Spatially resolved cell atlas of the teleost telencephalon and deep homology of the vertebrate forebrain.

The telencephalon has undergone remarkable diversification and expansion throughout vertebrate evolution, exhibiting striking variations in structural and functional complexity. Nevertheless, fundamental features are shared across vertebrate taxa, such as the presence of distinct regions including the pallium, subpallium, and olfactory structures. Teleost fishes have a uniquely "everted" telencephalon, which has confounded comparisons of their brain regions to other vertebrates. Here we combine spatial transcriptomics and single nucleus RNA-sequencing to generate a spatially-resolved transcriptional atlas of the Mchenga conophorus cichlid fish telencephalon. We then compare cell-types and anatomical regions in the cichlid telencephalon with those in amphibians, reptiles, birds, and mammals. We uncover striking transcriptional similarities between cell-types in the fish telencephalon and subpallial, hippocampal, and cortical cell-types in tetrapods, and find support for partial eversion of the teleost telencephalon. Ultimately, our work lends new insights into the organization and evolution of conserved cell-types and regions in the vertebrate forebrain.

Cell-type-resolved mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around the anatomical origins of specific brain cell subtypes and lineage relationships within the human forebrain1-7. Thus, direct observation in the mature human brain is critical for a complete understanding of its structural organization and cellular origins. Here we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific mosaic variant barcode analysis. From four hemispheres and two different human neurotypical donors, we identified 287 and 780 mosaic variants, respectively, that were used to deconvolve clonal dynamics. Clonal spread and allele fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted than resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome transcriptome analysis at both a cell-type-specific and a single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of mosaic variants across 17 locations within one parietal lobe reveals that restriction of clonal spread in the anterior-posterior axis precedes restriction in the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus, cell-type-resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Antisense oligonucleotide therapeutic approach for Timothy syndrome.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

A patterned human neural tube model using microfluidic gradients.

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.

Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Assembloid CRISPR screens reveal impact of disease genes in human neurodevelopment.

The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain1-3, which has been challenging to study at inaccessible stages of late gestation and early postnatal human development4. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development5, but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids6. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.

Nests of dividing neuroblasts sustain interneuron production for the developing human brain.

The human cortex contains inhibitory interneurons derived from the medial ganglionic eminence (MGE), a germinal zone in the embryonic ventral forebrain. How this germinal zone generates sufficient interneurons for the human brain remains unclear. We found that the human MGE (hMGE) contains nests of proliferative neuroblasts with ultrastructural and transcriptomic features that distinguish them from other progenitors in the hMGE. When dissociated hMGE cells are transplanted into the neonatal mouse brain, they reform into nests containing proliferating neuroblasts that generate young neurons that migrate extensively into the mouse forebrain and mature into different subtypes of functional interneurons. Together, these results indicate that the nest organization and sustained proliferation of neuroblasts in the hMGE provide a mechanism for the extended production of interneurons for the human forebrain.

S100B is selectively expressed by gray matter protoplasmic astrocytes and myelinating oligodendrocytes in the developing CNS.

Studies on the development of central nervous system (CNS) primarily rely on the use of specific molecular markers for different types of neural cells. S100B is widely being used as a specific marker for astrocytes in the CNS. However, the specificity of its expression in astrocyte lineage has not been systematically investigated and thus has remained a lingering issue. In this study, we provide several lines of molecular and genetic evidences that S100B is expressed in both protoplasmic astrocytes and myelinating oligodendrocytes. In the developing spinal cord, S100B is first expressed in the ventral neuroepithelial cells, and later in ALDH1L1+/GS+ astrocytes in the gray matter. Meanwhile, nearly all the S100B+ cells in the white matter are SOX10+/MYRF+ oligodendrocytes. Consistent with this observation, S100B expression is selectively lost in the white matter in Olig2-null mutants in which oligodendrocyte progenitor cells (OPCs) are not produced, and dramatically reduced in Myrf-conditional knockout mutants in which OPCs fail to differentiate. Similar expression patterns of S100B are observed in the developing forebrain. Based on these molecular and genetic studies, we conclude that S100B is not a specific marker for astrocyte lineage; instead, it marks protoplasmic astrocytes in the gray matter and differentiating oligodendrocytes.

GAS1 is required for NOTCH-dependent facilitation of SHH signaling in the ventral forebrain neuroepithelium.

Growth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.

Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination.

Thyroid hormones are messengers that bind to specific nuclear receptors and regulate a wide range of physiological processes in the early stages of vertebrate embryonic development, including neurodevelopment and myelogenesis. We here tested the effects of reduced T3 availability upon the myelination process by treating zebrafish embryos with low concentrations of iopanoic acid (IOP) to block T4 to T3 conversion. Black Gold II staining showed that T3 deficiency reduced the myelin density in the forebrain, midbrain, hindbrain and the spinal cord at 3 and 7 dpf. These observations were confirmed in 3 dpf mbp:egfp transgenic zebrafish, showing that the administration of IOP reduced the fluorescent signal in the brain. T3 rescue treatment restored brain myelination and reversed the changes in myelin-related gene expression induced by IOP exposure. NG2 immunostaining revealed that T3 deficiency reduced the amount of oligodendrocyte precursor cells in 3 dpf IOP-treated larvae. Altogether, the present results show that inhibition of T4 to T3 conversion results in hypomyelination, suggesting that THs are part of the key signaling molecules that control the timing of oligodendrocyte differentiation and myelin synthesis from very early stages of brain development.

Loss of all three APP family members during development impairs synaptic function and plasticity, disrupts learning, and causes an autism-like phenotype.

The key role of APP for Alzheimer pathogenesis is well established. However, perinatal lethality of germline knockout mice lacking the entire APP family has so far precluded the analysis of its physiological functions for the developing and adult brain. Here, we generated conditional APP/APLP1/APLP2 triple KO (cTKO) mice lacking the APP family in excitatory forebrain neurons from embryonic day 11.5 onwards. NexCre cTKO mice showed altered brain morphology with agenesis of the corpus callosum and disrupted hippocampal lamination. Further, NexCre cTKOs revealed reduced basal synaptic transmission and drastically reduced long-term potentiation that was associated with reduced dendritic length and reduced spine density of pyramidal cells. With regard to behavior, lack of the APP family leads not only to severe impairments in a panel of tests for learning and memory, but also to an autism-like phenotype including repetitive rearing and climbing, impaired social communication, and deficits in social interaction. Together, our study identifies essential functions of the APP family during development, for normal hippocampal function and circuits important for learning and social behavior.

Role of SHH in Patterning Human Pluripotent Cells towards Ventral Forebrain Fates.

The complexities of human neurodevelopment have historically been challenging to decipher but continue to be of great interest in the contexts of healthy neurobiology and disease. The classic animal models and monolayer in vitro systems have limited the types of questions scientists can strive to answer in addition to the technical ability to answer them. However, the tridimensional human stem cell-derived organoid system provides the unique opportunity to model human development and mimic the diverse cellular composition of human organs. This strategy is adaptable and malleable, and these neural organoids possess the morphogenic sensitivity to be patterned in various ways to generate the different regions of the human brain. Furthermore, recapitulating human development provides a platform for disease modeling. One master regulator of human neurodevelopment in many regions of the human brain is sonic hedgehog (SHH), whose expression gradient and pathway activation are responsible for conferring ventral identity and shaping cellular phenotypes throughout the neural axis. This review first discusses the benefits, challenges, and limitations of using organoids for studying human neurodevelopment and disease, comparing advantages and disadvantages with other in vivo and in vitro model systems. Next, we explore the range of control that SHH exhibits on human neurodevelopment, and the application of SHH to various stem cell methodologies, including organoids, to expand our understanding of human development and disease. We outline how this strategy will eventually bring us much closer to uncovering the intricacies of human neurodevelopment and biology.

Landmarks of human embryonic development inscribed in somatic mutations.

Although cell lineage information is fundamental to understanding organismal development, very little direct information is available for humans. We performed high-depth (250×) whole-genome sequencing of multiple tissues from three individuals to identify hundreds of somatic single-nucleotide variants (sSNVs). Using these variants as "endogenous barcodes" in single cells, we reconstructed early embryonic cell divisions. Targeted sequencing of clonal sSNVs in different organs (about 25,000×) and in more than 1000 cortical single cells, as well as single-nucleus RNA sequencing and single-nucleus assay for transposase-accessible chromatin sequencing of ~100,000 cortical single cells, demonstrated asymmetric contributions of early progenitors to extraembryonic tissues, distinct germ layers, and organs. Our data suggest onset of gastrulation at an effective progenitor pool of about 170 cells and about 50 to 100 founders for the forebrain. Thus, mosaic mutations provide a permanent record of human embryonic development at very high resolution.

Long-Range GABAergic Inhibition Modulates Spatiotemporal Dynamics of the Output Neurons in the Olfactory Bulb.

Local interneurons of the olfactory bulb (OB) are densely innervated by long-range GABAergic neurons from the basal forebrain (BF), suggesting that this top-down inhibition regulates early processing in the olfactory system. However, how GABAergic inputs modulate the OB output neurons, the mitral/tufted cells, is unknown. Here, in male and female mice acute brain slices, we show that optogenetic activation of BF GABAergic inputs produced distinct local circuit effects that can influence the activity of mitral/tufted cells in the spatiotemporal domains. Activation of the GABAergic axons produced a fast disinhibition of mitral/tufted cells consistent with a rapid and synchronous release of GABA onto local interneurons in the glomerular and inframitral circuits of the OB, which also reduced the spike precision of mitral/tufted cells in response to simulated stimuli. In addition, BF GABAergic inhibition modulated local oscillations in a layer-specific manner. The intensity of locally evoked θ oscillations was decreased on activation of top-down inhibition in the glomerular circuit, while evoked γ oscillations were reduced by inhibition of granule cells. Furthermore, BF GABAergic input reduced dendrodendritic inhibition in mitral/tufted cells. Together, these results suggest that long-range GABAergic neurons from the BF are well suited to influence temporal and spatial aspects of processing by OB circuits.SIGNIFICANCE STATEMENT Disruption of GABAergic inhibition from the basal forebrain (BF) to the olfactory bulb (OB) impairs the discrimination of similar odors, yet how this centrifugal inhibition influences neuronal circuits in the OB remains unclear. Here, we show that the BF GABAergic neurons exclusively target local inhibitory neurons in the OB, having a functional disinhibitory effect on the output neurons, the mitral cells. Phasic inhibition by BF GABAergic neurons reduces spike precision of mitral cells and lowers the intensity of oscillatory activity in the OB, while directly modulating the extent of dendrodendritic inhibition. These circuit-level effects of this centrifugal inhibition can influence the temporal and spatial dynamics of odor coding in the OB.

Basal forebrain mediates prosocial behavior via disinhibition of midbrain dopamine neurons.

Sociability is fundamental for our daily life and is compromised in major neuropsychiatric disorders. However, the neuronal circuit mechanisms underlying prosocial behavior are still elusive. Here we identify a causal role of the basal forebrain (BF) in the control of prosocial behavior via inhibitory projections that disinhibit the midbrain ventral tegmental area (VTA) dopamine (DA) neurons. Specifically, BF somatostatin-positive (SST) inhibitory neurons were robustly activated during social interaction. Optogenetic inhibition of these neurons in BF or their axon terminals in the VTA largely abolished social preference. Electrophysiological examinations further revealed that SST neurons predominantly targeted VTA GABA neurons rather than DA neurons. Consistently, optical inhibition of SST neuron axon terminals in the VTA decreased DA release in the nucleus accumbens during social interaction, confirming a disinhibitory action. These data reveal a previously unappreciated function of the BF in prosocial behavior through a disinhibitory circuitry connected to the brain's reward system.

Cell-type specific knockout of peptidylglycine α-amidating monooxygenase reveals specific behavioral roles in excitatory forebrain neurons and cardiomyocytes.

Neuropeptides and peptide hormones play a crucial role in integrating the many factors that affect physiologic and cognitive processes. The potency of many of these peptides requires an amidated amino acid at the C-terminus; a single enzyme, peptidylglycine α-amidating monooxygenase (PAM), catalyzes this modification. Anxiety-like behavior is known to be altered in mice with a single functional Pam allele (Pam+/- ) and in mice unable to express Pam in excitatory forebrain neurons (PamEmx1-cKO/cKO ) or in cardiomyocytes (PamMyh6-cKO/cKO ). Examination of PAM-positive and glutamic acid decarboxylase 67 (GAD)-positive cells in the amygdala of PamEmx1-cKO/cKO mice demonstrated the absence of PAM in pyramidal neurons and its continued presence in GAD-positive interneurons, suggestive of altered excitatory/inhibitory balance. Additional behavioral tests were used to search for functional alterations in these cell-type specific knockout mice. PamEmx1-cKO/cKO mice exhibited a less focused search pattern for the Barnes Maze escape hole than control or PamMyh6-cKO/cKO mice. While wildtype mice favor interacting with novel objects as opposed to familiar objects, both PamEmx1-cKO/cKO and PamMyh6-cKO/cKO mice exhibited significantly less interest in the novel object. Since PAM levels in the central nervous system of PamMyh6-cKO/cKO mice are unaltered, the behavioral effect observed in these mice may reflect their inability to produce atrial granules and the resulting reduction in serum levels of atrial natriuretic peptide. In the sociability test, male mice of all three genotypes spent more time with same-sex stranger mice; while control females showed no preference for stranger mice, female PamEmx1-cKO/cKO mice showed preference for same-sex stranger mice in all trials.

On the origin of ultraslow spontaneous Na+ fluctuations in neurons of the neonatal forebrain.

Spontaneous neuronal and astrocytic activity in the neonate forebrain is believed to drive the maturation of individual cells and their integration into complex brain-region-specific networks. The previously reported forms include bursts of electrical activity and oscillations in intracellular Ca2+ concentration. Here, we use ratiometric Na+ imaging to demonstrate spontaneous fluctuations in the intracellular Na+ concentration of CA1 pyramidal neurons and astrocytes in tissue slices obtained from the hippocampus of mice at postnatal days 2-4 (P2-4). These occur at very low frequency (∼2/h), can last minutes with amplitudes up to several millimolar, and mostly disappear after the first postnatal week. To further investigate their mechanisms, we model a network consisting of pyramidal neurons and interneurons. Experimentally observed Na+ fluctuations are mimicked when GABAergic inhibition in the simulated network is made depolarizing. Both our experiments and computational model show that blocking voltage-gated Na+ channels or GABAergic signaling significantly diminish the neuronal Na+ fluctuations. On the other hand, blocking a variety of other ion channels, receptors, or transporters including glutamatergic pathways does not have significant effects. Our model also shows that the amplitude and duration of Na+ fluctuations decrease as we increase the strength of glial K+ uptake. Furthermore, neurons with smaller somatic volumes exhibit fluctuations with higher frequency and amplitude. As opposed to this, larger extracellular to intracellular volume ratio observed in neonatal brain exerts a dampening effect. Finally, our model predicts that these periods of spontaneous Na+ influx leave neonatal neuronal networks more vulnerable to seizure-like states when compared with mature brain.NEW & NOTEWORTHY Spontaneous activity in the neonate forebrain plays a key role in cell maturation and brain development. We report spontaneous, ultraslow, asynchronous fluctuations in the intracellular Na+ concentration of neurons and astrocytes. We show that this activity is not correlated with the previously reported synchronous neuronal population bursting or Ca2+ oscillations, both of which occur at much faster timescales. Furthermore, extracellular K+ concentration remains nearly constant. The spontaneous Na+ fluctuations disappear after the first postnatal week.

Protocol for the Standardized Generation of Forward Programmed Cryopreservable Excitatory and Inhibitory Forebrain Neurons.

This protocol describes a highly standardized pipeline for transcription factor-mediated forward programming of human pluripotent stem cells into highly enriched glutamatergic or GABAergic neurons followed by a cryopreservation step that enables the generation of large quality-controlled batches. This approach is particularly useful for reducing interexperimental variability in the context of collaborative studies across different locations and time points. For complete details on the use and execution of this protocol, please refer to Meijer et al. (2019) and Rhee et al. (2019).

Infection of Brain Organoids and 2D Cortical Neurons with SARS-CoV-2 Pseudovirus.

Since the global outbreak of SARS-CoV-2 (COVID-19), infections of diverse human organs along with multiple symptoms continue to be reported. However, the susceptibility of the brain to SARS-CoV-2, and the mechanisms underlying neurological infection are still elusive. Here, we utilized human embryonic stem cell-derived brain organoids and monolayer cortical neurons to investigate infection of brain with pseudotyped SARS-CoV-2 viral particles. Spike-containing SARS-CoV-2 pseudovirus infected neural layers within brain organoids. The expression of ACE2, a host cell receptor for SARS-CoV-2, was sustained during the development of brain organoids, especially in the somas of mature neurons, while remaining rare in neural stem cells. However, pseudotyped SARS-CoV-2 was observed in the axon of neurons, which lack ACE2. Neural infectivity of SARS-CoV-2 pseudovirus did not increase in proportion to viral load, but only 10% of neurons were infected. Our findings demonstrate that brain organoids provide a useful model for investigating SARS-CoV-2 entry into the human brain and elucidating the susceptibility of the brain to SARS-CoV-2.

Cell-Type-Specific Metabolic Profiling Achieved by Combining Desorption Electrospray Ionization Mass Spectrometry Imaging and Immunofluorescence Staining.

Cell-type-specific metabolic profiling in tissue with heterogeneous composition has been of great interest across all mass spectrometry imaging (MSI) technologies. We report here a powerful new chemical imaging capability in desorption electrospray ionization (DESI) MSI, which enables cell-type-specific and in situ metabolic profiling in complex tissue samples. We accomplish this by combining DESI-MSI with immunofluorescence staining using specific cell-type markers. We take advantage of the variable frequency of each distinct cell type in the lateral septal nucleus (LSN) region of mouse forebrain. This allows computational deconvolution of the cell-type-specific metabolic profile in neurons and astrocytes by convex optimization-a machine learning method. Based on our approach, we observed 107 metabolites that show different distributions and intensities between astrocytes and neurons. We subsequently identified 23 metabolites using high-resolution mass spectrometry (MS) and tandem MS, which include small metabolites such as adenosine and N-acetylaspartate previously associated with astrocytes and neurons, respectively, as well as accumulation of several phospholipid species in neurons which have not been studied before. Overall, this method overcomes the relatively low spatial resolution of DESI-MSI and provides a new platform for in situ metabolic investigation at the cell-type level in complex tissue samples with heterogeneous cell-type composition.