Transcription profiling of mouse telencephalon primary cultures from Xbp1 knockouts and wild-type controls

ABSTRACT: XBP1 is a transcription factor that is induced by unconventional splicing associated with endoplasmic reticulum stress and plays a role in development of liver and plasma cells. We previously reported that brain derived neurotrophic factor (BDNF) leads to splicing of XBP1 mRNA in neurites, and that XBP1 is required for BDNF-induced neurite extension and branching. To search for the molecular mechanisms of how XBP1 plays a role in neural development, comprehensive gene expression analysis was performed in primary telencephalic neurons obtained from Xbp1 knockout mice at embryonic day 12.5. By searching for the genes induced by BDNF in wild type neurons but this induction was reduced in Xbp1 knockout mice, we found that upregulation of three GABAergic markers, somatostatin (Sst), neuropeptide Y (Npy), and calbindin (Calb1), were compromised in Xbp1 knockout neurons. Attenuated induction of Npy and Calb1 was confirmed by quantitative RT-PCR. In neurons lacking in Xbp1, upregulation of GABAergic markers was attenuated. Impaired BDNF-induced neurite extension in Xbp1 knockout neurons might be mediated by disturbed BDNF-induced differentiation of GABAergic interneurons. Experiment Overall Design: Brain derived neurotrophic factor (BDNF) effect examined in telencephalon primary cultures from Xbp1 knockouts and wild-type controls. Experiment Overall Design: Two female Xbp1 +/- mice were mated with male Xbp1 +/- mice, and at embryonic day 12.5 (E12.5) the embryos were dissected. Among the 25 embryos obtained from the 2 pregnant female mice, 6 were genotyped as Xbp1 -/- and 4 were Xbp1 +/+ by a rapid PCR assay using Z-Taq. Telencephalon was dissected from each embryo, and treated with collagenase and trypsin. Six Xbp1 -/- telencephalon samples and 4 Xbp1 +/+ samples were collected together, respectively. Each sample was divided into 15 aliquots, and the cells were subject to low density culture on plastic culture dishes. The neurons were maintained in a serum-free medium (Neurobasal medium supplemented with 0.5 mM glutamine and B27 supplement [Invitrogen]). On the third day in vitro (3 DIV) , neurons in 10 of 15 dishes in each group were stimulated with BDNF (100 ng/ml). On 4 DIV, neurons in 5 dishes stimulated with BDNF (24 hours BDNF treatment) and 5 dishes with no stimulation (0 hour) were lysed to extract total RNA. On 5 DIV, neurons in 5 dishes stimulated with BDNF (48 hours BDNF treatment) were lysed. Experiment Overall Design: Total RNA was extracted using RNAeasy Micro Kit (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer. The quantity and quality of RNA were measured using NanoDrop ND-1000. Biotin-labeled cRNA for DNA microarray analysis was synthesized using Two Cycle cDNA Synthesis and IVT labeling Kit. The integrity of the cRNA samples was verified using Test3 Array. Each biotin-labeled cRNA sample from one dish was hybridized to a single Affymetrix GeneChip Mouse Genome 430A 2.0 Array, and totally 30 arrays were used. The hybridization signal on the chip was scanned using an HP GeneArray scanner and processed by MAS5. After imported into GeneSpring software, data normalization was performed by dividing each microarray data set by its median value. Probes called as present in at least half of the 30 samples were selected.

ORGANISM(S): Mus musculus

SUBMITTER: Kazuya Iwamoto 

PROVIDER: E-GEOD-11322 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress