Human cerebellar organoids with functional Purkinje cells.

Research on human cerebellar development and disease has been hampered by the need for a human cell-based system that recapitulates the human cerebellum's cellular diversity and functional features. Here, we report a human organoid model (human cerebellar organoids [hCerOs]) capable of developing the complex cellular diversity of the fetal cerebellum, including a human-specific rhombic lip progenitor population that have never been generated in vitro prior to this study. 2-month-old hCerOs form distinct cytoarchitectural features, including laminar organized layering, and create functional connections between inhibitory and excitatory neurons that display coordinated network activity. Long-term culture of hCerOs allows healthy survival and maturation of Purkinje cells that display molecular and electrophysiological hallmarks of their in vivo counterparts, addressing a long-standing challenge in the field. This study therefore provides a physiologically relevant, all-human model system to elucidate the cell-type-specific mechanisms governing cerebellar development and disease.

Non-synaptic function of the autism spectrum disorder-associated gene SYNGAP1 in cortical neurogenesis.

Genes involved in synaptic function are enriched among those with autism spectrum disorder (ASD)-associated rare genetic variants. Dysregulated cortical neurogenesis has been implicated as a convergent mechanism in ASD pathophysiology, yet it remains unknown how 'synaptic' ASD risk genes contribute to these phenotypes, which arise before synaptogenesis. Here, we show that the synaptic Ras GTPase-activating (RASGAP) protein 1 (SYNGAP1, a top ASD risk gene) is expressed within the apical domain of human radial glia cells (hRGCs). In a human cortical organoid model of SYNGAP1 haploinsufficiency, we find dysregulated cytoskeletal dynamics that impair the scaffolding and division plane of hRGCs, resulting in disrupted lamination and accelerated maturation of cortical projection neurons. Additionally, we confirmed an imbalance in the ratio of progenitors to neurons in a mouse model of Syngap1 haploinsufficiency. Thus, SYNGAP1-related brain disorders may arise through non-synaptic mechanisms, highlighting the need to study genes associated with neurodevelopmental disorders (NDDs) in diverse human cell types and developmental stages.

Taming human brain organoids one cell at a time.

Human brain organoids are self-organizing three-dimensional structures that emerge from human pluripotent stem cells and mimic aspects of the cellular composition and functionality of the developing human brain. Despite their impressive self-organizing capacity, organoids lack the stereotypic structural anatomy of their in vivo counterpart, making conventional analysis techniques underpowered to assess cellular composition and gene network regulation in organoids. Advances in single cell transcriptomics have recently allowed characterization and improvement of organoid protocols, as they continue to evolve, by enabling identification of cell types and states along with their developmental origins. In this review, we summarize recent approaches, progresses and challenges in resolving brain organoid's complexity through single-cell transcriptomics. We then discuss emerging technologies that may complement single-cell RNA sequencing by providing additional readouts of cellular states to generate an organ-level view of developmental processes. Altogether, these integrative technologies will allow monitoring of global gene regulation in thousands of individual cells and will offer an unprecedented opportunity to investigate features of human brain development and disease across multiple cellular modalities and with cell-type resolution.

Distinct subnetworks of the thalamic reticular nucleus.

The thalamic reticular nucleus (TRN), the major source of thalamic inhibition, regulates thalamocortical interactions that are critical for sensory processing, attention and cognition1-5. TRN dysfunction has been linked to sensory abnormality, attention deficit and sleep disturbance across multiple neurodevelopmental disorders6-9. However, little is known about the organizational principles that underlie its divergent functions. Here we performed an integrative study linking single-cell molecular and electrophysiological features of the mouse TRN to connectivity and systems-level function. We found that cellular heterogeneity in the TRN is characterized by a transcriptomic gradient of two negatively correlated gene-expression profiles, each containing hundreds of genes. Neurons in the extremes of this transcriptomic gradient express mutually exclusive markers, exhibit core or shell-like anatomical structure and have distinct electrophysiological properties. The two TRN subpopulations make differential connections with the functionally distinct first-order and higher-order thalamic nuclei to form molecularly defined TRN-thalamus subnetworks. Selective perturbation of the two subnetworks in vivo revealed their differential role in regulating sleep. In sum, our study provides a comprehensive atlas of TRN neurons at single-cell resolution and links molecularly defined subnetworks to the functional organization of thalamocortical circuits.