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Cerebral organoids—3D cultures of human cerebral tissue derived from pluripotent stem cells—have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

Here, using further optimized 3D culture that allows highly selective induction and long-term growth of human ES cell (hESC)-derived cortical neuroepithelium, we demonstrate unique aspects of self-organization in human neocorticogenesis. Self-organized cortical tissue spontaneously forms a polarity along the dorsocaudal-ventrorostral axis and undergoes region-specific rolling morphogenesis that generates a semispherical structure. The neuroepithelium self-forms a multilayered structure including three neuronal zones (subplate, cortical plate, and Cajal-Retzius cell zones) and three progenitor zones (ventricular, subventricular, and intermediate zones) in the same apical-basal order as seen in the human fetal cortex in the early second trimester. In the cortical plate, late-born neurons tend to localize more basally to early-born neurons, consistent with the inside-out pattern seen in vivo. Furthermore, the outer subventricular zone contains basal progenitors that share characteristics with outer radial glia abundantly found in the human, but not mouse, fetal brain. Thus, human neocorticogenesis involves intrinsic programs that enable the emergence of complex neocortical features.

The primate cerebrum is characterized by a large expansion of cortical surface area, the formation of convolutions, and extraordinarily voluminous subcortical white matter. It was recently proposed that this expansion is primarily driven by increased production of superficial neurons in the dramatically enlarged outer subventricular zone (oSVZ). Here, we examined the development of the parietal cerebrum in macaque monkey and found that, indeed, the oSVZ initially adds neurons to the superficial layers II and III, increasing their thickness. However, as the oSVZ grows in size, its output changes to production of astrocytes and oligodendrocytes, which in primates outnumber cerebral neurons by a factor of three. After the completion of neurogenesis around embryonic day (E) 90, when the cerebrum is still lissencephalic, the oSVZ enlarges and contains Pax6⁺/Hopx⁺ outer (basal) radial glial cells producing astrocytes and oligodendrocytes until after E125. Our data indicate that oSVZ gliogenesis, rather than neurogenesis, correlates with rapid enlargement of the cerebrum and development of convolutions, which occur concomitantly with the formation of cortical connections via the underlying white matter, in addition to neuronal growth, elaboration of dendrites, and amplification of neuropil in the cortex, which are primary factors in the formation of cerebral convolutions in primates.

Neurodevelopmental diseases (NDDs), such as autism spectrum disorders, epilepsy, and schizophrenia, are characterized by diverse facets of neurological and psychiatric symptoms, differing in etiology, onset and severity. Such symptoms include mental delay, cognitive and language impairments, or restrictions to adaptive and social behavior. Nevertheless, all have in common that critical milestones of brain development are disrupted, leading to functional deficits of the central nervous system and clinical manifestation in child- or adulthood. To approach how the different development-associated neuropathologies can occur and which risk factors or critical processes are involved in provoking higher susceptibility for such diseases, a detailed understanding of the mechanisms underlying proper brain formation is required. NDDs rely on deficits in neuronal identity, proportion or function, whereby a defective development of the cerebral cortex, the seat of higher cognitive functions, is implicated in numerous disorders. Such deficits can be provoked by genetic and environmental factors during corticogenesis. Thereby, epigenetic mechanisms can act as an interface between external stimuli and the genome, since they are known to be responsive to external stimuli also in cortical neurons. In line with that, DNA methylation, histone modifications/variants, ATP-dependent chromatin remodeling, as well as regulatory non-coding RNAs regulate diverse aspects of neuronal development, and alterations in epigenomic marks have been associated with NDDs of varying phenotypes. Here, we provide an overview of essential steps of mammalian corticogenesis, and discuss the role of epigenetic mechanisms assumed to contribute to pathophysiological aspects of NDDs, when being disrupted.

The extracellular protein Reelin, expressed by Cajal–Retzius (CR) cells at early stages of cortical development and at late stages by GABAergic interneurons, regulates radial migration and the “inside-out” pattern of positioning. Current models of Reelin functions in corticogenesis focus on early CR cell–derived Reelin in layer I. However, developmental disorders linked to Reelin deficits, such as schizophrenia and autism, are related to GABAergic interneuron–derived Reelin, although its role in migration has not been established. Here we selectively inactivated the Reln gene in CR cells or GABAergic interneurons. We show that CR cells have a major role in the inside-out order of migration, while CR and GABAergic cells sequentially cooperate to prevent invasion of cortical neurons into layer I. Furthermore, GABAergic cell–derived Reelin compensates some features of the reeler phenotype and is needed for the fine tuning of the layer-specific distribution of cortical neurons. In the hippocampus, the inactivation of Reelin in CR cells causes dramatic alterations in the dentate gyrus and mild defects in the hippocampus proper. These findings lead to a model in which both CR and GABAergic cell–derived Reelin cooperate to build the inside-out order of corticogenesis, which might provide a better understanding of the mechanisms involved in the pathogenesis of neuropsychiatric disorders linked to abnormal migration and Reelin deficits.

Cerebral organoids, self-organizing structures with increased cellular diversity and longevity, have addressed shortcomings in mimicking human brain complexity and architecture. However, imaging intact organoids poses challenges due to size, cellular density, and light-scattering properties. Traditional one-photon microscopy faces limitations in resolution and contrast, especially for deep regions. Here, we first discuss the fundamentals of multiphoton microscopy (MPM) as a promising alternative, leveraging non-linear fluorophore excitation and longer wavelengths for improved imaging of live cerebral organoids. Then, we review recent applications of MPM in studying morphogenesis and differentiation, emphasizing its potential for overcoming limitations associated with other imaging techniques. Furthermore, our paper underscores the crucial role of cerebral organoids in providing insights into human-specific neurodevelopmental processes and neurological disorders, addressing the scarcity of human brain tissue for translational neuroscience. Ultimately, we envision using multimodal multiphoton microscopy for longitudinal imaging of intact cerebral organoids, propelling advancements in our understanding of neurodevelopment and related disorders.

The vision of astroglia as a bare scaffold to neuronal circuitry has been largely overturned. Astrocytes exert a neurotrophic function, but also take active part in supporting synaptic transmission and in calibrating blood circulation. Many aspects of their functioning have been unveiled from studies conducted in murine models, however evidence is showing many differences between mouse and human astrocytes starting from their development and encompassing morphological, transcriptomic and physiological variations when they achieve complete maturation. The evolutionary race toward superior cognitive abilities unique to humans has drastically impacted neocortex structure and, together with neuronal circuitry, astrocytes have also been affected with the acquisition of species-specific properties. In this review, we summarize diversities between murine and human astroglia, with a specific focus on neocortex, in a panoramic view that starts with their developmental origin to include all structural and molecular differences that mark the uniqueness of human astrocytes.

The Dmrt genes encode the transcription factor containing the DM (doublesex and mab-3) domain, an intertwined zinc finger-like DNA binding module. While Dmrt genes are mainly involved in the sexual development of various species, recent studies have revealed that Dmrt genes, which belong to the DmrtA subfamily, are differentially expressed in the embryonic brain and spinal cord and are essential for the development of the central nervous system. Herein, we summarize recent studies that reveal the multiple functions of the Dmrt genes in various aspects of vertebrate neural development, including brain patterning, neurogenesis, and the specification of neurons.

Sexual differences are prevalent in the brain. DMRT transcription factors have been postulated as important determinants of sex differences. Previous research focused on the DMA subfamily in the brain. Here, we reveal an unprecedented role for Dmrt2 in regulating the proliferation and development of cortical neurons in mice. Dmrt2 is expressed in deep-layer neurons of the cingulate cortex (CgCx) throughout development. Its downregulation in the CgCx primordium results in premature cell cycle exit of embryonic progenitors and subsequent reduction in cortical plate cellular density at later developmental stages. Dmrt2 expression is higher in male embryos during early development, potentially explaining their increased vulnerability to Dmrt2 depletion. As development progresses, Dmrt2 expression persists in deep-layer neurons, controlling processes like migration, axonal targeting, and neuronal-specific gene expression. This study broadens our understanding of Dmrt2 gene function in the brain and provides insights into the molecular basis of sexual differences in neurodevelopmental processes.

Intracellular calcium (Ca[sup.2+]) signaling is a central regulator of corticogenesis, governing haveneural stem cell behavior, fate transitions, neuronal migration, and circuit assembly. Beyond its canonical role as a second messenger, Ca[sup.2+] shapes cytoskeletal organization by modulating microtubule dynamics essential for mitotic spindle function, radial glial scaffold, nucleokinesis, and neurite extension. This review synthesizes evidence from in vivo, ex vivo, and in vitro studies to delineate Ca[sup.2+]-dependent pathways and Ca[sup.2+]-binding proteins that couple, within restricted Ca[sup.2+] microdomains in space and time, to microtubule regulation during mammalian cortical development. We highlight mechanistic nodes involving calmodulin, Ca[sup.2+]/calmodulin-dependent kinases (CaMKs), S100 proteins, cadherins/protocadherins, centrins (CENs), and Ca[sup.2+] sensors such as STIM1 and calneurons, which collectively coordinate spindle orientation, progenitor division modes, radial migration, and neurite outgrowth. Finally, we discuss how perturbations in Ca[sup.2+]-controlled cytoskeletal programs may contribute to abnormal cortical cytoarchitecture and neurodevelopmental disease. By integrating Ca[sup.2+] microdomain transients with microtubule control modules, this review provides a unified framework for understanding how Ca[sup.2+] orchestrates key cellular events during mammalian corticogenesis and propose that Ca[sup.2+] oscillatory codes are translated into direct or indirect microtubule/cytoskeletal remodeling transitions that determine neural stem cell fate, migration, and maturation, to accurately establish cortical architecture and function.