Zhirong Bao

The genome sequence of Caenorhabditis briggsae: A platform for comparative genomics

The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.

Pack-MULE transposable elements mediate gene evolution in plants

Mutator-like transposable elements (MULEs) are found in many eukaryotic genomes and are especially prevalent in higher plants. In maize, rice and Arabidopsis a few MULEs were shown to carry fragments of cellular genes. These chimaeric elements are called Pack-MULEs in this study. The abundance of MULEs in rice and the availability of most of the genome sequence permitted a systematic analysis of the prevalence and nature of Pack-MULEs in an entire genome. Here we report that there are over 3,000 Pack-MULEs in rice containing fragments derived from more than 1,000 cellular genes. Pack-MULEs frequently contain fragments from multiple chromosomal loci that are fused to form new open reading frames, some of which are expressed as chimaeric transcripts. About 5% of the Pack-MULEs are represented in collections of complementary DNA. Functional analysis of amino acid sequences and proteomic data indicate that some captured gene fragments might be functional. Comparison of the cellular genes and Pack-MULE counterparts indicates that fragments of genomic DNA have been captured, rearranged and amplified over millions of years. Given the abundance of Pack-MULEs in rice and the widespread occurrence of MULEs in all characterized plant genomes, gene fragment acquisition by Pack-MULEs might represent an important new mechanism for the evolution of genes in higher plants.

An active DNA transposon family in rice

The publication of draft sequences for the two subspecies of Oryza sativa (rice), japonica (cv. Nipponbare) and indica (cv. 93-11), provides a unique opportunity to study the dynamics of transposable elements in this important crop plant. Here we report the use of these sequences in a computational approach to identify the first active DNA transposons from rice and the first active miniature inverted-repeat transposable element (MITE) from any organism. A sequence classified as a Tourist-like MITE of 430 base pairs, called miniature Ping (mPing), was present in about 70 copies in Nipponbare and in about 14 copies in 93-11. These mPing elements, which are all nearly identical, transpose actively in an indica cell-culture line. Database searches identified a family of related transposase-encoding elements (called Pong), which also transpose actively in the same cells. Virtually all new insertions of mPing and Pong elements were into low-copy regions of the rice genome. Since the domestication of rice mPing MITEs have been amplified preferentially in cultivars adapted to environmental extremes—a situation that is reminiscent of the genomic shock theory for transposon activation.

The lineaging of fluorescently-labeled Caenorhabditis elegans embryos with StarryNite and AceTree

Lineage analysis of Caenorhabditis elegans is a powerful tool for characterizing developmental phenotypes and embryonic gene-expression patterns. We present a detailed protocol for the lineaging of embryos by computational analysis of 4D images of embryos that ubiquitously express histone–GFP (green fluorescent protein) fusion proteins through the 350 cell stage followed by manual editing. We describe how to optimize imaging settings for this purpose, the use of the lineage-extraction software, StarryNite, and the lineage-editing software, AceTree. In addition, we describe a useful polymer bead mounting technique for C. elegans embryos that has several advantages compared with the standard agar pad mounting technique. The protocol requires about 1 h of user time spread over 2 days to generate the raw lineage, and an additional 2 or 4 h to edit the lineage to the 194- or 350-cell stage, respectively.

AceTree: a tool for visual analysis of Caenorhabditis elegans embryogenesis

BackgroundThe invariant lineage of the nematode Caenorhabditis elegans has potential as a powerful tool for the description of mutant phenotypes and gene expression patterns. We previously described procedures for the imaging and automatic extraction of the cell lineage from C. elegans embryos. That method uses time-lapse confocal imaging of a strain expressing histone-GFP fusions and a software package, StarryNite, processes the thousands of images and produces output files that describe the location and lineage relationship of each nucleus at each time point.ResultsWe have developed a companion software package, AceTree, which links the images and the annotations using tree representations of the lineage. This facilitates curation and editing of the lineage. AceTree also contains powerful visualization and interpretive tools, such as space filling models and tree-based expression patterning, that can be used to extract biological significance from the data.ConclusionBy pairing a fast lineaging program written in C with a user interface program written in Java we have produced a powerful software suite for exploring embryonic development.

Comparative analysis of embryonic cell lineage between Caenorhabditis briggsae and Caenorhabditis elegans

Comparative genomic analysis of important signaling pathways in Caenorhabditis briggsae and Caenorhabditis elegans reveals both conserved features and also differences. To build a framework to address the significance of these features we determined the C. briggsae embryonic cell lineage, using the tools StarryNite and AceTree. We traced both cell divisions and cell positions for all cells through all but the last round of cell division and for selected cells through the final round. We found the lineage to be remarkably similar to that of C. elegans. Not only did the founder cells give rise to similar numbers of progeny, the relative cell division timing and positions were largely maintained. These lineage similarities appear to give rise to similar cell fates as judged both by the positions of lineally equivalent cells and by the patterns of cell deaths in both species. However, some reproducible differences were seen, e.g., the P4 cell cycle length is more than 40% longer in C. briggsae than that in C. elegans (p<0.01). The extensive conservation of embryonic development between such divergent species suggests that substantial evolutionary distance between these two species has not altered these early developmental cellular events, although the developmental defects of transpecies hybrids suggest that the details of the underlying molecular pathways have diverged sufficiently so as to not be interchangeable.

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 P(3) (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.

Mounting Caenorhabditis elegans Embryos for Live Imaging of Embryogenesis

Caenorhabditis elegans has been a key model organism for biomedical research. Light microscopy has played a central role in C. elegans biology. C. elegans is transparent throughout its life cycle, and its physical size, from 50 µm (embryos) to 1 mm (adults), is well suited for light microscopy. Furthermore, it has an invariant body plan that arises from an invariant cell lineage. A wide range of biological processes, from patterns of gene expression to cell migration to neuronal activity, can be readily observed in single cells with a well-defined developmental context. This protocol describes how to collect and mount young C. elegans embryos for live imaging throughout embryogenesis.

Automated Lineage and Expression Profiling in Live Caenorhabditis elegans Embryos

Describing gene expression during animal development requires a way to quantitatively measure expression levels with cellular resolution and to describe how expression changes with time. Fluorescent protein reporters make it possible to measure expression dynamics in live cells by time-lapse microscopy, but it can be challenging to identify expressing cells in complex tissues and to compare expression across organisms. This protocol describes how to use automated lineage analysis to identify cells in Caenorhabditis elegans embryos expressing fluorescent reporters and how to quantify that expression with cellular resolution. Because C. elegans develops through an invariant pattern of cell divisions, every cells identity and future fate can be predicted from its pattern of previous cell divisions. Automated analysis of images collected from embryos expressing a fluorescent histone transgene in all cells allows lineage tracing and cell identification. This provides a scaffold with which to describe expression of a second color reporter such as a fusion of a second fluorescent protein to a gene of interest or its regulatory sequences. These methods can also be used for analysis of reporter expression, cell division timing, and cell position in genetically perturbed embryos. The protocol describes how to prepare C. elegans strains containing nuclear-expressed fluorescent reporters, collect images of appropriate quality from embryos, perform automated lineage analysis, manually edit and curate the lineage, and, finally, extract and display reporter signals.

6 Genomics in Caenorhabditis elegans : So Many Genes, Such a Little Worm

In 1965 sydney brenner selected Caenorhabditis elegans for his studies of development and the nervous system because of its simple anatomy, its stereotyped behavior, and the ease of genetic manipulation. Even at inception, the goal of studying the worm was an understanding of how genes dictated form and behavior. This holistic view of the organism (now dubbed “systems biology”) stimulated the collection of comprehensive data sets. The anatomy was described through serial electron microscopic reconstruction with the nervous system defined at the level of the synapse (White et al. 1986). The complete cell lineage of the 959 adult somatic cells was determined (Sulston and Horvitz 1977; Kimble and Hirsh 1979; Sulston et al. 1983) and found to be remarkably consistent animal to animal. Investigators commonly sought to collect all genes affecting a certain trait through mutations (however illusory that completeness might be in retrospect). The construction of a clone-based physical map (Coulson et al. 1986Coulson et al. 1995; Sulston et al. 1988), one of the earliest genome projects, was undertaken in the early 1980s in the same spirit. The map of overlapping cosmids and later Yeast Artificial Chromosomes (YACs) (Coulson et al. 1988Coulson et al. 1991), along with efficient means of transformation, provided the community with the wherewithal to recover the DNA for any well-mapped mutant readily and rapidly. But perhaps more importantly, the existence of a nearly complete physical map in 1989 helped convince James D. Watson, head of the National Center for Human Genome Research at the time, that the worm should...