Evolutionary development of the human brain is characterized by the expansion of various brain regions. Here, we show that developmental processes specific to humans are responsible for malformations ...of cortical development (MCDs), which result in developmental delay and epilepsy in children. We generated a human cerebral organoid model for tuberous sclerosis complex (TSC) and identified a specific neural stem cell type, caudal late interneuron progenitor (CLIP) cells. In TSC, CLIP cells over-proliferate, generating excessive interneurons, brain tumors, and cortical malformations. Epidermal growth factor receptor inhibition reduces tumor burden, identifying potential treatment options for TSC and related disorders. The identification of CLIP cells reveals the extended interneuron generation in the human brain as a vulnerability for disease. In addition, this work demonstrates that analyzing MCDs can reveal fundamental insights into human-specific aspects of brain development.
To advance our understanding of the genetic programs that drive cell and tissue specialization, it is necessary to obtain a comprehensive overview of gene expression patterns. Here, we have used ...spatial transcriptomics to generate high-resolution, anteroposterior gene expression maps of C. elegans males and hermaphrodites. To explore these maps, we have developed computational methods for discovering region- and tissue-specific genes. We have found extensive sex-specific gene expression differences in the germline and sperm and discovered genes that are specifically expressed in the male reproductive tract. These include a group of uncharacterized genes that encode small secreted proteins that are required for male fertility. We conclude that spatial gene expression maps provide a powerful resource for identifying tissue-specific gene functions in C. elegans. Importantly, we found that expression maps from different animals can be precisely aligned, enabling transcriptome-wide comparisons of gene expression patterns.
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•20 μm resolution AP gene expression maps of C. elegans males and hermaphrodites•Resource for in silico identification of cell- and tissue-specific genes•Identification of sex-specific germline and sperm genes•Discovery of male reproductive tract genes required for male fertility
By combining cryo-sectioning with mRNA sequencing, Ebbing and Vértesy et al. have generated genome-wide gene expression maps of C. elegans males and hermaphrodites. These maps provide a powerful resource for identifying tissue- and sex-specific genes, as demonstrated by the discovery of reproductive tract genes required for male fertility.
In contrast to mouse, human female germ cells develop asynchronously. Germ cells transition to meiosis, erase genomic imprints, and reactivate the X chromosome. It is unknown if these events all ...appear asynchronously, and how they relate to each other. Here we combine exome sequencing of human fetal and maternal tissues with single-cell RNA-sequencing of five donors. We reconstruct full parental haplotypes and quantify changes in parental allele-specific expression, genome-wide. First we distinguish primordial germ cells (PGC), pre-meiotic, and meiotic transcriptional stages. Next we demonstrate that germ cells from various stages monoallelically express imprinted genes and confirm this by methylation patterns. Finally, we show that roughly 30% of the PGCs are still reactivating their inactive X chromosome and that this is related to transcriptional stage rather than fetal age. Altogether, we uncover the complexity and cell-to-cell heterogeneity of transcriptional and epigenetic remodeling in female human germ cells.
Organoids enable in vitro modeling of complex developmental processes and disease pathologies. Like most 3D cultures, organoids lack sufficient oxygen supply and therefore experience cellular stress. ...These negative effects are particularly prominent in complex models, such as brain organoids, and can affect lineage commitment. Here, we analyze brain organoid and fetal single‐cell RNA sequencing (scRNAseq) data from published and new datasets, totaling about 190,000 cells. We identify a unique stress signature in the data from all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined subpopulation of cells that is unique to organoids and does not affect neuronal specification or maturation. We have developed a computational algorithm, Gruffi, which uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. We validated our method using six additional datasets from different organoid protocols and early brains, and show its usefulness to other organoid systems including retinal organoids. Our data show that the adverse effects of cell stress can be corrected by bioinformatic analysis for improved delineation of developmental trajectories and resemblance to in vivo data.
Synopsis
Cellular stress in 3D organoids due to insufficient oxygen transport can affect faithful lineage commitment and disease modeling. This work identifies a subpopulation of stressed cells characterized by a distinct gene expression signature that can be removed from scRNAseq datasets for better evaluation of fetal developmental trajectories.
ER‐ and glycolytic stress are found accross organoid protocols.
Stressed cells in 3D organoids form a separate cell state that is not found in vivo and that can be separated bioinformatically.
The presence of stressed cells in organoids does not affect cell‐type specification or the maturation of non‐stressed neurons.
Granular functional filtering (Gruffi) removes stressed cells from the single‐cell datasets but retains cell types found in vivo.
Stress removal leads to clearer developmental trajectories.
A unique stress signature found in in brain organoid samples but not in fetal samples can be quantified and removed from scRNAseq datasets using a new algorithm.
Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that is characterized by progressive loss of myocardium that is replaced by fibro-fatty cells, arrhythmias, and sudden cardiac ...death. While myocardial degeneration and fibro-fatty replacement occur in specific locations, the underlying molecular changes remain poorly characterized. Here, we aim to delineate local changes in gene expression to identify new genes and pathways that are relevant for specific remodelling processes occurring during ACM.
Using Tomo-Seq, genome-wide transcriptional profiling with high spatial resolution, we created transmural epicardial-to-endocardial gene expression atlases of explanted ACM hearts to gain molecular insights into disease-driving processes. This enabled us to link gene expression profiles to the different regional remodelling responses and allowed us to identify genes that are potentially relevant for disease progression. In doing so, we identified distinct gene expression profiles marking regions of cardiomyocyte degeneration and fibro-fatty remodelling and revealed Zinc finger and BTB domain-containing protein 11 (ZBTB11) to be specifically enriched at sites of active fibro-fatty replacement of myocardium. Immunohistochemistry indicated ZBTB11 to be induced in cardiomyocytes flanking fibro-fatty areas, which could be confirmed in multiple cardiomyopathy patients. Forced overexpression of ZBTB11 induced autophagy and cell death-related gene programmes in human cardiomyocytes, leading to increased apoptosis.
Our study shows the power of Tomo-Seq to unveil new molecular mechanisms in human cardiomyopathy and uncovers ZBTB11 as a novel driver of cardiomyocyte loss.
The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders
. Cerebral organoids enable the study of ...neurodevelopmental disorders in a human context. We have developed the CRISPR-human organoids-single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR-Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.
Inhibitory GABAergic interneurons migrate over long distances from their extracortical origin into the developing cortex. In humans, this process is uniquely slow and prolonged, and it is unclear ...whether guidance cues unique to humans govern the various phases of this complex developmental process. Here, we use fused cerebral organoids to identify key roles of neurotransmitter signaling pathways in guiding the migratory behavior of human cortical interneurons. We use scRNAseq to reveal expression of GABA, glutamate, glycine, and serotonin receptors along distinct maturation trajectories across interneuron migration. We develop an image analysis software package, TrackPal, to simultaneously assess 48 parameters for entire migration tracks of individual cells. By chemical screening, we show that different modes of interneuron migration depend on distinct neurotransmitter signaling pathways, linking transcriptional maturation of interneurons with their migratory behavior. Altogether, our study provides a comprehensive quantitative analysis of human interneuron migration and its functional modulation by neurotransmitter signaling.
SYNOPSIS
Using an interneuron‐specific reporter to track migrating cortical interneurons in human cerebral organoid fusions, this study provides a comprehensive quantitative analysis of human interneuron migration and its functional modulation by diverse neurotransmitter signaling pathways.
Single‐cell human GABAergic lineage reconstruction identifies neurotransmitter receptor expression in migrating human cortical interneurons.
Newly developed software package, TrackPal, quantifies large‐scale, live‐imaging based tracking of migrating human cortical interneurons.
Small‐molecule screening identifies divergent roles of neurotransmitter receptors in regulating intrinsic interneuron migration behavior.
Neurotransmitter signaling modulates distinct modes of interneuron migration.
Live‐imaging in cerebral organoids combined with single‐cell transcriptomics and small‐molecule screening reveals distinct modes of human interneuron migration and their modulation by different neurotransmitters.
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