Project description:Cultured adult mouse dorsal root ganglia (DRG) cells exhibit glial sensory progenitor properties in vitro. Therefore, they might be a good starter cell for reprogramming into sensory neurons. Here, we infected them with the retroviral vector Neurog2-Neurog1-DsRed to induce sensory neuron development and analyzed by scRNAseq at 14 days post infection whether infected cells show properties of sensory neurons such as nociceptors. After 10x Genomics, data analysis of 4,549 individual cells indicated the generation of neurons, but at an immature cell state.
Project description:Single cell RNAseq was performed on naïve adult mouse lumbar dorsal root ganglia (DRG) cells. Neuronal and non-neuronal cell populations were identified.
Project description:The proneural NEUROG2 is essential for neuronal commitment, cell cycle exit and neuronal differentiation. Characterizing genes networks regulated downstream of NEUROG2 is therefore of prime importance. To identify NEUROG2 early response genes, we combined gain of function in the neural tube with a global detection of modified transcripts using microarrays. We included in our study a mutant form of NEUROG2 (NEUROG2AQ) that cannot bind DNA and cannot trigger neurogenesis. Using this approach, we identified 942 genes modified at the onset of NEUROG2 activation. The global analysis of functions regulated by NEUROG2 allowed unmasking its rapid impact on cell cycle control. We found that NEUROG2 specifically represses a subset of cyclins acting at the G1 and S phases of the cell cycle, thereby impeding S phase re-entry. This repression occurs before modification of p27kip1, indicating that the decision to leave the cell cycle precedes the activation of this Cyclin-dependant Kinase Inhibitor. Moreover, NEUROG2 down-regulates only one of the D-type cyclins, cyclinD1, and maintaining cyclinD1 blocks the ability of the proneural to trigger cell cycle exit, without altering its capacity to trigger neuronal differentiation. The fact that NEUROG2 represses a subset but not all cell cycle regulators indicates that cell cycle exit is not an indirect consequence of neuronal differentiation but is precisely controlled by NEUROG2. Altogether our findings indicate that NEUROG2, by specifically repressing G1 and S cyclins, allows committed neuronal precursors to perform their last mitosis but blocks their re-entry in the cell cycle, thus favouring cell cycle exit. Stage HH10-11 embryos (11 to 15 somites) were electroporated with a control vector (pGIG-GFP), a NEUROG2-expressing vector (pCIGNEUROG2-GFP), or a NEUROG2AQ-expressing vector (pCIGNEUROG2AQ-GFP). For each biological replicate, neural tubes from 20 embryos were pooled for GFP+ cells collection. GFP+ cells were collected 6h later using FACS sorting (Epics Altra HSS cell sorter, Toulouse Rio platform) and processed for RNA probe preparation and hybridization on Affymetrix microarrays. For each experimental condition, four biological replicates were processed.
Project description:To identify and purify NEUROG2-expressing cells and trace their short-term lineage, we engineered two NEUROG2-mCherry knock-in human embryonic stem cell (hESC) lines. Transcriptomic profiling of NEUROG2:mCherry knock-in hESC-derived cerebral organoids revealed an enrichment of neurogenic, oligodendrocyte precursor cell and extracellular matrix-associated gene transcripts in mCherry-high cells. Conversely, only neurogenic gene transcripts were enriched in mCherry-high cells from Neurog2:mCherry knock-in mouse cortices.
Project description:The proneural NEUROG2 is essential for neuronal commitment, cell cycle exit and neuronal differentiation. Characterizing genes networks regulated downstream of NEUROG2 is therefore of prime importance. To identify NEUROG2 early response genes, we combined gain of function in the neural tube with a global detection of modified transcripts using microarrays. We included in our study a mutant form of NEUROG2 (NEUROG2AQ) that cannot bind DNA and cannot trigger neurogenesis. Using this approach, we identified 942 genes modified at the onset of NEUROG2 activation. The global analysis of functions regulated by NEUROG2 allowed unmasking its rapid impact on cell cycle control. We found that NEUROG2 specifically represses a subset of cyclins acting at the G1 and S phases of the cell cycle, thereby impeding S phase re-entry. This repression occurs before modification of p27kip1, indicating that the decision to leave the cell cycle precedes the activation of this Cyclin-dependant Kinase Inhibitor. Moreover, NEUROG2 down-regulates only one of the D-type cyclins, cyclinD1, and maintaining cyclinD1 blocks the ability of the proneural to trigger cell cycle exit, without altering its capacity to trigger neuronal differentiation. The fact that NEUROG2 represses a subset but not all cell cycle regulators indicates that cell cycle exit is not an indirect consequence of neuronal differentiation but is precisely controlled by NEUROG2. Altogether our findings indicate that NEUROG2, by specifically repressing G1 and S cyclins, allows committed neuronal precursors to perform their last mitosis but blocks their re-entry in the cell cycle, thus favouring cell cycle exit.
Project description:To generate an unbiased view of changes to the retinal gene network in Neurog2 retinal mutants as well as understanding the effects of Mash1 ectopic expression in a Neurog2 retinal mutant, we generated and compared the E12.5 transcriptomes from Neurog2 heterozygote, Neurog2 mutant and Neurog2 mutant;Mash1 knock-in mice. A pair of E12.5 retinas from each biologic replicate underwent FACS sorting to isolate GFP+ cells. GFP+ cells were used to produce libraries for high throughput sequencing (n = 3 biologic replicates/genotype). Reads were aligned with BWA and Bowtie programs to the mm10 genome. Aligned reads were then analyzed for differentially expressed transcripts using the CuffDiff program in the Galaxy online bioinformatics package (www.usegalaxy.org).
Project description:To identify the target mRNAs of Ybx1 in mouse DRG, we first performed RNAseq to screen differentially expressed mRNAs after knockout of Ybx1. We generated Wnt1-cre+/-;Ybx1fl/fl cKO mice to specifically knockout Ybx1 in DRG. Then we collected E13.5 DRG from cKO mice and litermate Ybx1fl/fl control mice. After RNA extraction and library construction, RNA sequcencing was performed. Finally, we identified 1638 differentially expressed mRNAs. This study provides a gene list which shows mRNA that can be regulated by Ybx1 in mouse DRG.
Project description:Humans exemplify gyrencephalic species with folded cerebral cortices, contrasting with lissencephalic mammals such as mice. Here we investigated how proneural genes Neurog2 and Ascl1 control cortical folding by regulating neurogenic patterns. Cortical neural progenitor cells (NPCs) stratify into four pools (proneural negative, Neurog2+, Ascl1+, double+) that are distributed evenly in mouse cortices and modular in gyrencephalic macaque cortices and pseudo-folded human cerebral organoids. Each pool has distinct developmental potentials, transcriptomes, epigenomes, and gene regulatory networks. Neurog2-Ascl1 form a bistable toggle switch double+ NPCs to prevent lineage commitment observed in single+ NPCs. Neurog2 and Ascl1 act redundantly to control neurogenic timing, with NPCs precociously depleted in Neurog2-/-;Ascl1-/- cortices. Finally, selective killing of Neurog2/Ascl1 double+ NPCs using Neurog2/Ascl1 split-Cre;Rosa-DTA transgenics breaks neurogenic symmetry in mice by locally disrupting Notch signaling, leading to cortical folding. Our findings suggest that Neurog2/Ascl1 double+ NPCs are Notch-ligand expressing ‘niche’ cells that regulate neurogenic continuity and cortical gyrification.