ABSTRACT: Background. Nuclear factor I-A (NFI-A), a phylogenetically conserved transcription/replication protein, plays a crucial role in mouse brain development. Previous studies showed that disruption of the Nfia gene in mice leads to perinatal lethality, corpus callosum agenesis, and hydrocephalus. Results. To identify potential NFI-A target genes involved in the observed tissue malformations, we analyzed gene expression in brains from NFI-A-deficient and wild-type littermate control mice at the mRNA level using oligonucleotide microarrays. In young postnatal animals (P16), 356 genes were detected as differentially regulated, whereas at the late embryonic stage (E18), only 5 dysregulated genes were found. An in silico analysis identified phylogenetically conserved NFI binding sites in at least 70 of the differentially regulated genes. Moreover, assignment of gene function showed that marker genes for immature neural cells and neural precursors were expressed at elevated levels in young postnatal NFI-A mutants. By contrast, marker genes for differentiated neural cells were down-regulated at this stage. In particular, genes relevant for oligodendrocyte differentiation were affected. Conclusions. Our results suggest that a delay in early postnatal development, especially oligodendrocyte maturation, is exhibited by NFI-A knock-out mice, and at least partly accounts for their phenotype. The identification of potential NFI-A target genes by our study should both help to elucidate NFI-A-dependent transcriptional pathways and contribute to a better understanding of this period of brain formation, especially with regard to the function of NFI-A. Material and method Animals NFI-A-deficient mice were bred and maintained as described [20]. Animals were sacrificed by CO2 at embryonic stage E18.5 (E18) or postnatal day 16 (P16). Brains were dissected and snap frozen in liquid nitrogen. The respective KO and WT animals used for analysis at E18 and P16 were littermates. Only male mice were used. All of the animals were F1 hybrids of C57Bl/6 and 129S6 animals. Within these matings, males had a B6 and females had a 129S6 background. In a large scale test, 38.5% of the offspring survive until P30 using this kind of mating. In purebred B6, we observed 0% survival at P1 (R.M.G., unpublished observations). All animal use was performed under the approved protocol BCH05082N (RMG) of the University at Buffalo Institutional Animal Care and Use Committee in the UB Laboratory Animal Facility, an AAALAC licensed facility. RNA preparation Total RNA was extracted from the entire brain of NF-I-A-deficient and wild-type control mice (n=3 each) using TRIzol (Invitrogen) according to the manufacturer’s instructions. Further purification of RNA was performed with the RNeasy Mini Kit (Qiagen). Total RNA concentration was determined using a spectrophotometer at 260 nm and 280 nm wavelength. To check RNA integrity, total RNA was separated in a 1% agarose gel containing formaldehyde, and the intensity ratio of 28S and 18S ribosomal RNA bands was assessed after ethidium bromide staining. Microarray hybridization and signal detection Procedures for cDNA synthesis, labeling and hybridization were carried out according to the manufacturer's protocol (Affymetrix). All experiments were performed using Affymetrix mouse genome Genechip U74A version 2. Briefly, 15mg of total RNA were used for first strand cDNA synthesis with an HPLC-purified T7-(dT)24 primer. Synthesis of biotin-labeled cRNA was carried out using the ENZO RNA transcript labeling kit (Affymetrix). For hybridization, 15 µg of fragmented cRNA were incubated with the chip in 200 µl of hybridization solution in Hybridization Oven 640 (Affymetrix) at 45oC for 16 hours. Genechips were washed and stained with streptavidin-phycoerythrin using the microfluidic workstation™ (Affymetrix) and scanned with a laser scanner (Agilent Technologies). Microarray quantification and statistical analysis Quality controls were performed using Affymetrix Microarray Suite 5.0 (MAS 5.0) software. Data and statistical analysis and data visualization were performed with GeneSpring™ (Agilent Technologies). Expression values were extracted from the CEL file, and imported to GeneSpring using Robust Multi-array Average (RMA) for further analysis [24]. Two criteria were used to select for significant changes in gene expression. First, probe sets with less than 1.2 fold change comparing the KO to WT (control) were removed in order to reduce the number of genes undergoing statistical analysis. Second, genes were then tested statistically for significant changes in all three wild-type or NFI-A-deficient samples, using Student's t-test. Finally, genes were grouped based on their biological function using Affymetrix NetAffx, and NCBI Locuslink. Keywords: biotinylated cRNA template
