Mountain View
biomedical and healthCAre Data Discovery Index Ecosystem
help Advanced Search
Title: RNAi knock down of C.elegans mex-3 (zu155) mutant worms (JJ518) reveals pairing competitive and topologically distinct regulatory modules enhances patterned gene expression      
availability:
available
aggregation:
instance of dataset
privacy:
not applicable
refinement:
curated
dateReleased:
06-10-2011
ID:
E-GEOD-9665
description:
Biological networks are inherently modular, yet little is known about how modules are assembled to enable coordinated and complex functions. We used RNAi and time-series, whole-genome microarray analyses to systematically perturb and characterize components of a C. elegans lineage-specific transcriptional regulatory network. These data are supported by select reporter gene analyses and comprehensive yeast-one-hybrid and promoter sequence analyses. Based on these results we define and characterize two modules composed of muscle- and epidermal-specifying transcription factors that function together within a single cell lineage to robustly specify multiple cell types. The expression of these two modules, although positively regulated by a common factor, is reliably segregated among daughter cells. Our analyses indicate that these modules repress each other, and we propose that this cross-inhibition coupled with their relative time of induction function to enhance the initial asymmetry in their expression patterns, thus leading to the observed invariant gene expression patterns and cell lineage. The coupling of asynchronous and topologically distinct modules may be a general principle of module assembly that functions to potentiate genetic switches. Experiment Overall Design: To study the function of the C-lineage gene regulatory network we used RNAi to knockdown expression of the 13 TFs expressed early in the C lineage, and then used whole-genome microarrays to assess each perturbation for changes in mRNA abundance. RNAi has several advantages as a method to perturb gene function: it reduces both maternal and zygotic transcripts, thus avoiding maternal rescue effects; for essential genes it allows collection of fully affected progeny; and it enables comparison between perturbations. Measuring transcript levels on microarrays is also advantageous, because it is highly parallel, allowing direct comparisons between genes within each perturbation, and it is comprehensive, thus enabling discovery of regulated genes. In addition, it allows direct comparison to previous efforts to characterize the pal-1 regulatory network (Baugh et al, 2005a; Baugh et al, 2005b). To increase sensitivity and specificity, we used mex-3 mutant mothers whose progeny are primarily composed of C-like lineages (Draper et al, 1996). In each case, RNA was collected from embryos two and three cell cycles after the initial zygotic expression of the targeted TF mRNA (Figure 1). We note that our sampling of only two time points might underestimate the magnitude of the observed effect of RNAi on mRNA levels, particularly if the expression level of the assayed mRNA peaks before or after the selected time points. The paralogous tbx-8 and tbx-9 genes, which were previously demonstrated to be functionally redundant (Pocock et al, 2004b; Baugh et al, 2005a), were simultaneously targeted by RNAi in order to assay a more penetrant effect on gene expression. Experiment Overall Design: mex-3 (zu155) mutant worms (JJ518) were used in this experiment. RNAi was administered by soaking and RNA was collected from embryos, both as previously described (Baugh 2005; Baugh et al, 2005b). Cohorts of ten embryos were used for each RNA preparation, and half of the RNA was used for linear amplification. The Artus “ExpressArt mRNA Amplification Kit” was used for amplification. The manufacturer’s protocol was modified in round one with a ten-fold increase in concentration of primers A, B, and C and a four-fold decrease in cDNA synthesis reaction volumes. Microarray hybridization onto Affymetrix C. elegans GeneChip, scanning, data reduction and analysis were done as previously described (Baugh et al, 2003). Expression values were normalized using RMA (Bolstad et al, 2003).
keywords:
RNAi profiling by array
format:
HTML
storedIn:
Array Express
qualifier:
not compressed
accessType:
landing page
authorization:
none
authentication:
none
primary:
true
accessURL: https://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-9665
format:
JSON
storedIn:
OmicsDI
qualifier:
not compressed
accessType:
download
authorization:
none
authentication:
none
primary:
false
accessURL: www.omicsdi.org/ws/dataset/arrayexpress-repository/E-GEOD-9665.json
format:
XML
storedIn:
OmicsDI
qualifier:
not compressed
accessType:
download
authorization:
none
authentication:
none
primary:
false
accessURL: http://www.omicsdi.org/ws/dataset/arrayexpress-repository/E-GEOD-9665.xml
ID:
SCR:014747
name:
Omics Discovery Index
abbreviation:
OmicsDI
homePage: http://www.omicsdi.org/