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Nature Jun 2019Experimental models of the human brain are needed for basic understanding of its development and disease. Human brain organoids hold unprecedented promise for this...
Experimental models of the human brain are needed for basic understanding of its development and disease. Human brain organoids hold unprecedented promise for this purpose; however, they are plagued by high organoid-to-organoid variability. This has raised doubts as to whether developmental processes of the human brain can occur outside the context of embryogenesis with a degree of reproducibility that is comparable to the endogenous tissue. Here we show that an organoid model of the dorsal forebrain can reliably generate a rich diversity of cell types appropriate for the human cerebral cortex. We performed single-cell RNA-sequencing analysis of 166,242 cells isolated from 21 individual organoids, finding that 95% of the organoids generate a virtually indistinguishable compendium of cell types, following similar developmental trajectories and with a degree of organoid-to-organoid variability comparable to that of individual endogenous brains. Furthermore, organoids derived from different stem cell lines show consistent reproducibility in the cell types produced. The data demonstrate that reproducible development of the complex cellular diversity of the central nervous system does not require the context of the embryo, and that establishment of terminal cell identity is a highly constrained process that can emerge from diverse stem cell origins and growth environments.
Topics: Cell Line; Cerebral Cortex; Female; Fetus; Humans; Induced Pluripotent Stem Cells; Male; Organoids; Prosencephalon; RNA-Seq; Reproducibility of Results; Single-Cell Analysis; Time Factors; Tissue Culture Techniques; Transcriptome
PubMed: 31168097
DOI: 10.1038/s41586-019-1289-x -
Nature Protocols Sep 2018The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both...
The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region-specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively. We also describe how to combine the neural spheroids in vitro to assemble forebrain assembloids that recapitulate the interactions of glutamatergic and GABAergic neurons seen in vivo. Astrocytes are also present in the human forebrain-specific spheroids, and these undergo maturation when the forebrain spheroids are cultured long term. The initial generation of neural spheroids from hPSCs occurs in <1 week, with regional patterning occurring over the subsequent 5 weeks. After the maturation stage, brain region-specific spheroids are amenable to a variety of assays, including live-cell imaging, calcium dynamics, electrophysiology, cell purification, single-cell transcriptomics, and immunohistochemistry studies. Once generated, forebrain spheroids can also be matured for >24 months in culture.
Topics: Humans; Models, Biological; Organ Culture Techniques; Organogenesis; Organoids; Pluripotent Stem Cells; Prosencephalon; Tissue Engineering
PubMed: 30202107
DOI: 10.1038/s41596-018-0032-7 -
Nature Oct 2023The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain, which has been challenging to study at...
The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain, which has been challenging to study at inaccessible stages of late gestation and early postnatal human development. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development, but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.
Topics: Female; Humans; Infant, Newborn; Pregnancy; Cell Movement; CRISPR-Cas Systems; Gene Editing; Interneurons; Neurodevelopmental Disorders; Organoids; Endoplasmic Reticulum; Prosencephalon; Active Transport, Cell Nucleus
PubMed: 37758944
DOI: 10.1038/s41586-023-06564-w -
Cell Nov 2017N-methyladenosine (mA), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether mA regulates mammalian brain...
N-methyladenosine (mA), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether mA regulates mammalian brain development is unknown. Here, we show that mA depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages. mA depletion by Mettl3 knockdown also leads to a prolonged cell cycle and maintenance of radial glia cells. mA sequencing of embryonic mouse cortex reveals enrichment of mRNAs related to transcription factors, neurogenesis, the cell cycle, and neuronal differentiation, and mA tagging promotes their decay. Further analysis uncovers previously unappreciated transcriptional prepatterning in cortical neural stem cells. mA signaling also regulates human cortical neurogenesis in forebrain organoids. Comparison of mA-mRNA landscapes between mouse and human cortical neurogenesis reveals enrichment of human-specific mA tagging of transcripts related to brain-disorder risk genes. Our study identifies an epitranscriptomic mechanism in heightened transcriptional coordination during mammalian cortical neurogenesis.
Topics: Animals; Cell Cycle; Gene Expression Regulation; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Humans; Methylation; Methyltransferases; Mice; Mice, Knockout; Neural Stem Cells; Neurogenesis; Organoids; Prosencephalon; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger
PubMed: 28965759
DOI: 10.1016/j.cell.2017.09.003 -
Nature Neuroscience Feb 2006The developmental origin of oligodendrocyte progenitors (OLPs) in the forebrain has been controversial. We now show, by Cre-lox fate mapping in transgenic mice, that the...
The developmental origin of oligodendrocyte progenitors (OLPs) in the forebrain has been controversial. We now show, by Cre-lox fate mapping in transgenic mice, that the first OLPs originate in the medial ganglionic eminence (MGE) and anterior entopeduncular area (AEP) in the ventral forebrain. From there, they populate the entire embryonic telencephalon including the cerebral cortex before being joined by a second wave of OLPs from the lateral and/or caudal ganglionic eminences (LGE and CGE). Finally, a third wave arises within the postnatal cortex. When any one population is destroyed at source by the targeted expression of diphtheria toxin, the remaining cells take over and the mice survive and behave normally, with a normal complement of oligodendrocytes and myelin. Thus, functionally redundant populations of OLPs compete for space in the developing brain. Notably, the embryonic MGE- and AEP-derived population is eliminated during postnatal life, raising questions about the nature and purpose of the competition.
Topics: Animals; Animals, Newborn; Cell Lineage; Embryo, Mammalian; Immunohistochemistry; In Situ Hybridization; Mice; Mice, Transgenic; Oligodendroglia; Prosencephalon; Stem Cells
PubMed: 16388308
DOI: 10.1038/nn1620 -
International Journal of Molecular... May 2021A prolonged developmental timeline for GABA (γ-aminobutyric acid)-expressing inhibitory neurons (GABAergic interneurons) is an amplified trait in larger, gyrencephalic... (Review)
Review
A prolonged developmental timeline for GABA (γ-aminobutyric acid)-expressing inhibitory neurons (GABAergic interneurons) is an amplified trait in larger, gyrencephalic animals. In several species, the generation, migration, and maturation of interneurons take place over several months, in some cases persisting after birth. The late integration of GABAergic interneurons occurs in a region-specific pattern, especially during the early postnatal period. These changes can contribute to the formation of functional connectivity and plasticity, especially in the cortical regions responsible for higher cognitive tasks. In this review, we discuss GABAergic interneuron development in the late gestational and postnatal forebrain. We propose the protracted development of interneurons at each stage (neurogenesis, neuronal migration, and network integration), as a mechanism for increased complexity and cognitive flexibility in larger, gyrencephalic brains. This developmental feature of interneurons also provides an avenue for environmental influences to shape neural circuit formation.
Topics: Animals; Animals, Newborn; Female; Gestational Age; Interneurons; Pregnancy; Prosencephalon; gamma-Aminobutyric Acid
PubMed: 34066025
DOI: 10.3390/ijms22105113 -
Developmental Biology Jan 2017Striatal cholinergic interneurons and basal forebrain cholinergic projection neurons, which together comprise the forebrain cholinergic system, regulate attention,... (Review)
Review
Striatal cholinergic interneurons and basal forebrain cholinergic projection neurons, which together comprise the forebrain cholinergic system, regulate attention, memory, reward pathways, and motor activity through the neuromodulation of multiple brain circuits. The importance of these neurons in the etiology of neurocognitive disorders has been well documented, but our understanding of their specification during embryogenesis is still incomplete. All forebrain cholinergic projection neurons and interneurons appear to share a common developmental origin in the embryonic ventral telencephalon, a region that also gives rise to GABAergic projection neurons and interneurons. Significant progress has been made in identifying the key intrinsic and extrinsic factors that promote a cholinergic fate in this precursor population. However, how cholinergic interneurons and projection neurons differentiate from one another during development, as well as how distinct developmental programs contribute to heterogeneity within those two classes, is not yet well understood. In this review we summarize the transcription factors and signaling molecules known to play a role in the specification and early development of striatal and basal forebrain cholinergic neurons. We also discuss the heterogeneity of these populations and its possible developmental origins.
Topics: Animals; Body Patterning; Cholinergic Neurons; Humans; Neural Stem Cells; Prosencephalon; Signal Transduction; Transcription Factors
PubMed: 27847324
DOI: 10.1016/j.ydbio.2016.11.007 -
Journal of Neuroscience Research Mar 2017Cholinergic activation regulates cognitive function, particularly long-term memory consolidation. This Review presents an overview of the anatomical, neurochemical, and... (Review)
Review
Cholinergic activation regulates cognitive function, particularly long-term memory consolidation. This Review presents an overview of the anatomical, neurochemical, and pharmacological evidence supporting the cholinergic regulation of Pavlovian contextual and cue-conditioned fear learning and extinction. Basal forebrain cholinergic neurons provide inputs to neocortical regions and subcortical limbic structures such as the hippocampus and amygdala. Pharmacological manipulations of muscarinic and nicotinic receptors support the role of cholinergic processes in the amygdala, hippocampus, and prefrontal cortex in modulating the learning and extinction of contexts or cues associated with threat. Additional evidence from lesion studies and analysis of in vivo acetylcholine release with microdialysis similarly support a critical role of cholinergic neurotransmission in corticoamygdalar or corticohippocampal circuits during acquisition of fear extinction. Although a few studies have suggested a complex role of cholinergic neurotransmission in the cellular plasticity essential for extinction learning, more work is required to elucidate the exact cholinergic mechanisms and physiological role of muscarinic and nicotinic receptors in these fear circuits. Such studies are important for elucidating the role of cholinergic neurotransmission in disorders such as posttraumatic stress disorder that involve deficits in extinction learning as well as for developing novel therapeutic approaches for such disorders. © 2016 Wiley Periodicals, Inc.
Topics: Acetylcholine; Animals; Extinction, Psychological; Fear; Humans; Learning; Prosencephalon
PubMed: 27704595
DOI: 10.1002/jnr.23840 -
Neurotoxicology and Teratology 2017The majority of commonly used anesthetic agents induce widespread neuronal degeneration in the developing mammalian brain. Downstream, the process appears to involve... (Review)
Review
The majority of commonly used anesthetic agents induce widespread neuronal degeneration in the developing mammalian brain. Downstream, the process appears to involve activation of the oxidative stress-associated mitochondrial apoptosis pathway. Targeting this pathway could result in prevention of anesthetic toxicity in the immature brain. Carbon monoxide (CO) is a gas that exerts biological activity in the developing brain and low dose exposures have the potential to provide neuroprotection. In recent work, low concentration CO exposures limited isoflurane-induced neuronal apoptosis in a dose-dependent manner in newborn mice and modulated oxidative stress within forebrain mitochondria. Because infants and children are routinely exposed to low levels of CO during low-flow general endotracheal anesthesia, such anti-oxidant and pro-survival cellular effects are clinically relevant. Here we provide an overview of anesthesia-related CO exposure, discuss the biological activity of low concentration CO, detail the effects of CO in the brain during development, and provide evidence for CO-mediated inhibition of anesthesia-induced neurotoxicity.
Topics: Animals; Apoptosis; Brain; Carbon Monoxide; Dose-Response Relationship, Drug; Humans; Isoflurane; Mitochondria; Neurotoxicity Syndromes; Oxidative Stress; Prosencephalon
PubMed: 27616667
DOI: 10.1016/j.ntt.2016.09.002 -
Wiley Interdisciplinary Reviews.... Jan 2017The adult human brain is arguably the most complex of biological systems. It contains 86 billion neurons (the information processing cells of the brain) and many more... (Review)
Review
The adult human brain is arguably the most complex of biological systems. It contains 86 billion neurons (the information processing cells of the brain) and many more support cells. The neurons, with the assistance of the support cells, form trillions of connections creating complex, interconnected neural networks that support all human thought, feeling, and action. A challenge for modern neuroscience is to provide a model that accounts for this exquisitely complex and dynamic system. One fundamental part of this model is an account of how the human brain develops. This essay describes two important aspects of this developmental story. The first part of the story focuses on the remarkable and dynamic set of events that unfold during the prenatal period to give rise to cell lineage that form the essential substance of the brain, particularly the structures of the cerebral hemispheres. The second part of the story focuses on the formation of the major brain pathways of the cerebrum, the intricate fiber bundles that connect different populations of neurons to form the information processing systems that support all human thought and action. These two aspects of early brain development provide an essential foundation for understanding how the structure, organization, and functioning of the human brain emerge. WIREs Cogn Sci 2017, 8:e1409. doi: 10.1002/wcs.1409 For further resources related to this article, please visit the WIREs website.
Topics: Humans; Neural Pathways; Neural Stem Cells; Neuroglia; Neurons; Prosencephalon
PubMed: 27906520
DOI: 10.1002/wcs.1409