Project description:A hallmark of cortical evolution is the high dynamic subventricular zone (SVZ) expansion, where basal progenitors (BPs) amplify and neuronal transcriptional programs unfold. How non-coding molecular factors such as microRNAs influence these developmental trajectories and regulate the acquisition of cortical type identities is largely unknown. Here we demonstrate that miR-137 and miR-122 regulate the positioning and identity features of superficial layer cortical neurons by acting at distinct steps of their developmental trajectories. MiR-137 sustains basal progenitor amplification by reverting their neurogenic commitment and inducing high proliferative state upregulating Cd63 and inhibiting Myt1l. Cd63 is an extra-cellular matrix (ECM) receptor which interacts with b3- and 1-integrin pathways to promote proliferation, while Myt1l is a transcription factor that promotes and sustains neuronal fate. The BPs amplification by miR-137 is converted in the promotion of intracortical projecting neuron (ICPN) identity and L2/3 expansion. As opposed to miR-137, miR-122 acts postmitotically, affecting the bioelectrical properties, the calcium and cytoskeleton dynamics of newborn neurons as well as their transcriptional program, leading to a persistent molecular immaturity across time. Overall, these findings reveal that miR-137 and miR-122 are key regulators of the developmental trajectory of cortical neurons across evolution.
Project description:The mammalian cerebral cortex contains an extraordinary diversity of cell types that emerge through the implementation of different developmental programs. Delineating when and how cellular diversification occurs is particularly challenging for cortical inhibitory neurons, as they represent a relatively small proportion of all cortical cells, migrate tangentially from their embryonic origin to the cerebral cortex, and have a protracted development. Here we combine single-cell RNA sequencing and spatial transcriptomics to characterize the emergence of neuronal diversity among somatostatin-expressing (SST+) cells, the most diverse subclass of inhibitory neurons in the mouse cerebral cortex. We found that SST+ inhibitory neurons segregate during embryonic stages into long-range projection (LRP) neurons and two types of interneurons, Martinotti cells and non-Martinotti cells, following distinct developmental trajectories. Two main subtypes of LRP neurons and several subtypes of interneurons are readily distinguishable in the embryo, although interneuron diversity is further refined during early postanal life. Our results suggest that the timing for cellular diversification is unique for different subtypes of SST+ neurons and particularly divergent for LRP neurons and interneurons. Thus, the diversification of SST+ inhibitory neurons involves a temporal cascade of unique molecular programs driving their divergent developmental trajectories.
Project description:Chromodomain helicase DNA-binding 8 (CHD8) is one of the most frequently mutated genes causative of autism spectrum disorder (ASD). While its phenotypic spectrum often encompasses macrocephaly and hence implicates cortical abnormalities in this form of ASD, the neurodevelopmental impact of human CHD8 haploinsufficiency remains unexplored. Here we combined human cerebral organoids and single cell transcriptomics to define the effect of ASD-linked CHD8 mutations on human cortical development. We found that CHD8 haploinsufficiency causes a major disruption of neurodevelopmental trajectories with an accelerated generation of inhibitory neurons and a delayed production of excitatory neurons alongside the ensuing protraction of the proliferation phase. This imbalance may contribute to the significant enlargement of cerebral organoids in line with the macrocephaly observed in patients with CHD8 mutations. By adopting an isogenic design of patient-specific mutations and mosaic cerebral organoids, we define genotype-phenotype relationships and uncover their cell-autonomous nature. Finally, our results assign different CHD8-dependent molecular defects to particular cell types, pointing to an abnormal and extended program of proliferation and alternative splicing specifically affected in, respectively, the radial glial and immature neuronal compartments. By identifying temporally restricted cell-type specific effects of human CHD8 mutations, our study uncovers developmental alterations as reproducible endophenotypes for neurodevelopmental disease modelling.
Project description:This SuperSeries is composed of the following subset Series: GSE24440: Sprouting transcriptome in cortical neurons: young GSE24441: Sprouting transcriptome in cortical neurons: aged Refer to individual Series
Project description:A SHAPE-MaP structure probing experiment was performed on 40 eRNAs. The eRNA transcription start sites (TSSs) were identified by 5'-ExoSeq. The 5' end fragment (1-200 nucleotides) of each eRNA was cloned from mouse cortical neurons and the eRNAs were produced by in vitro transcription. Each eRNA was treated in one sample with DMSO (control) and in a second sample with the SHAPE reagent 1-Methyl-7-nitroisatoic anhydride (1M7). The 1M7 chemical will react preferentially with the ribose 2'OH of the flexible nucleotides in single stranded regions and the produced adduct will lead to mutations during reverse transcription.
Project description:Cortical neural progenitor cells (NPCs) change their competency over time during development, giving rise to distinct cell types sequentially. Many genes that govern cortical development are now known, but it remains elusive how their temporal expression is controlled. Recently, long non-coding RNAs are found to be essential for cell-fate specification and precise gene regulation in many developmental events. In this study, strand-specific RNA sequencing studies unveil large amount of long non-coding RNAs are actively and differentially expressed across mouse cortical development. Integration of RNA sequencing data from key stages of developing mouse cortex enables us to cluster coding and non-coding transcripts into co-expression “modules” to infer functional relationships. Intriguingly, the cortical transcriptome undergoes significant changes in early mouse neurogenesis. Cortical long non-coding RNAs tends to be transcribed from genomic loci adjacent to protein-coding genes related to neural development. Finally, we found large amount of predicted enhancer regions are able to transcribe RNAs. This study will help us better understand molecularly how cortical NPCs specify their fates during development, especially roles of lncRNAs in this process.
Project description:During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branchpoints contained cells expressing genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework to reconstruct complex developmental trees from single-cell transcriptomes. This SuperSeries is composed of the SubSeries listed below.
Project description:Biologists rely on morphology, function, and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis (PCA) of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three cell lineage trajectories, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast, and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ (EG) cells and induced pluripotent (iPS) cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast (MEF) and fibroblast cell lines are mapped near the far end of the trajectories. We propose that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. Keywords: cell type comparison design,reference design,replicate design,time series design Most of the cells and RNA samples used in this study were described in detail previously (See paper's citation associated with this dataset). To maximize the uniformity of the microarray data, all the samples, including ones analyzed by DNA microarray previously, were hybridized to the same platform (the NIA Mouse 44K Microarray manufactured by Agilent Technologies: AMADID #015087). The intensity of each gene feature per array was extracted from scanned microarray images using Feature Extraction Software V9.5.
Project description:E18 embryonic rat cortical neurons cultured in vitro are infected with lentivirus expressing control or PHF6shRNA-2, and harvested 5 days after infection pLL3.7 lentivirus expressing control or PHF6shRNA-2 was generated in 293T cells and concentrated using ultracentrifuge. In vitro cultured cortical neurons were infected and RNA was harvested 5 days after infection. PHF6 knockdown was validated by QPCR before sample was processed for microarray analysis.