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Series GSE4230

Query DataSets for GSE4230

Status

Public on Feb 13, 2006

Title

Gene expression profiles in developing nephrons using Lim1 metanephric mesenchyme-specific conditional mutant mice

Organism

Mus musculus

Experiment type

Expression profiling by array

Summary

BACKGROUND: Lim1 is a homeobox gene that is essential for nephrogenesis. During metanephric kidney development, Lim1 is expressed in the nephric duct, ureteric buds, and the induced metanephric mesenchyme. Conditional ablation of Lim1 in the metanephric mesenchyme blocks the formation of nephrons at the nephric vesicle stage, leading to the production of small, non-functional kidneys that lack nephrons.
METHODS: In the present study, we used Affymetrix probe arrays to screen for nephron-specific genes by comparing the expression profiles of control and Lim1 conditional mutant kidneys. Kidneys from two developmental stages, embryonic day 14.5 (E14.5) and 18.5 (E18.5), were examined.
RESULTS: Comparison of E18.5 kidney expression profiles generated a list of 465 nephron-specific gene candidates that showed a more than 2-fold increase in their expression level in control kidney versus the Lim1 conditional mutant kidney. Computational analysis confirmed that this screen enriched for kidney-specific genes. Furthermore, at least twenty-eight of the top fifty (56%) candidates (or their vertebrate orthologs) were previously reported to have a nephron-specific expression pattern. Our analysis of E14.5 expression data yielded 41 candidate genes that are up-regulated in the control kidneys compared to the conditional mutants. Three of them are related to the Notch signaling pathway that is known to be important in cell fate determination and nephron patterning.
CONCLUSIONS: Therefore, we demonstrate that Lim1 conditional mutant kidneys serve as a novel tissue source for comprehensive expression studies and provide a means to identify nephron-specific genes.
Keywords: tissue specificity, time course, development, kidney, metanephric mesenchyme, nephron, Lim1, knockout mice, conditional knockout

Overall design

Microarray experiment on each time point and genotype was performed in technical duplicates (ie. a single RNA preparation of pooled kidneys of one genotype used for two separate target preparations). Data from a total of 8 independent arrays were used in this study, 2 arrays were used for RNA samples from E18.5 control kidneys, 2 for E18.5 Lim1 conditional mutant kidneys, 2 for E14.5 control kidneys, and the other 2 for E14.5 Lim1 conditional mutant kidneys. Data generated from all arrays that satisfied the preliminary analysis were exported and loaded into DNA-Chip Analyzer (dChip2004), where statistical and comparative analyses were performed to verify the data. The data were normalized using the default normalization method. Briefly, an iterative procedure was used to identify an invariant set of probes, which presumably consisted of non-differentially expressed genes. A piecewise-linear running median curve was then calculated and used as the normalization curve. After normalization, all arrays had similar brightness. Median intensities around 155 (between 155 to 158) were obtained after normalization. Percent gene present (P call%) values between 55.7% and 63.6% were observed using default detection p-value cut offs (a1=0.04 and a2=0.06). Array outlier (%) and single outlier (%) were detected at ranges from 0.016% to 0.080% and from 0.009% to 0.045%. Expression data obtained from all arrays used are provided.
Normalized data were exported in a tab delimited text format. Fold changes of each transcript from different samples were calculated and sorted using Microsoft Excel 5.0 software. Signal obtained from control kidney samples were used as an experiment to compare to the signal obtained from Lim1 conditional mutant kidneys that was designated as a baseline. A 2-fold change in the means of signal obtained from experimental duplicates and those from baseline duplicates was used as the criterion to identify differentially expressed transcripts. To ensure the quality of the data, probe sets that showed a fold change between duplicates greater than between the experimental mean and baseline mean were removed. To study only genes that showed consistent expression on experimental chips, probe sets that did not show consistent present calls in the experimental duplicates were removed. To focus on genes with a significant fold change between the experiment and the baseline, only probe sets that the product of their experimental mean and fold change were more than 100 were retained. To produce a compact differentially expressed gene list, the probe set list was sorted within Microsoft Excel based on Locus Link number and redundant entries were removed. Our experimental design description and the data format provided in the Additional files fulfill the MIAME (minimum information about a microarray experiment) standards.

Contributor(s)

Chen Y, Kobayashi A, Kwan K, Johnson RL, Behringer RR

Citation(s)

16464245

Submission date

Feb 10, 2006

Last update date

Feb 11, 2019

Contact name

You-Tzung Chen

E-mail(s)

ytchen@mdanderson.org