The C. elegans genome has been completely sequenced, and the developmental anatomy of this model organism is described at single-cell resolution. Here we utilize strategies that exploit this precisely defined architecture to link gene expression to cell type. We obtained RNAs from specific cells and from each developmental stage using tissue-specific promoters to mark cells for isolation by FACS or for mRNA extraction by the mRNA-tagging method. We then generated gene expression profiles of more than 30 different cells and developmental stages using tiling arrays. Machine-learning–based analysis detected transcripts corresponding to established gene models and revealed novel transcriptionally active regions (TARs) in noncoding domains that comprise at least 10% of the total C. elegans genome. Our results show that about 75% of transcripts with detectable expression are differentially expressed among developmental stages and across cell types. Examination of known tissue- and cell-specific transcripts validates these data sets and suggests that newly identified TARs may exercise cell-specific functions. Additionally, we used self-organizing maps to define groups of coregulated transcripts and applied regulatory element analysis to identify known transcription factor– and miRNA-binding sites, as well as novel motifs that likely function to control subsets of these genes. By using cell-specific, whole-genome profiling strategies, we have detected a large number of novel transcripts and produced high-resolution gene expression maps that provide a basis for establishing the roles of individual genes in cellular differentiation.

Previous studies demonstrated that a subset of synMuv B mutants ectopically misexpress germline-specific P-granule proteins in their somatic cells, suggesting a failure to properly orchestrate a soma/germline fate decision. Surprisingly, this fate confusion does not affect viability at low to ambient temperatures. Here, we show that, when grown at high temperature, a majority of synMuv B mutants irreversibly arrest at the L1 stage. High temperature arrest (HTA) is accompanied by upregulation of many genes characteristic of germ line, including genes encoding components of the synaptonemal complex and other meiosis proteins. HTA is suppressed by loss of global regulators of germline chromatin, including MES-4, MRG-1, ISW-1 and the MES-2/3/6 complex, revealing that arrest is caused by somatic cells possessing a germline-like chromatin state. Germline genes are preferentially misregulated in the intestine, and necessity and sufficiency tests demonstrate that the intestine is the tissue responsible for HTA. We propose that synMuv B mutants fail to erase or antagonize an inherited germline chromatin state in somatic cells during embryonic and early larval development. As a consequence, somatic cells gain a germline program of gene expression in addition to their somatic program, leading to a mixed fate. Somatic expression of germline genes is enhanced at elevated temperature, leading to developmentally compromised somatic cells and arrest of newly hatched larvae.

The use of fluorescent protein tags has had a huge impact on cell biological studies in virtually every experimental system. Incorporation of coding sequence for fluorescent proteins such as green fluorescent protein (GFP) into genes at their endogenous chromosomal position is especially useful for generating GFP-fusion proteins that provide accurate cellular and subcellular expression data. We tested modifications of a transposon-based protein trap screening procedure in Drosophila to optimize the rate of recovering useful protein traps and their analysis. Transposons carrying the GFP-coding sequence flanked by splice acceptor and donor sequences were mobilized, and new insertions that resulted in production of GFP were captured using an automated embryo sorter. Individual stocks were established, GFP expression was analyzed during oogenesis, and insertion sites were determined by sequencing genomic DNA flanking the insertions. The resulting collection includes lines with protein traps in which GFP was spliced into mRNAs and embedded within endogenous proteins or enhancer traps in which GFP expression depended on splicing into transposon-derived RNA. We report a total of 335 genes associated with protein or enhancer traps and a web-accessible database for viewing molecular information and expression data for these genes. A S a model organism, perhaps the most important advantage of Drosophila is the extensive range of very sophisticated genetic tools available. Central among these are transposons, including P elements and piggyBac (PBac) elements, which can be used to create easily mapped insertion mutations and to analyze gene expression with a variety of reporter molecules. P elements are naturally occurring Drosophila transposable elements that were first modified to provide vectors for efficient DNA-mediated gene transfer in Drosophila (Rubin and Spradling 1982) and then to create collections of random, single-element insertions in the genome (Robertson et al. 1988;Cooley et al. 1989;Spradling et al. 1995Spradling et al. , 1999Bellen et al. 2004). Although very useful for mutagenesis, P elements also have limitations including preference for the 59 region of genes (O'Hare and Rubin 1983;Tsubota et al. 1985), bias toward particular sequence motifs (O'Hare and Rubin 1983), and ''hot spots'' that have been hit at a high frequency (Spradling et al. 1999).The more recent use of transposons based on the Lepidopteran PBac element has expanded the number of genes disrupted by single transposon insertions (Horn et al. 2003;Bellen et al. 2004;Bonin and Mann 2004;Thibault et al. 2004). First introduced into the Drosophila melanogaster germline by Handler and Harrell (1999), the PBac elements were shown to transpose and insert at TTAA sequences. Like the P element, PBac contains a single open reading frame encoding transposase and is bounded by short terminal inverted repeats. PBac elements have been demonstrated to insert into new genes that have not previously been hit using P-element techniques.Engineered tr...

An essential component of normal development is controlling the transition from cell proliferation to differentiation. One such transition occurs during Drosophila oogenesis. In early oogenesis, germ cells undergo mitotic proliferation and contain a specialized organelle called a fusome, whereas later post-mitotic cells differentiate and lose the fusome as F-actin-rich ring canals form. The hts gene encodes the only Drosophila Adducin, and is a female-sterile mutant that affects both the fusome and ring canals. We show that one Hts protein, Ovhts, is a polyprotein that is cleaved to produce two products, Ovhts-Fus and Ovhts-RC. Whereas Ovhts-Fus localizes to the fusome in mitotic cells, Ovhts-RC localizes to ring canals throughout later oogenesis. We demonstrate that an uncleavable version of Ovhts delays the transition from fusome-containing cells to those that have ring canals. Ovhts is the first polyprotein shown to produce proteins that function in separate structures.

The temperature sensitivity of the germ line is conserved from nematodes to mammals. Previous studies in C. briggsae and Drosophila showed that isolates originating from temperate latitudes lose fertility at a lower temperature than strains originating from tropical latitudes. In order to investigate these relationships in C. elegans, analysis of the fertility of 22 different wild-type isolates of C. elegans isolated from equatorial, tropical and temperate regions was undertaken. It was found that there are significant temperature, genotype and temperature × genotype effects on fertility but region of isolation showed no significant effect on differences in fertility. For most isolates 100% of the population maintained fertility from 20°C to 26°C, but there was a precipitous drop in the percentage of fertile hermaphrodites at 27°C. In contrast, all isolates show a progressive decrease in brood size as temperature increases from 20°C to 26°C, followed by a brood size near zero at 27°C. Temperature shift experiments were performed to better understand the causes of high temperature loss of fertility. Males up-shifted to high temperature maintained fertility, while males raised at high temperature lost fertility. Down-shifting males raised at high temperature generally did not restore fertility. This result differs from that observed in Drosophila and suggested that in C. elegans spermatogenesis or sperm function is irreversibly impaired in males that develop at high temperature. Mating and down-shifting experiments with hermaphrodites were performed to investigate the relative contributions of spermatogenic and oogenic defects to high temperature loss of fertility. It was found that the hermaphrodites of all isolates demonstrated loss in both spermatogenic and oogenic germ lines that differed in their relative contribution by isolate. These studies uncovered unexpectedly high variation in both the loss of fertility and problems with oocyte function in natural variants of C. elegans at high temperature.