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)

Characterization of the octamer, a cis-regulatory element that modulates excretory cell gene-expression in Caenorhabditis elegans

Background: We have previously demonstrated that the POU transcription factor CEH-6 is required for driving aqp-8 expression in the C. elegans excretory (canal) cell, an osmotic regulatory organ that is functionally analogous to the kidney. This transcriptional regulation occurs through a CEH-6 binding to a cis-regulatory element called the octamer (ATTTGCAT), which is located in the aqp-8 promoter. Results Here, we further characterize octamer driven transcription in C. elegans. First, we analyzed the positional requirements of the octamer. To do so, we assayed the effects on excretory cell expression by placing the octamer within the well-characterized promoter of vit-2. Second, using phylogenetic footprinting between three Caenorhabditis species, we identified a set of 165 genes that contain conserved upstream octamers in their promoters. Third, we used promoter::GFP fusions to examine the expression patterns of 107 of the 165 genes. This analysis demonstrated that conservation of octamers in promoters increases the likelihood that the gene is expressed in the excretory cell. Furthermore, we found that the sequences flanking the octamers may have functional importance. Finally, we altered the octamer using site-directed mutagenesis. Thus, we demonstrated that some nucleotide substitutions within the octamer do not affect the expression pattern of nearby genes, but change their overall expression was changed. Therefore, we have expanded the core octamer to include flanking regions and variants of the motif. Conclusions Taken together, we have demonstrated that octamer-containing regions are associated with excretory cell expression of several genes that have putative roles in osmoregulation. Moreover, our analysis of the octamer sequence and its sequence variants could aid in the identification of additional genes that are expressed in the excretory cell and that may also be regulated by CEH-6.Medical Genetics, Department ofMedicine, Faculty ofMolecular Medicine and Therapeutics, Centre forReviewedFacult

Touch receptor neuron (TRN) morphology, position and number defects in the <i>coel-1</i> overexpression and deletion strains.

<p>A. Schematic representation of wild-type morphology of the 6 mechanosensory neurons of <i>C. elegans</i>. B. ALM and AVM cell bodies are misplaced posteriorly in <i>coel-1</i> mutants compared to wild-type animals. Only AVM cell bodies are misplaced in <i>coel-1XS</i> animals. Each data point represents the distance from each cell body to the back of the pharynx divided by the length of the anterior body, (i.e., from the tip of the nose to the vulva). C. PLM neurites in <i>coel-1</i> mutants are statistically significantly longer than in wild-type. Values indicated represent the length of each PLM neurite divided by the length of the posterior body (i.e., from the vulva to the tail). In panels B and C, the horizontal bar corresponds to the mean of all data points. D. Typical anterior neurite termination site in wild-type worms expressing <i>zdIs5</i> (<i>mec-4</i>::GFP), a TRN-specific reporter. (i) example of AVM neurite premature termination (arrowhead) at the nerve ring observed in <i>coel-1XS</i> animals; (ii) asterisks show crosstalk into the GFP channel from <i>dpy-30</i>::dsRED, a co-marker used for the <i>coel-1</i> overexpression strain. E. Quantitative analysis of the AVM termination site defect in animals overexpressing <i>coel-1</i>. F. In wild-type animals, one AVM and one PVM are observed and AVM is localized anterior to ALM cell bodies. (i) example of AVM mispositioning in <i>coel-1XS</i> animals, where AVM is found posterior to the vulva; (ii) Quantification of AVM position defect is presented in G. H. AVM/PVM cell number defect in <i>coel-1XS</i> animals. Images shown are of animals with two cells or no cell in AVM (i, iii) and/or PVM (ii, iv); positions are shown and the quantification is reported in I. Scale bar represents 20 µm. V, vulva; WT, wild-type. Brackets indicate the number of neurons measured or scored. *, <i>p</i>≤0.05, Fisher exact test.</p

Identification and established functions/interactions of a set of 526 ortholog groups strictly conserved in metazoans.

<p>A. Phylogeny of species used to identify the metazoan-specific genes. Grey lines represent possible paraphyletic groups. Dotted lines represent groups of uncertain phylogenetic position. Metazoan groups are colored blue or orange, non-metazoan groups are colored red, and groups that were not included in the analysis are colored grey. All included metazoan species are shown in parentheses; for a complete list of non-metazoan genomes used see Ortho-MCL website. Tree not drawn to scale. B. Comparative genomics approach used to identify the metazoan-specific genes (the ‘metazoanome’). 526 ortholog groups (dashed green oval) were identified on the basis of being conserved in 24 well-sequenced metazoan genomes and absent from 112 well-sequenced non-metazoan genomes. Of these 526 ortholog groups, 346 contained a <i>T. adhaerens</i> ortholog (orange circle) and 180 did not. As a comparison to the small number of core metazoan-specific genes, the size of the gene space (total numbers of homologous protein families with at least two members, paralogs included) in metazoan (blue circle) and non-metazoan (red circle) organisms are indicated. Not drawn to scale. C. KEGG functional categories containing disproportionately high or low numbers of human metazoan-specific orthologs. The proportion of metazoan-specific genes belonging to each category is compared with the proportion of human genes in the KEGG database belonging to the same functional category. Categories for which there is a statistically significant difference within a functional category are shown (<i>p</i><0.05 hypergeometric test with Bejamini-Hochburg multiple hypothesis correction). D. RNAi phenotypes of the metazoan-specific genes in <i>C. elegans</i>. Fractions of metazoan-specific genes and conserved eukaryotic genes associated with each RNAi phenotype group, are compared. Phenotypic groups are described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003804#s4" target="_blank">Materials and Methods</a> section. Any phenotype shown relative to the total number of genes with RNAi data. Other phenotypes shown as proportional to the number of genes that display any phenotype. For all four phenotypic groups there is a statistically significant difference between the two sets of genes (Fisher exact test, <i>p</i><0.001). E. Functional interactions analysis. Network representing interactions between metazoan-specific genes, conserved eukaryotic genes and between those two groups. Nodes (representing genes) are colored according to their phylogenetic class: metazoan-specific (blue) and conserved core-eukaryotic (red); edges represent interactions. Distribution of the average proportion of interactions for the two sets of genes. Metazoan-specific and conserved eukaryotic genes interact approximately equally with each other.</p

Expression patterns of metazoan-specific genes in <i>C. elegans</i>.

<p>A. Global analysis of expression patterns of metazoan and widely conserved eukaryotic genes. Expression patterns were categorized as neuronal, muscle, intestinal, secretory/excretory, hypodermal or reproductive, and quantified using GExplore <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003804#pgen.1003804-Hutter1" target="_blank">[14]</a>. The left panel shows only tissue-specific expression and the right panel shows all expression. Grey bars show novel metazoan-specific expression patterns determined in this study, blue lines show expression patterns of metazoan-specific genes (previously characterized plus the 43 novel expression patterns collected in this study), and red lines show expression patterns of widely conserved eukaryotic genes. Asterisks indicate statistically-significant differences between metazoan (blue) and conserved eukaryotic (red) genes (<i>p</i><0.05, Fischer's exact test). B–F. Sampling of novel expression patterns of genes investigated in this study. (B) Pan-neuronal expression of D2092.5 <i>(maco-1)</i>, ortholog of macoilin, a transmembrane and coiled-coil domain-containing protein. Our data are consistent with studies published during the course of our investigation that showed neuronal-specific expression pattern for this protein and an involvement in neuronal functions in <i>C. elegans </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003804#pgen.1003804-Miyara1" target="_blank">[42]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003804#pgen.1003804-ArellanoCarbajal1" target="_blank">[43]</a>, motoneurons (mns) in the ventral nerve cord are indicated. (C) Neuronal-specific expression of C15C8.4, homolog of LRPAP1 a low-density lipoprotein receptor-related protein tentatively associated with degenerative dementia <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003804#pgen.1003804-Pandey1" target="_blank">[77]</a>. Cells expressing C15C8.4 includes RIF or RIG, RIS, 8–10 additional head neurons, and PVT/ALN (a pair of tail neurons), (intestine staining here is unspecific). (D) W09G3.7, homolog of WBSCR16 a predicted RCC1-like nucleotide exchange factor is expressed in a few tissues, hypoderm, intestine and a pair of sensory neurons. (E) C34C12.4, homolog of human C4orf34, is expressed in a subset of neurons in the head, body wall muscle (bwm), intestinal cells, gland cells (gc), vulva muscle and anal depressor (adp). (F) Nearly ubiquitous expression of F09G2.2, a cyclin domain-containing protein homolog to human C2orf34; neuronal and non-neuronal cells in head and tail, pharyngeal muscle (pm), body wall muscle, hypoderm, intestinal cells. Except for D2092.5, the genes are functionally uncharacterized.</p

The feasibility of task-sharing the identification, emergency treatment, and referral for women with pre-eclampsia by community health workers in India

Background: Hypertensive disorders are the second highest direct obstetric cause of maternal death after haemorrhage, accounting for 14% of maternal deaths globally. Pregnancy hypertension contributes to maternal deaths, particularly in low- and middle-income countries, due to a scarcity of doctors providing evidence-based emergency obstetric care. Task-sharing some obstetric responsibilities may help to reduce the mortality rates. This study was conducted to assess acceptability by the community and other healthcare providers, for task-sharing by community health workers (CHW) in the identification and initial care in hypertensive disorders in pregnancy. Methods: This study was conducted in two districts of Karnataka state in south India. A total of 14 focus group discussions were convened with various community representatives: women of reproductive age (N = 6), male decision-makers (N = 2), female decision-makers (N = 3), and community leaders (N = 3). One-to-one interviews were held with medical officers (N = 2), private healthcare OBGYN specialists (N = 2), senior health administrators (N = 2), Taluka (county) health officers (N = 2), and obstetricians (N = 4). All data collection was facilitated by local researchers familiar with the setting and language. Data were subsequently transcribed, translated and analysed thematically using NVivo 10 software. Results: There was strong community support for home visits by CHW to measure the blood pressure of pregnant women; however, respondents were concerned about their knowledge, training and effectiveness. The treatment with oral antihypertensive agents and magnesium sulphate in emergencies was accepted by community representatives but medical practitioners and health administrators had reservations, and insisted on emergency transport to a higher facility. The most important barriers for task-sharing were concerns regarding insufficient training, limited availability of medications, the questionable validity of blood pressure devices, and the ability of CHW to correctly diagnose and intervene in cases of hypertensive disorders of pregnancy. Conclusion: Task-sharing to community-based health workers has potential to facilitate early diagnosis of the hypertensive disorders of pregnancy and assist in the provision of emergency care. We identified some facilitators and barriers for successful task-sharing of emergency obstetric care aimed at reducing mortality and morbidity due to hypertensive disorders of pregnancy.Medicine, Faculty ofOther UBCNon UBCObstetrics and Gynaecology, Department ofReviewedFacult