ABSTRACT
The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders1-4. 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.
Subject(s)
Autism Spectrum Disorder , Brain , Developmental Disabilities , Organoids , Single-Cell Gene Expression Analysis , Humans , Autism Spectrum Disorder/complications , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/pathology , Autistic Disorder/complications , Autistic Disorder/genetics , Autistic Disorder/pathology , Brain/cytology , Brain/metabolism , Cell Lineage/genetics , Chromatin/genetics , CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems , Developmental Disabilities/complications , Developmental Disabilities/genetics , Developmental Disabilities/pathology , Gene Editing , Loss of Function Mutation , Mosaicism , Neurons/metabolism , Neurons/pathology , Organoids/cytology , Organoids/metabolism , RNA, Guide, CRISPR-Cas Systems , Transcription, GeneticABSTRACT
The establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in neural organoids, the effects of different ECM sources in tissue morphogenesis remain underexplored. Here, we show that exposure to a solubilized basement membrane matrix substrate, Matrigel, at early neuroepithelial stages causes rapid tissue polarization and rearrangement of neuroepithelial architecture. In cultures exposed to pure ECM components or unexposed to any exogenous ECM, polarity acquisition is slower and driven by endogenous ECM production. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source, but Matrigel exposure has long-lasting effects on tissue patterning. These results advance the knowledge on mechanisms of exogenously and endogenously guided morphogenesis, demonstrating the self-sustainability of neuroepithelial cultures by endogenous processes.
Subject(s)
Extracellular Matrix , Organoids , Humans , MorphogenesisABSTRACT
Cerebral organoids recapitulate human brain development at a considerable level of detail, even in the absence of externally added signaling factors. The patterning events driving this self-organization are currently unknown. Here, we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions, we demonstrate the presence of a variety of discrete ventral and dorsal regions. Clearing and subsequent 3D reconstruction of entire organoids reveal that many of these regions are interconnected, suggesting that the entire range of dorso-ventral identities can be generated within continuous neuroepithelia. Consistent with this, we demonstrate the presence of forebrain organizing centers that express secreted growth factors, which may be involved in dorso-ventral patterning within organoids. Furthermore, we demonstrate the timed generation of neurons with mature morphologies, as well as the subsequent generation of astrocytes and oligodendrocytes. Our work provides the methodology and quality criteria for phenotypic analysis of brain organoids and shows that the spatial and temporal patterning events governing human brain development can be recapitulated in vitro.
Subject(s)
Brain/embryology , Cell Differentiation , Cell Proliferation , Organoids/growth & development , Body Patterning , Humans , Spatio-Temporal AnalysisABSTRACT
Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B+/- neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an in vitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2+ neurons as a cause of ACC in ARID1B patients.
Subject(s)
Axons , Corpus Callosum , DNA-Binding Proteins , Organoids , Transcription Factors , Humans , Corpus Callosum/metabolism , Transcription Factors/metabolism , Transcription Factors/genetics , Organoids/metabolism , Axons/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Matrix Attachment Region Binding Proteins/metabolism , Matrix Attachment Region Binding Proteins/genetics , Transcription, Genetic , Neurons/metabolismABSTRACT
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.
Subject(s)
Brain Neoplasms/pathology , Brain/pathology , Interneurons/cytology , Neural Stem Cells/physiology , Tuberous Sclerosis/genetics , Tuberous Sclerosis/pathology , Brain/embryology , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Carcinogenesis , Cell Lineage , Cell Proliferation , Disease Progression , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Gene Expression Profiling , Humans , Induced Pluripotent Stem Cells , Interneurons/physiology , Loss of Heterozygosity , Neural Stem Cells/cytology , Organoids , RNA-Seq , TOR Serine-Threonine Kinases/metabolism , Tuberous Sclerosis/drug therapy , Tuberous Sclerosis/metabolism , Tuberous Sclerosis Complex 1 Protein/genetics , Tuberous Sclerosis Complex 1 Protein/metabolism , Tuberous Sclerosis Complex 2 Protein/genetics , Tuberous Sclerosis Complex 2 Protein/metabolismABSTRACT
Stem cell-specific transcriptional networks are well known to control pluripotency, but constitutive cellular processes such as mRNA splicing and protein synthesis can add complex layers of regulation with poorly understood effects on cell-fate decisions. Here, we show that the RNA binding protein HTATSF1 controls embryonic stem cell differentiation by regulating multiple aspects of RNA processing during ribosome biogenesis. HTATSF1, in a complex with splicing factor SF3B1, controls intron removal from ribosomal protein transcripts and regulates ribosomal RNA transcription and processing, thereby controlling 60S ribosomal abundance and protein synthesis. HTATSF1-dependent protein synthesis is essential for naive pre-implantation epiblast to transition into post-implantation epiblast, a stage with transiently low protein synthesis, and further differentiation toward neuroectoderm. Together, these results identify coordinated regulation of ribosomal RNA and protein synthesis by HTATSF1 and show that this essential mechanism controls protein synthesis during early mammalian embryogenesis.