Project description:We have examined the nuclear (nuc) and cytoplasmic (cyt) polyA+ transcriptomes of undifferentiated mouse embryonic stem cells (un) and cells differentiated to neural precursors (d5) using strand-specific RNA-Seq. The 46C mouse embryonic stem cell line was used for this study.
Project description:We have examined the nuclear (nuc) and cytoplasmic (cyt) polyA+ transcriptomes of undifferentiated mouse embryonic stem cells (un) and cells differentiated to neural precursors (d5) using strand-specific RNA-Seq. The 46C mouse embryonic stem cell line was used for this study. Two cell types were examined: undifferentiated mouse embryonic stem cells (un) and cells differentiated to neural precursors (d5). For each cell type, cells were fractionated to nuclear and cytoplasmic components. RNAs were extracted from each component and were fragmented enzymatically for library construction. For each cell type and component, strand-specific RNA-Seq libraries were generated using at least two different fragmentation protocols.
Project description:The activation of quiescent neural stem cells (qNSCs) in the dentate gyrus is required for lifelong neurogenesis. However, the mechanisms that promote the exit of neural stem cells (NSCs) from quiescence remain elusive. We demonstrate that the expression of plant homeodomain finger protein 2 (Phf2) activates the exit of postnatal mouse NSC from shallow quiescence. Loss of Phf2 prevents NSC activation and neurogenesis in postnatal 30 (P30) mice but does not decrease the label-retaining NSC pool, indicating that Phf2 is not required for the exit of NSC from quiescence. NSC-specific deletion of Phf2 modestly compromises embryonic mouse NSC proliferation without increasing apoptosis, indicating that Phf2 is crucial for embryonic development. Moreover, human cortical organoids reveal that Phf2 promotes NPC proliferation via a lysine demethylase-independent manner. Mechanistically, Phf2 directly binds to the cohesion complex via Rad21 and regulates the DNA replication in mouse NSC by associating with the cohesion complex releasing protein Wapl activity. Our study identifies the Phf2-cohesin complex mediated DNA replication for neural stem cell activation in a lysine demethylase-independent manner.
Project description:Comparison of genomic data from neural progenitor cells derived from mouse embryonic stem cells under different experimetnal conditions in vitro and invivo. We conducted genome-wide RNA sequencing of immunoprecipitated specific ribosome-associated mRNA using RiboTag methods from: (i) mouse embryonic stem cell (ESC), (ii) derived neural progenitor cells, (iii) differentiated neural progenitor cells (in vitro), (iv) grafted neural progenitor cells (recovered from different in vivo tissue enivornments - healthy spinal cord, spinal cord injury lesions) and (v) host astrocytes using GFAp-Cre RiboTag mice.
Project description:Long non-coding RNAs (lncRNAs) comprise a diverse class of transcripts that can regulate molecular and cellular processes in brain development and diseasee. LncRNAs exhibit cell type- and tissue-specific expression, but little is known about the expression and function of lncRNAs in the developing human brain. Here, we deeply profiled lncRNAs from polyadenylated and total RNA obtained from human neocortex at different stages of development and integrated this resource to analyze the transcriptomes of single cells. While lncRNAs were generally detected at low levels in whole tissues, single cell transcriptomics revealed that many lncRNAs are abundantly expressed in individual cells and are cell type-specific. Furthermore, we used CRISRPi to show that LOC646329, a lncRNA enriched in radial glia but detected at low abundance in tissues, regulates cell proliferation. The discrete and abundant expression of lncRNAs among individual cells has important implications for both their biological function and utility for distinguishing neural cell types. 16 Bulk Tissue Samples from GW13-23; 226 Single Cells from GW19.5-23.5 ------------------ bulk_tpm.polya.txt: bulk RNA-seq expression; using polyA full reference scell_ncounts.genes.thresh.txt: single cell RNA-seq expression; using polyA stringent reference; includes 50 GW16, GW21, GW21p3 cells previously analyzed (Pollen et. al. 2014) polya_RNA_stringent_ref.gtf: bulk RNA-seq polyA stringent transcriptome reference polya_RNA_full_ref.gtf: bulk RNA-seq polyA full transcriptome reference total_RNA_stringent_ref.gtf: bulk RNA-seq total stringent transcriptome reference total_RNA_full_ref.gtf: bulk RNA-seq total full transcriptome reference GW13_1_polya_minus.bw: strand-specific bulk RNA-seq alignment signal GW13_1_polya_plus.bw: strand-specific bulk RNA-seq alignment signal GW13_1_total_minus.bw: strand-specific bulk RNA-seq alignment signal GW13_1_total_plus.bw: strand-specific bulk RNA-seq alignment signal GW14.5_1_polya_minus.bw: strand-specific bulk RNA-seq alignment signal GW14.5_1_polya_plus.bw: strand-specific bulk RNA-seq alignment signal GW14.5_1_total_minus.bw: strand-specific bulk RNA-seq alignment signal GW14.5_1_total_plus.bw: strand-specific bulk RNA-seq alignment signal GW16_1_polya_minus.bw: strand-specific bulk RNA-seq alignment signal GW16_1_polya_plus.bw: strand-specific bulk RNA-seq alignment signal GW16_1_total_minus.bw: strand-specific bulk RNA-seq alignment signal GW16_1_total_plus.bw: strand-specific bulk RNA-seq alignment signal GW16_2_polya_minus.bw: strand-specific bulk RNA-seq alignment signal GW16_2_polya_plus.bw: strand-specific bulk RNA-seq alignment signal GW16_2_total_minus.bw: strand-specific bulk RNA-seq alignment signal GW16_2_total_plus.bw: strand-specific bulk RNA-seq alignment signal GW21_1_polya_minus.bw: strand-specific bulk RNA-seq alignment signal GW21_1_polya_plus.bw: strand-specific bulk RNA-seq alignment signal GW21_1_total_minus.bw: strand-specific bulk RNA-seq alignment signal GW21_1_total_plus.bw: strand-specific bulk RNA-seq alignment signal GW21_2_polya_minus.bw: strand-specific bulk RNA-seq alignment signal GW21_2_polya_plus.bw: strand-specific bulk RNA-seq alignment signal GW21_2_total_minus.bw: strand-specific bulk RNA-seq alignment signal GW21_2_total_plus.bw: strand-specific bulk RNA-seq alignment signal GW23_1_polya_minus.bw: strand-specific bulk RNA-seq alignment signal GW23_1_polya_plus.bw: strand-specific bulk RNA-seq alignment signal GW23_1_total_minus.bw: strand-specific bulk RNA-seq alignment signal GW23_1_total_plus.bw: strand-specific bulk RNA-seq alignment signal GW23_2_polya_minus.bw: strand-specific bulk RNA-seq alignment signal GW23_2_polya_plus.bw: strand-specific bulk RNA-seq alignment signal GW23_2_total_minus.bw: strand-specific bulk RNA-seq alignment signal GW23_2_total_plus.bw: strand-specific bulk RNA-seq alignment signal
Project description:We have generated RNA-sequencing datasets of the regulome of mouse neural stem and progenitor cells derived from embryonic stem cells, with allele-specific deletions of Sox2 enhancer cluster regions. RNA-seq experiments were conducted to evaluate the regulatory function of Sox2 candidate enhancers in neural stem and progenitor cells.
Project description:Melanocytes are pigment-producing cells of neural crest origin responsible for protecting the skin against UV-irradiation. Melanocyte dysfunction leads to pigmentation defects including albinism, vitiligo, and piebaldism and is a key feature of systemic pathologies such as Hermansky-Pudlak (HP) and Chediak-Higashi (CH) Syndromes. Pluripotent stem cell technology offers a novel approach for studying human melanocyte development and disease. Here we report that timed exposure to activators of WNT, BMP and EDN3 signaling triggers the sequential induction of neural crest and melanocyte precursor fates under dual-SMAD inhibition conditions. Using a SOX10::GFP hESC reporter line, we demonstrate that the temporal onset of WNT activation is particularly critical for human neural crest induction. Surprisingly, suppression of BMP signaling does reduce neural crest yield. Subsequent differentiation of hESC-derived melanocyte precursors under defined conditions yields pure populations of pigmented cells matching the molecular and functional properties of adult melanocytes. Melanocytes from patient-specific iPSCs faithfully reproduce the ultrastructural features of the HP- and CH-specific pigmentation defects with minimal variability across lines. Our data define a highly specific requirement for WNT signaling during neural crest induction and enable the generation of pure populations of hiPSC-derived melanocytes for faithful modeling of human pigmentation disorders. Total RNA obtained from embryonic stem cells (ESCs), ESC-derived melanocyte progenitors, ESC-derived mature melanocytes, primary melanocytes, and disease-specific induced pluripotent stem cell-derived melanocytes.
Project description:Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into 4 subtypes based on different genomic alterations, gene expression profiles and response to treatment: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. The extensive heterogeneity has made it difficult to assess the relevance of genes to malignant progression. For example, expression of the transcription factor, OTX2, is frequently dysregulated in multiple MB variants; however, it's role may be subtype specific. Here, we utilized human embryonic stem cell-derived neural precursors to determine the role of OTX2 in MB tumor progression using gain and loss of function studies. We used global gene expression profiling to determine what transcripts and pathways were differentially expressed following knockdown of OTX2 in transformed or neoplastic human embryonic neural precursor cells. OTX2 was knocked down in transformed human embyronic neural precursors (trans-hEN) using Silencer select siRNAs. trans-hEN OTX2 KD and scrambled control trans-hENs were then grown as neurospheres in defined medium and collected at passage 1. RNA was extracted using the Norgen All-in-One kit.