Multiscale 3D Genome Rewiring during Mouse Neural Development.

Chromosome conformation capture technologies have revealed important insights into genome folding. Yet, how spatial genome architecture is related to gene expression and cell fate remains unclear. We comprehensively mapped 3D chromatin organization during mouse neural differentiation in vitro and in vivo, generating the highest-resolution Hi-C maps available to date. We found that transcription is correlated with chromatin insulation and long-range interactions, but dCas9-mediated activation is insufficient for creating TAD boundaries de novo. Additionally, we discovered long-range contacts between gene bodies of exon-rich, active genes in all cell types. During neural differentiation, contacts between active TADs become less pronounced while inactive TADs interact more strongly. An extensive Polycomb network in stem cells is disrupted, while dynamic interactions between neural transcription factors appear in vivo. Finally, cell type-specific enhancer-promoter contacts are established concomitant to gene expression. This work shows that multiple factors influence the dynamics of chromatin interactions in development.

Mammalian CDC14 phosphatases control exit from stemness in pluripotent cells.

Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal.

Heterozygous Mutations in SMARCA2 Reprogram the Enhancer Landscape by Global Retargeting of SMARCA4.

Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase (either SMARCA4 or SMARCA2). Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that the AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate that SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.

A wound-induced differentiation trajectory for neurons.

Animals capable of whole-body regeneration can replace any missing cell type and regenerate fully functional new organs, including new brains, de novo. The regeneration of a new brain requires the formation of diverse neural cell types and their assembly into an organized structure with correctly wired circuits. Recent work in various regenerative animals has revealed transcriptional programs required for the differentiation of distinct neural subpopulations, however, how these transcriptional programs are initiated in response to injury remains unknown. Here, we focused on the highly regenerative acoel worm, Hofstenia miamia, to study wound-induced transcriptional regulatory events that lead to the production of neurons and subsequently a functional brain. Footprinting analysis using chromatin accessibility data on a chromosome-scale genome assembly revealed that binding sites for the Nuclear Factor Y (NFY) transcription factor complex were significantly bound during regeneration, showing a dynamic increase in binding within one hour upon amputation specifically in tail fragments, which will regenerate a new brain. Strikingly, NFY targets were highly enriched for genes with neuronal function. Single-cell transcriptome analysis combined with functional studies identified soxC+ stem cells as a putative progenitor population for multiple neural subtypes. Further, we found that wound-induced soxC expression is likely under direct transcriptional control by NFY, uncovering a mechanism for the initiation of a neural differentiation pathway by early wound-induced binding of a transcriptional regulator.

Translational landscape in human early neural fate determination.

Gene expression regulation in eukaryotes is a multi-level process, including transcription, mRNA translation and protein turnover. Many studies have reported sophisticated transcriptional regulation during neural development, but the global translational dynamics are still ambiguous. Here, we differentiate human embryonic stem cells (ESCs) into neural progenitor cells (NPCs) with high efficiency and perform ribosome sequencing and RNA sequencing on both ESCs and NPCs. Data analysis reveals that translational controls engage in many crucial pathways and contribute significantly to regulation of neural fate determination. Furthermore, we show that the sequence characteristics of the untranslated region (UTR) might regulate translation efficiency. Specifically, genes with short 5'UTR and intense Kozak sequence are associated with high translation efficiency in human ESCs, whereas genes with long 3'UTR are related to high translation efficiency in NPCs. In addition, we have identified four biasedly used codons (GAC, GAT, AGA and AGG) and dozens of short open reading frames during neural progenitor differentiation. Thus, our study reveals the translational landscape during early human neural differentiation and provides insights into the regulation of cell fate determination at the translational level.

Neurogenesis redirects ß-catenin from adherens junctions to the nucleus to promote axonal growth.

Here, we show that, in the developing spinal cord, after the early Wnt-mediated Tcf transcription activation that confers dorsal identity to neural stem cells, neurogenesis redirects ß-catenin from the adherens junctions to the nucleus to stimulate Tcf-dependent transcription in a Wnt-independent manner. This new ß-catenin activity regulates genes implicated in several aspects of contralateral axon growth, including axon guidance and adhesion. Using live imaging of ex-vivo chick neural tube, we showed that the nuclear accumulation of ß-catenin and the rise in Tcf-dependent transcription both initiate before the dismantling of the adherens junctions and remain during the axon elongation process. Notably, we demonstrated that ß-catenin activity in post-mitotic cells depends on TCF7L2 and is central to spinal commissural axon growth. Together, our results reveal Wnt-independent Tcf/ß-catenin regulation of genes that control the growth and guidance of commissural axons in chick spinal cord.

Dissociative Effects of Age on Neural Differentiation at the Category and Item Levels.

Increasing age is associated with age-related neural dedifferentiation, a reduction in the selectivity of neural representations, which has been proposed to contribute to cognitive decline in older age. Recent findings indicate that when operationalized in terms of selectivity for different perceptual categories, age-related neural dedifferentiation and the apparent age-invariant association of neural selectivity with cognitive performance are largely restricted to the cortical regions typically recruited during scene processing. It is currently unknown whether this category-level dissociation extends to metrics of neural selectivity defined at the level of individual stimulus items. Here, we examined neural selectivity at the category and item levels using multivoxel pattern similarity analysis (PSA) of fMRI data. Healthy young and older male and female adults viewed images of objects and scenes. Some items were presented singly, while others were either repeated or followed by a "similar lure." In agreement with recent findings, category-level PSA revealed robustly lower differentiation in older than in younger adults in scene-selective, but not object-selective, cortical regions. By contrast, at the item level, robust age-related declines in neural differentiation were evident for both stimulus categories. Additionally, we identified an age-invariant association between category-level scene selectivity in the parahippocampal place area and subsequent memory performance, but no such association was evident for item-level metrics. Lastly, category- and item-level neural metrics were uncorrelated. Thus, the present findings suggest that age-related category- and item-level dedifferentiation depend on distinct neural mechanisms.

Human cytomegalovirus infection impairs neural differentiation via repressing sterol regulatory element binding protein 2-mediated cholesterol biosynthesis.

Congenital human cytomegalovirus (HCMV) infection is a major cause of abnormalities and disorders in the central nervous system (CNS) and/or the peripheral nervous system (PNS). However, the complete pathogenesis of neural differentiation disorders caused by HCMV infection remains to be fully elucidated. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells (MSCs) with a high proliferation and neurogenic differentiation capacity. Since SHEDs originate from the neural crest of the early embryonic ectoderm, SHEDs were hypothesized to serve as a promising cell line for investigating the pathogenesis of neural differentiation disorders in the PNS caused by congenital HCMV infection. In this work, SHEDs were demonstrated to be fully permissive to HCMV infection and the virus was able to complete its life cycle in SHEDs. Under neurogenic inductive conditions, HCMV infection of SHEDs caused an abnormal neural morphology. The expression of stem/neural cell markers was also disturbed by HCMV infection. The impairment of neural differentiation was mainly due to a reduction of intracellular cholesterol levels caused by HCMV infection. Sterol regulatory element binding protein-2 (SREBP2) is a critical transcription regulator that guides cholesterol synthesis. HCMV infection was shown to hinder the migration of SREBP2 into nucleus and resulted in perinuclear aggregations of SREBP2 during neural differentiation. Our findings provide new insights into the prevention and treatment of nervous system diseases caused by congenital HCMV infection.

Transcription factor SOX15 regulates stem cell pluripotency and promotes neural fate during differentiation by activating the neurogenic gene Hes5.

SOX2 and SOX15 are Sox family transcription factors enriched in embryonic stem cells (ESCs). The role of SOX2 in activating gene expression programs essential for stem cell self-renewal and acquisition of pluripotency during somatic cell reprogramming is well-documented. However, the contribution of SOX15 to these processes is unclear and often presumed redundant with SOX2 largely because overexpression of SOX15 can partially restore self-renewal in SOX2-deficient ESCs. Here, we show that SOX15 contributes to stem cell maintenance by cooperating with ESC-enriched transcriptional coactivators to ensure optimal expression of pluripotency-associated genes. We demonstrate that SOX15 depletion compromises reprogramming of fibroblasts to pluripotency which cannot be compensated by SOX2. Ectopic expression of SOX15 promotes the reversion of a postimplantation, epiblast stem cell state back to a preimplantation, ESC-like identity even though SOX2 is expressed in both cell states. We also uncover a role of SOX15 in lineage specification, by showing that loss of SOX15 leads to defects in commitment of ESCs to neural fates. SOX15 promotes neural differentiation by binding to and activating a previously uncharacterized distal enhancer of a key neurogenic regulator, Hes5. Together, these findings identify a multifaceted role of SOX15 in induction and maintenance of pluripotency and neural differentiation.

Laminin-Augmented Decellularized Extracellular Matrix Ameliorating Neural Differentiation and Neuroinflammation in Human Mini-Brains.

Non-neural extracellular matrix (ECM) has limited application in humanized physiological neural modeling due to insufficient brain-specificity and safety concerns. Although brain-derived ECM contains enriched neural components, certain essential components are partially lost during the decellularization process, necessitating augmentation. Here, it is demonstrated that the laminin-augmented porcine brain-decellularized ECM (P-BdECM) is xenogeneic factor-depleted as well as favorable for the regulation of human neurons, astrocytes, and microglia. P-BdECM composition is comparable to human BdECM regarding brain-specificity through the matrisome and gene ontology-biological process analysis. As augmenting strategy, laminin 111 supplement promotes neural function by synergic effect with laminin 521 in P-BdECM. Annexin A1(ANXA1) and Peroxiredoxin(PRDX) in P-BdECM stabilized microglial and astrocytic behavior under normal while promoting active neuroinflammation in response to neuropathological factors. Further, supplementation of the brain-specific molecule to non-neural matrix also ameliorated glial cell inflammation as in P-BdECM. In conclusion, P-BdECM-augmentation strategy can be used to recapitulate humanized pathophysiological cerebral environments for neurological study.

Lethal Phenotype-Based Database Screening Identifies Ceramide as a Negative Regulator of Primitive Streak Formation.

In early embryogenesis, the primitive streak (PrS) generates the mesendoderm and is essential for organogenesis. However, because the PrS is a minute and transient tissue, elucidating the mechanism of its formation has been challenging. We performed comprehensive screening of 2 knockout mouse databases based on the fact that failure of PrS formation is lethal. We identified 812 genes involved in various cellular functions and responses that might be linked to PrS formation, with the category of greatest abundance being "Metabolism." In this study, we focused on genes of sphingolipid metabolism and investigated their roles in PrS formation using an in vitro mouse ES cell differentiation system. We show here that elevated intracellular ceramide negatively regulates gene expression essential for PrS formation and instead induces neurogenesis. In addition, sphingosine-1-phosphate (a ceramide derivative) positively regulates neural maturation. Our results indicate that ceramide regulates both PrS formation and the induction of neural differentiation.

Electroacupuncture Ameliorates Depression-Like Behaviors Comorbid to Chronic Neuropathic Pain via Tet1-Mediated Restoration of Adult Neurogenesis.

Although electroacupuncture (EA) stimulation is a widely used therapy for chronic pain and comorbid psychiatric disorders, its long-term effects on chronic neuropathic pain-induced depression and the underlying mechanisms remain elusive. In the present study, we found that EA stimulation was able to restore adult neurogenesis in the ventral dentate gyrus (DG), by both increasing neuronal differentiation and restoring the normal morphology of newborn dendrites, in mice with spared nerve injury surgery. By ablating the Nestin+ neural stem cells (NSCs) via diphtheria toxin fragment A expression, we further proved that neurogenesis in the ventral DG was crucial to the long-term, but not the immediate antidepressant effect of EA, nor was it associated with nociception. Furthermore, we found that the restoration of neurogenesis was dependent on Tet1-mediated epigenetic modification upon EA treatment. Tet1 could bind to the promoter of the Prox1 gene, thus catalyzing its demethylation and facilitating its expression, which finally contributed to the restoration of neurogenesis and amelioration of depression-like behaviors induced by chronic neuropathic pain. Thus, we conclude that EA stimulation restores inhibited Tet1 expression in hippocampal NSCs of mice with chronic neuropathic pain, and increased Tet1 expression ameliorates hypermethylation of Prox1 and restores normal adult neurogenesis in the ventral DG, which contributes to the long-term antidepressant effect of EA.

Extracellular vesicles from UTX-knockout endothelial cells boost neural stem cell differentiation in spinal cord injury.

BACKGROUND: Vascular endothelial cells are pivotal in the pathophysiological progression following spinal cord injury (SCI). The UTX (Ubiquitously Transcribed Tetratripeptide Repeat on Chromosome X) serves as a significant regulator of endothelial cell phenotype. The manipulation of endogenous neural stem cells (NSCs) offers a compelling strategy for the amelioration of SCI. METHODS: Two mouse models were used to investigate SCI: NSCs lineage-traced mice and mice with conditional UTX knockout (UTX KO) in endothelial cells. To study the effects of UTX KO on neural differentiation, we harvested extracellular vesicles (EVs) from both UTX KO spinal cord microvascular endothelial cells (SCMECs) and negative control SCMECs. These EVs were then employed to modulate the differentiation trajectory of endogenous NSCs in the SCI model. RESULTS: In our NSCs lineage-traced mice model of SCI, a marked decrease in neurogenesis was observed post-injury. Notably, NSCs in UTX KO SCMECs mice showed enhanced neuronal differentiation compared to controls. RNA sequencing and western blot analyses revealed an upregulation of L1 cell adhesion molecule (L1CAM), a gene associated with neurogenesis, in UTX KO SCMECs and their secreted EVs. This aligns with the observed promotion of neurogenesis in UTX KO conditions. In vivo administration of L1CAM-rich EVs from UTX KO SCMECs (KO EVs) to the mice significantly enhanced neural differentiation. Similarly, in vitro exposure of NSCs to KO EVs resulted in increased activation of the Akt signaling pathway, further promoting neural differentiation. Conversely, inhibiting Akt phosphorylation or knocking down L1CAM negated the beneficial effects of KO EVs on NSC neuronal differentiation. CONCLUSIONS: In conclusion, our findings substantiate that EVs derived from UTX KO SCMECs can act as facilitators of neural differentiation following SCI. This study not only elucidates a novel mechanism but also opens new horizons for therapeutic interventions in the treatment of SCI. Video Abstract.

3-Amino-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carbonitrile: A fluorescent molecule that induces differentiation in PC12 cells.

Neural differentiation is triggered by the activation of multiple signaling pathways initiated by various neurotrophic factors. An elucidation of these mechanisms is anticipated to facilitate the prevention of diseases and the development of novel therapeutic approaches. Alternative small-molecule inducers for neuroscience studies are required instead of protein-based reagents for more efficient and convenient experiments. We demonstrated that small molecules of thieno[2,3-b]pyridine derivatives that induce neural differentiation, compounds 3a and 9a in particular, exhibited significant neuritogenic activity in rat pheochromocytoma (PC12) cells. Moreover, 3a displayed pronounced fluorescence and a discernible Stokes shift. Furthermore, the outcome of the experiment conducted on the NGF-insensitive clones of rat PC12 cells, and the results of the intercellular uptake analyses suggested that the 3a-mediated activation of neural differentiation occurred independently of the TrkA receptor. Therefore, 3a portrays potential applicability both as a small molecule reagent to replace novel neurotrophic factors and as a potent fluorescent reagent for various techniques, including bioimaging.

The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2.

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.

Comparison of Extracellular Vesicles from Induced Pluripotent Stem Cell-Derived Brain Cells.

The pathophysiology of many neuropsychiatric disorders is still poorly understood. Identification of biomarkers for these diseases could benefit patients due to better classification and stratification. Exosomes excreted into the circulatory system can cross the blood-brain barrier and carry a cell type-specific set of molecules. Thus, exosomes are a source of potential biomarkers for many diseases, including neuropsychiatric disorders. Here, we investigated exosomal proteins produced from human-induced pluripotent stem cells (iPSCs) and iPSC-derived neural stem cells, neural progenitors, neurons, astrocytes, microglia-like cells, and brain capillary endothelial cells. Of the 31 exosome surface markers analyzed, a subset of biomarkers were significantly enriched in astrocytes (CD29, CD44, and CD49e), microglia-like cells (CD44), and neural stem cells (SSEA4). To identify molecular fingerprints associated with disease, circulating exosomes derived from healthy control (HC) individuals were compared against schizophrenia (SCZ) patients and late-onset Alzheimer's disease (LOAD) patients. A significant epitope pattern was identified for LOAD (CD1c and CD2) but not for SCZ compared to HC. Thus, analysis of cell type- and disease-specific exosome signatures of iPSC-derived cell cultures may provide a valuable model system to explore proteomic biomarkers for the identification of novel disease profiles.

The involvement of Neuregulin-1 in the process of facial nerve injury repair through the utilization of dental pulp stem cells.

BACKGROUND: Facial nerve injury often results in poor prognosis due to the challenging process of nerve regeneration. Neuregulin-1, a human calmodulin, is under investigation in this study for its impact on the reparative capabilities of Dental Pulp Stem Cells (DPSCs) in facial nerve injury. METHODS: Lentivirus was used to transfect and construct Neuregulin-1 overexpressed DPSCs. Various techniques assessed the effects of Neuregulin-1: osteogenic induction, lipid induction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Counting Kit-8 assay, wound healing, immunofluorescence, Phalloidin staining, nerve stem action potential, Hematoxylin-eosin staining, transmission electron microscopy, and immunohistochemistry. RESULTS: Neuregulin-1 effectively enhanced the proliferation, migration, and cytoskeletal rearrangement of DPSCs, while simultaneously suppressing the expression of Ras homolog gene family member A (RhoA) and Microfilament actin (F-actin). These changes facilitated the neural differentiation of DPSCs. Additionally, in vivo experiments showed that Neuregulin-1 expedited the restoration of action potential in the facial nerve trunk, increased the thickness of the myelin sheath, and stimulated axon regeneration. CONCLUSION: Neuregulin-1 has the capability to facilitate the repair of facial nerve injuries by promoting the regenerative capacity of DPSCs. Thus, Neuregulin-1 is a significant potential gene in the reparative processes of nerve damage.

Neuronal and Muscle Differentiation of Mammalian Cells Is Accompanied by a Change in PHF10 Isoform Expression.

The PBAF chromatin remodeling complex of the SWI/SNF family plays a critical role in the regulation of gene expression during tissue differentiation and organism development. The subunits of the PBAF complex have domains responsible for binding to N-terminal histone sequences. It determines the specificity of binding of the complex to chromatin. PHF10, a specific subunit of the PBAF complex, contains a DPF domain, which is a unique chromatin interaction domain. A PHF10 isoform that lacks the DPF domain is also present in vertebrate cells. This work shows that during neuronal and muscle differentiation of human and mouse cells, the expression of PHF10 isoforms changes: the form that does not have DPF replaces the form in which it is present. Replacement of PHF10 isoforms in the PBAF complex may affect its selectivity in the regulation of genes in differentiating cells.

E2A regulates neural ectoderm fate specification in human embryonic stem cells.

E protein transcription factors are crucial for many cell fate decisions. However, the roles of E proteins in the germ-layer specification of human embryonic stem cells (hESCs) are poorly understood. We disrupted the TCF3 gene locus to delete the E protein E2A in hESCs. E2A knockout (KO) hESCs retained key features of pluripotency, but displayed decreased neural ectoderm coupled with enhanced mesoendoderm outcomes. Genome-wide analyses showed that E2A directly regulates neural ectoderm and Nodal pathway genes. Accordingly, inhibition of Nodal or E2A overexpression partially rescued the neural ectoderm defect in E2A KO hESCs. Loss of E2A had little impact on the epigenetic landscape of hESCs, whereas E2A KO neural precursors displayed increased accessibility of the gene locus encoding the Nodal agonist CRIPTO. Double-deletion of both E2A and HEB (TCF12) resulted in a more severe neural ectoderm defect. Therefore, this study reveals critical context-dependent functions for E2A in human neural ectoderm fate specification.

Nitric Oxide Attenuates Human Cytomegalovirus Infection yet Disrupts Neural Cell Differentiation and Tissue Organization.

Human cytomegalovirus (HCMV) is a prevalent betaherpesvirus that is asymptomatic in healthy individuals but can cause serious disease in immunocompromised patients. HCMV is also the leading cause of virus-mediated birth defects. Many of these defects manifest within the central nervous system and include microcephaly, sensorineural hearing loss, and cognitive developmental delays. Nitric oxide is a critical effector molecule produced as a component of the innate immune response during infection. Congenitally infected fetal brains show regions of brain damage, including necrotic foci with infiltrating macrophages and microglia, cell types that produce nitric oxide during infection. Using a 3-dimensional cortical organoid model, we demonstrate that nitric oxide inhibits HCMV spread and simultaneously disrupts neural rosette structures, resulting in tissue disorganization. Nitric oxide also attenuates HCMV replication in 2-dimensional cultures of neural progenitor cells (NPCs), a prominent cell type in cortical organoids that differentiate into neurons and glial cells. The multipotency factor SOX2 was decreased during nitric oxide exposure, suggesting that early neural differentiation is affected. Nitric oxide also reduced maximal mitochondrial respiration in both uninfected and infected NPCs. We determined that this reduction likely influences neural differentiation, as neurons (Tuj1+ GFAP- Nestin-) and glial populations (Tuj1- GFAP+ Nestin-) were reduced following differentiation. Our studies indicate a prominent, immunopathogenic role of nitric oxide in promoting developmental defects within the brain despite its antiviral activity during congenital HCMV infection. IMPORTANCE Human cytomegalovirus (HCMV) is the leading cause of virus-mediated congenital birth defects. Congenitally infected infants can have a variety of symptoms manifesting within the central nervous system. The use of 3-dimensional (3-D) cortical organoids to model infection of the fetal brain has advanced the current understanding of development and allowed broader investigation of the mechanisms behind disease. However, the impact of the innate immune molecule nitric oxide during HCMV infection has not been explored in neural cells or cortical 3-D models. Here, we investigated the effect of nitric oxide on cortical development during HCMV infection. We demonstrate that nitric oxide plays an antiviral role during infection yet results in disorganized cortical tissue. Nitric oxide contributes to differentiation defects of neuron and glial cells from neural progenitor cells despite inhibiting viral replication. Our results indicate that immunopathogenic consequences of nitric oxide during congenital infection promote developmental defects that undermine its antiviral activity.