Human Corticogenesis Research Articles

The people behind the papers - Juan Moriano and Cedric Boeckx.

It is well known that the human cortex is expanded compared with other mammalian species, but the molecular mechanisms underpinning the evolution of human corticogenesis are not well understood. A new paper in Development takes a novel computational approach to screen for genetic changes that could have played an important role in human brain evolution. To learn more about the story behind the paper, we caught up with first author Juan Moriano and corresponding author Cedric Boeckx, a Research Professor at Catalan Institute for Research and Advanced Studies (ICREA).

ARID1B controls transcriptional programs of axon projection in an organoid model of the human corpus callosum

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 invitro 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.

Endogenous mutant Huntingtin alters the corticogenesis via lowering Golgi recruiting ARF1 in cortical organoid

Pathogenic mutant huntingtin (mHTT) infiltrates the adult Huntington’s disease (HD) brain and impairs fetal corticogenesis. However, most HD animal models rarely recapitulate neuroanatomical alterations in adult HD and developing brains. Thus, the human cortical organoid (hCO) is an alternative approach to decode mHTT pathogenesis precisely during human corticogenesis. Here, we replicated the altered corticogenesis in the HD fetal brain using HD patient-derived hCOs. Our HD-hCOs had pathological phenotypes, including deficient junctional complexes in the neural tubes, delayed postmitotic neuronal maturation, dysregulated fate specification of cortical neuron subtypes, and abnormalities in early HD subcortical projections during corticogenesis, revealing a causal link between impaired progenitor cells and chaotic cortical neuronal layering in the HD brain. We identified novel long, oriented, and enriched polyQ assemblies of HTTs that hold large flat Golgi stacks and scaffold clathrin+ vesicles in the neural tubes of hCOs. Flat Golgi stacks conjugated polyQ assemblies by ADP-ribosylation factor 1 (ARF1). Inhibiting ARF1 activation with Brefeldin A (BFA) disassociated polyQ assemblies from Golgi. PolyQ assembles with mHTT scaffolded fewer ARF1 and formed shorter polyQ assembles with fewer and shorter Golgi and clathrin vesicles in neural tubes of HD-hCOs compared with those in hCOs. Inhibiting the activation of ARF1 by BFA in healthy hCOs replicated impaired junctional complexes in the neural tubes. Together, endogenous polyQ assemblies with mHTT reduced the Golgi recruiting ARF1 in the neuroepithelium, impaired the Golgi structure and activities, and altered the corticogenesis in HD-hCO.

TREX tetramer disruption alters RNA processing necessary for corticogenesis in THOC6 Intellectual Disability Syndrome

THOC6 variants are the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 is critical for mammalian Transcription Export complex (TREX) tetramer formation, which is composed of four six-subunit THO monomers. The TREX tetramer facilitates mammalian RNA processing, in addition to the nuclear mRNA export functions of the TREX dimer conserved through yeast. Human and mouse TIDS model systems revealed novel THOC6-dependent, species-specific TREX tetramer functions. Germline biallelic Thoc6 loss-of-function (LOF) variants result in mouse embryonic lethality. Biallelic THOC6 LOF variants reduce the binding affinity of ALYREF to THOC5 without affecting the protein expression of TREX members, implicating impaired TREX tetramer formation. Defects in RNA nuclear export functions were not detected in biallelic THOC6 LOF human neural cells. Instead, mis-splicing was detected in human and mouse neural tissue, revealing novel THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for key signaling pathways known to regulate the transition from proliferative to neurogenic divisions during human corticogenesis. Together, these findings implicate altered RNA processing in the developmental biology of TIDS neuropathology.

Apically localized PANX1 impacts neuroepithelial expansion in human cerebral organoids

Dysfunctional paracrine signaling through Pannexin 1 (PANX1) channels is linked to several adult neurological pathologies and emerging evidence suggests that PANX1 plays an important role in human brain development. It remains unclear how early PANX1 influences brain development, or how loss of PANX1 alters the developing human brain. Using a cerebral organoid model of early human brain development, we find that PANX1 is expressed at all stages of organoid development from neural induction through to neuroepithelial expansion and maturation. Interestingly, PANX1 cellular distribution and subcellular localization changes dramatically throughout cerebral organoid development. During neural induction, PANX1 becomes concentrated at the apical membrane domain of neural rosettes where it co-localizes with several apical membrane adhesion molecules. During neuroepithelial expansion, PANX1−/− organoids are significantly smaller than control and exhibit significant gene expression changes related to cell adhesion, WNT signaling and non-coding RNAs. As cerebral organoids mature, PANX1 expression is significantly upregulated and is primarily localized to neuronal populations outside of the ventricular-like zones. Ultimately, PANX1 protein can be detected in all layers of a 21–22 post conception week human fetal cerebral cortex. Together, these results show that PANX1 is dynamically expressed by numerous cell types throughout embryonic and early fetal stages of human corticogenesis and loss of PANX1 compromises neuroepithelial expansion due to dysregulation of cell-cell and cell-matrix adhesion, perturbed intracellular signaling, and changes to gene regulation.

Integrated transcriptome and proteome analysis reveals posttranscriptional regulation of ribosomal genes in human brain organoids.

During development of the human cerebral cortex, multipotent neural progenitors generate excitatory neurons and glial cells. Investigations of the transcriptome and epigenome have revealed important gene regulatory networks underlying this crucial developmental event. However, the posttranscriptional control of gene expression and protein abundance during human corticogenesis remains poorly understood. We addressed this issue by using human telencephalic brain organoids grown using a dual reporter cell line to isolate neural progenitors and neurons and performed cell class and developmental stage-specific transcriptome and proteome analysis. Integrating the two datasets revealed modules of gene expression during human corticogenesis. Investigation of one such module uncovered mTOR-mediated regulation of translation of the 5'TOP element-enriched translation machinery in early progenitor cells. We show that in early progenitors partial inhibition of the translation of ribosomal genes prevents precocious translation of differentiation markers. Overall, our multiomics approach proposes novel posttranscriptional regulatory mechanisms crucial for the fidelity of cortical development.

Benchmarking brain organoid recapitulation of fetal corticogenesis

Brain organoids are becoming increasingly relevant to dissect the molecular mechanisms underlying psychiatric and neurological conditions. The in vitro recapitulation of key features of human brain development affords the unique opportunity of investigating the developmental antecedents of neuropsychiatric conditions in the context of the actual patients’ genetic backgrounds. Specifically, multiple strategies of brain organoid (BO) differentiation have enabled the investigation of human cerebral corticogenesis in vitro with increasing accuracy. However, the field lacks a systematic investigation of how closely the gene co-expression patterns seen in cultured BO from different protocols match those observed in fetal cortex, a paramount information for ensuring the sensitivity and accuracy of modeling disease trajectories. Here we benchmark BO against fetal corticogenesis by integrating transcriptomes from in-house differentiated cortical BO (CBO), other BO systems, human fetal brain samples processed in-house, and prenatal cortices from the BrainSpan Atlas. We identified co-expression patterns and prioritized hubs of human corticogenesis and CBO differentiation, highlighting both well-preserved and discordant trends across BO protocols. We evaluated the relevance of identified gene modules for neurodevelopmental disorders and psychiatric conditions finding significant enrichment of disease risk genes especially in modules related to neuronal maturation and synapsis development. The longitudinal transcriptomic analysis of CBO revealed a two-step differentiation composed of a fast-evolving phase, corresponding to the appearance of the main cell populations of the cortex, followed by a slow-evolving one characterized by milder transcriptional changes. Finally, we observed heterochronicity of differentiation across BO models compared to fetal cortex. Our approach provides a framework to directly compare the extent of in vivo/in vitro alignment of neurodevelopmentally relevant processes and their attending temporalities, structured as a resource to query for modeling human corticogenesis and the neuropsychiatric outcomes of its alterations.

Proper acquisition of cell class identity in organoids allows definition of fate specification programs of the human cerebral cortex

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 over610,000 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 invitro as a resource to prime investigation into the mechanisms of human cortical development.

The ciliary gene INPP5E confers dorsal telencephalic identity to human cortical organoids by negatively regulating Sonic hedgehog signaling.

Defects in primary cilia, cellular antennas that control multiple intracellular signaling pathways, underlie several neurodevelopmental disorders, but it remains unknown how cilia control essential steps in human brain formation. Here, we show that cilia are present on the apical surface of radial glial cells in human fetal forebrain. Interfering with cilia signaling in human organoids by mutating the INPP5E gene leads to the formation of ventral telencephalic cell types instead of cortical progenitors and neurons. INPP5E mutant organoids also show increased Sonic hedgehog (SHH) signaling, and cyclopamine treatment partially rescues this ventralization. In addition, ciliary expression of SMO, GLI2, GPR161, and several intraflagellar transport (IFT) proteins is increased. Overall, these findings establish the importance of primary cilia for dorsal and ventral patterning in human corticogenesis, indicate a tissue-specific role of INPP5E as a negative regulator of SHH signaling, and have implications for the emerging roles of cilia in the pathogenesis of neurodevelopmental disorders.

Cellular and Molecular Mechanisms Linking Human Cortical Development and Evolution.

The cerebral cortex is at the core of brain functions that are thought to be particularly developed in the human species. Human cortex specificities stem from divergent features of corticogenesis, leading to increased cortical size and complexity. Underlying cellular mechanisms include prolonged patterns of neuronal generation and maturation, as well as the amplification of specific types of stem/progenitor cells. While the gene regulatory networks of corticogenesis appear to be largely conserved among all mammals including humans, they have evolved in primates, particularly in the human species, through the emergence of rapidly divergent transcriptional regulatory elements, as well as recently duplicated novel genes. These human-specific molecular features together control key cellular milestones of human corticogenesis and are often affected in neurodevelopmental disorders, thus linking human neural development, evolution, and diseases.

Human intermediate progenitor diversity during cortical development

Studies of the spatiotemporal, transcriptomic, and morphological diversity of radial glia (RG) have spurred our current models of human corticogenesis. In the developing cortex, neural intermediate progenitor cells (nIPCs) are a neuron-producing transit-amplifying cell type born in the germinal zones of the cortex from RG. The potential diversity of the nIPC population, that produces a significant portion of excitatory cortical neurons, is understudied, particularly in the developing human brain. Here we explore the spatiotemporal, transcriptomic, and morphological variation that exists within the human nIPC population and provide a resource for future studies. We observe that the spatial distribution of nIPCs in the cortex changes abruptly around gestational week (GW) 19/20, marking a distinct shift in cellular distribution and organization during late neurogenesis. We also identify five transcriptomic subtypes, one of which appears at this spatiotemporal transition. Finally, we observe a diversity of nIPC morphologies that do not correlate with specific transcriptomic subtypes. These results provide an analysis of the spatiotemporal, transcriptional, and morphological diversity of nIPCs in developing brain tissue and provide an atlas of nIPC subtypes in the developing human cortex that can benchmark in vitro models of human development such as cerebral organoids and help inform future studies of how nIPCs contribute to cortical neurogenesis.

Dynamic properties of mitochondria during human corticogenesis.

ABSTRACTMitochondria are signaling hubs responsible for the generation of energy through oxidative phosphorylation, the production of key metabolites that serve the bioenergetic and biosynthetic needs of the cell, calcium (Ca2+) buffering and the initiation/execution of apoptosis. The ability of mitochondria to coordinate this myriad of functions is achieved through the exquisite regulation of fundamental dynamic properties, including remodeling of the mitochondrial network via fission and fusion, motility and mitophagy. In this Review, we summarize the current understanding of the mechanisms by which these dynamic properties of the mitochondria support mitochondrial function, review their impact on human cortical development and highlight areas in need of further research.

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

BackgroundThe inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes—in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points.ResultsConventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes.ConclusionWe present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.

Neuronal fate acquisition and specification: time for a change.

During embryonic development, neural stem/progenitor cells generate hundreds of different cell types through the combination of intrinsic and extrinsic cues. Recent data obtained in mouse and human cortical neurogenesis provide novel views about this interplay and how it evolves with time, whether during irreversible cell fate transitions that neural stem cells undergo to become neurons, or through gradual temporal changes of competence that lead to increased neuronal diversity from a common stem cell pool. In each case the temporal changes result from a dynamic balance between intracellular states and extracellular signalling factors. The underlying mechanisms are mostly conserved across species, but some display unique features in human corticogenesis, thereby linking temporal features of neurogenesis and human brain evolution.

Cell-type-specific 3D epigenomes in the developing human cortex.

Lineage-specific epigenomic changes during human corticogenesis have remained elusive due to challenges with sample availability and tissue heterogeneity. For example, previous studies used single-cell RNA sequencing to identify at least nine major cell types and up to 26 distinct subtypes in the dorsal cortex alone1,2. Here, we characterize cell type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational human cortex samples. We show that chromatin interactions underlie multiple aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell type-specific manner. In addition, promoters with significantly increased levels of chromatin interactivity, termed super interactive promoters, are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a novel technique integrating immunostaining, CRISPRi, RNAscope, and image analysis for validating cell type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our study presents the first cell type-specific characterization of 3D epigenomes in the developing human cortex, advancing our understanding of gene regulation and lineage specification during this critical developmental window.

Foxp1 Regulates Neural Stem Cell Self-Renewal and Bias Toward Deep Layer Cortical Fates

The laminar architecture of the mammalian neocortex depends on the orderly generation of distinct neuronal subtypes by apical radial glia (aRG) during embryogenesis. Here, we identify critical roles for the autism risk gene Foxp1 in maintaining aRG identity and gating the temporal competency for deep-layer neurogenesis. Early in development, aRG express high levels ofFoxp1 mRNA and protein, which promote self-renewingcell divisions and deep-layer neuron production. Foxp1 levels subsequently decline during the transitionto superficial-layer neurogenesis. Sustained Foxp1 expression impedes this transition, preserving a population of cells with aRG identity throughout development and extending the early neurogenic period into postnatal life. FOXP1 expression is further associated with the initial formation and expansion of basal RG (bRG) during human corticogenesis and can promote the formation of cells exhibiting characteristics of bRG when misexpressed in the mouse cortex. Together, these findings reveal broad functions for Foxp1 in cortical neurogenesis.

Cortical Malformations: Lessons in Human Brain Development.

Creating a functional cerebral cortex requires a series of complex and well-coordinated developmental steps. These steps have evolved across species with the emergence of cortical gyrification and coincided with more complex behaviors. The presence of diverse progenitor cells, a protracted timeline for neuronal migration and maturation, and diverse neuronal types are developmental features that have emerged in the gyrated cortex. These factors could explain how the human brain has expanded in size and complexity. However, their complex nature also renders new avenues of vulnerability by providing additional cell types that could contribute to disease and longer time windows that could impact the composition and organization of the cortical circuit. We aim to discuss the unique developmental steps observed in human corticogenesis and propose how disruption of these species-unique processes could lead to malformations of cortical development.

Human Cortical Organoids Expose a Differential Function of GSK3 on Cortical Neurogenesis.

SummaryThe regulation of the proliferation and polarity of neural progenitors is crucial for the development of the brain cortex. Animal studies have implicated glycogen synthase kinase 3 (GSK3) as a pivotal regulator of both proliferation and polarity, yet the functional relevance of its signaling for the unique features of human corticogenesis remains to be elucidated. We harnessed human cortical brain organoids to probe the longitudinal impact of GSK3 inhibition through multiple developmental stages. Chronic GSK3 inhibition increased the proliferation of neural progenitors and caused massive derangement of cortical tissue architecture. Single-cell transcriptome profiling revealed a direct impact on early neurogenesis and uncovered a selective role of GSK3 in the regulation of glutamatergic lineages and outer radial glia output. Our dissection of the GSK3-dependent transcriptional network in human corticogenesis underscores the robustness of the programs determining neuronal identity independent of tissue architecture.

Distinct Vulnerability and Resilience of Human Neuroprogenitor Subtypes in Cerebral Organoid Model of Prenatal Hypoxic Injury.

Prenatal hypoxic injury (HI) is a leading cause of neurological disability. The immediate and long-term effects of hypoxia on progenitor homeostasis and developmental progression during early human brain development remain unclear. This gap is due to difficulty to access human fetal brain tissues and inadequate animal models to study human corticogenesis. Recent optimizations of cerebral organoid models derived from human embryonic stem (ES) cells present new opportunities to investigate pathophysiology of prenatal HI. Here, we implemented a transient HI model using human cerebral organoids with dorsal forebrain specification. We demonstrated that transient hypoxia resulted in immediate and prolonged apoptosis in cerebral organoids, with outer radial glia (oRG), a progenitor population more prominent in primates, and differentiating neuroblasts/immature neurons suffering larger losses. In contrast, neural stem cells in ventricular zone displayed relative resilience to HI and exhibited a shift of cleavage plane angle favoring symmetric division, thereby providing a mechanism to replenish the stem cell pool. Furthermore, we defined the vulnerable window and neurodifferentiation stages that are particularly sensitive to HI. Understanding cell type-specific and stage-dependent effects of prenatal HI on survival and mitotic behavior of human neuroprogenitor subtypes during early human corticogenesis helps elucidate the etiology of neurodevelopmental disorders, and provides a therapeutic starting point to protect the vulnerable populations at critical timeframes.

Using brain organoids to study human neurodevelopment, evolution and disease.

The brain is one of the most complex organs, responsible for the advanced intellectual and cognitive ability of humans. Although primates are to some extent capable of performing cognitive tasks, their abilities are less evolved. One of the reasons for this is the vast differences in the brain of humans compared to other mammals, in terms of shape, size and complexity. Such differences make the study of human brain development fascinating. Interestingly, the cerebral cortex is by far the most complex brain region resulting from its selective evolution within mammals over millions of years. Unraveling the molecular and cellular mechanisms regulating brain development, as well as the evolutionary differences seen across species and the need to understand human brain disorders, are some of the reasons why scientists are interested in improving their current knowledge on human corticogenesis. Toward this end, several animal models including primates have been used, however, these models are limited in their extent to recapitulate human-specific features. Recent technological achievements in the field of stem cell research, which have enabled the generation of human models of corticogenesis, called brain or cerebral organoids, are of great importance. This review focuses on the main cellular and molecular features of human corticogenesis and the use of brain organoids to study it. We will discuss the key differences between cortical development in human and nonhuman mammals, the technological applications of brain organoids and the different aspects of cortical development in normal and pathological conditions, which can be modeled using brain organoids. This article is categorized under: Comparative Development and Evolution > Regulation of Organ Diversity Nervous System Development > Vertebrates: General Principles.