RIKEN Center for Developmental Biology

2003 Annual Report

Interactions between genes at the network level are of fundamental importance in instructing the development and function of multicellular organisms. While the characterization of genomes at the level of the solitary gene remains an important challenge in many yet unsequenced organisms, the analysis of how genes function in networks is now in increasing demand for those genomes that are already available. In the post-genome era, scientists seek to understand the roles of genes in evolution through comparative genomics studies as well as to characterize genes in isolation and understand their functions in the context of interactive networks, a field of investigation known as functional genomics.

Asako Sugimoto has adopted the nematode Caenorhabditis elegans as an experimental model to take advantage of its tractability to the systematic functional analysis of its genome using unique high-throughput screening techniques. By studying the interactions between the approximately 19,000 genes predicted for C. elegans, the Sugimoto research team seeks to identify the means by which sets of genes working in combination help to establish and direct developmental processes. The lab also looks to take the findings from these studies as a base for advancing the understanding of developmentally important mechanisms. Sugimoto hopes that by opening windows into the role of networked genes in guiding development in a simple worm, new light will be shed on universal mechanisms in the genetic regulation of the developmental program.

RNAi-based profiling of gene function

The wild-type nematode is built from precisely 959 somatic cells, yet this simple organism exhibits a wide range of the specialized cell types, such as muscles and neurons, that characterize more highly derived species. And, thanks to the complete knowledge of the lineage of every cell in the C. elegans body, the differentiative pathway of every one of those cells can be followed from its origin in the fertilized egg to its role in the fully-grown adult. The amenability of this worm's sequenced genome to reverse-genetic techniques has also served to make it one of the standard model organisms in the world of genetics research. The discovery in the late 1990s that the introduction of double stranded RNA (dsRNA) could be used to knock down the expression of specific genes in the nematode has only added to its appeal, giving scientists the ability to inhibit gene function without disturbing its underlying DNA. Sugimoto has refined this technique of RNA interference (RNAi) by developing a method in which nematodes directly uptake dsRNA in solution. This process of 'RNAi by soaking' offers greater efficiency and ease-of-use than other RNAi methods and has enabled systematic high-throughput studies of gene suppression to be conducted more rapidly than ever before possible.

Starting with a cDNA library of approximately 10,000 genes expressed in developmental processes, the Sugimoto lab used RNAi to knock down each gene's function and has started to construct a database in which the resulting phenotypes are sorted by developmental outcome. To date, nearly 6,000 phenotypes have been categorized, and it was found that loss of function in more than 25% of these genes resulted in lethal, morphologically altered or sterile phenotypes. Many lethal phenotypes result in developmental arrest and death at very early stages, which typically prevents the study of the underlying gene's function in later development. By exposing worms to RNAi-inducing dsRNA at the L1 stage of larval development, Sugimoto is able to study the post-embryonic function of genes that are lethal when knocked down in embryos. This is critically important to the understanding of genetic networks in post-embryonic development, as more than one out of every ten genes analyzed by the lab so far is essential for embryogenesis. Sugimoto's studies to date show that approximately 50% of all such genes play post-embryonic roles as well, supporting the concept that a great number of genes play multiple roles at different stages in development.

Phenotype analysis

The data sets produced by high-throughput phenotype analyses can be formidably large and unwieldy, and the lack of standardized descriptive terms and categories can limit the accessibility to the wealth of information they contain. To improve the value, ease-of-use and distributability of her RNAi phenotyping results, Sugimoto has developed a taxonomic system and nomenclature to enable the precise and consistent description of embryonic and post-embryonic phenotypes using a checklist of more than 50 identifying traits, such as abnormalities in cell division, cellular differentiation, organogenesis, morphogenesis, growth, and movement. She plans to make these RNAi phenotype profiles available to the C. elegans research community, in the hopes of establishing a universal taxonomy for phenotype analysis. Phenotypes described using this system can be subjected to hierarchical clustering analysis, which makes it possible to categorize genes based on relatedness between phenotypes. The genes clustered by this method are likely to be involved in the same developmental process, thus this analysis will provide pivotal information to uncover genetic networks involved in the regulation of development. Members of the Sugimoto team are now utilizing these RNAi analysis data to investigate developmental processes such as microtubule dynamics in mitosis, morphogenesis, and programmed cell death.

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