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Development and RNA Interference in C. elegans
Summary: Craig Mello uses the nematode worm C. elegans as a model organism to investigate how embryonic cells differentiate and communicate during development. In addition, he is investigating the mechanism of RNA interference, a form of sequence-specific gene silencing triggered by double-stranded RNA.
Embryogenesis
Embryogenesis in Caenorhabditis elegans is a remarkably reproducible process mapped out to the resolution of every single cell division in the wild-type embryo (a total of only 558 cells at hatching). Despite the small number of cells, the newly hatched larvae contain all of the major types of differentiated tissues found in other animals (e.g., intestine, muscles, germline, epidermis, and a nervous system). Because of its relative simplicity, the C. elegans embryo provides a valuable opportunity to understand the process of animal development at the level of individual cells.
The C. elegans embryo utilizes several mechanisms to produce early blastomeres that are committed to distinct patterns of development. These mechanisms include position-dependent cell-cell interactions as well as the asymmetric expression of maternally provided transcriptional activators and repressors. We are interested in the mechanisms that lead to the initial diversification of cell fates during the first several divisions of the embryo. One key protein involved in this process is the PIE-1 protein, a small Cys3His–zinc finger protein that functions in the specification of certain somatic cell fates and in maintaining the germline fate. After each round of cell division in the early embryo, the PIE-1 protein is localized to one and only one cell until the 28-cell stage of embryogenesis, when it is expressed in the two primordial germ cells. During early embryogenesis, PIE-1 appears to act as a repressor that protects the germline lineage from differentiating in response to transcriptional activators that are present in the germline. Our recent work suggests that PIE-1 not only prevents transcription but also blocks chromatin remodeling in the germline. We are exploring the mechanism of PIE-1 localization and function.
In addition to the various mechanisms that lead to the distinct fates of certain early blastomeres, several studies have indicated that cells throughout the embryo share a common mechanism for linking the axis of cell division to cell fate. For example, most of the cell divisions after the eight-cell stage of embryogenesis are oriented along the anterior/posterior (a/p) axis, and essentially all of these divisions result in a/p daughter cells with different fates. Using a combination of reverse and forward genetics, we identified several components of the signaling pathways that control cell fate and cell polarity in early C. elegans embryos.
Several genes involved in polarity signaling in the embryo encode highly conserved proteins related to Wnt-signaling components. Wnt genes comprise a large family, including numerous vertebrate and invertebrate members, and encode secreted proteins important in cell-cell interactions. In the early C. elegans embryo, Wnt signaling acts in parallel with a phosphotyrosine-signaling pathway mediated by SRC-1, a C. elegans homolog of tyrosine kinase pp60 Src. Wnt and SRC signaling converge to regulate WRM-1, a homolog of human β-catenin. Mutations that stabilize β-catenin are found in numerous forms of cancer in humans. Genetic tests suggest that signaling overcomes, or down-regulates, a WRM-1 nuclear-export pathway that is mediated by IMB-4, a homolog of the conserved exportin CRM-1. Our ongoing studies involve a biochemical dissection of signaling as well as extensive genetic screening, with a focus on understanding how these and other signaling pathways function in concert to provide the positional information necessary for patterning the developing embryo.
Reverse Genetics and RNA Interference
During our developmental studies, we became intrigued by some strange properties of the reverse genetic method we were using. This method was analogous to “antisense,‿ wherein the microinjection of RNA complementary to the messenger RNA is used to block the activity of specific genes. However, although antisense methodology generally causes a partial block of gene activity, we observed a remarkably robust interference effect that could spread from tissue to tissue in the animal. In most experiments, nearly 100 percent of the progeny of an injected animal showed phenotypic effects consistent with blocking the targeted gene's activity. Even more surprising, interference could be transmitted for multiple generations in the germline as an extrachromosomal element.
The remarkable potency and long-lasting effects of RNA interference (RNAi) prompted us to investigate the RNAi mechanism. In a collaborative study with Andrew Fire (Carnegie Institution of Washington), we showed that double-stranded RNA (dsRNA) was 10 to 100 times more efficient than antisense RNA at inducing interference. This exciting discovery soon led to the application of RNAi via dsRNA in other organisms. To date, dsRNA-induced interference has been reported to one degree or another in a variety of animals and plants. In C. elegans, dsRNA is so potent at causing interference that simply soaking worms in RNA, or feeding the worms bacteria expressing a dsRNA, is sufficient to induce interference.
We have identified several mutant strains that are resistant to RNAi. One class of mutants exhibits an activation of the endogenous transposons, selfish DNA elements, in the genome of the animal. This suggests that one in vivo function of the silencing mechanism may be to silence the transposons. Molecular and genetic studies of RNAi and of similar gene-silencing mechanisms in other organisms have revealed striking similarities. For example, the rde-1 gene, required for RNAi in C. elegans, has homologs required for gene-silencing mechanisms in Drosophila, plants, and fungi. These and other findings indicate that an ancient mechanism underlies the RNAi process, and we now know that related forms of gene regulation are important in development and disease in humans.
Consistent with this latter idea, we have found that C. elegans genes related to rde-1 are required for development and for proper chromosome segregation. The genomes of plants and animals, including humans, contain hundreds of small dsRNA-encoding genes, which have recently been named micro-RNA genes (miRNAs). The miRNAs are similar in size to the small-interfering RNAs (siRNAs) that mediate RNAi and like siRNAs are processed from dsRNA precursors through the action of an RNase III–related enzyme, Dicer. Understanding the RNAi mechanism will not only lead to better reverse genetic tools for manipulating and studying gene function but will also shed light on an ancient and highly conserved gene regulatory mechanism. Ultimately, it may be possible to harness this natural gene-regulatory mechanism to reduce the expression of disease-related mutant genes.
This work was supported in part by a Pew Scholarship and by grants from the National Institutes of Health, the American Cancer Society, and the March of Dimes Birth Defects Foundation.
Last updated: February 6, 2007
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