expressed genes, ego-1 mutants were resistant to a form of PTGS called RNA interference. Conclusions: The ego-1 gene is the first example of a gene encoding an RdRP-related protein with an essential developmental function. The ego-1 gene is also required for a robust response to RNA interference by certain genes. Hence, a protein required for germ-line development in C. elegans may be a component of the RNA interference/PTGS machinery.

Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins

The switch gene, Sex-lethal (Sxl), controls sexual development and dosage compensation. It must be active in females and inactive in males throughout development. Analysis of Sxl cDNAs shows that this on/off regulation may be explained by differential RNA splicing; only female transcripts appear to encode functional products, whereas all male transcripts contain an exon that truncates the open reading frame. The functional female product shows sequence similarities with ribonucleoproteins, suggesting that it is an RNA binding protein. Thus, we propose that Sxl encodes a factor that interacts with both its own pre-mRNA (accounting for positive autoregulation) and that of downstream genes to confer female-specific splicing. In this way, a single, simple mechanism could account for both the maintenance and expression of the sexually determined state.

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.

Regulation of Heterochromatin Assembly on Unpaired Chromosomes during Caenorhabditis elegans Meiosis by Components of a Small RNA-Mediated Pathway

Many organisms have a mechanism for down regulating the expression of non-synapsed chromosomes and chromosomal regions during meiosis. This phenomenon is thought to function in genome defense. During early meiosis in Caenorhabditis elegans, unpaired chromosomes (e.g., the male X chromosome) become enriched for a modification associated with heterochromatin and transcriptional repression, dimethylation of histone H3 on lysine 9 (H3K9me2). This enrichment requires activity of the cellular RNA-directed RNA polymerase, EGO-1. Here we use genetic mutation, RNA interference, immunofluorescence microscopy, fluorescence in situ hybridization, and molecular cloning methods to identify and analyze three additional regulators of meiotic H3K9me2 distribution: CSR-1 (a Piwi/PAZ/Argonaute protein), EKL-1 (a Tudor domain protein), and DRH-3 (a DEAH/D-box helicase). In csr-1, ekl-1, and drh-3 mutant males, we observed a reduction in H3K9me2 accumulation on the unpaired X chromosome and an increase in H3K9me2 accumulation on paired autosomes relative to controls. We observed a similar shift in H3K9me2 pattern in hermaphrodites that carry unpaired chromosomes. Based on several assays, we conclude that ectopic H3K9me2 accumulates on paired and synapsed chromosomes in these mutants. We propose alternative models for how a small RNA-mediated pathway may regulate H3K9me2 accumulation during meiosis. We also describe the germline phenotypes of csr-1, ekl-1, and drh-3 mutants. Our genetic data suggest that these factors, together with EGO-1, participate in a regulatory network to promote diverse aspects of development.

Grassland root communities: species distributions and how they are linked to aboveground abundance

There is little comprehensive information on the distribution of root systems among coexisting species, despite the expected importance of those distributions in determining the composition and diversity of plant communities. This gap in knowledge is particularly acute for grasslands, which possess large numbers of species with morphologically indistinguishable roots. In this study we adapted a molecular method, fluorescent fragment length polymorphism, to identify root fragments and determine species root distributions in two grasslands in Yellowstone National Park (YNP). Aboveground biomass was measured, and soil cores (2 cm in diameter) were collected to depths of 40 cm and 90 cm in an upland, dry grassland and a mesic, slope-bottom grassland, respectively, at peak foliar expansion. Cores were subdivided, and species that occurred in each 10-cm interval were identified. The results indicated that the average number of species in 10-cm intervals (31 cm3) throughout the sampled soil profile was 3.9 and 2.8 species at a dry grassland and a mesic grassland, respectively. By contrast, there was an average of 6.7 and 14.1 species per 0.5 m2, determined by the presence of shoot material, at dry and mesic sites, respectively. There was no relationship between soil depth and number of species per 10-cm interval in either grassland, despite the exponential decline of root biomass with soil depth at both sites. There also was no relationship between root frequency (i.e., the percentage of samples in which a species occurred) and soil depth for the vast majority of species at both sites. The preponderance of species were distributed throughout the soil profile at both sites. Assembly analyses indicated that species root occurrences were randomly assorted in all soil intervals at both sites, with the exception that Festuca idahoensis segregated from Artemisia tridentata and Pseudoroegnaria spicata in 10-20 cm soil at the dry grassland. Root frequency throughout the entire sampled soil profile was positively associated with shoot biomass among species. Together these results indicated the importance of large, well-proliferated root systems in establishing aboveground dominance. The findings suggest that spatial belowground segregation of species probably plays a minor role in fostering resource partitioning and species coexistence in these YNP grasslands.

The Sex-lethal gene of drosophila: DNA alterations associated with sex-specific lethal mutations

Genomic DNA encoding Sex-lethal, a developmental switch gene in Drosophila melanogaster that regulates sex determination and dosage compensation has been isolated. Wild-type DNA sequence organization of the gene has been compared at the restriction level with those of 17 female-specific, loss-of-function and five male-specific, gain-of-function mutant alleles. DNA lesions associated with 12 of these mutations delimit an 11 kb DNA region that is necessary for proper Sex-lethal function in females. Males who are deleted for this region are both viable and fertile. Loss-of-function alleles are associated with gross DNA alterations as well as true point mutations; the former are located throughout the region. In contrast, all five gain-of-function alleles are associated with DNA insertions that are clustered within a 1 kb portion of the Sxl gene region.

A conserved mechanism for post-transcriptional gene silencing?

Proteins with homology to RNA-directed RNA polymerases function in post-transcriptional gene silencing: in quelling in the fungus Neurospora crassa, RNAi in the nematode Caenorhabditis elegans, and co-suppression in the mustard plant Arabidopsis thaliana. These findings are consistent with a conserved mechanism operating in these diverse species.

Studying gene function in Caenorhabditis elegans using RNA-mediated interference

The RNA interference (RNAi) method for targeted gene silencing is widely used in Caenorhabditis elegans for large-scale functional genomic studies, analysis of limited gene sets and detailed analysis of individual gene function. The application of RNAi has identified genes that participate in various aspects of development, physiology and cell biology. In addition, RNAi has been used to identify interacting genes and to study functionally redundant genes. This review discusses the various applications of RNAi in C. elegans, focusing particularly on the analysis of developmental processes.

Fine-scale belowground species associations in temperate grassland

Evaluating how belowground processes contribute to plant community dynamics is hampered by limited information on the spatial structure of root communities at the scale that plants interact belowground. In this study, roots were mapped to the nearest one mm and molecularly identified by species on vertical (0–15 cm deep) surfaces of soil blocks excavated from dry and mesic grasslands in Yellowstone National Park (YNP) to examine the spatial relationships among species at the scale that roots interact. Our results indicated that average interspecific root – root distances for the majority of species were within a distance (3 mm) that roots have been shown to compete for resources. Most species placed their roots at random, although low root numbers for many species probably led to overestimating the occurrence of random patterns. According to theory, we expected that most of the remaining species would segregate their root systems to avoid competition. Instead we found that more species aggregated than segregated from others. Based on previous investigations, we hypothesize that species aggregate to increase uptake of water, nitrogen and/or phosphorus made available by neighbouring roots, or as a consequence of a reduction in the pathogenicity of soil biota growing in multispecies mixtures. Our results indicate that YNP grassland root communities are organized as closely interdigitating networks of species that potentially can support strong interactions among many species combinations. Future root research should address the prevalence and functional consequences of species aggregation across plant communities.

The Bro1-Domain Protein, EGO-2, Promotes Notch Signaling in Caenorhabditis elegans

In Caenorhabditis elegans, as in other animals, Notch-type signaling mediates numerous inductive events during development. The mechanism of Notch-type signaling involves proteolytic cleavage of the receptor and subsequent transport of the receptor intracellular domain to the nucleus, where it acts as a transcriptional regulator. Notch-type signaling activity is modulated by post-translational modifications and endocytosis of ligand and receptor. We previously identified the ego-2 (enhancer of glp-1) gene as a positive regulator of germline proliferation that interacts genetically with the GLP-1/Notch signaling pathway in the germline. Here, we show that ego-2 positively regulates signaling in various tissues via both GLP-1 and the second C. elegans Notch-type receptor, LIN-12. ego-2 activity also promotes aspects of development not known to require GLP-1 or LIN-12. The EGO-2 protein contains a Bro1 domain, which is known in other systems to localize to certain endosomal compartments. EGO-2 activity in the soma promotes GLP-1 signaling in the germline, consistent with a role for EGO-2 in production of active ligand. Another C. elegans Bro1-domain protein, ALX-1, is known to interact physically with LIN-12/Notch. We document a complex phenotypic interaction between ego-2 and alx-1, consistent with their relationship being antagonistic with respect to some developmental processes and agonistic with respect to others.