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Human-derived cortical organoids (hCOs) recapitulate cell diversity and 3D structure found in the human brain and offer a promising model for discovery of new gene therapies targeting neurological disorders. Adeno-associated viruses (AAVs) are the most promising vehicles for non-invasive gene delivery to the central nervous system (CNS), but reliable and reproduciblein vitromodels to assess their clinical potential are lacking. hCOs can take on these issues as they are a physiologically relevant model to assess AAV transduction efficiency, cellular tropism, and biodistribution within the tissue parenchyma, all of which could significantly modulate therapeutic efficacy. Here, we examine a variety of naturally occurring AAV serotypes and measure their ability to transduce neurons and glia in hCOs from multiple donors. We demonstrate cell tropism driven by AAV serotype and hCO donor and quantify fractions of neurons and astrocytes transduced with GFP as well as overall hCO health.

Elevated expression of the complement component 4A (C4A) protein has been linked to an increased risk of schizophrenia (SCZ). However, there are few human models available to study the mechanisms by which C4A contributes to the development of SCZ. In this study, we established a C4A overexpressing neuroimmune cortical organoid (NICO) model, which includes mature neuronal cells, astrocytes, and functional microglia. The C4A NICO model recapitulated several neuroimmune endophenotypes observed in SCZ patients, including modulation of inflammatory genes and increased cytokine secretion. C4A expression also increased microglia-mediated synaptic uptake in the NICO model, supporting the hypothesis that synapse and brain volume loss in SCZ patients may be due to excessive microglial pruning. Our results highlight the role of C4A in the immunogenetic risk factors for SCZ and provide a human model for phenotypic discovery and validation of immunomodulating therapies.

The convolutions of the mammalian cerebral cortex allow the enlargement of its surface and addition of novel functional areas during evolution while minimizing expansion of the cranium. Cognitive neurodevelopmental disorders in humans, including microcephaly and lissencephaly, are often associated with impaired gyrification. In the classical model of gyrification, surface area is initially set by the number of radial units, and the forces driving cortical folding include neuronal growth, formation of neuropil, glial cell intercalation, and the patterned growth of subcortical white matter. An alternative model proposes that specified neurogenic hotspots in the outer subventricular zone (oSVZ) produce larger numbers of neurons that generate convexities in the cortex. This directly contradicts reports showing that cortical neurogenesis and settling of neurons into the cortical plate in primates, including humans, are completed well prior to the formation of secondary and tertiary gyri and indeed most primary gyri. In addition, during the main period of gyrification, the oSVZ produces mainly astrocytes and oligodendrocytes. Here we describe how rapid growth of intracortical neuropil, addition of glial cells, and enlargement of subcortical white matter in primates are the primary forces responsible for the post-neurogenic expansion of the cortical surface and formation of gyri during fetal development. Using immunohistochemistry for markers of proliferation and glial and neuronal progenitors combined with transcriptomic analysis, we show that neurogenesis in the ventricular zone and oSVZ is phased out and transitions to gliogenesis prior to gyral development. In summary, our data support the classical model of gyrification and provide insight into the pathogenesis of congenital cortical malformations.

Cerebral organoids provide unparalleled access to human brain development in vitro. However, variability induced by current culture methodologies precludes using organoids as robust disease models. To address this, we developed an automated Organoid Culture and Assay (ORCA) system to support longitudinal unbiased phenotyping of organoids at scale across multiple patient lines. We then characterized organoid variability using novel machine learning methods and found that the contribution of donor, clone, and batch is significant and remarkably consistent over gene expression, morphology, and cell-type composition. Next, we performed multi-factorial protocol optimization, producing a directed forebrain protocol compatible with 96-well culture that exhibits low variability while preserving tissue complexity. Finally, we used ORCA to study tuberous sclerosis, a disease with known genetics but poorly representative animal models. For the first time, we report highly reproducible early morphological and molecular signatures of disease in heterozygous TSC+/− forebrain organoids, demonstrating the benefit of a scaled organoid system for phenotype discovery in human disease models.

The primary stem cells of the cerebral cortex are the radial glial cells (RGCs), and disturbances in their operation lead to myriad brain disorders in all mammals from mice to humans. Here, we found in mice that maternal gestational obesity and hyperglycemia can impair the maturation of RGC fibers and delay cortical neurogenesis. To investigate potential mechanisms, we used optogenetic live-imaging approaches in embryonic cortical slices. We found that Ca(2+) signaling regulates mitochondrial transport and is crucial for metabolic support in RGC fibers. Cyclic intracellular Ca(2+) discharge from localized RGC fiber segments detains passing mitochondria and ensures their proper distribution and enrichment at specific sites such as endfeet. Impairment of mitochondrial function caused an acute loss of Ca(2+) signaling, while hyperglycemia decreased Ca(2+) activity and impaired mitochondrial transport, leading to degradation of the RGC scaffold. Our findings uncover a physiological mechanism indicating pathways by which gestational metabolic disturbances can interfere with brain development.

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.

SUMMARY Better understanding of the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here, we use RNA sequencing, cell imaging, and lineage tracing of mouse and human in vitro NSCs and monkey brain sections to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs develop from pluripotency in vitro, they transition through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines vary in these early patterning states, leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analyses of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon., In Brief Micali et al. report that human telencephalic NSCs in vitro transition through the organizer states that pattern the neocortex. Human pluripotent lines vary in organizer formation, generating divergent neuronal differentiation trajectories biased toward dorsal or ventral telencephalic fates and opening further analysis of the earliest cortical specification events., Graphical Abstract

Division of the telencephalic vesicle into hemispheres and specification of the cerebral cortex are key stages in forebrain development. We investigate the interplay in these processes of Sonic hedgehog (Shh), fibroblast growth factors (Fgfs), and the transcription factor Gli3, which in its repressor form (Gli3R) antagonizes Shh signaling and downregulates expression of severalFgfgenes.Contrary to previous reports, Shh is not required for dorsal hemisphere separation. Mice lacking Shh develop a dorsal telencephalic midline, a cortical hem, and two cortical hemispheres. The hemispheres do not divide rostrally, probably because of reduced localFgfgene expression, resulting from the loss of Shh inhibition of Gli3R. Removing one functional copy ofGli3substantially rescuesFgfexpression and rostral telencephalic morphology.In mice lacking Gli3 function, cortical development is arrested, and ventral gene expression invades the dorsal telencephalon. These defects are potentially explained by disinhibition of Shh activity. However, when both copies ofShhare removed from Gli3-null mice, dorsal telencephalic defects persist. One such defect is a large dorsal expansion of the expression ofFgfgenes.Fgf15expression, for example, expands from a discrete ventral domain throughout the dorsal telencephalon. We propose that Fgf signaling, known to ventralize the telencephalon in a Shh-independent manner, suppresses cortical fate in the absence of Gli3. Our findings point away from Shh involvement in dorsal telencephalic patterning and encourage additional exploration of Fgf signaling and Gli3 repression in corticogenesis.

Calcium activity along radial glial cell fibers influences neurogenesis during early cortical column development., Cortical columns are basic cellular and functional units of the cerebral cortex that are malformed in many brain disorders, but how they initially develop is not well understood. Using an optogenetic sensor in the mouse embryonic forebrain, we demonstrate that Ca2+ fluxes propagate bidirectionally within the elongated fibers of radial glial cells (RGCs), providing a novel communication mechanism linking the proliferative and postmitotic zones before the onset of synaptogenesis. Our results indicate that Ca2+ activity along RGC fibers provides feedback information along the radial migratory pathway, influencing neurogenesis and migration during early column development. Furthermore, we find that this columnar Ca2+ propagation is induced by Notch and fibroblast growth factor activities classically implicated in cortical expansion and patterning. Thus, cortical morphogens and growth factors may influence cortical column assembly in part by regulating long-distance Ca2+ communication along the radial axis of cortical development.

Two anatomical patterns characterize the neocortex, and both are essential for normal cortical function. First, neocortex is divided into anatomically distinct and functionally specialized areas that form a species-specific map. Second, neocortex is composed of layers that organize cortical connectivity. Recent studies of layer and area development have used time-lapse microscopy to follow cortical cell division and migration, gene arrays to find layer- or area- specific regulatory genes, time- and region- specific manipulations of candidate genes, and optical imaging to compare area maps in wild type with genetically altered mice. New observations clarify the molecular and cellular mechanisms that generate each pattern, and stress the links between layer and area formation.

In many vertebrate and invertebrate systems, pioneering axons play a crucial role in establishing large axon tracts. Previous studies have addressed whether the first axons to cross the midline to from the corpus callosum arise from neurons in either the cingulate cortex (Koester and O'Leary [1994] J. Neurosci. 11:6608-6620) or the rostrolateral neocortex (Ozaki and Wahlsten [1998] J. Comp. Neurol. 400:197-206). However, these studies have not provided a consensus on which populations pioneer the corpus callosum. We have found that neurons within the cingulate cortex project axons that cross the midline and enter the contralateral hemisphere at E15.5. By using different carbocyanine dyes injected into either the cingulate cortex or the neocortex of the same brain, we found that cingulate axons crossed the midline before neocortical axons and projected into the contralateral cortex. Furthermore, the first neocortical axons to reach the midline crossed within the tract formed by these cingulate callosal axons, and appeared to fasciculate with them as they crossed the midline. These data indicate that axons from the cingulate cortex might pioneer a pathway for later arriving neocortical axons that form the corpus callosum. We also found that a small number of cingulate axons project to the septum as well as to the ipsilateral hippocampus via the fornix. In addition, we found that neurons in the cingulate cortex projected laterally to the rostrolateral neocortex at least 1 day before the neocortical axons reach the midline. Because the rostrolateral neocortex is the first neocortical region to develop, it sends the first neocortical axons to the midline to form the corpus callosum. We postulate that, together, both laterally and medially projecting cingulate axons may pioneer a path for the medially directed neocortical axons, thus helping to guide these axons toward and across the midline during the formation of the corpus callosum. (C) 2001 Wiley-Liss, Inc.

Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is the first enzyme in the posttranslational sequence of reactions resulting in the lipid modification of lipoproteins in bacteria. A previous comparison of the primary sequences of the Lgt enzymes from phylogenetically distant bacterial species revealed several highly conserved amino acid sequences throughout the molecule; the most extensive of these was the region 103HGGLIG108 in the Escherichia coli Lgt (H.-Y. Qi, K. Sankaran, K. Gan, and H. C. Wu, J. Bacteriol. 177:6820-6824, 1995). These studies also revealed that the kinetics of inactivation of E. coli Lgt with diethylpyrocarbonate were consistent with the modification of a single essential histidine or tyrosine residue. The current study was conducted in an attempt to identify this essential amino acid residue in order to further define structure-function relationships in Lgt. Accordingly, all of the histidine residues and seven of the tyrosine residues of E. coli Lgt were altered by site-directed mutagenesis, and the in vitro activities of the altered enzymes, as well the abilities of the respective mutant lgt alleles to complement the temperature-sensitive phenotype of E. coli SK634 defective in Lgt activity, were determined. The data obtained from these studies, in conjunction with additional chemical inactivation studies, support the conclusion that His-103 is essential for Lgt activity. These studies also indicated that Tyr-235 plays an important role in the function of this enzyme. Although other histidine and tyrosine residues were not found to be essential for Lgt activity, alterations of His-196 resulted in a significant reduction of in vitro activity.

Gyrification allows an expanded cortex with greater functionality to fit into a smaller cranium. However, the mechanisms of gyrus formation have been elusive. We show that ventricular injection of FGF2 protein at embryonic day 11.5-before neurogenesis and before the formation of intrahemispheric axonal connections-altered the overall size and shape of the cortex and induced the formation of prominent, bilateral gyri and sulci in the rostrolateral neocortex. We show increased tangential growth of the rostral ventricular zone (VZ) but decreased Wnt3a and Lef1 expression in the cortical hem and adjacent hippocampal promordium and consequent impaired growth of the caudal cortical primordium, including the hippocampus. At the same time, we observed ectopic Er81 expression, increased proliferation of Tbr2-expressing (Tbr2(+)) intermediate neuronal progenitors (INPs), and elevated Tbr1(+) neurogenesis in the regions that undergo gyrification, indicating region-specific actions of FGF2 on the VZ and subventricular zone (SVZ). However, the relative number of basal radial glia-recently proposed to be important in gyrification-appeared to be unchanged. These findings are consistent with the hypothesis that increased radial unit production together with rapid SVZ growth and heightened localized neurogenesis can cause cortical gyrification in lissencephalic species. These data also suggest that the position of cortical gyri can be molecularly specified in mice. In contrast, a different ligand, FGF8b, elicited surface area expansion throughout the cortical primordium but no gyrification. Our findings demonstrate that individual members of the diverse Fgf gene family differentially regulate global as well as regional cortical growth rates while maintaining cortical layer structure.

The processes regulating cortical surface area expansion during development and evolution are unknown. We show that loss of function of all fibroblast growth factor receptors (FgfRs) expressed at the earliest stages of cortical development causes severe deficits in surface area growth by embryonic day 12.5 (E12.5) in the mouse. In FgfR mutants, accelerated production of neurons led to severe loss of radial progenitors and premature termination of neurogenesis. Nevertheless, these mutants showed remarkably little change in cortical layer structure. Birth-dating experiments indicated that a greater proportion of layer fates was generated during early neurogenic stages, revealing that FgfR activity normally slows the temporal progression of cortical layer fates. Electroporation of a dominant-negative FgfR at E11.5 increased cortical neurogenesis in normal mice—an effect that was blocked by simultaneous activation of the Notch pathway. Together with changes in the expression of Notch pathway genes in FgfR mutant embryos, these findings indicate that Notch lies downstream of FgfR signaling in the same pathway regulating cortical neurogenesis and begin to establish a mechanism for regulating cortical surface expansion.

There is increasing appreciation for the neurodevelopmental underpinnings of many psychiatric disorders. Disorders that begin in childhood such as autism, language disorders or mental retardation as well as adult-onset mental disorders may have origins early in neurodevelopment. Neural stem cells (NSCs) can be defined as self-renewing, multipotent cells that are present in both the embryonic and adult brain. Several recent research findings demonstrate that psychiatric illness may begin with abnormal specification, growth, expansion and differentiation of embryonic NSCs. For example, candidate susceptibility genes for schizophrenia, autism and major depression include the signaling molecule Disrupted In Schizophrenia-1 (DISC-1), the homeodomain gene engrailed-2 (EN-2), and several receptor tyrosine kinases (RTKs), including MET, brain-derived growth factor (BDNF) and fibroblast growth factors (FGF), all of which have been shown to play important roles in NSCs or neuronal precursors. We will discuss here stem cell biology, signaling factors that affect these cells, and the potential contribution of these processes to the etiology of neuropsychiatric disorders. Hypotheses about how some of these factors relate to psychiatric disorders will be reviewed.

Cortical convolutions—prominent folds on the surface of the human brain—have a long history of speculation ( 1 ). The claims range from their function as a bodily cooling system to the attribution of Einstein's genius to the unusual shape of a single gyrus (the ridge of a cortical fold). Only recently, with advances in molecular genetics and brain imaging techniques, has it become possible to study the development, evolution, and abnormalities of cerebral convolutions in a scientifically rigorous manner ( 2 ). On page 764 of this issue, Bae et al. ( 3 ) show that a specific gene controls the number of gyri that form in a region of the cerebral cortex that includes Broca's area (the major language area). This begins to pinpoint mechanisms that underlie the development of specialized regions of the human brain and may be relevant to understanding human brain evolution.

In human holoprosencephaly (HPE), the forebrain does not separate fully into two hemispheres. Further, the border between the telencephalon and diencephalon, the telencephalic/diencephalic junction (TDJ), is often indistinct, and the ventricular system can be blocked at the third ventricle, creating a forebrain ‘holosphere’. Mice deficient in Sonic Hedgehog (Shh) have previously been described to show HPE and associated cyclopia. Here we report that the third ventricle is blocked in Shh null mutants, similar to human HPE, and that characteristic telencephalic and diencephalic signaling centers, the cortical hem and zona limitans intrathalamica (ZLI), are merged, obliterating the TDJ. The resulting forebrain holosphere comprises Foxg1-positive telencephalic- and Foxg1-negative diencephalic territories. Loss of one functional copy of Gli3 in Shh nulls rescues ventricular collapse and substantially restores the TDJ. Characteristic regional gene expression patterns are rescued on the telencephalic side of the TDJ but not in the diencephalon. Further analysis of compound Shh;Gli3 mutants revealed an unexpected type of signaling center deregulation. In Shh;Gli3 mutants, adjacent rings of Fgf8 and Wnt3a expression are induced in the diencephalon at the ZLI, reminiscent of the Fgf8/Wnt1-expressing isthmic organizer. Neither Shh nor Gli3 single mutants show this forebrain double ring of Fgf/Wnt expression; thus both Shh and Gli3 are independently required to suppress it. Adjacent tissue is not respecified to a midbrain/hindbrain fate, but shows overgrowth, consistent with ectopic mitogen expression. Our observations indicate that the separation of the telencephalon and diencephalon depends on interactions between Shh and Gli3, and, moreover, demonstrate that both Shh and Gli3 suppress a potential Fgf/Wnt signaling source in the forebrain. That optional signaling centers are actively repressed in normal development is a striking new insight into the processes of vertebrate brain development.