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

Abstract

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Figure 1. qRT-PCR validation of expression changes identified by microarray.

Expression values were normalized to the housekeeping gene Sdha, and are depicted as a ratio of expression in Gtf2ird1^−/− mice relative to WT littermates. * p<0.05, ** p<0.005 using Student's t-test. A. Expression in whole brain from P0 mice. RNA from 9 mice of the same genotype was pooled together to make cDNA (n = 3 pools/genotype). B. Expression in heads of E15.5 mouse embryos (n = 5/genotype).

Figure 2. qRT-PCR of candidate genes in P0 brain from mice of different genetic backgrounds.

Expression values are shown relative to the housekeeping gene Sdha. (For presentation purposes, some values were scaled as indicated). WT 129S1/SvImJ (n = 7), Gtf2ird1^−/− (n = 5), WT CD1 (n = 6). * p<0.05, ** p<0.005 using Student's t-test.

Figure 3. Knockdown of Gtf2ird1 in neuronal cell lines Neuro2A and N1E-115.

Expression values are shown relative to the housekeeping gene Sdha. A. Three different pools of Gtf2ird1-siRNAs were tested, each in two separate transfections. Expression of the housekeeping gene Hmbs was not affected by siRNA treatment. B. Expression of candidate genes in Gtf2ird1-siRNA treated neuronal cell lines. Expression of genes transfected with each of the different Gtf2ird1-siRNA pools were averaged together (n = 6). No statistically significant changes in expression were detected between Gtf2ird1-siRNA treated cells and non-targeting siRNA treated or untreated cells using Student's t-test.

Figure 4. Expression of Stx3 and Mrpl16 show natural inter-individual variation.

Expression is shown relative to the housekeeping gene Sdha. A. Expression in the heads of individual E15.5 mouse embryos. Considerable variation in expression within the 3′ UTR can be seen for both genes within each genotype group. Each bar on the graph represents the expression level of an individual mouse, and the same 10 mice are shown in the same order for each primer pair. B. Expression of Stx3 in P0 and adult brain from Gtf2ird1^−/− mice and control littermates. Each dot represents the expression of Stx3 in an individual mouse, and horizontal bars represent the mean expression level for the group.

Figure 5. qRT-PCR of previously identified in vitro targets of GTF2IRD1.

Expression values were normalized to the housekeeping gene Sdha, and are depicted as a ratio of expression in Gtf2ird1^−/− or Gtf2ird1^+/− mice relative to WT. No statistically significant differences in expression were detected between genotypes using Student's t-test. A. Expression of Bmpr1b and Fgf15 in whole brain of P0 mice (n = 3/genotype). B. Expression of previously identified in vitro targets of GTF2IRD1 in primary MEFs. MEFs were cultured from WT (n = 3), Gtf2ird1^+/− (n = 2), and Gtf2ird1^−/− (n = 1) E15.5 mouse embryos. Hmbs is a housekeeping gene and was used as a control. C. Expression of previously identified in vitro targets of GTF2IRD1 in whole brains. E18.5 (n = 3/genotype), adult (WT, n = 3; Gtf2ird1^−/−, n = 2) mice. Hprt is a housekeeping gene and was used as a control.