14 research outputs found
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
Identification of 526 Conserved Metazoan Genetic Innovations Exposes a New Role for Cofactor E-like in Neuronal Microtubule Homeostasis
The evolution of metazoans from their choanoflagellate-like unicellular ancestor coincided with the acquisition of novel biological functions to support a multicellular lifestyle, and eventually, the unique cellular and physiological demands of differentiated cell types such as those forming the nervous, muscle and immune systems. In an effort to understand the molecular underpinnings of such metazoan innovations, we carried out a comparative genomics analysis for genes found exclusively in, and widely conserved across, metazoans. Using this approach, we identified a set of 526 core metazoan-specific genes (the ‘metazoanome’), approximately 10% of which are largely uncharacterized, 16% of which are associated with known human disease, and 66% of which are conserved in Trichoplax adhaerens, a basal metazoan lacking neurons and other specialized cell types. Global analyses of previously-characterized core metazoan genes suggest a prevalent property, namely that they act as partially redundant modifiers of ancient eukaryotic pathways. Our data also highlights the importance of exaptation of pre-existing genetic tools during metazoan evolution. Expression studies in C. elegans revealed that many metazoan-specific genes, including tubulin folding cofactor E-like (TBCEL/coel-1), are expressed in neurons. We used C. elegans COEL-1 as a representative to experimentally validate the metazoan-specific character of our dataset. We show that coel-1 disruption results in developmental hypersensitivity to the microtubule drug paclitaxel/taxol, and that overexpression of coel-1 has broad effects during embryonic development and perturbs specialized microtubules in the touch receptor neurons (TRNs). In addition, coel-1 influences the migration, neurite outgrowth and mechanosensory function of the TRNs, and functionally interacts with components of the tubulin acetylation/deacetylation pathway. Together, our findings unveil a conserved molecular toolbox fundamental to metazoan biology that contains a number of neuronally expressed and disease-related genes, and reveal a key role for TBCEL/coel-1 in regulating microtubule function during metazoan development and neuronal differentiation
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
<i>coel-1</i> interacts genetically with regulators of tubulin acetylation.
<p>A–B. Loss of tubulin acetylation in the <i>mec-17</i>;<i>atat-2</i> double mutant suppresses the effect of <i>coel-1</i> deletion on AVM cell body positioning, and enhances the phenotype caused by overexpression of <i>coel-1</i>. C. Images showing that the normally small or non-existent ALM posterior process (top panel) extends abnormally in <i>mec-17;atat-2</i> animals (middle panel), a phenotype that is rescued by the overexpression of <i>coel-1</i> (bottom panel). The graph shows the quantitation of the average length of the process expressed in cell body equivalent +/− SEM. D. PLM length defect in <i>coel-1</i> animals is suppressed when combined with <i>mec-17;atat-2</i> mutations. E–F. <i>hdac-6</i> and <i>mec-17;atat-2</i> mutations respectively suppress, or enhance, the AVM neurite termination defect (E) and the touch sensitivity response (F) observed in <i>coel-1XS</i> animals. The mean of touch sensitivity responses ± SEM is represented (n≥30 worms/genotype). G. AVM/PVM cell number defect (extra = 2 or missing = 0) in the <i>coel-1XS</i> animals are reduced significantly by the <i>hdac-6</i>, but not the <i>mec-17;atat-2</i> mutations. H. Quantitative western blot analyses of total acetylated α-tubulin (Acet-tub) using the 6-11B-1 antibody on lysates from young adults. Actin served as loading control and was used to normalize the amounts of protein loaded between samples. Data are normalized to WT (wild-type) intensity (n = 3) and error bars are SEM. No statistically significant difference is observed between <i>coel-1</i>, <i>coel-1XS</i> or <i>hdac-6</i> compared to wild-type. The positive control strain, <i>mec-17;atat-2</i>, as expected lacks acetylated tubulin. Brackets indicate the total number of neurons scored. Statistical significances were determined using Student's <i>t</i>-test for all panels except B, E and G for which Fisher's exact test was used. *, <i>p</i>≤0.05, **; <i>p</i>≤0.005; ***, <i>p</i>≤0.0005; n.s = not significant.</p
<i>coel-1</i> activity influences microtubule number and length, but not protofilament count in PLM touch receptor neurons.
<p>A. High-resolution electron micrographs of thin (50 nm) sections of PLM touch receptor neurons in wild-type (WT), <i>coel-1XS</i> and <i>coel-1(tm2136)</i> animals. Insets show a single microtubule profile, revealing the protofilaments. Scale bar, 100 nm. B. Number of MTs per section as a function of genotype. Bars are the mean values and filled circles are the average values in each dataset. C. Microtubule length as a function of genotype. Bars are the mean and filled circles are the number of serial section datasets tested. Microtubule (MT) length computed from: <i>L</i> = 2<i>Na</i>/T, where <i>N</i> = the average number of MTs/section, <i>a</i> = total length of serial reconstruction and <i>T</i> = number of MT endpoints observed <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003804#pgen.1003804-Chalfie2" target="_blank">[25]</a>. In panels B and C, a total of at least 7 µm was reconstructed for each genotype. *, p<0.05, Wilcoxon-Rank test compared to wild-type; n.s, not significantly different.</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