RESUMO
Realizing the full utility of brain organoids to study human development requires understanding whether organoids precisely replicate endogenous cellular and molecular events, particularly since acquisition of cell identity in organoids can be impaired by abnormal metabolic states. We present a comprehensive single-cell transcriptomic, epigenetic, and spatial atlas of human cortical organoid development, comprising over 610,000 cells, from generation of neural progenitors through production of differentiated neuronal and glial subtypes. We show that processes of cellular diversification correlate closely to endogenous ones, irrespective of metabolic state, empowering the use of this atlas to study human fate specification. We define longitudinal molecular trajectories of cortical cell types during organoid development, identify genes with predicted human-specific roles in lineage establishment, and uncover early transcriptional diversity of human callosal neurons. The findings validate this comprehensive atlas of human corticogenesis in vitro as a resource to prime investigation into the mechanisms of human cortical development.
Assuntos
Córtex Cerebral , Organoides , Diferenciação Celular , Córtex Cerebral/metabolismo , Humanos , Neurogênese , Neurônios , Organoides/metabolismoRESUMO
Interindividual genetic variation affects the susceptibility to and progression of many diseases1,2. However, efforts to study how individual human brains differ in normal development and disease phenotypes are limited by the paucity of faithful cellular human models, and the difficulty of scaling current systems to represent multiple people. Here we present human brain Chimeroids, a highly reproducible, multidonor human brain cortical organoid model generated by the co-development of cells from a panel of individual donors in a single organoid. By reaggregating cells from multiple single-donor organoids at the neural stem cell or neural progenitor cell stage, we generate Chimeroids in which each donor produces all cell lineages of the cerebral cortex, even when using pluripotent stem cell lines with notable growth biases. We used Chimeroids to investigate interindividual variation in the susceptibility to neurotoxic triggers that exhibit high clinical phenotypic variability: ethanol and the antiepileptic drug valproic acid. Individual donors varied in both the penetrance of the effect on target cell types, and the molecular phenotype within each affected cell type. Our results suggest that human genetic background may be an important mediator of neurotoxin susceptibility and introduce Chimeroids as a scalable system for high-throughput investigation of interindividual variation in processes of brain development and disease.
Assuntos
Córtex Cerebral , Quimera , Predisposição Genética para Doença , Neurotoxinas , Organoides , Feminino , Humanos , Masculino , Linhagem da Célula/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Quimera/genética , Etanol/efeitos adversos , Etanol/toxicidade , Variação Genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurotoxinas/toxicidade , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Fenótipo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Doadores de Tecidos , Ácido Valproico/efeitos adversos , Ácido Valproico/toxicidade , Predisposição Genética para Doença/genéticaRESUMO
The striatum is a large brain nucleus with an important role in the control of movement and emotions. Medium spiny neurons (MSNs) are striatal output neurons forming prominent descending axon tracts that target different brain nuclei. However, how MSN axon tracts in the forebrain develop remains poorly understood. Here, we implicate the Wnt binding receptor Frizzled3 in several uncharacterized aspects of MSN pathway formation [i.e., anterior-posterior guidance of MSN axons in the striatum and their subsequent growth into the globus pallidus (GP), an important (intermediate) target]. In Frizzled3 knock-out mice, MSN axons fail to extend along the anterior-posterior axis of the striatum, and many do not reach the GP. Wnt5a acts as an attractant for MSN axons in vitro, is expressed in a posterior high, anterior low gradient in the striatum, and Wnt5a knock-out mice phenocopy striatal anterior-posterior defects observed in Frizzled3 mutants. This suggests that Wnt5a controls anterior-posterior guidance of MSN axons through Frizzled3. Axons that reach the GP in Frizzled3 knock-out mice fail to enter this structure. Surprisingly, entry of MSN axons into the GP non-cell-autonomously requires Frizzled3, and our data suggest that GP entry may be contingent on the correct positioning of "corridor" guidepost cells for thalamocortical axons by Frizzled3. Together, these data dissect MSN pathway development and reveal (non)cell-autonomous roles for Frizzled3 in MSN axon guidance. Further, they are the first to identify a gene that provides anterior-posterior axon guidance in a large brain nucleus and link Frizzled3 to corridor cell development. SIGNIFICANCE STATEMENT: Striatal axon pathways mediate complex physiological functions and are an important therapeutic target, underscoring the need to define how these connections are established. Remarkably, the molecular programs regulating striatal pathway development remain poorly characterized. Here, we determine the embryonic ontogeny of the two main striatal pathways (striatonigral and striatopallidal) and identify novel (non)cell-autonomous roles for the axon guidance receptor Frizzled3 in uncharacterized aspects of striatal pathway formation (i.e., anterior-posterior axon guidance in the striatum and axon entry into the globus pallidus). Further, our results link Frizzled3 to corridor guidepost cell development and suggest that an abnormal distribution of these cells has unexpected, widespread effects on the development of different axon tracts (i.e., striatal and thalamocortical axons).
Assuntos
Axônios/fisiologia , Polaridade Celular/genética , Corpo Estriado/citologia , Receptores Frizzled/metabolismo , Vias Neurais/embriologia , Neurônios/citologia , Animais , Células Cultivadas , Corpo Estriado/embriologia , Embrião de Mamíferos , Feminino , Receptores Frizzled/genética , Globo Pálido/citologia , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The mammalian brain's somatosensory cortex is a topographic map of the body's sensory experience. In mice, cortical barrels reflect whisker input. We asked whether these cortical structures require sensory input to develop or are driven by intrinsic activity. Thalamocortical columns, connecting the thalamus to the cortex, emerge before sensory input and concur with calcium waves in the embryonic thalamus. We show that the columnar organization of the thalamocortical somatotopic map exists in the mouse embryo before sensory input, thus linking spontaneous embryonic thalamic activity to somatosensory map formation. Without thalamic calcium waves, cortical circuits become hyperexcitable, columnar and barrel organization does not emerge, and the somatosensory map lacks anatomical and functional structure. Thus, a self-organized protomap in the embryonic thalamus drives the functional assembly of murine thalamocortical sensory circuits.
Assuntos
Neurônios/fisiologia , Córtex Somatossensorial/embriologia , Tálamo/embriologia , Potenciais de Ação , Animais , Mapeamento Encefálico , Sinalização do Cálcio , Estimulação Elétrica , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Plasticidade Neuronal , Canais de Potássio Corretores do Fluxo de Internalização/genéticaRESUMO
The developmental programs that control the specification of cortical and thalamic territories are maintained largely as independent processes. However, bulk of evidence demonstrates the requirement of the reciprocal interactions between cortical and thalamic neurons as key for the correct development of functional thalamocortical circuits. This reciprocal loop of connections is essential for sensory processing as well as for the execution of complex sensory-motor tasks. Here, we review recent advances in our understanding of how mutual collaborations between both brain regions define area patterning and cell differentiation in the thalamus and cortex.
Assuntos
Padronização Corporal/fisiologia , Diferenciação Celular/fisiologia , Córtex Cerebral/fisiologia , Neurônios/fisiologia , Tálamo/fisiologia , Animais , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Humanos , Neurônios/metabolismo , Tálamo/crescimento & desenvolvimento , Tálamo/metabolismoRESUMO
The cerebral cortex is organized into specialized sensory areas, whose initial territory is determined by intracortical molecular determinants. Yet, sensory cortical area size appears to be fine tuned during development to respond to functional adaptations. Here we demonstrate the existence of a prenatal sub-cortical mechanism that regulates the cortical areas size in mice. This mechanism is mediated by spontaneous thalamic calcium waves that propagate among sensory-modality thalamic nuclei up to the cortex and that provide a means of communication among sensory systems. Wave pattern alterations in one nucleus lead to changes in the pattern of the remaining ones, triggering changes in thalamic gene expression and cortical area size. Thus, silencing calcium waves in the auditory thalamus induces Rorß upregulation in a neighbouring somatosensory nucleus preluding the enlargement of the barrel-field. These findings reveal that embryonic thalamic calcium waves coordinate cortical sensory area patterning and plasticity prior to sensory information processing.