Project description: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. Keywords: time-cource, genetic modification
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other
Project description:Rationale: A previous transcriptome meta-analysis revealed significantly lower levels of corticotropin-releasing hormone (CRH) mRNA in corticolimbic brain regions in major depressive disorder (MDD) subjects. Rodent studies show that cortical CRH is mostly expressed in GABAergic neurons; however, the characteristic features of CRH+ cells in human brain cortex and their association with MDD are largely unknown. Methods: Subgenual anterior cingulate cortex (sgACC) of human subjects without brain disorders were labeled using fluorescent in situ hybridization (FISH) for CRH and markers of excitatory (SLC17A7), inhibitory (GAD1) neurons, as well as markers of other interneuron subpopulations (PVALB, SST, VIP). MDD-associated changes in CRH+ cell density and cellular CRH expression (n=6/group) were analyzed. RNA-sequencing was performed on sgACC CRH+ neurons from comparison and MDD subjects (n=6/group), and analyzed for group differences. Results: About 80% of CRH+ cells were GABAergic and 17.5% were glutamatergic. CRH+ GABAergic neurons co-expressed VIP (52%), SST (7%), or PVALB (7%). MDD subjects displayed lower CRH mRNA levels in GABAergic neurons relative to comparison subjects without changes in cell density. CRH+ neurons show transcriptomic profile suggesting lower excitability and less GABA release and reuptake. Further analyses suggested that these molecular changes are not mediated by altered glucocorticoid feedback and potentially occur downstream for a common modulator of neurotrophic function. Summary: CRH+ cells in human sgACC are a heterogeneous population of GABAergic neurons, although largely co-expressing VIP. MDD is associated with reduced markers of inhibitory function of CRH+ neurons.
Project description:Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of growth factors, which generally tend to result in mixed populations of neurons. Here we report that over-expression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron types. Analysis of published data on gene expression changes early (two days) after induction of each of 185 induced TFs implicated candidate TFs for further ESC differentiation studies. After induction for 6 days four of them (Ascl1, Smad7, Nr2f1, and Ascl2) generated a high proportion (>35%) of cells with neural progenitor marker PSA-NCAM and clear neural morphology on day 14. The capacity of these TFs to induce neural differentiation is inferred to be most likely linked to early activation of the Notch signaling pathway. Among the neuron-like cells, GABA-positive cells were most abundant (32-97% for 4 top TFs), whereas Isl1-positive cells and TH-positive cells were less abundant (<12% and <5%, respectively). Enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using beads with anti-PSA-NCAM antibody resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and highly expressed markers of GABAergic neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific and GABAergic-specific mRNA and miRNAs. We identified mRNA and miRNAs, whose expression depended on the induction of Ascl1, and showed that they were enriched in Ascl1 target genes. In summary, this study indicates that induction of transcription factors is a promising approach to generate candidate specific neural cell types from ESCs. Transcription factor Ascl1 was induced in mouse ESCs to facilitate neural differentiation. Expression of transgenic Ascl1 was repressed by doxycycline (Dox); thus, it were induced in Dox- conditions, whereas Dox+ conditions represent control cells with no expression of Ascl1 transgene. For neural differentiation, cells were cultured 3 days in alpha-MEM medium and then - in NeuroCult neural differentiation medium for 2-11 days (total up to 14 days). RNA was extracted with mirVana kit (Thermo Fisher Scientific).
Project description:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.
Project description:Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of growth factors, which generally tend to result in mixed populations of neurons. Here we report that over-expression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron types. Analysis of published data on gene expression changes early (two days) after induction of each of 185 induced TFs implicated candidate TFs for further ESC differentiation studies. After induction for 6 days four of them (Ascl1, Smad7, Nr2f1, and Ascl2) generated a high proportion (>35%) of cells with neural progenitor marker PSA-NCAM and clear neural morphology on day 14. The capacity of these TFs to induce neural differentiation is inferred to be most likely linked to early activation of the Notch signaling pathway. Among the neuron-like cells, GABA-positive cells were most abundant (32-97% for 4 top TFs), whereas Isl1-positive cells and TH-positive cells were less abundant (<12% and <5%, respectively). Enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using beads with anti-PSA-NCAM antibody resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and highly expressed markers of GABAergic neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific and GABAergic-specific mRNA and miRNAs. We identified mRNA and miRNAs, whose expression depended on the induction of Ascl1, and showed that they were enriched in Ascl1 target genes. In summary, this study indicates that induction of transcription factors is a promising approach to generate candidate specific neural cell types from ESCs.
Project description:Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of growth factors, which generally tend to result in mixed populations of neurons. Here we report that over-expression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron types. Analysis of published data on gene expression changes early (two days) after induction of each of 185 induced TFs implicated candidate TFs for further ESC differentiation studies. After induction for 6 days four of them (Ascl1, Smad7, Nr2f1, and Ascl2) generated a high proportion (>35%) of cells with neural progenitor marker PSA-NCAM and clear neural morphology on day 14. The capacity of these TFs to induce neural differentiation is inferred to be most likely linked to early activation of the Notch signaling pathway. Among the neuron-like cells, GABA-positive cells were most abundant (32-97% for 4 top TFs), whereas Isl1-positive cells and TH-positive cells were less abundant (<12% and <5%, respectively). Enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using beads with anti-PSA-NCAM antibody resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and highly expressed markers of GABAergic neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific and GABAergic-specific mRNA and miRNAs. We identified mRNA and miRNAs, whose expression depended on the induction of Ascl1, and showed that they were enriched in Ascl1 target genes. In summary, this study indicates that induction of transcription factors is a promising approach to generate candidate specific neural cell types from ESCs.