Developing Mouse Cerebral Cortex Research Articles

N4BP1 mediates RAM domain-dependent notch signaling turnover during neocortical development.

Notch signaling pathway activity, particularly fluctuations in the biologically active effector fragment NICD, is required for rapid and efficient dynamic regulation of proper fate decisions in stem cells. In this study, we identified NEDD4-binding protein 1 (N4BP1), which is highly expressed in the developing mouse cerebral cortex, as a negative modulator of Notch signaling dynamics in neural progenitor cells. Intriguingly, N4BP1 regulated NICD stability specifically after Notch1 S3 cleavage through ubiquitin-mediated degradation that depended on its RAM domain, not its PEST domain, as had been extensively and previously described. The CoCUN domain in N4BP1, particularly the "Phe-Pro" motif (862/863 amino acid), was indispensable for mediating NICD degradation. The Ring family E3 ligase Trim21 was, in contrast to other NEDD4 family members, required for N4BP1-regulated NICD degradation. Overexpression of N4BP1 in cortical neural progenitors promoted neural stem cell differentiation, whereas neural progenitor cells lacking N4BP1 were sensitized to Notch signaling, resulting in the maintenance of stem-like properties in neural progenitor cells and lower production of cortical neurons.

Calnexin controls TrkB cell surface transport and ER-phagy in mouse cerebral cortex development.

Transactivation of Tropomyosin receptor kinase B (TrkB) by EGF leads to cell surface transport of TrkB, promoting its signaling responsiveness to brain-derived neurotrophic factor (BDNF), a critical process for proper cortical plate development. However, the mechanisms that regulate the transport of TrkB to the cell surface are not fully understood. Here, we identified Calnexin as a regulator for targeting TrkB either to the cell surface or toward autophagosomal processing. Calnexin-deficient mouse embryos show impaired cortical plate formation and elevated levels of transactivated TrkB. In Calnexin-depleted mouse neuronal precursor cells, we detected an impaired cell surface transport of TrkB in response to EGF and an impaired delivery to autophagosomes. Mechanistically, we show that Calnexin facilitates the interaction of TrkB with the ER-phagy receptor Fam134b, thereby targeting TrkB to ER-phagy. This mechanism appears as a critical process for fine-tuning the sensitivity of neurons to BDNF.

Krüppel-like factor 7 deficiency disrupts corpus callosum development and neuronal migration in the developing mouse cerebral cortex.

Krüppel-like Factor 7 (KLF7) is a zinc finger transcription factor that has a critical role in cellular differentiation, tumorigenesis, and regeneration. Mutations in Klf7 are associated with autism spectrum disorder, which is characterized by neurodevelopmental delay and intellectual disability. Here we show that KLF7 regulates neurogenesis and neuronal migration during mouse cortical development. Conditional depletion of KLF7 in neural progenitor cells resulted in agenesis of the corpus callosum, defects in neurogenesis, and impaired neuronal migration in the neocortex. Transcriptomic profiling analysis indicated that KLF7 regulates a cohort of genes involved in neuronal differentiation and migration, including p21 and Rac3. These findings provide insights into our understanding of the potential mechanisms underlying neurological defects associated with Klf7 mutations.

Infection of Mouse Neural Progenitor Cells by Toxoplasma gondii Reduces Proliferation, Migration, and Neuronal Differentiation in Vitro

Congenital toxoplasmosis constitutes a major cause of pre- and postnatal complications. Fetal infection with Toxoplasma gondii influences development and can lead to microcephaly, encephalitis, and neurologic abnormalities. Systematic studies concerning the effects of neural progenitor cell infection with T. gondii are unavailable. Cortical intermediate progenitor cells cultivated as neurospheres obtained from E16.5 Swiss Webster mice were infected with T. gondii (ME49 strain) tachyzoites to mimic the developing mouse cerebral cortex invitro. Infection was associated with decreased cell proliferation, detected by Ki-67 staining at 48 and 72 hours after infection in floating neurospheres, and reduced cellularity at 96 hours. Transient decreases in the expression of the neurogenesis-related transcription factors T-box brain protein 1, mouse atonal homolog protein 1, and hairy and enhancer of split protein 1 were found in infected cultures, while the level of transcription factor SOX-2 remained unaltered. Neurogenic potential, assessed in plated neurospheres, was impaired in infected cultures, as indicated by decreased late neuronal marker neurofilament heavy chain immunoreactivity. Infected cultures exhibited decreased overall migration rates at 48 and 120 hours. These findings indicate that T. gondii infection of neural progenitor cells may lead to reduced neurogenesis due to an imbalance in cell proliferation alongside an altered migratory profile. If translated to the invivo situation, these data could explain, in part, cortical malformations in congenitally infected individuals.

Npas3 regulates stemness maintenance of radial glial cells and neuronal migration in the developing mouse cerebral cortex.

The neuronal PAS domain 3 (NPAS3) is a member of the basic helix-loop-helix (bHLH) PAS family of transcription factors and is implicated in psychiatric and neurodevelopmental disorders. NPAS3 is robustly expressed in the cortical ventricle zone (VZ), a transient proliferative zone containing progenitor cells, mainly radial glial cells, destined to give rise to cortical excitatory neurons. However, the role of NPAS3 in corticogenesis remains largely unknown. In this study, we knocked down Npas3 expression in the neural progenitor cells residing in the cortical VZ to investigate the role of Npas3 in cerebral cortical development in mice. We demonstrated that Npas3 knockdown profoundly impaired neuronal radial migration and changed the laminar cell fate of the cells detained in the deep cortical layers. Furthermore, the downregulation of Npas3 led to the stemness maintenance of radial glial cells and increased the proliferation rate of neural progenitor cells residing in the VZ/subventricular zone (SVZ). These findings underline the function of Npas3 in the development of the cerebral cortex and may shed light on the etiology of NPAS3-related disorders.

Requirement of CRMP2 Phosphorylation in Neuronal Migration of Developing Mouse Cerebral Cortex and Hippocampus and Redundant Roles of CRMP1 and CRMP4.

The mammalian cerebral cortex is characterized by a 6-layer structure, and proper neuronal migration is critical for its formation. Cyclin-dependent kinase 5 (Cdk5) has been shown to be a critical kinase for neuronal migration. Several Cdk5 substrates have been suggested to be involved in ordered neuronal migration. However, in vivo loss-of-function studies on the function of Cdk5 phosphorylation substrates in neuronal migration in the developing cerebral cortex have not been reported. In this study, we demonstrated that Cdk5-mediated phosphorylation of collapsing mediator protein (CRMP) 2 is critical for neuronal migration in the developing cerebral cortex with redundant functions of CRMP1 and CRMP4. The cerebral cortices of triple-mutant CRMP1 knock-out (KO); CRMP2 knock-in (KI)/KI; and CRMP4 KO mice showed disturbed positioning of layers II-V neurons in the cerebral cortex. Further experiments using bromodeoxyuridine birthdate-labeling and in utero electroporation implicated radial migration defects in cortical neurons. Ectopic neurons were detected around the CA1 region and dentate gyrus in CRMP1 KO; CRMP2 KI/KI; and CRMP4 KO mice. These results suggest the importance of CRMP2 phosphorylation by Cdk5 and redundancy of CRMP1 and CRMP4 in proper neuronal migration in the developing cerebral cortex and hippocampus.

Long Non-coding RNA T-uc.189 Modulates Neural Progenitor Cell Fate by Regulating Srsf3 During Mouse Cerebral Cortex Development.

Neurogenesis is a complex process that depends on the delicate regulation of spatial and temporal gene expression. In our previous study, we found that transcribed ultra-conserved regions (T-UCRs), a class of long non-coding RNAs that contain UCRs, are expressed in the developing nervous systems of mice, rhesus monkeys, and humans. In this study, we first detected the full-length sequence of T-uc.189, revealing that it was mainly concentrated in the ventricular zone (VZ) and that its expression decreased as the brain matured. Moreover, we demonstrated that knockdown of T-uc.189 inhibited neurogenesis. In addition, we found that T-uc.189 positively regulated the expression of serine-arginine-rich splicing factor 3 (Srsf3). Taken together, our results are the first to demonstrate that T-uc.189 regulates the expression of Srsf3 to maintain normal neurogenesis during cortical development.

Single-cell transcriptomics of the early developing mouse cerebral cortex disentangle the spatial and temporal components of neuronal fate acquisition

In the developing cerebral cortex, how progenitors that seemingly display limited diversity end up producing a vast array of neurons remains a puzzling question. The prevailing model suggests that temporal maturation of progenitors is a key driver in the diversification of the neuronal output. However, temporal constraints are unlikely to account for all diversity, especially in the ventral and lateral pallium where neuronal types significantly differ from their dorsal neocortical counterparts born at the same time. In this study, we implemented single-cell RNAseq to sample the diversity of progenitors and neurons along the dorso-ventral axis of the early developing pallium. We first identified neuronal types, mapped them on the tissue and determined their origin through genetic tracing. We characterised progenitor diversity and disentangled the gene modules underlying temporal versus spatial regulations of neuronal specification. Finally, we reconstructed the developmental trajectories followed by ventral and dorsal pallial neurons to identify lineage-specific gene waves. Our data suggest a model by which discrete neuronal fate acquisition from a continuous gradient of progenitors results from the superimposition of spatial information and temporal maturation.

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling.

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

Cell-type and fetal-sex-specific targets of prenatal alcohol exposure in developing mouse cerebral cortex

SummaryPrenatal alcohol exposure (PAE) results in cerebral cortical dysgenesis. Single-cell RNA sequencing was performed on murine fetal cerebral cortical cells from six timed pregnancies, to decipher persistent cell- and sex-specific effects of an episode of PAE during early neurogenesis. We found, in an analysis of 38 distinct neural subpopulations across 8 lineage subtypes, that PAE altered neural maturation and cell cycle and disrupted gene co-expression networks. Whereas most differentially regulated genes were inhibited, particularly in females, PAE also induced sex-independent neural expression of fetal hemoglobin, a presumptive epigenetic stress adaptation. PAE inhibited Bcl11a, Htt, Ctnnb1, and other upstream regulators of differentially expressed genes and inhibited several autism-linked genes, suggesting that neurodevelopmental disorders share underlying mechanisms. PAE females exhibited neural loss of X-inactivation, with correlated activation of autosomal genes and evidence for spliceosome dysfunction. Thus, episodic PAE persistently alters the developing neural transcriptome, contributing to sex- and cell-type-specific teratology.

MACF1, Involved in the 1p34.2p34.3 Microdeletion Syndrome, is Essential in Cortical Progenitor Polarity and Brain Integrity.

1p34.2p34.3 deletion syndrome is characterized by an increased risk for autism. Microtubule Actin Crosslinking Factor 1 (MACF1) is one candidate gene for this syndrome. It is unclear, however, how MACF1 deletion is linked to brain development and neurodevelopmental deficits. Here we report on Macf1 deletion in the developing mouse cerebral cortex, focusing on radial glia polarity and morphological integrity, as these are critical factors in brain formation. We found that deleting Macf1 during cortical development resulted in double cortex/subcortical band heterotopia as well as disrupted cortical lamination. Macf1-deleted radial progenitors showed increased proliferation rates compared to control cells but failed to remain confined within their defined proliferation zone in the developing brain. The overproliferation of Macf1-deleted radial progenitors was associated with elevated cell cycle speed and re-entry. Microtubule stability and actin polymerization along the apical ventricular area were decreased in the Macf1 mutant cortex. Correspondingly, there was a disconnection between radial glial fibers and the apical and pial surfaces. Finally, we observed that Macf1-mutant mice exhibited social deficits and aberrant emotional behaviors. Together, these results suggest that MACF1 plays a critical role in cortical progenitor proliferation and localization by promoting glial fiber stabilization and polarization. Our findings may provide insights into the pathogenic mechanism underlying the 1p34.2p34.3 deletion syndrome.

Sevoflurane inhibits neuronal migration and axon growth in the developing mouse cerebral cortex.

The highly organized laminar structure of the mammalian brain is dependent on successful neuronal migration, and migration deficits can cause lissencephaly and behavioral and cognitive defects. Here, we investigated the contribution of neuronal migration dysregulation to anesthesia-induced neurotoxicity in the fetal brain. Pregnant C57BL/6 mice at embryonic day 14.5 received 2.5% sevoflurane daily for two days. Cortical neuron migration and axon lengths were evaluated using GFP immunostaining. Morris water maze tests were performed to assess the effects of sevoflurane exposure on spatial memory in offspring. We found that sevoflurane exposure decreased axon length and caused cognitive defects in young mice. RNA sequencing revealed that these defects were associated with reduced neuro-oncological ventral antigen 2 (Nova2) expression. In utero electroporation experiments using Nova2 shRNA recapitulated this finding. Nova2 shRNA inhibited neuronal migration and decreased axon lengths. Finally, we found that Netrin-1/Deleted in Colorectal Cancer (Dcc) proteins acted downstream of Nova2 to suppresses neuronal migration. These findings describe a novel mechanism by which prenatal anesthesia exposure affects embryonic neural development and postnatal behavior.

FGF Signaling Directs the Cell Fate Switch from Neurons to Astrocytes in the Developing Mouse Cerebral Cortex.

During mammalian neocortical development, neural precursor cells generate neurons first and astrocytes later. The cell fate switch from neurons to astrocytes is a key process generating proper numbers of neurons and astrocytes. Although the intracellular mechanisms regulating this cell fate switch have been well characterized, extracellular regulators are still largely unknown. Here, we uncovered that fibroblast growth factor (FGF) regulates the cell fate switch from neurons to astrocytes in the developing cerebral cortex using mice of both sexes. We found that the FGF signaling pathway is activated in radial glial cells of the ventricular zone at time points corresponding to the switch in cell fate. Our loss- and gain-of-function studies using in utero electroporation indicate that activation of FGF signaling is necessary and sufficient to change cell fates from neurons to astrocytes. We further found that the FGF-induced neuron-astrocyte cell fate switch is mediated by the MAPK pathway. These results indicate that FGF is a critical extracellular regulator of the cell fate switch from neurons to astrocytes in the mammalian cerebral cortex.SIGNIFICANCE STATEMENT Although the intracellular mechanisms regulating the neuron-astrocyte cell fate switch in the mammalian cerebral cortex during development have been well studied, their upstream extracellular regulators remain unknown. By using in utero electroporation, our study provides in vivo data showing that activation of FGF signaling is necessary and sufficient for changing cell fates from neurons to astrocytes. Manipulation of FGF signaling activity led to drastic changes in the numbers of neurons and astrocytes. These results indicate that FGF is a key extracellular regulator determining the numbers of neurons and astrocytes in the mammalian cerebral cortex, and is indispensable for the establishment of appropriate neural circuitry.

Role of Per3, a circadian clock gene, in embryonic development of mouse cerebral cortex

Per3 is one of the primary components of circadian clock system. While circadian dysregulation is known to be involved in the pathogenesis of several neuropsychiatric diseases. It remains largely unknown whether they participate in embryonic brain development. Here, we examined the role of clock gene Per3 in the development of mouse cerebral cortex. In situ hybridization analysis revealed that Per3 is expressed in the developing mouse cortex. Acute knockdown of Per3 with in utero electroporation caused abnormal positioning of cortical neurons, which was rescued by RNAi-resistant Per3. Per3-deficient cells showed abnormal migration phenotypes, impaired axon extension and dendritic arbor formation. Taken together, Per3 was found to play a pivotal role in corticogenesis via regulation of excitatory neuron migration and synaptic network formation.

A role for polycystin-1 and polycystin-2 in neural progenitor cell differentiation.

Polycystin-1 (PC1) and polycystin-2 (PC2) are transmembrane proteins encoded by the Pkd1 and Pkd2 genes, respectively. Mutations in these genes are causative for the development of autosomal-dominant polycystic kidney disease. A prominent feature of this disease is an unbalanced cell proliferation. PC1 and PC2 physically interact to form a complex, which localizes to the primary cilia of renal epithelial cells. Recently, PC1 and PC2 have also been described to be present in primary cilia of radial glial cells (RGCs) and to contribute to the planar cell polarity of late RGCs and E1 ependymal cells. As neural progenitor cells (NPCs), early RGCs have to balance proliferation for expansion, or for self-renewal and differentiation to generate neurons. It is not known whether the polycystins play a role in this process. Here, we show that PC1 and PC2 are expressed in RGCs of the developing mouse cerebral cortex during neurogenesis. Loss-of-function analysis and cell-based assays reveal that a reduction of PC1 or PC2 expression leads to increased NPC proliferation, while the differentiation to neurons becomes impaired. The increased NPC proliferation is preceded by enhanced Notch signaling and accompanied by a rise in the number of symmetric cell divisions. The transcription factor STAT3 seems to be mechanistically important for polycystin signaling in NPCs as either STAT3 knockdown or inhibition of STAT3 function abrogates the increased proliferation driven by reduced polycystin expression. Our findings indicate that PC1 and PC2 are critical for maintaining a balance between proliferation and differentiation of NPCs.

Loss of Lgl1 Disrupts the Radial Glial Fiber-guided Cortical Neuronal Migration and Causes Subcortical Band Heterotopia in Mice

Radial glial cells (RGCs) are neuronal progenitors and function as scaffolds for neuronal radial migration in the developing cerebral cortex. These functions depend on a polarized radial glial scaffold, which is of fundamental importance for brain development. Lethal giant larvae 1 (Lgl1), a key regulator for cell polarity from Drosophila to mammals, plays a key role in tumorigenesis and brain development. To overcome neonatal lethality in Lgl1-null mice and clarify the role of Lgl1 in mouse cerebral cortex development and function, we created Lgl1 dorsal telencephalon-specific knockout mice mediated by Emx1-Cre. Lgl1Emx1 conditional knockout (CKO) mice had normal life spans and could be used for function research. Histology results revealed that the mutant mice displayed an ectopic cortical mass in the dorsolateral hemispheric region between the normotopic cortex and the subcortical white matter, resembling human subcortical band heterotopia (SBH). The Lgl1Emx1 CKO cortex showed disrupted adherens junctions (AJs), which were accompanied by ectopic RGCs and intermediate progenitors, and disorganization of the radial glial fiber system. The early- and late-born neurons failed to reach the destined position along the disrupted radial glial fiber scaffold and instead accumulated in ectopic positions and formed SBH. Additionally, the absence of Lgl1 led to severe abnormalities in RGCs, including hyperproliferation, impaired differentiation, and increased apoptosis. Lgl1Emx1 CKO mice also displayed deficiencies in anxiety-related behaviors. We concluded that Lgl1 is essential for RGC development and neural migration during cerebral cortex development.

Drebrin-like (Dbnl) Controls Neuronal Migration via Regulating N-Cadherin Expression in the Developing Cerebral Cortex.

The actin cytoskeleton is crucial for neuronal migration in the mammalian developing cerebral cortex. The adaptor protein Drebrin-like (Dbnl) plays important roles in reorganization of the actin cytoskeleton, dendrite formation, and endocytosis by interacting with F-actin, cobl, and dynamin. Although Dbnl is known to be expressed in the brain, the functions of this molecule during brain development are largely unknown. In this study, to examine the roles of Dbnl in the developing cerebral cortex, we conducted experiments using mice of both sexes with knockdown of Dbnl, effected by in utero electroporation, in the migrating neurons of the embryonic cortex. Time-lapse imaging of the Dbnl-knockdown neurons revealed that the presence of Dbnl is a prerequisite for appropriate formation of processes in the multipolar neurons in the multipolar cell accumulation zone or the deep part of the subventricular zone, and for neuronal polarization and entry into the cortical plate. We found that Dbnl knockdown decreased the amount of N-cadherin protein expressed on the plasma membrane of the cortical neurons. The defect in neuronal migration caused by Dbnl knockdown was rescued by moderate overexpression of N-cadherin and αN-catenin or by transfection of the phospho-mimic form (Y337E, Y347E), but not the phospho-resistant form (Y337F, Y347F), of Dbnl. These results suggest that Dbnl controls neuronal migration, neuronal multipolar morphology, and cell polarity in the developing cerebral cortex via regulating N-cadherin expression.SIGNIFICANCE STATEMENT Disruption of neuronal migration can cause neuronal disorders, such as lissencephaly and subcortical band heterotopia. During cerebral cortical development, the actin cytoskeleton plays a key role in neuronal migration; however, the mechanisms of regulation of neuronal migration by the actin cytoskeleton still remain unclear. Herein, we report that the novel protein Dbnl, an actin-binding protein, controls multiple events during neuronal migration in the developing mouse cerebral cortex. We also showed that this regulation is mediated by phosphorylation of Dbnl at tyrosine residues 337 and 347 and αN-catenin/N-cadherin, suggesting that the Dbnl-αN-catenin/N-cadherin pathway is important for neuronal migration in the developing cortex.

The Pace of Neurogenesis Is Regulated by the Transient Retention of the Apical Endfeet of Differentiating Cells.

The development of the mammalian cerebral cortex involves a variety of temporally organized events such as successive waves of neuronal production and the transition of progenitor competence for each neuronal subtype generated. The number of neurons generated in a certain time period, that is, the rate of neuron production, varies across the regions of the brain and the specific developmental stage; however, the underlying mechanism of this process is poorly understood. We have recently found that nascent neurons communicate with undifferentiated progenitors and thereby regulate neurogenesis, through a transiently retained apical endfoot that signals via the Notch pathway. Here, we report that the retention time length of the neuronal apical endfoot correlates with the rate of neuronal production in the developing mouse cerebral cortex. We further demonstrate that a forced reduction or extension of the retention period through the disruption or stabilization of adherens junction, respectively, resulted in the acceleration or deceleration of neurogenesis, respectively. Our results suggest that the apical endfeet of differentiating cells serve as a pace controller for neurogenesis, thereby assuring the well-proportioned laminar organization of the neocortex.

Rer1-mediated quality control system is required for neural stem cell maintenance during cerebral cortex development.

Rer1 is a retrieval receptor for endoplasmic reticulum (ER) retention of various ER membrane proteins and unassembled or immature components of membrane protein complexes. However, its physiological functions during mammalian development remain unclear. This study aimed to investigate the role of Rer1-mediated quality control system in mammalian development. We show that Rer1 is required for the sufficient cell surface expression and activity of γ-secretase complex, which modulates Notch signaling during mouse cerebral cortex development. When Rer1 was depleted in the mouse cerebral cortex, the number of neural stem cells decreased significantly, and malformation of the cerebral cortex was observed. Rer1 loss reduced γ-secretase activity and downregulated Notch signaling in the developing cerebral cortex. In Rer1-deficient cells, a subpopulation of γ-secretase complexes and components was transported to and degraded in lysosomes, thereby significantly reducing the amount of γ-secretase complex on the cell surface. These results suggest that Rer1 maintains Notch signaling by maintaining sufficient expression of the γ-secretase complex on the cell surface and regulating neural stem cell maintenance during cerebral cortex development.

De novo PHACTR1 mutations in West syndrome and their pathophysiological effects.

Trio-based whole exome sequencing identified two de novo heterozygous missense mutations [c.1449T > C/p.(Leu500Pro) and c.1436A > T/p.(Asn479Ile)] in PHACTR1, encoding a molecule critical for the regulation of protein phosphatase 1 (PP1) and the actin cytoskeleton, in unrelated Japanese individuals with West syndrome (infantile spasms with intellectual disability). We then examined the role of Phactr1 in the development of mouse cerebral cortex and the pathophysiological significance of these two mutations and others [c.1561C > T/p.(Arg521Cys) and c.1553T > A/p.(Ile518Asn)], which had been reported in undiagnosed patients with intellectual disability. Immunoprecipitation analyses revealed that actin-binding activity of PHACTR1 was impaired by the p.Leu500Pro, p.Asn479Ile and p.Ile518Asn mutations while the p.Arg521Cys mutation exhibited impaired binding to PP1. Acute knockdown of mouse Phactr1 using in utero electroporation caused defects in cortical neuron migration during corticogenesis, which were rescued by an RNAi-resistant PHACTR1 but not by the four mutants. Experiments using knockdown combined with expression mutants, aimed to mimic the effects of the heterozygous mutations under conditions of haploinsufficiency, suggested a dominant negative effect of the mutant allele. As for dendritic development in vivo, only the p.Arg521Cys mutant was determined to have dominant negative effects, because the three other mutants appeared to be degraded with these experimental conditions. Electrophysiological analyses revealed abnormal synaptic properties in Phactr1-deficient excitatory cortical neurons. Our data show that the PHACTR1 mutations may cause morphological and functional defects in cortical neurons during brain development, which is likely to be related to the pathophysiology of West syndrome and other neurodevelopmental disorders.