Body-wall muscle formation in Caenorhabditis elegans embryos that lack the MyoD homolog hlh-1.

The myoD family of DNA binding proteins has been implicated in the control of myogenesis in a variety of organisms. Searches for homologs in the nematode Caenorhabditis elegans yielded only one gene, designated hlh-1, expressed in body-wall muscle cells and their precursors. To assess the role of hlh-1 in C. elegans myogenesis, genetic deficiencies spanning the hlh-1 locus were isolated after gamma irradiation. Embryos homozygous for these deficiencies exhibited extensive body-wall muscle differentiation, including expression of several characteristic myofilament proteins and weak contracile behavior. Thus, zygotic hlh-1 expression was not required for body-wall muscle precursors to adopt muscle cell fates.

Regulation of apoptosis by C. elegans CED-9 in the absence of the C-terminal transmembrane domain.

Bcl-2 proteins regulate apoptosis in organisms as diverse as mammals and nematodes. These proteins are often localized at mitochondria by a C-terminal transmembrane domain. Although the transmembrane domain and mitochondrial localization are centrally involved in specific cases of vertebrate Bcl-2 activity, the significance of this localization is not clear for all species. Studying the Caenorhabditis elegans Bcl-2 homolog CED-9, we found that the transmembrane domain was both necessary and sufficient for localization at mitochondrial outer membranes. Furthermore, we found that in our assays, ced-9 transgenes lacking the transmembrane domain, although somewhat less active than equivalent transgenes derived from wild-type ced-9, rescued embryonic lethality of ced-9(lf) animals and responded properly to upstream signals in controlling the fate of Pn.aap neurons. Both of these apoptotic activities were retained in a construct where CED-9 lacking the transmembrane domain was targeted to the cytosolic surface of the endoplasmic reticulum and derived organelles, suggesting that in wild-type animals, accumulation at mitochondria is not essential for CED-9 to either inhibit or promote apoptosis in C. elegans. Taken together, these data are consistent with a multimodal character of CED-9 action, with an ability to regulate apoptosis through interactions in the cytosol coexisting with additional evolutionarily conserved role(s) at the membrane.

RNA-triggered gene silencing.

Double-stranded RNA (dsRNA) has recently been shown to trigger sequence-specific gene silencing in a wide variety of organisms, including nematodes, plants, trypanosomes, fruit flies and planaria; meanwhile an as yet uncharacterized RNA trigger has been shown to induce DNA methylation in several different plant systems. In addition to providing a surprisingly effective set of tools to interfere selectively with gene function, these observations are spurring new inquiries to understand RNA-triggered genetic-control mechanisms and their biological roles.

Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes.

Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.

A screen for genetic loci required for body-wall muscle development during embryogenesis in Caenorhabditis elegans.

We have used available chromosomal deficiencies to screen for genetic loci whose zygotic expression is required for formation of body-wall muscle cells during embryogenesis in Caenorhabditis elegans. To test for muscle cell differentiation we have assayed for both contractile function and the expression of muscle-specific structural proteins. Monoclonal antibodies directed against two myosin heavy chain isoforms, the products of the unc-54 and myo-3 genes, were used to detect body-wall muscle differentiation. We have screened 77 deficiencies, covering approximately 72% of the genome. Deficiency homozygotes in most cases stain with antibodies to the body-wall muscle myosins and in many cases muscle contractile function is observed. We have identified two regions showing distinct defects in myosin heavy chain gene expression. Embryos homozygous for deficiencies removing the left tip of chromosome V fail to accumulate the myo-3 and unc-54 products, but express antigens characteristic of hypodermal, pharyngeal and neural development. Embryos lacking a large region on chromosome III accumulate the unc-54 product but not the myo-3 product. We conclude that there exist only a small number of loci whose zygotic expression is uniquely required for adoption of a muscle cell fate.

Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene.

In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues. We have named this product nucampholin. Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes. Nucampholin resides in all nuclei of C. elegans and is essential in early development and in differentiating tissue. Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression. Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline. The broad requirements allowed investigation of soma-germline differences in gene expression. When introduced into standard transgene arrays, let-858 (like many other C. elegans genes) functions well in soma but poorly in germline. We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like. To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA. The resulting transgenes show robust expression in both germline and soma. Our results suggest the possibility of concerted mechanisms for silencing unwanted germiline expression of repetitive sequences.

Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans.

Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers.

Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.

Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.

A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans.

We describe a series of plasmid vectors which contain modular features particularly useful for studying gene expression in eukaryotic systems. The vectors contain the Escherichia coli beta-galactosidase (beta Gal)-encoding region (the lacZ gene) flanked by unique polylinker segments on the 5' and 3' ends, and several combinations of a variety of modules: a selectable marker (an amber suppressor tRNA), a translational initiation region, a synthetic intron segment, the early polyadenylation signal from SV40, and 3' regions from two nematode genes. A segment encoding the nuclear localization peptide from the SV40 T antigen is incorporated into many of the constructs, leading to beta Gal accumulation in nuclei, which can facilitate identification of producing cells in complex tissues. To make functional beta Gal fusions to secreted proteins, we constructed plasmids with an alternate module encoding a synthetic transmembrane domain upstream from lacZ. This domain is designed to stop transfer of secreted proteins across the membrane during secretion, allowing the beta Gal domain of the fusion polypeptide to remain in the cytoplasm and thus function in enzymatic assays. We have used the vectors to analyze expression of several genes in the nematode Caenorhabditis elegans, and have demonstrated in these studies that lacZ can be expressed in a wide variety of different tissues and cell types. These vectors should be useful in studying gene expression both in C. elegans and in other experimental systems.

dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference.

RNA interference (RNAi) is a form of post-transcriptional gene silencing that has been described in a number of plant, nematode, protozoan, and invertebrate species. RNAi is characterized by a number of features: induction by double stranded RNA (dsRNA), a high degree of specificity, remarkable potency and spread across cell boundaries, and a sustained down-regulation of the target gene. Previous studies of RNAi have examined this effect in whole organisms or in extracts thereof; we have now examined the induction of RNAi in tissue culture. A screen of mammalian cells from three different species showed no evidence for the specific down-regulation of gene expression by dsRNA. By contrast, RNAi was observed in Drosophila Schneider 2 (S2) cells. Green fluorescent protein (GFP) expression in S2 cells was inhibited in a dose-dependent manner by transfection of dsRNA corresponding to gfp when GFP was expressed either transiently or stably. This effect was structure- and sequence-specific in that: (1) little or no effect was seen when antisense (or sense) RNA was transfected; (2) an unrelated dsRNA did not reduce GFP expression; and (3) dsRNA corresponding to gfp had no effect on the expression of an unrelated target transgene. This invertebrate tissue culture model should allow facile assays for loss of function in a well-defined cellular system and facilitate further understanding of the mechanism of RNAi and the genes involved in this process.

Two-color GFP expression system for C. elegans.

We describe the use of modified versions of the Aequora victoria green fluorescent protein (GFP) to simultaneously follow the expression and distribution of two different proteins in the nematode, Caenorhabditis elegans. A cyan-colored GFP derivative, designated CFP, contains amino acid (aa) substitutions Y66W, N146I, M153T and V163A relative to the original GFP sequence and is similar to the previously reported "W7" form. A yellow-shifted GFP derivative, designated YFP, contains aa substitutions S65G, V68A, S72A and T203Y and is similar to the previously described "I0C" variant. Coding regions for CFP and YFP were constructed in the context of a high-activity C. elegans expression system. Previously characterized promoters and localization signals have been used to express CFP and YFP in C. elegans. Filter sets designed to distinguish YFP and CFP fluorescence spectra allowed visualization of the two distinct forms of GFP in neurons and in muscle cells. A series of expression vectors carrying CFP and YFP have been constructed and are being made available to the scientific community.

Regulation of adenovirus mRNA synthesis.

The lytic cycle of adenovirus is a tightly regulated sequence of stages. When this regulation is studied at the level of mRNA production, the most significant step in controlling gene expression is initiation of transcription. Thus in preceding from one stage of expression to another, viral factors seem to turn on transcription of new sets of genes. At the moment, it is thought that viral mRNA synthesis involves initiation of transcription at ten different promoter sites. It is likely that in some manner the frequency of an initiation of transcription at nine of these sites is affected by one or more viral gene products. With the recent development of soluble in vitro transcription systems that respond to exogenously added DNA, it should be possible to begin to study regulation of gene expression at this stage of transcription. At present, these systems yield the paradoxical observation that extracts prepared from uninfected human cells more efficiently recognize the late promoter as compared to the early promoter of adenovirus. As more is learned about regulation of synthesis of viral mRNAs, examples will surely be found where RNA processing and RNA turnover play a critical role in determining the level of mRNAs. Such cases are more likely to appear in the balancing of synthesis of different mRNAs derived from one transcriptional unit. Few experiments have been directed to this possibility and the study of adenovirus molecular biology is only now entering the age of maturity where these experiments are feasible.

MicroRNA expression signature of human sarcomas.

MicroRNAs (miRNAs) are approximately 22 nucleotide-long noncoding RNAs involved in several biological processes including development, differentiation and proliferation. Recent studies suggest that knowledge of miRNA expression patterns in cancer may have substantial value for diagnostic and prognostic determinations as well as for eventual therapeutic intervention. We performed comprehensive analysis of miRNA expression profiles of 27 sarcomas, 5 normal smooth muscle and 2 normal skeletal muscle tissues using microarray technology and/or small RNA cloning approaches. The miRNA expression profiles are distinct among the tumor types as demonstrated by an unsupervised hierarchical clustering, and unique miRNA expression signatures were identified in each tumor class. Remarkably, the miRNA expression patterns suggested that two of the sarcomas had been misdiagnosed and this was confirmed by reevaluation of the tumors using histopathologic and molecular analyses. Using the cloning approach, we also identified 31 novel miRNAs or other small RNA effectors in the sarcomas and normal skeletal muscle tissues examined. Our data show that different histological types of sarcoma have distinct miRNA expression patterns, reflecting the apparent lineage and differentiation status of the tumors. The identification of unique miRNA signatures in each tumor type may indicate their role in tumorigenesis and may aid in diagnosis of soft tissue sarcomas.

A four-dimensional digital image archiving system for cell lineage tracing and retrospective embryology.

The paper describes a digital image archiving system for time-lapse microscopy. The system uses an MS-DOS compatible computer to store video images while simultaneously controlling a stepping motor. In a typical experiment, images might be taken at 30 s intervals in each of 25 consecutive focal planes. A system with 2.5 Gbyte disk capacity can store approximately 18,000 full frame images: 6 h recording at maximum resolution. Once recorded, images series stored on disk can be 'played back' in any order. Generally, images from a single focal plane are displayed consecutively in either forward or reverse time. The focal plane can be shifted during playback, allowing individual cells to be followed as they move between focal planes. To facilitate the annotation and interpretation of the real-time images, a mouse-driven interface allows users to define and follow individual objects (e.g. cells). The recorded image series can be achieved inexpensively using standard digital tape backup hardware. In this laboratory, the system has been particularly useful for tracing embryonic cell lineages and cell migrations. Detailed system specifications, including source code, compiled programs, hardware requirements and users manual are available directly from the author or by anonymous FTP (ciw1.ciwemb.edu).

Histochemical techniques for locating Escherichia coli beta-galactosidase activity in transgenic organisms.

Escherichia coli beta-galactosidase is a commonly used reporter molecule for analyzing gene expression. Recently, beta-galactosidase fusions have been applied to a variety of eukaryotic systems. The techniques for constructing and introducing beta-galactosidase fusion constructs as well as soluble assays for total enzyme function have been described in detail elsewhere. This article describes histochemical techniques for analyzing organisms that contain a functional beta-galactosidase fusion construct. The object is to determine semiquantitatively which cells are expressing the beta-galactosidase fusion protein, as well as the subcellular localization of the protein. Due to its prevalence in the author's laboratory, Caenorhabditis elegans is used as a canonical organism for the detailed methods described.

Integrative transformation of Caenorhabditis elegans.

A technique for introducing exogenous DNA into the chromosomes of the nematode Caenorhabditis elegans is presented. A cloned C. elegans amber suppressor tRNA gene, sup-7, is used as a selectable marker. The activity of this amber suppressor is selected for by injecting worms which carry an amber termination mutation in a gene (tra-3) whose function is required for fertility. Transient expression of sup-7 is evidenced by the presence of fertile (rescued) animals in the generation after injection. In a fraction of cases, these fertile animals give rise to stable suppressor lines (eight have been characterized so far). Each of the stable suppressor lines carries injected DNA sequences. The suppressor activities have been mapped to chromosomal loci, indicating that the exogenous DNA has integrated into the genome. This technique has been used to introduce a chimeric gene containing a Drosophila heat shock promoter element fused to coding sequences from the Escherichia coli beta-galactosidase gene. This chimeric gene functions and is heat inducible in the resulting stably transformed lines.

Soma-germline asymmetry in the distributions of embryonic RNAs in Caenorhabditis elegans.

Early embryogenesis in Caenorhabditis elegans is characterized by a series of unequal cleavages that mark the stepwise separation of somatic and germ lineages. We have developed an in situ hybridization protocol to examine the localization of specific maternal and embryonically transcribed messenger RNAs during these early cleavages. We detected three classes of maternal RNAs: RNAs that are maintained in all cells, RNAs that are maintained in germline cells but are lost from somatic cells, and a population of RNAs that are associated with the germline-specific P granules. We observed embryonically transcribed RNAs in somatic cells as early as the 4-cell stage. These transcripts were not detected in germline cells. These observations suggest that mechanisms which distinguish between soma and germline cause asymmetries in mRNA stability and transcription within the first few cleavages of C. elegans embryogenesis.