Robert C. Johnsen

Basal body dysfunction is a likely cause of pleiotropic Bardet–Biedl syndrome

Bardet–Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized primarily by retinal dystrophy, obesity, polydactyly, renal malformations and learning disabilities. Although five BBS genes have been cloned, the molecular basis of this syndrome remains elusive. Here we show that BBS is probably caused by a defect at the basal body of ciliated cells. We have cloned a new BBS gene, BBS8, which encodes a protein with a prokaryotic domain, pilF, involved in pilus formation and twitching mobility. In one family, a homozygous null BBS8 mutation leads to BBS with randomization of left–right body axis symmetry, a known defect of the nodal cilium. We have also found that BBS8 localizes specifically to ciliated structures, such as the connecting cilium of the retina and columnar epithelial cells in the lung. In cells, BBS8 localizes to centrosomes and basal bodies and interacts with PCM1, a protein probably involved in ciliogenesis. Finally, we demonstrate that all available Caenorhabditis elegans BBS homologues are expressed exclusively in ciliated neurons, and contain regulatory elements for RFX, a transcription factor that modulates the expression of genes associated with ciliogenesis and intraflagellar transport.

High-throughput in vivo analysis of gene expression in Caenorhabditis elegans.

Using DNA sequences 5′ to open reading frames, we have constructed green fluorescent protein (GFP) fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5′ DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index) and through WormBase (http://www.wormbase.org).

Identification of ciliary and ciliopathy genes in Caenorhabditis elegans through comparative genomics

BackgroundThe recent availability of genome sequences of multiple related Caenorhabditis species has made it possible to identify, using comparative genomics, similarly transcribed genes in Caenorhabditis elegans and its sister species. Taking this approach, we have identified numerous novel ciliary genes in C. elegans, some of which may be orthologs of unidentified human ciliopathy genes.ResultsBy screening for genes possessing canonical X-box sequences in promoters of three Caenorhabditis species, namely C. elegans, C. briggsae and C. remanei, we identified 93 genes (including known X-box regulated genes) that encode putative components of ciliated neurons in C. elegans and are subject to the same regulatory control. For many of these genes, restricted anatomical expression in ciliated cells was confirmed, and control of transcription by the ciliogenic DAF-19 RFX transcription factor was demonstrated by comparative transcriptional profiling of different tissue types and of daf-19(+) and daf-19(-) animals. Finally, we demonstrate that the dye-filling defect of dyf-5(mn400) animals, which is indicative of compromised exposure of cilia to the environment, is caused by a nonsense mutation in the serine/threonine protein kinase gene M04C9.5.ConclusionOur comparative genomics-based predictions may be useful for identifying genes involved in human ciliopathies, including Bardet-Biedl Syndrome (BBS), since the C. elegans orthologs of known human BBS genes contain X-box motifs and are required for normal dye filling in C. elegans ciliated neurons.

The RNA-binding protein SUP-12 controls muscle-specific splicing of the ADF/cofilin pre-mRNA in C. elegans

Tissue-specific alternative pre-mRNA splicing is essential for increasing diversity of functionally different gene products. In Caenorhabditis elegans, UNC-60A and UNC-60B, nonmuscle and muscle isoforms of actin depolymerizing factor (ADF)/cofilin, are expressed by alternative splicing of unc-60 and regulate distinct actin-dependent developmental processes. We report that SUP-12, a member of a new family of RNA recognition motif (RRM) proteins, including SEB-4, regulates muscle-specific splicing of unc-60. In sup-12 mutants, expression of UNC-60B is decreased, whereas UNC-60A is up-regulated in muscle. sup-12 mutations strongly suppress muscle defects in unc-60B mutants by allowing expression of UNC-60A in muscle that can substitute for UNC-60B, thus unmasking their functional redundancy. SUP-12 is expressed in muscle and localized to the nuclei in a speckled pattern. The RRM domain of SUP-12 binds to several sites of the unc-60 pre-mRNA including the UG repeats near the 3′-splice site in the first intron. Our results suggest that SUP-12 is a novel tissue-specific splicing factor and regulates functional redundancy among ADF/cofilin isoforms.

Characterization of the Caenorhabditis elegans G protein-coupled serotonin receptors.

Serotonin (5-HT) regulates a wide range of behaviors in Caenorhabditis elegans, including egg laying, male mating, locomotion and pharyngeal pumping. So far, four serotonin receptors have been described in the nematode C. elegans, three of which are G protein-coupled receptors (GPCR), (SER-1, SER-4 and SER-7), and one is an ion channel (MOD-1). By searching the C. elegans genome for additional 5-HT GPCR genes, we identified five further genes which encode putative 5-HT receptors, based on sequence similarities to 5-HT receptors from other species. Using loss-of-function mutants and RNAi, we performed a systematic study of the role of the eight GPCR genes in serotonin-modulated behaviors of C. elegans (F59C12.2, Y22D7AR.13, K02F2.6, C09B7.1, M03F4.3, F16D3.7, T02E9.3, C24A8.1). We also examined their expression patterns. Finally, we tested whether the most likely candidate receptors were able to modulate adenylate cyclase activity in transfected cells in a 5-HT-dependent manner. This paper is the first comprehensive study of G protein-coupled serotonin receptors of C. elegans. It provides a direct comparison of the expression patterns and functional roles for 5-HT receptors in C. elegans.

Comprehensive analysis of gene expression patterns of hedgehog -related genes

BackgroundThe Caenorhabditis elegans genome encodes ten proteins that share sequence similarity with the Hedgehog signaling molecule through their C-terminal autoprocessing Hint/Hog domain. These proteins contain novel N-terminal domains, and C. elegans encodes dozens of additional proteins containing only these N-terminal domains. These gene families are called warthog, groundhog, ground-like and quahog, collectively called hedgehog (hh)-related genes. Previously, the expression pattern of seventeen genes was examined, which showed that they are primarily expressed in the ectoderm.ResultsWith the completion of the C. elegans genome sequence in November 2002, we reexamined and identified 61 hh-related ORFs. Further, we identified 49 hh-related ORFs in C. briggsae. ORF analysis revealed that 30% of the genes still had errors in their predictions and we improved these predictions here. We performed a comprehensive expression analysis using GFP fusions of the putative intergenic regulatory sequence with one or two transgenic lines for most genes. The hh-related genes are expressed in one or a few of the following tissues: hypodermis, seam cells, excretory duct and pore cells, vulval epithelial cells, rectal epithelial cells, pharyngeal muscle or marginal cells, arcade cells, support cells of sensory organs, and neuronal cells. Using time-lapse recordings, we discovered that some hh-related genes are expressed in a cyclical fashion in phase with molting during larval development. We also generated several translational GFP fusions, but they did not show any subcellular localization. In addition, we also studied the expression patterns of two genes with similarity to Drosophila frizzled, T23D8.1 and F27E11.3A, and the ortholog of the Drosophila gene dally-like, gpn-1, which is a heparan sulfate proteoglycan. The two frizzled homologs are expressed in a few neurons in the head, and gpn-1 is expressed in the pharynx. Finally, we compare the efficacy of our GFP expression effort with EST, OST and SAGE data.ConclusionNo bona-fide Hh signaling pathway is present in C. elegans. Given that the hh-related gene products have a predicted signal peptide for secretion, it is possible that they constitute components of the extracellular matrix (ECM). They might be associated with the cuticle or be present in soluble form in the body cavity. They might interact with the Patched or the Patched-related proteins in a manner similar to the interaction of Hedgehog with its receptor Patched.

An antibiotic selection marker for nematode transgenesis.

We have developed a nematode transformation vector carrying the bacterial neomycin resistance gene (NeoR) and shown that it could confer resistance to G-418 on both wild-type Caenorhabditis elegans and C. briggsae. This selection system allows hands-off maintenance and enrichment of transgenic worms carrying non-integrated transgenes on selective plates. We also show that this marker can be used for Mos1-mediated single-copy insertion in wild-type genetic backgrounds (MosSCI-biotic).

Genomic organization in Caenorhabditis elegans : deficiency mapping on linkage group V(left)

In this study we genetically analyse a large autosomal region (23 map units) in Caenorhabditis elegans. The region comprises the left half of linkage group V [LGV(left)] and is recombinationally balanced by the translocation eT1 ( III; V ). We have used rearrangement breakpoints to subdivide the region from the left end of LGV to daf-11 into a set of 23 major zones. Twenty of these zones are balanced by eT1. To establish the zones we examined a total of 110 recessive lethal mutations derived from a variety of screening protocols. The mutations identified 12 deficiencies, 1 duplication, as well as 98 mutations that fell into 59 complementation groups, significantly increasing the number of available genetic sites on LGV. Twenty-six of the latter had more than 1 mutant allele. Significant differences were observed among the alleles of only 6 genes, 3 of which have at least one ‘visible’ allele. Several deficiencies and 3 alleles of let-336 were demonstrated to affect recombination. The duplication identified in this study is sDp30 ( V;X ). Lethal mutations covered by sDp30 were not suppressed uniformly in hermaphrodites. The basis for this non-uniformity may be related to the mechanism of X chromosome dosage compensation in C. elegans.

Distinct regulatory elements mediate similar expression patterns in the excretory cell of Caenorhabditis elegans.

Identification of cis-regulatory elements and their binding proteins constitutes an important part of understanding gene function and regulation. It is well accepted that co-expressed genes tend to share transcriptional elements. However, recent findings indicate that co-expression data show poor correlation with co-regulation data even in unicellular yeast. This motivates us to experimentally explore whether it is possible that co-expressed genes are subject to differential regulatory control using the excretory cell of Caenorhabditis elegans as an example. Excretory cell is a functional equivalent of human kidney. Transcriptional regulation of gene expression in the cell is largely unknown. We isolated a 10-bp excretory cell-specific cis-element, Ex-1, from a pgp-12 promoter. The significance of the element has been demonstrated by its capacity of converting an intestine-specific promoter into an excretory cell-specific one. We also isolated a cDNA encoding an Ex-1 binding transcription factor, DCP-66, using a yeast one-hybrid screen. Role of the factor in regulation of pgp-12 expression has been demonstrated both in vitro and in vivo. Search for occurrence of Ex-1 reveals that only a small portion of excretory cell-specific promoters contain Ex-1. Two other distinct cis-elements isolated from two different promoters can also dictate the excretory cell-specific expression but are independent of regulation by DCP-66. The results indicate that distinct regulatory elements are able to mediate the similar expression patterns.

Formaldehyde mutagenesis of the eT1 balanced region in Caenorhabditis elegans: dose-response curve and the analysis of mutational events

In this study we have generated a dose-response curve for the formaldehyde induction of recessive lethal mutations in the eT1(III;V)-balanced region of C. elegans. We have mapped 96 out of 112 formaldehyde-induced lesions to either LGIII or LGV and genetically analyzed 31 lesions that mapped to LGV. Our findings showed that a 4-h treatment with 0.1% formaldehyde gave the best mutation induction frequency with the least side effects. We found that formaldehyde induced putative point mutations, deficiencies and more complex lesions in C. elegans. We isolated 11 putative point mutations, 3 of which defined new genes and 8 were alleles of known genes. One of the new genes, let-450, is currently the left-most known gene on LGV. We also isolated 5 deficiencies. Our formaldehyde-induced lesions increased the number of zones in the eT1-balanced region of LGV from 22 to 34.