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Global analysis of gene expression in the developing brain of Gtf2ird1 knockout mice.

ABSTRACT: Williams-Beuren Syndrome (WBS) is a neurodevelopmental disorder caused by a hemizygous deletion of a 1.5 Mb region on chromosome 7q11.23 encompassing 26 genes. One of these genes, GTF2IRD1, codes for a putative transcription factor that is expressed throughout the brain during development. Genotype-phenotype studies in patients with atypical deletions of 7q11.23 implicate this gene in the neurological features of WBS, and Gtf2ird1 knockout mice show reduced innate fear and increased sociability, consistent with features of WBS. Multiple studies have identified in vitro target genes of GTF2IRD1, but we sought to identify in vivo targets in the mouse brain.We performed the first in vivo microarray screen for transcriptional targets of Gtf2ird1 in brain tissue from Gtf2ird1 knockout and wildtype mice at embryonic day 15.5 and at birth. Changes in gene expression in the mutant mice were moderate (0.5 to 2.5 fold) and of candidate genes with altered expression verified using real-time PCR, most were located on chromosome 5, within 10 Mb of Gtf2ird1. siRNA knock-down of Gtf2ird1 in two mouse neuronal cell lines failed to identify changes in expression of any of the genes identified from the microarray and subsequent analysis showed that differences in expression of genes on chromosome 5 were the result of retention of that chromosome region from the targeted embryonic stem cell line, and so were dependent upon strain rather than Gtf2ird1 genotype. In addition, specific analysis of genes previously identified as direct in vitro targets of GTF2IRD1 failed to show altered expression.We have been unable to identify any in vivo neuronal targets of GTF2IRD1 through genome-wide expression analysis, despite widespread and robust expression of this protein in the developing rodent brain.

SUBMITTER: O'Leary J

PROVIDER: S-EPMC3166129 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Global analysis of gene expression in the developing brain of Gtf2ird1 knockout mice.

O'Leary Jennifer J Osborne Lucy R LR

PloS one 20110831 8

<h4>Background</h4>Williams-Beuren Syndrome (WBS) is a neurodevelopmental disorder caused by a hemizygous deletion of a 1.5 Mb region on chromosome 7q11.23 encompassing 26 genes. One of these genes, GTF2IRD1, codes for a putative transcription factor that is expressed throughout the brain during development. Genotype-phenotype studies in patients with atypical deletions of 7q11.23 implicate this gene in the neurological features of WBS, and Gtf2ird1 knockout mice show reduced innate fear and inc ...[more]

PMID: 21909369

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Project description:PurposeWe found earlier that 42 day-old Egr-1 knockout mice had longer eyes and a more myopic refractive error compared to their wild-types. To identify genes that could be responsible for the temporarily enhanced axial eye growth, a microarray analysis was performed in knockout and wild-type mice at the postnatal ages of 30 and 42 days.MethodsThe retinas of homozygous and wild-type Egr-1 knockout mice (Taconic, Ry, Denmark) were prepared for RNA isolation (RNeasy Mini Kit, Qiagen) at the age of 30 or 42 days, respectively (n=12 each). Three retinas were pooled and labeled cRNA was made. The samples were hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Hybridization signals were calculated using GC-RMA normalization. Genes were identified as differentially expressed if they showed a fold-change (FC) of at least 1.5 and a p-value <0.05. A false-discovery rate of 5% was applied. Ten genes with potential biologic relevance were examined further with semiquantitative real-time RT-PCR.ResultsComparing mRNA expression levels between wild-type and homozygous Egr-1 knockout mice, we found 73 differentially expressed genes at the age of 30 days and 135 genes at the age of 42 days. Testing for differences in gene expression between the two ages (30 versus 42 days), 54 genes were differently expressed in wild-type mice and 215 genes in homozygous animals. Based on three networks proposed by Ingenuity pathway analysis software, nine differently expressed genes in the homozygous Egr-1 knockout mice were chosen for further validation by real-time RT-PCR, three genes in each network. In addition, the gene that was most prominently regulated in the knockout mice, compared to wild-type, at both 30 days and 42 days of age (protocadherin beta-9 [Pcdhb9]), was tested with real-time RT-PCR. Changes in four of the ten genes could be confirmed by real-time RT-PCR: nuclear prelamin A recognition factor (Narf), oxoglutarate dehydrogenase (Ogdh), selenium binding protein 1 (Selenbp1), and Pcdhb9. Except for Pcdhb9, the genes whose mRNA expression levels were validated were listed in one of the networks proposed by Ingenuity pathway analysis software. In addition to these genes, the software proposed several key-regulators which did not change in our study: retinoic acid, vascular endothelial growth factor A (VEGF-A), FBJ murine osteosarcoma viral oncogene homolog (cFos), and others.ConclusionsIdentification of genes that are differentially regulated during the development period between postnatal day 30 (when both homozygous and wild-type mice still have the same axial length) and day 42 (where the difference in eye length is apparent) could improve the understanding of mechanisms for the control of axial eye growth and may lead to potential targets for pharmacological intervention. With the aid of pathway-analysis software, a coarse picture of possible biochemical pathways could be generated. Although the mRNA expression levels of proteins proposed by the software, like VEGF, FOS, retinoic acid (RA) receptors, or cellular RA binding protein, did not show any changes in our experiment, these molecules have previously been implicated in the signaling cascades controlling axial eye growth. According to the pathway-analysis software, they represent links between several proteins whose mRNA expression was changed in our study.

Dataset Information

Global analysis of gene expression in the developing brain of Gtf2ird1 knockout mice.

Publications

Global analysis of gene expression in the developing brain of Gtf2ird1 knockout mice.

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