Epilepsy-associated FOXJ3 variants link a transcriptional program of the PTEN-mTOR pathway to neuronal specification and cortical lamination

Domain: chemistry
Published: 2026-03-09
Editorial outcome: minor_revision

Abstract
Focal cortical dysplasia (FCD), a major cause of drug-resistant epilepsy, involves abnormal neuronal migration and cortical architecture, yet its molecular basis remains poorly defined. Here, we identify FOXJ3 pathogenic variants in patients with autosomal dominant focal epilepsy and FCD. In the developing mouse cortex, FOXJ3 declines sharply in neural progenitors after embryonic day 15.5. In utero electroporation-mediated Foxj3 knockdown in mouse brains impairs neuronal migration, disrupts cortical lamination, and alters neuronal specification, promoting upper-layer neuron production at the expense of deeper-layer neurons. ChIP-seq and scRNA-seq analyses identify Pten as a key FOXJ3 target. Notably, Pten overexpression rescues cortical defects caused by FOXJ3 deficiency. FCD-associated variant fails to upregulate Pten, leading to dysregulated mTOR signaling and enlarged neuronal soma, a hallmark of FCD. These findings suggest that mutations in FOXJ3 may cause epilepsy and FCD and define a transcriptional program that regulates the PTEN-mTOR pathway for neuronal specification and cortical lamination.

Introduction
The development of the cerebral cortex relies on a precisely regulated sequence of events, and disruptions in this complex process can lead to malformations of cortical development (MCD). Among these, focal cortical dysplasia (FCD) is one of the most frequently diagnosed forms in pediatric and young adult patients with epilepsy.

FCD is characterized by abnormal neuronal organization and cortical architecture and classified based on histopathological criteria established by the International League Against Epilepsy (ILAE)12–14. FCD type II is defined by disrupted cortical lamination and distinctive cytological abnormalities, distinguishing type IIa (dysmorphic neurons without balloon cells) from type IIb (dysmorphic neurons with balloon cells)13,14. Magnetic resonance Imaging (MRI) remains the primary noninvasive diagnostic tool for FCD; however, subtle lesions may be overlooked, and FCD type II is still the most common etiology of drug-resistant epilepsy (DRE), even in MRI-negative cases13,15–17.

Advances in molecular genetics have revealed that epileptogenesis in FCD II is strongly linked to aberrant hyperactivation of the PI3K (phosphatidylinositol-3-kinase)-PTEN (phosphatase and tensin homolog)-mTOR (mechanistic target of rapamycin) pathway18. The PTEN-mTOR Pathway is a master regulator of cell proliferation, survival, and growth and plays a critical role in cerebral cortex development and synaptic plasticity. Genetically, FCD type II has been linked to mutations in genes that regulate the PI3K-PTEN-mTOR pathway, with PTEN acting as a key inhibitory factor. Dysregulation of this pathway, which can be due to pathogenic variations of PTEN or other genes involved in mTOR signaling, disrupts normal cortical development, leading to abnormal neuronal migration and differentiation19–23.

Transcription factors are pivotal regulators of cortical development, orchestrating neurogenesis, migration, and maturation. Among them, members of the forkhead box (FOX) family have been implicated in diverse biological processes, including neural development. The FOX family consists of approximately 50 genes in humans and 44 in mice, encoding transcription factors with a conserved winged-helix DNA binding domain, known as the FOX domain, and are divided into 19 subfamilies, from FOXA to FOXS24. Previous studies have shown that pathogenic variations in FOX genes can impact embryogenesis, including neural development25. For example, FOXG1 loss of function leads to FOXG1 syndrome, characterized by severe intellectual disability and abnormal movements26–30. The FOXJ family, comprising FOXJ1, FOXJ2, and FOXJ3 (Gene ID: 22887; OMIM: 616035), is involved in various biological processes. FOXJ1 regulates ciliary and spinal cord development and controls left-right asymmetry31,32. FOXJ2 promotes meiosis in spermatogenesis, affects cell migration in glioma, and influences embryo development, leading to embryonic lethality33–35. FOXJ3 is primarily expressed in the neuroectoderm, neural crest, and telencephalic vesicle of mice during early development, but its precise function remains unclear36. Studies suggest FOXJ3 prevents embryonic stem cells from neural differentiation and regulates cell cycle in different cell lines37–39. FOXJ3 also plays a role in cell proliferation, migration, and the regulation of Snail expression and has been implicated in various cancers, including lung, prostate, breast, and colorectal cancer40–42. These findings highlight FOXJ3’s role in early embryonic development, particularly in regulating differentiation and proliferation.

Here, we identified heterozygous variants in FOXJ3 associated with autosomal dominant focal epilepsy linked to FCD. We found that FOXJ3 plays a crucial role in specifying deep-layer versus upper-layer neurons in mice. Foxj3 (Gene ID: 230700) is expressed in both NPCs within the VZ and neurons in the CP, with expression in NPCs showing a sharp decline after embryonic day (E) 15.5. FOXJ3 appears to regulate transcriptional programs and cell cycle dynamics, likely by modulating genes involved in cortical development. Notably, Pten emerged as a key downstream target of FOXJ3 in regulating neuronal fate determination during corticogenesis. Together, our data suggest that FOXJ3 orchestrates transcriptional programs and cell cycle regulation to control neuronal migration and deep-layer neuron specification via the PTEN-mTOR pathway, representing a pathogenic mechanism underlying focal epilepsy and FCD.

Results
Heterozygous FOXJ3 missense variants are associated with focal epilepsy related to focal cortical dysplasia
The index patient (DNA No. 50) presented with focal seizures at the age of 6 (Fig. 1A), with brain MRI revealing FCD in the right frontal gyrus (Fig. 1B). Due to DRE, the patient underwent resection surgery, and histopathology confirmed FCD type IIa (Fig. 1C). Notably, the patient’s mother (DNA No. 51) and cousin (DNA No. 55) also had focal epilepsy and suspected FCD on MRI (right frontal FCD in the mother and right temporal FCD in the cousin). To identify the underlying genetic cause, whole-genome sequencing (WGS) was performed on the index patient (DNA No. 50), her cousin (DNA No. 55), and their asymptomatic grandmother (DNA No. 57). Through WGS and Sanger sequencing for intrafamilial segregation, we identified a heterozygous FOXJ3 variant (NM_014947.5:c.1052A>G, p.Asn351Ser [p.N351S]) shared by the index patient, her mother and her cousin, which was considered the candidate causative variant (Fig. 1D, E). According to the SNP database (https://www.ncbi.nlm.nih.gov/snp/), the FOXJ3:p.Asn351Ser variant (rs1471785278) has a very low minor allele frequency (MAF) in public databases (0.000008/2 in TOPMED, and C=0.00002/5 in GnomAD_exomes). In the Taiwan View database, which is specific to the Han-Taiwanese population (https://taiwanview.twbiobank.org.tw/), the MAF is 0.0003 in 1,492 WGS cases. In ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), it has been submitted only once, classified as a variant of uncertain significance, without associated clinical details.

We further screened FOXJ3 mutations in 170 patients with MCD and 76 patients with epilepsy without MCD on brain MRI at Taipei Veterans General Hospital in Taiwan. Additionally, we analyzed 85,000 WGS datasets from the Genomics England dataset, which primarily includes patients with rare diseases and their parents (https://www.genomicsengland.co.uk/initiatives/100000-genomes-project), as well as 30,000 whole-exome sequencing (WES) datasets from neurodevelopmental or neuromuscular patients registered at the Neurogenetics lab of UCL Institute of Neurology. The p.N351S variant was not detected in these datasets, but two additional heterozygous FOXJ3 missense variants were identified in two unrelated patients with focal epilepsy. FOXJ3 (NM_014947.5:c.1861A>G, p.Ile621Val [p.I621V]) was found in a male patient with focal epilepsy of unknown etiology, while FOXJ3 (NM_014947.5:c.757C>A, p.Pro253Thr [p.P253T]) was identified in a male patient who developed focal epilepsy in his second decade. Brain MRI at 1.5 T in both patients was reported as essentially normal; however, it is important to note that subtle FCD lesions can be overlooked on standard MRI16.

Although in silico prediction tools provided conflicting evidence regarding the pathogenicity of these three FOXJ3 missense variants (Supplementary Fig. 1A), all are rare and affect highly conserved residues across multiple species (Supplementary Fig. 1B). These clinical-genetic observations suggest that heterozygous pathogenic variants in FOXJ3 are associated with focal epilepsy, potentially related to FCD. This prompted us to investigate the functional role of FOXJ3 in cortical development and its contribution to FCD.

FOXJ3 is expressed in cortical progenitors and neurons during cortical development
To investigate FOXJ3 expression during neocortical development, we collected protein lysates from mouse brain tissue at E11.5 to E18.5. FOXJ3 expression was consistently detected across these developmental stages, with a progressive increase from E11.5 to E18.5 (Fig. 2A, B). To further delineate its spatial expression, we performed immunofluorescence staining (IF) of FOXJ3 in mouse brain slices from E12.5 to E18.5, followed by confocal microscopy (Fig. 2C). FOXJ3 was localized to the nuclei of cells within the CP and the VZ. Notably, in the CP, FOXJ3 was highly expressed from E12.5 to E18.5. In contrast, in the VZ, FOXJ3 expression was strong from E12.5 to E14.5 but markedly declined after E15.5. Quantitative analysis of fluorescence intensity corroborated these observations, demonstrating sustained FOXJ3 expression in the CP and a significant reduction in the VZ post-E15.5 (Fig. 2D). Additionally, FOXJ3 immunostaining of primary cortical neurons from E14.5 mice at 8 days in vitro (DIV) confirmed the nuclear localization of FOXJ3 in neurons (Fig. 2E). Considering the pathogenesis of FCD mainly in early corticogenesis43, these findings suggest that FOXJ3 may play a crucial role in cortical progenitors during cortical development.

Foxj3 knockdown at E14.5 mildly impaired neuronal migration but did not affect cortical lamination or cell fate determination
To elucidate the roles of FOXJ3 in cortical development, especially in cortical progenitors, we utilized RNA interference (RNAi) to knock down Foxj3 expression (Fire et al., 1998). Short hairpin RNA (shRNA) targeting the Foxj3 coding region (shFoxj3) was delivered via lentivirus to primary cortical cells isolated from E16 mouse embryos at DIV3. After 6 days in culture, shFoxj3-infected cortical neurons exhibited significantly reduced FOXJ3 protein levels compared to control neurons infected with scrambled shRNA (shCtrl) (shFoxj3: 29.1±6.8 %, n=3; p<0.001, Student’s t-test), indicating effective (> 70 %) Foxj3 knockdown (Supplementary Fig. 2A).

Next, we assessed the impact of Foxj3 knockdown on the developing neocortex using in utero electroporation (IUE) of shRNAs together with a GFP reporter into mouse embryonic neocortex at E14.5 (Supplementary Fig. 2B). Four days post electroporation, the majority of the GFP+ cells were distributed in the CP (69.4% ± 2.4%, n=5), with only a few cells in the IZ (19.3% ± 1.4%, n=5) and VZ/SVZ (11.3% ± 1.5%, n=5) in control brains electroporated with shCtrl. However, in the brains with Foxj3 knockdown, a significant migration delay was observed, with fewer GFP+ cells in the CP (53.1% ± 1.3%, n=3; p<0.01, Student’s t-test) and more in the IZ (38.0% ± 2.2%, p<0.001; n=3, Student’s t-test), while the proportion of GFP+ cells in the VZ/SVZ remained unchanged (8.8% ± 0.9%, n=3; Supplementary Fig. 2B).

To further evaluate FOXJ3’s role during late cortical development, we analyzed brains at postnatal day (P) 7 following IUE at E14.5 (Supplementary Fig. 2C). In control brains electroporated with shCtrl, the majority of GFP+ cells localized to Layer 2/3. Similarly, GFP+ cells in shFoxj3 brains were positioned in Layer 2/3 (Supplementary Fig. 2D, E). Since neurons born at E14.5 are destined for Layer 2/3 and primarily differentiated as BRN2+ and SATB2+ callosal projection neurons8, we examined whether Foxj3 knockdown affected this molecular profile. Immunostaining revealed that the majority of GFP+ cells expressed upper-layer gene BRN2 and SATB2 in both control and Foxj3 knockdown brains (BRN2+: 93.0% ± 1.5%, n=3 control vs. 97.3% ± 0.9%, n=3 shFoxj3; SATB2+: 84.3% ± 6.7%, n=3 control vs. 98.0% ± 1.1%, n=3 shFoxj3) (Supplementary Fig. 2D, E). These results suggest that while FOXJ3 dysfunction may impair neuronal migration during early cortical development, it does not appear to influence the generation or fate of upper-layer callosal projection neurons.

FOXJ3’s critical role in cell fate determination and cortical lamination in deep-layer and Layer 4 neurons
Given that FOXJ3 dysfunction affects neuronal migration without altering the fate of later-born neurons, we explored whether similar phenotypes manifest in earlier-born neurons. Therefore, we conducted IUE of shFoxj3 along with GFP at E12.5. Samples were collected at P7 to examine the identity of the targeted cells (Fig. 3A). In control brains, GFP+ cells electroporated at E12.5 predominantly populated Layer 5/6 of the neocortex at P7. Strikingly, in Foxj3 knockdown brains, most GFP+ cells were found in the superficial layers of the CP. To assess the fates of these GFP+ cells, we performed immunofluorescence staining using cortical layer markers, including TBR1 (Layer 6 neuron marker), CTIP2 (layer 5 neuron marker), and SATB2 (upper layer neuron marker). In control brains, ~40% of GFP+ cells expressed TBR1 (38.1±0.5 %, n=3), and around 20% expressed CTIP2 (17.8±1.8 %, n=3; Fig. 3A, B). Additionally, GFP signals in the ventral cerebrum indicated the presence of subcortical projections (Fig. 3C), confirming the deep-layer neuronal identity of these GFP+ cells. Foxj3 knockdown resulted in a significant reduction in TBR1+ and CTIP2+ cells compared to control brains (TBR1: 16.4±2.6%, n=3, p<0.01; CTIP2: 0.8% ± 0.4%, n=3, p<0.001, Student’s t-test). Conversely, the SATB2+ cell population significantly increased under Foxj3 knockdown conditions (Ctrl: 26.7±1.0%, n=3; shFoxj3: 46.6±3.3%, n=3; p<0.01, Student’s t-test; Fig. 3A, B). Supporting these molecular expression changes, subcortical projections were reduced (Fig. 3C). These findings suggest that FOXJ3 dysfunction impairs the acquisition of deep-layer neuronal fate and induces the production of SATB2+ callosal projection neurons.

To further investigate the effects of FOXJ3 dysfunction on Layer 4 neurons, we performed IUE to knock down Foxj3 at E13.75. Samples were collected at P7 and subjected to immunofluorescence staining using RORB (Layer 4 neuron marker) and BRN2 antibodies (Fig. 3D). In control brains, the majority of GFP+ cells localized in Layer 4 and expressed RORB (69.9±3.1 %, n=3), consistent with previous findings44. In brains electroporated with shFoxj3, the GFP+ cells shifted to the superficial Layer 2/3, with a significant reduction in the RORB+ cell population (9.5±1.0%, n=3; p<0.0001, Student’s t-test). Instead, most of these Foxj3-knockdown GFP+ cells expressed BRN2 (84.1±2.2 %, n=3), compared to control brains (21.9±5.9 %, n=3; p<0.001, Student’s t-test; Fig. 3D, E). Consistent with these molecular expression changes, we observed a 3-fold increase in GFP signals in the corpus callosum of brains with Foxj3 knockdown (Fig. 3F, G). These results indicate that FOXJ3 dysfunction impairs Layer 4 cell fate acquisition and promotes the production of BRN2+ callosal projection neurons. Collectively, our findings suggest that FOXJ3 dysfunction promotes callosal neuron production, regardless of the timing the genetic manipulation.

FOXJ3 deficiency leads to severe delays in neuronal migration
Cortical lamination defects often arise from dysregulated neuronal migration in postmitotic neurons45,46. Given that Foxj3 knockdown induces the formation of upper callosal projection neurons (Fig. 3), we investigated whether Foxj3 knockdown also impairs neuronal migration, thereby affecting cell fate. We collected brain sections 3 days after electroporation at E12.5 and stained them with TBR1 antibody (Fig. 4A). In control brains, most GFP+ cells had entered the IZ and reached the CP (CP: 50.7% ± 1.9%, IZ: 23.6% ± 1.5%, VZ: 25.7% ± 0.9%, n=3). However, in Foxj3-knockdown brains, a significant proportion of GFP+ cells failed to enter the CP and remained restricted to the IZ (CP: 24.7±1.7%, p<0.001; IZ: 47.9±3.6%, p<0.01, VZ: 27.4% ± 1.9%; n=3, Student’s t-test). This migration defect persisted in the brains 5 days after electroporation (E12.5-E17.5; Fig. 4B), where the majority of GFP+ cells in control brains had entered the CP (CP: 49.6±0.8%; IZ: 29.5±1.1%; VZ: 20.9±1.9% in shCtrl vs. CP: 28.8±3.9%, p<0.01; IZ: 37.3±2.9%, VZ: 33.9±6.7% in shFoxj3; n=3 in each group, Student’s t-test). Similar migration defects were observed in E16.75 brains following electroporation at E13.75 (Fig. 4C). In control brains electroporated with shCtrl, most GFP+ neurons had entered the CP (CP: 84.1±3.4%, IZ: 12.0±1.8%, VZ: 3.9±1.7%, n=3). In contrast, GFP+ cells in Foxj3-knockdown brains were still confined to the IZ and VZ (CP: 22.1±4.8%, p<0.001; IZ: 67.6±6.6 %, p<0.01; VZ: 10.3±1.9 %; n=3, Student’s t-test). Collectively, these results indicate that FOXJ3 deficiency induces significant neuronal migration delays, ultimately leading to changes in cell fate during early cortical development.

The essential role of FOXJ3 in neural stem cells for cell fate determination and cortical lamination
Given that FOXJ3 is expressed in both progenitors and cortical neurons (Fig. 2), we sought to determine whether FOXJ3 exerts its regulatory effects during the progenitor stage or the postmitotic neuron stage. To address this question, we employed a doxycycline-inducible knockdown system to temporally control FOXJ3 expression during cortical development (Supplementary Fig. 3A, B). By electroporating inducible shRNA constructs into mouse brains at E12.5 or E13.75 and subsequently administering doxycycline at E15 or E15.75, respectively (Supplementary Fig. 3C, D), we selectively knocked down Foxj3 once the targeted cells had exited the cell cycle. Samples were harvested at P7 for analysis (Supplementary Fig. 4A, C). As anticipated, GFP+ cells in control brains differentiated into deeper-layer or Layer 4 neurons expressing TBR1 (47.0±3.0%, n=3) and RORB (86.0±1.4%, n=3). Induced Foxj3 knockdown in postmitotic cells did not alter their cell fate, as these cells maintained their identities as deeper-layer (48.5±1.6%, n=3) and Layer 4 (85.4±2.2%, n=3) neurons similar to controls (Supplementary Fig. 4). These findings indicate that FOXJ3 regulates cell fate acquisition during the progenitor stage.

Foxj3 knockdown increases neural stem cell maintenance and delays cell cycle exit
Previous studies have highlighted the critical role of FOXJ3 in regulating cell cycle progression38,39,47. Nonetheless, its specific role in controlling the cell cycle of cortical progenitors during cortical development remains unclear. To explore this, we electroporated shCtrl or shFoxj3 constructs into embryonic brains at E12.5 and analyzed the proportion of SOX2+ cortical progenitors among GFP+ cells two days post-electroporation (Fig. 5A). In control brains electroporated at E12.5, approximately 25% of GFP+ cells remained undifferentiated SOX2+ progenitors at E14.5 (18.4±0.6%, n=4). However, in the Foxj3 knockdown brains, we observed a significant increase in the proportion of SOX2+ cells (30.1±4.6%, n=3; p<0.05, Student’s t-test; Fig. 5A). A similar increase in the SOX2+ progenitor population was observed in brains electroporated with shFoxj3 at E13.75 and collected at E15.75 (shCtrl: 24.6±1.1%, n=3; shFoxj3: 34.2±2.5%, n=4; p<0.05, Student’s t-test; Fig. 5B). In addition, we access whether this effect of delayed neuronal differentiation extended to IPCs by performing EOMES/TBR2 immunostaining at E15.5 in embryonic brains electroporated at E12.5 (Supplementary Fig. 5). Foxj3 knockdown significantly increased the proportion of EOMES/TRB2+ IPCs. These results together indicate that FOXJ3 delays neuronal differentiation not only in RGCs but also in IPCs.

Given that an increased progenitor population could result from cell cycle re-entry, we next assessed this possibility by administering EdU intraperitoneally one day post-electroporation and collecting brains one day later for EdU and Ki67 staining (Fig. 5C, D). As the cortical progenitor cell cycle lasts approximately 14 hours48, the presence of EdU+ cells that are negative for Ki67 one day after EdU administration suggests that these cells have exited the cell cycle. In control brains electroporated at E12.5 and E13.75, we found that most GFP+ cells were EdU- (GFP+EdU-/GFP+: E12.5, 60.75±7.2 %, n=3; E13.75, 58.0±2.6%, n=3), and most GFP+EdU+ cells were ki67- (GFP+EdU+Ki67-/GFP+EdU+: E12.5, 62.6±5.1%, n=3; E13.75, 57.0±1.8%, n=3), consistent with previous reports indicating cell cycle exit48,49. In contrast, shFoxj3-electroporated brains exhibited a significant decrease in the proportion of GFP+EdU+Ki67- cells (GFP+EdU+Ki67-/GFP+EdU+: E12.5, 36.6±1.2%, n=3; E13.75, 41.4±2.5%, n=3; p<0.01, Student’s t-test), indicating that Foxj3 knockdown impairs cell cycle exit. In contrast, Foxj3 knockdown at E14.5 did not lead to significant changes in neuronal differentiation or cell cycle re-entry (Supplementary Fig. 6), suggesting a specific role for FOXJ3 in progenitors during earlier corticogenesis. Furthermore, FOXJ3 overexpression in progenitors electroporated at E12.5 and E13.75 significantly reduced the percentage of SOX2+ cells (Supplementary Fig. 7), indicating its influence on neuronal differentiation. Collectively, our results suggest that FOXJ3 regulates progenitor cell fate by modulating cell cycle progression.

FOXJ3 is involved in transcriptional programs and cell cycle regulation
The FOX family of transcriptional factors is known to play critical roles in regulating cortical development through their transcriptional regulatory functions27,29,50. To further understand FOXJ3’s role in this process, we sought to identify potential downstream genes directly targeted by FOXJ3 that could regulate cortical development. Using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in FOXJ3-stable expressing Neuro2A cell lines (clones #1 and #2; Supplementary Fig. 8), we identified 39,576 and 41,853 peaks in FOXJ3 clones #1 and #2, respectively (Fig. 6A). To identify potential FOXJ3-targeted genes active in NPCs, we applied two criteria: First, we cross-referenced FOXJ3-occupied genomic loci with potential active regulatory elements marked by H3K27ac51. Second, we focused on FOXJ3 binding sites close to RNA polymerase II (Pol-II) targeted transcription start site (TSS, from −500 bp to +500 bp; Supplementary Fig. 8). We identified 14,341 FOXJ3-H3K27ac co-occupied peaks (Fig. 6B), of which 6,244 peaks were located near TSS regions (Supplementary Fig. 8C).

To explore the functions of these 6,244 FOXJ3-targeted genes, we conducted a gene ontology (GO) term analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID; https://david.ncifcrf.gov/). The top 10 GO terms in biological processes included Cell cycle; Protein transport; Transcription, DNA-templated; Cellular response to DNA damage stimulus; mRNA processing; DNA repair; Cell division; Regulation of transcription, DNA-templated; RNA splicing; and Mitotic nuclear division (Fig. 6C). These findings suggest that FOXJ3 predominantly regulates transcriptional programs and cell cycle, both of which are crucial for cortical development.

Potential FOXJ3 target genes are involved in cerebral cortex development
Our findings suggest that FOXJ3 plays a role in regulating the cell cycle during cortical development. To identify potential FOXJ3 target genes implicated in this process, we curated a candidate gene list encompassing genes annotated to both the Cell cycle (GO:0007049) and Pallium development (GO:00219543) Gene Ontology terms. This identified 29 genes associated with cerebral cortex development, including Tsc1 (tuberous sclerosis 1), Pten (phosphatase and tensin homolog), Wdr62 (WD repeat domain), Cep120 (centrosomal protein 120), Mcph1 (microcephaly, primary autosomal recessive 1), Pafah1b1 (platelet-activating factor acetylhydrolase, isoform 1b, subunit 1; also known as Lis1), Dicer1 (dicer1, ribonuclease type III), Bax (BCL2-associated X protein), and Nde1 (nudeneurodevelopment protein 1) among others (Supplementary Table 1).

If these genes are indeed regulated by FOXJ3 and involved in the FOXJ3-mediated pathway during cortical development, their expression profiles should theoretically be co-expressed in similar cell populations. To access this, we utilized our single-cell RNA sequencing (scRNA-seq) dataset on cells isolated from the dorsal pallium at E13.5 and E15.5 (Supplementary Fig. 9)52. Cells were categorized into progenitors (i.e., RGC), precursors (i.e., IP), and neurons (Supplementary Fig. 9A) based on the expression of neural cell markers, such as Pax6, Emx1, Eomes, Neurod6, Tbr1, and Satb2 (Supplementary Fig. 9B). Examination of FOXJ3-targeted gene expression profiles revealed conserved expression patterns in either progenitor or neuron cell types for several genes, including Pten, Tsc1, Pafah1b1, Cep120, Dicer1, Nde1, Wdr62, and Mcph1 (Fig. 6D, Supplementary Fig. 10). Taken together, these analyses refined the list of potential FOXJ3 target genes to the 5 most confident candidates: Pten, Tsc1, Pafah1b1, Cep120, and Dicer1. These findings suggest that FOXJ3 may play a significant role in cortical development through the regulation of its targeted genes involved in cell cycle control, providing insights into the FOXJ3-deficient phenotypes observed at the progenitor stage (Figs. 4, 5).

To validate that FOXJ3 binding sites from ChIP-seq align with active promoter regions, we visualized the peaks using Integrative Genomics Viewer (IGV) software (Fig. 6E). The sharpness of the peaks indicated specific DNA binding, as observed when transcription factors interact with their binding sites53. Active promoter and enhancer regions, marked by histone modification H3K27ac, typically exhibit sharp peaks54, while active promoter regions bound by RNA Pol-II appear as broader regions55,56. Applying these criteria, we identified peaks within the same loci as potential FOXJ3 target genes. Among the 29 candidate FOXJ3 targets selected using GO analysis, Pten, Tsc1, Pafah1b1, Cep120, Dicer1, Nde1, Wdr62, and Mcph1 met these criteria (Fig. 6E). To prevent potential mislabeling, we further analyzed whether FOXJ3 directly binds to genes commonly used as markers for cell identity, including Tbr1, Ctip2, Satb2, Rorb, Brn2, Sox2, and Mki67. Our analysis revealed no significant peaks in the promoter regions of these genes (Supplementary Fig. 11A), indicating that FOXJ3 does not directly regulate their expression.

To further confirm the association between FOXJ3 and the candidate downstream genes PTEN and TSC1 in developing human neocortical cells, we performed an in silico scRNA-seq analysis using a published dataset57. The analysis revealed that the expression pattern of FOXJ3 in human cortical cells mirrors that observed in mouse cortical cells, with enriched expression in progenitors and specific neuron cell types (Fig. 6H). Additionally, PTEN and TSC1 displayed conserved expression patterns consistent with those identified in mice (Fig. 6H). These findings suggest that FOXJ3 plays conserved roles in regulating cortical development in both mice and humans.

PTEN’s role in FOXJ3-mediated functions during cortical development
To confirm FOXJ3’s actual regulation of gene transcription within the identified target list, we employed RT-qPCR to assess the mRNA expression of Pten, Tsc1, Pafah1b1, Cep120, and Dicer1 following FOXJ3 overexpression in vivo. IUE of FOXJ3 fused with mCherry was conducted at E12.5, and the mCherry+ region in the dorsal pallium was isolated at E14.5 for RNA extraction. A significantly increased mRNA level of FOXJ3 was confirmed (Supplementary Fig. 11B). RT-qPCR analysis of the candidate FOXJ3 target genes revealed significant upregulation in Pten, Tsc1, and Pafah1b1 upon FOXJ3 overexpression, suggesting that FOXJ3 can activate the expression of Pten and Tsc1 (Fig. 6F). Notably, the FCDII-associated variant p.N351S abolished this transcriptional activation, indicating a loss of regulatory function (Fig. 6F). To further validate FXOJ3’s role in regulating Pten and Tsc1 expression, we performed RT-qPCR following CRISPR Cas9-mediated Foxj3 knockdown in mouse embryonic stem cells. Under these conditions, the mRNA level of Pten, but not Tsc1, showed a significant decrease under Foxj3 knockdown conditions (Fig. 6G).

Given that PTEN is a critical inhibitor of the mTOR pathway19–23, we next investigated whether FOXJ3 loss influences mTOR signaling. We assessed the phosphorylation of ribosomal protein S6 (pS6), a key downstream effector of the mTOR pathway (Supplementary Fig. 12). Neuronal cells transfected with shFoxj3 exhibited reduced PTEN expression accompanied by a robust increase in S6 phosphorylation. These findings demonstrate that FOJX3 regulates the mTOR pathway through transcriptional control of Pten, and that FOXJ3 dysfunction results in aberrant mTOR hyperactivation.

Next, we investigated the involvement of PTEN and TSC1 in FOXJ3-mediated mechanisms regulating cortical development. We performed a rescue experiment to determine whether the expression of Pten or Tsc1 could mitigate the phenotypes caused by FOXJ3 dysfunction. First, we evaluated the maintenance of progenitor identity and the percentages of cell cycle exit two days after IUE at E12.5 (Fig. 7A, B). Co-electroporation with shFoxj3 and a Pten-expressing construct resulted in a reduced proportion of cells remaining as SOX2+ cortical progenitors (shFoxj3: 30.9±4.6%; shFoxj3+Pten: 12.3±0.9%; Fig. 7A, C) and an increased proportion of cells exiting the cell cycle at E14.5 (shFoxj3: 36.6±1.2 %; shFoxj3+Pten: 68.3±2.3 %; Fig. 7B, D), compared to non-rescued Foxj3 knockdown brains.

Moreover, the neuronal migration defect observed in Foxj3-knockdown brains was dramatically reversed by Pten overexpression, with significantly more GFP+ cells reaching the CP by E15.5 (CP: 24.7±1.7%; IZ: 48.5±3.0%; VZ: 27.4±1.9% in shFoxj3 versus CP: 37.3±0.5%; p<0.01, one-way ANOVA, IZ: 42.2±1.5%, VZ: 20.6±1.0% in shFoxj3 + Pten; n=3 in each group; Fig. 7E, F). Additionally, Pten overexpression in the Foxj3 knockdown condition reversed the cell fate alterations observed at P7 (Fig. 7G, H). In Pten-rescued Foxj3 knockdown brains, the proportion of GFP+ cells expressing TBR1 was significantly increased compared to Foxj3-knockdown brains (shFoxj3: 16.4±2.6%, n=3; shFoxj3+Pten: 41.2±0.4%, n=3; p<0.0001, one-way ANOVA), while the proportion of GFP+ cells expressing SATB2 was significantly decreased (shFoxj3: 46.6±3.3%, n=3; shFoxj3 + Pten: 25.8±1.3%, n=3; p<0.001, one-way ANOVA; Fig. 7G, H).

Consistent with these findings, Pten knockdown alone recapitulated the neuronal migration delay observed in Foxj3-deficient brains (Supplementary Fig. 13A, B), further supporting that FOXJ3 and PTEN act within the same pathway during cortical development. In contrast, overexpression of Tsc1 in the Foxj3-knockdown condition did not result in significant changes in cell fate (Supplementary Fig. 13C, D). Taken together, these results demonstrate that Pten, rather than Tsc1, is a critical downstream gene of FOXJ3, playing a pivotal role in regulating cortical progenitors during cortical development.

p.N351S variant increased neuronal soma size in adolescent mice
Because FCDII pathology is characterized by dysmorphic neurons and balloon cells, we next investigated the effect of the FCDII-associated FOXJ3 variant on neuronal morphology. We electroporated embryonic brains at E14.5 with either wild-type (WT) FOXJ3 or the p.N351S variant and allowed the mice to develop until P28, corresponding to adolescence (Fig. 8). Neuronal morphology was then examined, and somal dimensions were quantified. Neurons expressing the p.N351S mutant exhibited significantly enlarged somata, as evidenced by increased somal area, diameter, and perimeter, compared with WT FOXJ3-expressing neurons (Fig. 8B, C), while both WT and mutant FOXJ3 localized properly to the nucleus (Fig. 8D). These findings suggest that the p.N351S variant disrupts FOXJ3 function, potentially through dysregulation of the mTOR pathway, thereby driving abnormal neuronal hypertrophy reminiscent of the dysmorphic neurons and balloon cells observed in FCDII.

Methods
Ethical approval
The human study was performed under the approval of the Institutional Review Board (IRB) of Taipei Veterans General Hospital (IRB 2018-01-003C, 2020-02-007A, 2023-02-014B). All animal experiments and maintenance were carried out under the guidelines and protocols approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang Ming Chiao Tung University.

Human genomic DNA extraction and purification
Genomic DNA was extracted from the peripheral blood with the approval of the patients and their family members. 10 c.c. of blood was drawn from each subject into 10 c.c. sodium citrate tubes containing Monovettes (Sarstedt, Germany). Whole blood samples were gently mixed under room temperature on the same day of blood draw. Then, the blood samples were submitted for DNA extraction and genotyping. Genomic DNA will be isolated by using the PUREGENE DNA purification systems (Gentra Systems, Minnesota, USA).

Whole genome sequencing and Sanger sequencing
The library preparation of WGS was performed using Nextera DNA Flex Library Prep Kit (Illumina) according to the manufacturer’s protocol. The quality and size distribution of each exome-enriched DNA library were analyzed by Bioanalyzer (Agilent). The amount of each library was quantified using both Qubit Fluorometer (Invitrogen) and quantitative PCR. The enriched DNA library was subjected to massively parallel sequencing with 500-bp paired-end reads. The WGS was performed by using the Illumina NovaSeq 6000 sequencers at a read depth of 30x at the Institute of Molecular and Genomic Medicine, National Health Research Institutes. Sanger sequencing was repeated to validate the identified variants.

DNA extraction from fresh frozen brain tissues
Thin sections (10–30 μm) were cut from fresh frozen brain tissue using a microtome, ensuring a total tissue weight of no more than 25 mg. DNA was extracted with the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s instructions with minor modifications. Briefly, tissue samples were homogenized with a pestle in a 1.5ml microcentrifuge tube containing 180 μl Buffer ATL, followed by the addition of 20 μl Proteinase K (Thermo Scientific, Waltham, MA). Samples were vortexed and incubated at 56°C until complete lysis. Subsequently, 200 μl Buffer ATL was added, and samples were incubated at 70°C for 10 min. After addition of 200 μl ethanol (96-100%), the mixture was applied to a QIAamp Mini Spin Column and centrifuged at 6000 x g for 1 min. Columns were washed subsequently with 500 μl Buffer AW1 (6000 x g, 1 min) and 500 μl Buffer AW2 (20,000 x g, 3 min). DNA was eluted with 200 μl Buffer AE after 1 min incubation at room temperature for 1 min, followed by centrifugation at 6000 x g for 1 min.

Identifying somatic mutations by WES
WES was performed on DNA extracted from both fresh frozen brain tissue (lesion) and peripheral blood leukocytes (matched normal control). Sequencing data were analyzed using the Sentieon® Genomics – software suite (Sentieon Inc.), following a Lesion-Normal analysis pipeline. Raw FASTQ files were aligned to the human reference genome (GRCh38) using DRAGEN (v4.2.4) with the corresponding reference bundle (hg38 v3, Illumina). Duplicate reads were marked, and base quality scores were recalibrated as part of the DRAGEN pipeline. Somatic single nucleotide variants and structural variants were called using DRAGEN’s tumor-normal analysis mode, restricted to the target regions defined by the SureSelect Clinical Research Exome V2 (GRCh38) BED file. Annotation was performed with VEP (v112.0), incorporating ClinVar, gnomAD, dbSNP, and the Taiwan Biobank. Variants were filtered and prioritized based on functional impact and allele frequency. All genes were considered in the analysis, with focused interrogation of FOXJ3 and genes within the PTEN-mTOR pathway (Supplementary Table 2).

Protein extraction and western blot
The samples were treated with RIPA lysis buffer containing 10% protease and 10% phosphatase inhibitor, followed by sonication to lyse the cells and brain tissues. The resulting protein lysate was then centrifuged at 4°C, 15,000 x g for 15 min, and the supernatant was collected. Protein quantification was performed using the BCA assay kit (Thermo Fisher Scientific, USA), after which equal amounts of protein were separated by electrophoresis on 8% SDS-PAGE and transferred to a PVDF membrane (Immobilon, Millipore). The membrane was incubated in the blocking buffer (0.1% T-TBS containing 5% skim milk and 3% Bovine Serum Albumin, BSA) for 1 h. To detect specific proteins, the membrane was then incubated with primary antibodies overnight at 4°C. The antibodies used, along with their concentrations, were as follows: FOXJ3 (Affinity Biosciences, AF0602, 1:1000, https://www.affbiotech.com/goods-1139-AF0602-FOXJ3_Antibody.html), PTEN (Cell Signaling Technology, 9559S, 1:3000, https://www.cellsignal.com/products/primary-antibodies/pten-138g6-rabbit-mab/9559), TSC1 (Cell Signaling Technology, 4906S, 1:3000, https://www.cellsignal.com/products/primary-antibodies/hamartin-tsc1-antibody/4906), S6 (Cell Signaling Technology, 2217S, 1:3000, https://www.cellsignal.com/products/primary-antibodies/s6-ribosomal-protein-5g10-rabbit-mab/2217), pS6(235/236) (Cell Signaling Technology, 4858S, 1:3000, https://www.cellsignal.com/products/primary-antibodies/phospho-s6-ribosomal-protein-ser235-236-d57-2-2e-xp-rabbit-mab/4858), GAPDH (Proteintech, 60004-1-Ig, 1:10,000, https://www.ptglab.com/products/GAPDH-Antibody-60004-1-Ig.htm) and Beta-actin (Proteintech, 66009-1- Ig, 1:10,000, https://www.ptglab.com/products/Pan-Actin-Antibody-66009-1-Ig.htm). After washing the membrane three times, it was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse (GeneTex, 1:10,000) and anti-rabbit (Sigma Aldrich, 1:10,000). Protein signals on the membrane were visualized using ECL-Plus reagent (Millipore) and the Amersham Imager 680 (GE). The intensities of protein bands were analyzed using ImageJ software (version 2.17.0, NIH).

Animal model and in utero electroporation (IUE)
IUE was performed following established protocols in previous reports52,77–83. Timed pregnant ICR mice (BioLASCO, Taiwan) at E12.5, E13.75, and E14.5 were anesthetized with isoflurane. The skin and muscular tissue were incised to expose the uterine horns from the abdomen. Plasmid DNA was injected into one side of the lateral ventricles of the embryos using a glass micropipette. Forceps-type electrodes (5 mm in diameter, Harvard Apparatus) were used for electroporation. Electric pulses delivered at different embryonic stages were as follows: 4 × 30 V pulses, 50 ms duration, 950 ms interval at E12.5; 5 × 35 V pulses, 50 ms duration, 950 ms interval at E13.75; 5 × 40 V pulses, 50 ms duration, 450 ms interval at E14.5. Following electroporation, embryos were carefully returned to the abdominal cavity, and the incision was closed using a surgical suture. Brains of electroporated mice were harvested at E14.5, E15.5, E15.75, E16.75, E17.5, E18.5 or P7. In each animal, multiple embryos were electroporated to minimize individual variations. Thus, even with n=3 animals, the dataset provides sufficient statistical power, while minimizing animal use is consistent with ethical guidelines. The welfare of the pregnant mice was carefully monitored in accordance with the guidelines from the Institutional Animal Care and Use Committee (IACUC) of National Yang Ming Chiao Tung University (No. 1080626).

Mouse brain sectioning
To obtain brain samples, mice were first perfused with phosphate-buffered saline (PBS, PH 7.4) and then fixed with 4% paraformaldehyde (PFA). The brains were collected immediately and immersed in 1% or 2% PFA at 4°C overnight. For vibratome sectioning, the brains were embedded in 4% low-melting agarose (AMRESCO) dissolved in PBS. The brains were then coronally sectioned into 100 μm-thick slices using a vibrating microtome (Leica, VT1000S) and stored in PBS with 0.05% sodium azide (Sigma) at 4°C. For cryostat sectioning, the brains were placed in 30% sucrose at 4°C overnight before being embedded in Tissue-Tek® O.C.T. Compound (Sakura Finetek). The brains were then sectioned coronally into 12 μm-thick slices using a cryostat machine (Thermo Fisher Scientific) and stored at -80°C.

Immunofluorescence staining
For immunostaining of cryosections, the slices underwent three 10 min washes each in 0.05% PBST. Brain sections were then blocked by incubating them in 0.1% PBST containing 10% normal goat serum (NGS) for 1 h. The sections were then immersed with primary antibodies in the blocking solution at 4°C overnight. After three washes with 0.05% PBST, the sections were incubated with secondary antibodies in the blocking solution at room temperature for 2 h. Nuclear staining with 4’,6-diamidino-2-phenylindole (DAPI, Molecular Probes) was conducted at room temperature for 15 min. All steps were carried out in the dark. The coverslips were mounted with antifade mounting medium (VECTASHIELD® Vibrance™) and stored at 4°C until imaging. For immunostaining of vibratome sections, the slices were permeabilized in PBS containing 0.2% Triton X-100 (PBST) for 30 minutes. Then, the brain sections were soaked in 0.2% PBST with 10% NGS and 5% BSA, followed by incubation with primary antibodies in 0.2% PBST containing 5% NGS and 5% BSA at 4°C for 2–3 days. After six PBS washes, the sections were incubated with secondary antibodies in 0.2% PBST containing 2.5% NGS and 2.5% BSA at room temperature for 2 hours. DAPI was used for nuclear staining, and the sections were incubated overnight at 4°C. All steps were performed in the dark, and the coverslips were mounted with antifade mounting medium (VECTASHIELD® Vibrance™) and stored at 4°C until imaging.

The primary antibodies used for staining were: FOXJ3 (1:500; Affinity Biosciences, AF0602, https://www.affbiotech.com/goods-1139-AF0602-FOXJ3_Antibody.html), PTEN (1:500; CST, 9559S, https://www.cellsignal.com/products/primary-antibodies/pten-138g6-rabbit-mab/9559), TSC1 (1:500; CST, 4906S, https://www.cellsignal.com/products/primary-antibodies/hamartin-tsc1-antibody/4906), TBR1 (1:500; Abcam, ab31940, https://www.abcam.com/tbr1-antibody-ab31940.html), TBR2 (1:500; Abcam, ab23345, https://www.abcam.com/tbr2--eomes-antibody-ab23345.html), CTIP2 (1:500; abcam, ab18465, https://www.abcam.com/ctip2-antibody-25b6-ab18465.html), SATB2 (1:500; Santa Cruz, sc-81376, https://www.scbt.com/p/satb2-antibody-satba4b10?srsltid=AfmBOoplp-XvgR2LrqcLV7-bOVwlS5fcz1YvopqXU5SArKzYQdh3kwRG), RORB (1:500; R&D, PP-N7927, https://www.rndsystems.com/products/human-ror-beta-antibody-clone-n7927-n7927_pp-n7927-00), BRN2 (1:500; Santa Cruz, sc-393324, https://www.scbt.com/p/brn-2-antibody-b-2?srsltid=AfmBOoqF0knd1AuP6tdlihlYYGTy9nZs42L90W0Uig6ejDTOejhy7rG3), Ki-67 (1:500; Abcam, ab15580, https://www.abcam.com/ki67-antibody-ab15580.html), SOX2 (1:500; Millipore, AB5603, https://www.merckmillipore.com/TW/zh/product/Anti-Sox2-Antibody,MM_NF-AB5603?ReferrerURL=https%3A%2F%2Fwww.google.com%2F), GFP (1:1000; Abcam, ab13970, https://www.abcam.com/gfp-antibody-ab13970.html). The secondary antibodies used were as follows: goat anti-mouse, anti-rabbit, anti-chicken or anti-rat Alexa Fluor-546, Alexa Fluor-488, and Alexa Fluor-647 conjugated antibodies (1:500; Invitrogen).

Cell culture and transfection
The Neuro-2a (N2A, purchased from Taiwan Bioresource Collection and Research Center, BCRC) cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and low glucose (Life Technologies, USA) and maintained at 37°C in a humidified atmosphere with 5% CO2. For transfection of constructs, N2A cells were seeded in 3.5 cm Petri dishes at 70% density. Transfection was performed using the T-pro Non-liposomal Transfection Reagent II (T-Pro Biotechnology, Taiwan) following the manufacturer’s instruction, and cells were then incubated for 24 to 48 h to conduct further experiments.

To assess the knockdown efficiency of shRNA, primary cultured cortical neurons were isolated from the cerebral cortex of ICR mouse embryos at E16. These cultured neurons were infected by lentivirus packaged with shRNA constructs designed and produced by the RNAi core of Academia Sinica at DIV2, and the virus was removed after 24 h by replacing the medium. Proteins from primary cultures were extracted at DIV6 based on a previous report84.

Constructs
Knockdown constructs of Foxj3 were based on the pLKO-TRC011 vectors. A sequence targeting Foxj3 CDS (shFoxj3; CCACAGTAACCTTCCAGAA) was used for Foxj3 knockdown and a non-targeting sequence scramble (shCtrl; CCTAAGGTTAAGTCGCCCTCG) as a control. All knockdown constructs were purchased from the Academia Sinica RNAi core (Taiwan). For conditional Foxj3 knockdown experiments, we utilized an inducible knockdown system setup in a previous study85. Control (shCtrl) and Foxj3 (shFoxj3) shRNA sequence was subcloned into pInducer10-mir-RUP-PheS (addgene #44011) vector. The US2-GFP construct was a generous gift from Dr. Jenn-Yah Yu (National Yang Ming Chiao Tung University, Taiwan). For overexpression of FOXJ3, we subcloned the human FOXJ3 gene (NCBI ref seq: NM_014947.5) from a CMV-promoter driven plasmid (Sino Biological Inc.) into the pCAGGS vector (Addgene #41583). The pCAGGS vector is driven by the CMV enhancer chicken β-actin promoter, with mCherry or Flag tagged on the N terminus of FOXJ3. Site-directed mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, USA) to generate the human FOXJ3 p.N351S variant. The forward primer (5’-CAACCAAAGCAGCCTGTCCAGCAGTCATGGCAGTGGCCTCA-3’) and reverse primer (5’-GAGGCCACTGCCATGACTGCTGGACAGGCTGCTTTGGTTG-3’) were designed to introduce the nucleotide substitution.

To disrupt Foxj3 gene expression in mouse embryonic stem cells, we induced double-strand breaks in the Foxj3 gene locus using the CRISPR/Cas9 system through the following sgRNAs (http://chopchop.cbu.uib.no)86: two pairs of sgRNAs for Foxj3 knockout: gRNA1: GCTAGACAGATAAATCACATAGG, gRNA3: CTCATCTGTTAAATATTATGCGG targeting sequence before Foxj3 start codon and gRNA2: GAGTCTCACCTTGAAACTCACGG, gRNA4: AGTGCAGTTTGGTGGAGTAGGGG targeting sequence after Foxj3 start codon. All sgRNAs were cloned into the backbone from the Multiplex CRISPR/Cas9 Assembly System (addgene, #1000000055) following the manufacturer’s instructions87. To generate Pten overexpression constructs, we subcloned the mouse Pten gene (NCBI ref seq: NM_008960.2) from the TRE promoter plasmid (add gene #135663) into the pCAGGS vector. For the overexpression of Tsc1 (NCBI ref seq: NM_001289575.1), we extracted genomic DNA from mouse tissues and used RT-PCR to amplify the Tsc1 coding sequence (CDS). The full-length Tsc1 CDS was then purified by gel extraction and cloned into the pCAGG vector. The sequences were verified by the protein BLAST algorithm from the National Center for Biotechnology Information (NCBI).

Cell-cycle exit analysis
24 h after the IUE experiment, EdU (5-ethynyl-2’-deoxyuridine, Thermo Fisher Scientific) was intraperitoneally (IP) injected into the maternal mice, with a dosage of 50 mg per kilogram of mouse body weight. Embryonic brains were then harvested and fixed. After immunostaining for Ki-67, the brain slices were stained with Click-iT™ EdU Alexa Fluor™ 647 imaging Kit (C10339, Thermo Scientific) for 30 minutes. The cell-cycle exit index (%) was calculated as the ratio of Ki67-/EdU+/GFP+ signals to the number of electroporated GFP+/EdU+ cells.

Image acquisition and quantification
Fixed brain slices were imaged using Zeiss LSM880 or LSM700 inverted confocal microscopes equipped with a 20× Plan-Apochromat NA 0.8 air objective for tissue imaging and a 100× NA 1.4 oil-immersion objective for cultured cells or high-resolution imaging. DAPI, GFP/Alexa Fluor 488, Alexa Fluor 546, and Alexa Fluor 633 were excited at 405, 488, 555, and 633 nm lasers, respectively. z-stacks were acquired at intervals determined by the microscope’s optimal acquisition settings, and maximum-intensity projections were generated for figure preparation. Images were processed using ZEN (version 3.7, Zeiss) and ImageJ (version 2.17.0, NIH) for contrast adjustment, montage assembly, and pseudocolor rendering. Quantification was performed on at least three slices per animal, with manual cell counting using the ImageJ Cell Counter plugin.

Chromatin immunoprecipitation (ChIP)
108 cells were fixed with 0.95% formaldehyde at room temperature for 9 min and quenched with glycine at a final concentration of 0.125 M at room temperature for 10 min. The cells were then rinsed three times and then centrifuged in ice-cold PBS with 0.05% Triton X-100 and protease inhibitor at 350 x g at 4°C for 5 min. Cell pellets were resuspended in 3 mL FA lysis buffer (50 mM HEPES-KOH [pH 7.5] / 140 mM NaCl / 1 mM EDTA [pH 8.0] / 1% (v/v) Triton X-100 / 0.1% (w/v) Sodium Deoxycholate / 0.1% (w/v) SDS / 0.5% Sarcosyl / Protease inhibitor). Lysates were sonicated for 12 min (40% amplitude, 20 sec-ON/30 sec-OFF) for chromatin shearing. Then, 10 μg of rabbit anti-HA (Abcam: ab9110, https://www.abcam.com/ha-tag-antibody-chip-grade-ab9110.html) and rabbit anti-RNA Polymerase II, and 1 μg rabbit anti-H3K27ac (Abcam: ab4729, https://www.abcam.com/en-us/products/primary-antibodies/histone-h3-acetyl-k27-antibody-chip-grade-ab4729) antibodies were conjugated to protein G beads (PureProteome™ Protein A/G Mix Magnetic Beads, Millipore), which had been washed with antibody blocking reagent (0.5% BSA / 0.05% Triton X-100 / PBS). The mixtures were incubated with rotation at 4°C for 2 h. 2 mL of chromatin solutions after centrifugation were diluted with 8 mL dilution buffer (50 mM HEPES [pH 7.5] / 1 mM EDTA / 150 mM NaCl / 1% (v/v) Triton X-100 / Protease inhibitor). 1% diluted chromatin was taken as input. The diluted chromatins were incubated with antibody-protein G matrix at 4°C overnight. The beads were washed 6 times with high salt buffer (20 mM Tris [pH 8.0] / 2 mM EDTA / 0.1% SDS / 1% Triton X-100 / 500 mM NaCl) and 1 time with TE buffer. The beads were transferred to a new tube after every wash. To elute DNA, beads were incubated with elution buffer (90 μL TE buffer [pH 8.0] / 10 μL 10% SDS) at 65°C for 15 min. Then, solutions were incubated while shaking at 650 rpm at 65°C overnight after adding 4 μL 5 M NaCl to reverse the covalent histone-DNA bonds. RNase A and Protease K (Sigma) were treated for 2 h at 45°C and 1 h at 55°C, respectively. After adding 4 μL 1 M Tris pH 6.5, DNA purification was proceeded following the protocol of the purification kit (Qiagen PCR Purification kit, Cat. no. 28106). The DNA concentrations were measured by using a Qubit fluorometer.

ChIP-sequencing and bioinformatics analysis
High-throughput sequencing was performed on the Illumina NextSeq 550 platform following the manufacturer’s protocols by the Sequencing Core Facility of National Yang Ming Chiao Tung University Genome Center. Library size construction was targeted for 250-350 bp. The experiments achieved an average depth of 40 million reads, with a minimum of 32 million. Raw reads were exported in FASTQ formats and subsequently subjected to bioinformatics analysis of ChIP-seq data. FASTQ files were converted into BAM files, and alignment to mouse reference genome mm9 was carried out by Bowtie 2. Peak calling was executed with MACS version 1.4.2. (P ≤ 1e−5). Enriched peaks were annotated to identify their nearest genes using Hypergeometric Optimization of Motif EnRichment (HOMER) software, with peaks assigned to a gene if they spanned from -500 to +500 bp from its transcription start site. The quality score of ChIP-seq is defined as -10 log10 (P). For gene enrichment analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to conduct Gene Ontology (GO) analysis.

Single‑cell RNA sequencing (scRNA-seq) and analysis
Mouse scRNA‑seq data from E13.5 and E15.5 mouse embryos were acquired from a previous study52 and human scRNA-seq data from embryonic week 8 to week 28 were from a previous study57. The dataset was analyzed using Seurat v4.0 R toolkit88 with R Studio software. The number of principal components was calculated using the ElbowPlot function. In mouse data, three developmental states, i.e., progenitors, transient stage, and post-mitotic neurons, were designated based on marker genes for dorsal pallium cells (Emx1), progenitors (Pax6), transient stage (Eomes), and post-mitotic cells (Neurod6), as well as cortical genes (Tbr1 and Satb2). In human data, cell identities were defined according to the gene expression profiles based on previous reports57,89.

Cell body size quantification
To quantify cortical neuronal soma size, segmentation was performed using Cellpose (version 2.0)90, a deep learning-based segmentation tool. The cyto model was applied to delineate soma boundaries, with the estimated cell diameter set at 23.1 μm based on visual inspection. Both the graphical user interface (GUI) and command-line interface (CLI) modes were used depending on batch size and computational requirements. Segmentation masks were manually corrected when necessary to improve accuracy. Following segmentation, regions of interest (ROIs) were exported from Cellpose and imported into ImageJ (version 2.17.0, NIH) for quantitative analysis. Morphological parameters, including soma area, diameter, and perimeter, were measured using the ImageJ Measure function. Only neurons with complete boundaries fully contained within the field of view were included in the analysis.

Statistical analysis
Statistical analyses were conducted using Excel (Office 365, version 3511, Microsoft) and Prism (version 10.6.1, Graphpad). Student’s t-test and one-way ANOVA were used to compare differences between groups. The significant difference levels were denoted as follows: * for P<0.05, ** for P<0.01, *** for P<0.001, and **** for P<0.0001. Error bars in the bar graphs represent means ± S.E.M.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
All data and materials are contained within the paper and the files or available on reasonable request. The mouse scRNA-seq data52 is acquired from https://doi.org/10.57770/IWASQL. The spatial gene transcriptome data52 from mouse E15.5 is acquired from https://doi.org/10.57770/P3TK72. The human scRNA-seq data57 is acquired from https://tanglab.shinyapps.io/Human_Fetal_Brain_Cell/. Further information and requests for resources and reagents should be directed to and will be fulfilled by Jin-Wu Tsai (tsaijw@nycu.edu.tw). Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The present study originates from the discovery of a FOXJ3 mutation segregating in family members affected by focal cortical dysplasia (FCD) and autosomal dominant focal epilepsy. FCD is characterized by abnormal neuronal organization and cortical architecture, which can be visualized through high-resolution magnetic resonance imaging (MRI). These alterations contribute to epileptogenesis.\nBy screening 170 patients with malformations of cortical development (MCD) and 76 individuals with epilepsy without MCD, the authors identified two additional FOXJ3 mutations in individuals with focal epilepsy without MCD. Based on these findings, they investigated the role of FOXJ3 in cortical development. They found that FOXJ3 is expressed in cortical progenitors and neurons during development. Through FOXJ3 knockdown during early cortical development (E12.5 and E13.75), they demonstrated that FOXJ3 impacts neuronal migration, leading to disorganized cortical lamination.\nUsing ChIP-seq and scRNA-seq analysis, they showed that FOXJ3 regulates genes involved in cortical development, with PTEN emerging as the key downstream target. Since the PI3K-PTEN-mTOR pathway plays a critical role in cerebral cortex development, the authors showed that FOXJ3 dysfunction appears to be rescued by PTEN overexpression.\nThe manuscript is well written, providing strong experimental evidence for the role of FOXJ3 in neuronal migration, cortical lamination, and neuronal specification. Their findings align with previous studies showing the role of FOXJ3 in neuronal differentiation of embryonic stem cells and its involvement in cell proliferation and migration in various cancers. However, the precise function of FOXJ3 and the underlying mechanisms remain unclear. Thus, this study offers important insights into the role of the FOXJ3 gene.\nConcerns and Suggestions:\n1. Starting Hypothesis:\noThe study identifies only one family with a missense FOXJ3 variant associated with FCD, whereas the other two variants were found in individuals with focal epilepsy without cortical malformations.\noSince the investigation mainly focuses on cortical malformations, the authors should justify why patients with FOXJ3 mutations did not present such malformations.\n2. Variant Reporting:\nThe three identified FOXJ3 variants should be reported using the reference sequence for isoform 1 of the gene (Uniprot code Q9UPW0-1).\n3. Structural and Functional Analysis of Variants:\nThe three variants occur in disordered regions of the protein with unclear function. The authors should:\nAssess the conservation of these residues across species.\nUse state-of-the-art pathogenicity prediction tools to evaluate the potential impact of these mutations.\nInvestigate the potential structural or functional consequences, for example, by searching for functional linear motifs involved in protein interactions, post-translational modifications, or protein localization.\n4. Functional Impact of Missense Variants:\nThe experiments rely on gene knockdown, but the exact effect of the missense variants identified in patients was not addressed.\nThe authors should demonstrate whether these patient-specific mutations affect FOXJ3 function in a similar manner to gene silencing.", "reviewer_2": "In the manuscript “Epilepsy-associated FOXJ3 variants link a novel transcriptional program of PTEN-mTOR pathway to neuronal specification and cortical lamination” Cheng and colleagues identify a new gene potentially linked to epilepsy and focal cortical dysplasia (FCD), and show that this gene, FOXJ3, plays a role in cortical development by performing knockdown experiments in the mouse. The authors performed a series of experiments that nicely lead to propose a mechanism for which FOXJ3 knockdown in early cortical progenitors prevents them to exit the cell cycle and produce lower/middle-layer neurons, favoring a change of fate towards upper-layer neurons. Furthermore, the authors PTEN as one of the genes regulated by FOXJ3, and propose a scenario where FOXJ3 mutation would cause FCD by dysregulating the AKT-mTOR pathway.\nThis is an interesting study that spans patient genetics and functional work performed in the mouse. The study provides mechanistic insights into the consequence of FOXJ3 loss for cortical development. However, some points, especially in relation to the relevance for FCD pathology, need to be addressed before it can be accepted for publication.\n1. The authors mention that FCD type IIb was confirmed in the patient included in this study. The authors should mention in the text how diagnosis was performed. Since FCD type IIb is defined by the presence of cortical dyslamination, as well as cellular hallmarks such as dysmorphic neurons and balloon cells, the authors should provide, if available, histological images supporting diagnosis. Also, did histopathology reveal mTOR hyperactivity in the patient brain tissue such as by pS6 staining?\n2. The authors mention that three individuals were sequenced whole-genome to identify the causative variant. They should mention in main text what type of DNA was sent for sequencing (blood?) and the depth of the sequencing. Also, they should mention the Sanger sequencing reported in Fig.1 since it is unclear from the main text how the variant was identified in the mother (she did not undergo WGS).\n3. Since FCD type II is recognized as a mosaic brain disorder and an mTORopathy, the authors should discuss the possibility that the variable penetrance of the FCD and epilepsy phenotypes may involve somatic mosaic variants. Do the authors think that second hit variants in FOXJ3 may be present in the brain of the patient? Could this be assessed by deep targeted sequencing of DNA extracted from the surgically resected tissue? This is particularly relevant in view of the fact that PTEN is an mTOR pathway inhibitor, and it has been shown that double-hit somatic variants in other mTOR inhibitors lead to FCD type II.\n4. The authors propose FOXJ3 as a new gene potentially implicated in FCD type II, and show that FOXJ3 interacts with PTEN, an mTOR pathway regulator. However, there is no FCD and mTOR hallmark assessment performed in the mouse model. The authors should check for presence of mTOR hyperactivity for example by pS6 staining. Furthermore, they could perform immunostaining to assess whether their model recapitulates the generation of dysmorphic neurons or balloon cells in the electroporated area.\n5. It would be interesting to know whether the mice develop seizures postnatally following FOXJ3 knockdown?\n6. The authors nicely show that FOXJ3 knockdown in progenitors producing lower/middle-layer neurons causes a depletion of these lower/middle-layer neurons in favor of upper-layer neurons. They also show a delay in neuronal migration that may or may not contribute to this fate change. They then claim that knocking-down FOXJ3 in postmitotic cells does not cause a fate change and suggest that prevention of cell cycle exit may be at the origin of the fate change. It would be interesting for the authors to perform EOMES staining to assess the effect of FOXJ3 knockdown on intermediate progenitors and whether there is any change in direct versus indirect neurogenesis.\n7. The schematic in Figure 8 is not super interesting as it is. The authors should rather show the two scenarios with and without functional FOXJ3.\n8. There are few writing errors: line 244 (in both controls and Foxj3 knockdown brains); line 276 (samples were collected at P7); line 508 (AKT instead of ATK).", "reviewer_3": "Nature Communications manuscript NCOMMS-25-06773-T\nMajor findings\nThe authors have conducted an extensive series of experiments implicating mutations in the transcription factor FOXJ3 as a casual mutation in focal cortical dysplasia type II (FCDII). The investigators identified a point mutation in a family with FCDII and epilepsy, and different mutation in two additional unrelated patients with epilepsy. To establish the functional role of FOXJ3 in the development of cortical malformations, a series of in utero electroporation (IUE) experiments were conducted in mice to knockdown (KD) FoxJ3. KD impaired migration, disrupted cortical structure and shifted neuronal differentiation from the production of deep layer to upper layer neurons. Elegant developmental time course experiments were conducted to establish when FoxJ3 expression was critical. Use of a dox-inducible shFoxJ3 construct supports the conclusion that downregulation during the progenitor cell stage regulates cell fate. CHiP-seq and scRNA-seq experiments implicated the mTOR negative regulator Pten as a key target of FoxJ3. FoxJ3 overexpression and KD experiments showed that the transcription factor acts to increase Pten levels. To demonstrate that Pten mediates FoxJ3 effects on cortical development, FoxJ3 KD was combined with Pten overexpression, which rescued cortical development.\nFindings implicate FoxJ3 as a novel pathogenic gene in the development of FCDII. The proposed mechanism, that reduced FoxJ3 leads to reduced Pten, which in turn increases mTOR signaling, aligns perfectly with known mechanisms of FCDII. Findings also provide new insights into the role of FoxJ3 in cortical development.\nOverall, experiments are viewed as being rigorous and provide good support for the key conclusions. A noted limitation is that more work could be done to link FoxJ3 KD to mTOR hyperactivation, which would strengthen the overall model. Additional points and edits that will strengthen the paper are outlined below. Findings make a significant advance to the field and will be of broad interest to scientists interested in cortical development and focal epilepsy.\nKey Points\n• Pten is a major negative regulator of mTOR signaling. Assessment of mTOR signaling would strengthen the evidence for Pten involvement. One would predict increased mTOR signaling (i.e. phosphorylated S6 and others) in the shFoxJ3 treated tissues, and reduced signaling with Pten overexpression. Hyperactivating mTOR pathway mutations are proposed to be the key mechanism of FCDII, so these studies are important to better link FOXJ3 to FCDII.\n• Although not viewed as a required experiment, downregulation of Pten should replicate the shFoxJ3 phenotype if the effect is mediated by Pten.\n• Inclusion of data to assess the impact of the N351S mutation on protein function would strengthen the study. Is this a loss of function mutation, as the study assumes with the KD model?\n• Have the authors considered a second-hit mechanism to account for the focality of brain lesions in FOXJ3 patients? It is interesting that some patients with heterozygous FOXJ3 mutations present with focal brain malformations, while other carriers do not have epilepsy or a focal lesion at all. The pattern is suggestive of a second hit mechanism, as has been proposed for tuber and SEGA formation in tuberous sclerosis complex (TSC). In TSC, patients have germline heterozygous mutations in TSC1/TSC2, while some tubers and most SEGAs show somatic second hits. By a similar mechanism, FOXJ3 heterozygosity combined with a second hit in a neural progenitor would explain the appearance of focal malformations. Notably, the shFoxJ3 produces 70% KD, so the model is closer to a two-hit/KO rather than a 50% reduction that might occur in patients. This would explain why the mice consistently show a phenotype while some family carriers are unaffected.\n• Fig.5: The panels are too small/low resolution to discern whether GFP+ cells are co-labeled or not. Higher resolution micrographs are needed.\n• Fig.5: The arrangement and description of figure 5 is confusing. This figure should be extensively reworked and checked for accuracy. Specifically, the figure lettering and arrangement of panels is confusing. A,C,E,G describe the first experiment and are discussed in lines 334-346, and B,D,F,H describe a second experiment and are discussed in lines 347-366. Line 359 appears to refer to the wrong panels, and the description of the findings in the text uses different group names than on the y-axis of panels D and H. As presented, the result is hard to interpret.\n• Animals studies, for the most part, use n’s of 3. The data appear convincing, in that data is generally well clustered for a group, but use of small groups sizes reduces experimental rigor.\nMinor points\n• Line 113: revised terminology for mTOR is “mechanistic” target of rapamycin.\n• Line 117; the implication that FCD is “often” caused by Pten mutations misrepresents their frequency. Mutations in mTOR itself are most common, while Pten mutations tend to be relatively rare. A better characterization would be “can be”.\n• Family member #56 is described as “affected”, but the phenotype is not described. The pedigree also indicates another family member had epilepsy, but no details are provided. Was this also FCDII?\n• Fig.1A; a line is missing between the 3rd and 4th gen in the pedigree\n• Figure 2: The text describes FOXJ3 levels as “peaking” at E14.5, but levels are similar at E12.5 and E13.5, so this is not really a peak and should be rephrased.\n• Fig.4E. The y-axis label in the figure does not match the text (E12.5 – E17.5)\n• Line 323: text refers to panel S2E, but no E panel is labeled in the figure. Figure S2 labels appear to be incorrect.\n• Figure 8 does not appear to be referenced in the text\n• Figure 8 could be further developed to better convey the details of the model. Colors are used to denote different layers, but are not referenced in the figure text to help explain the model. The impact of FOXJ3 dysfunction is described in the figure legend, but is not shown in the model. Pten, shown in the figure, is not described in the figure legend and this part of the figure is not well annotated (e.g. is pten increased, decreased? Is mTOR signaling increased?). In its current form the figure is not particularly helpful, and should either be eliminated or substantially reworked.\n• Suppl Figure 5: Microscope images in panel A for FOXJ3 data do not appear to be representative of the data in B. Large numbers of GFP+,SOX2+ cells appear in the image.\n• Suppl Figure 7A: key is misaligned in the figure; Neurod6 is misspelled.\n• Suppl Figure 10: Addition of an experimental timeline, as in other figures, would help with interpretation\n• Mouse source is not reported"}, "author_response": "Response to Reviewers\nReviewer #1 (Remarks to the Author):\n1. Starting Hypothesis:\noThe study identifies only one family with a missense FOXJ3 variant associated with FCD, whereas the other two variants were found in individuals with focal epilepsy without cortical malformations.\noSince the investigation mainly focuses on cortical malformations, the authors should justify why patients with FOXJ3 mutations did not present such malformations.\nResponse: We agree with the reviewer’s comment. Subtle FCD lesions are frequently overlooked on routine MRI, particularly when using standard 1.5T scanners without dedicated high-resolution protocols. Indeed, multiple studies have shown that FCD type II remains the most common etiology of drug-resistant epilepsy (DRE), even in cases with “MRI-negative” findings. Two patients in our study underwent 1.5T MRI, which may have lacked sufficient sensitivity to detect subtle FCD lesions. We have revised the discussion and added references to clarify that negative MRI findings do not exclude the possibility of FCD:\n “Magnetic resonance Imaging (MRI) remains the primary noninvasive diagnostic tool for FCD; however, subtle lesions may be overlooked, and FCD type II is still the most common etiology of drug-resistant epilepsy (DRE), even in MRI-negative cases(Najm, Lal et al. 2022, Urbach, Kellner et al. 2022, Balestrini, Barba et al. 2023, Chiarello, Cognolato et al. 2024) ).” (p5, lines 112-122)\n “Brain MRI at 1.5T in both patients were reported as essentially normal; however, it is important to note that subtle FCD lesions can be overlooked on standard MRI.” (p9, lines 202-204)\n “Some carriers of heterozygous FOXJ3 variants exhibited non-lesional brain MRI or did not present with clinical seizures. These observations suggest that FOXJ3-associated FCD may be underdiagnosed due to incomplete penetrance of the autosomal-dominant variants and the presence of “invisible” lesions on conventional MRI. Notably, FCD represents the most common etiology among individuals with focal epilepsy and negative MRI findings (Chiarello, Cognolato et al. 2024 ). Advances in imaging, including optimized sequences, post-processing approaches, and ultra-high-field 7T MRI, have improved detection of subtle FCD lesions, though many remain undetectable. A deeper understanding of the FOXJ3-mediated transcriptional regulation on cortical development may help explain epileptogenesis in cases where epileptogenic lesions are not visible on MRI.” (p26, line 616-626)\n2. Variant Reporting:\nThe three identified FOXJ3 variants should be reported using the reference sequence for isoform 1 of the gene (Uniprot code Q9UPW0-1).\nResponse: We thank the reviewer for the point. We have corrected the reports of all three FOXJ3 variants according to the reference sequence for isoform 1 (Uniprot code Q9UPW0-1) (p9, lines 199-202).\n3. Structural and Functional Analysis of Variants:\nThe three variants occur in disordered regions of the protein with unclear function. The authors should:\nAssess the conservation of these residues across species.\nUse state-of-the-art pathogenicity prediction tools to evaluate the potential impact of these mutations.\nInvestigate the potential structural or functional consequences, for example, by searching for functional linear motifs involved in protein interactions, post-translational modifications, or protein localization.\nResponse: The clinical significance and functional predictions of the three FOXJ3 missense variants are provided in Supplementary Figure 1. Although in silico prediction tools yielded conflicting evidence regarding their pathogenicity, all three variants are extremely rare and affect highly conservated residues across multiple species. These clinical-genetic observations suggest that heterozygous pathogenic variants in FOXJ3 are associated with focal epilepsy, potentially related to FCD type II (p9, lines 199-204). Furthermore, we provided evidence for the p.N351S variant, showing that it leads to increased neuronal soma size in adolescent mice (Supplementary Figure 14), consistent with dysmorphic neuronal phenotypes observed in FCD (p22, lines 515-528).\n4. Functional Impact of Missense Variants:\nThe experiments rely on gene knockdown, but the exact effect of the missense variants identified in patients was not addressed.\nThe authors should demonstrate whether these patient-specific mutations affect FOXJ3 function in a similar manner to gene silencing.\nResponse: We thank the reviewer for the comment. To address this, we provided the clinical significance and functional predictions of the three FOXJ3 missense variants in Supplementary Figure 1. In addition, we performed RT-qPCR on FOXJ3 target genes and found that the patient-associate variant failed to induce the expected upregulation of downstream genes, such as Pten and Tsc1, confirming functional impairment of the variant (Figure 6F; p20, lines 470-471). We further demonstrated the pathological consequence of the p.N351S variant by showing that it leads to increased neuronal soma size in adolescent mice (Supplementary Figure 14; p22, lines 515-528). Together, these results indicate that the patient-specific missense mutations disrupt FOXJ3 activity in a manner consistent with the functional defects observed upon gene knockdown.\nReviewer #2 (Remarks to the Author):\n1. The authors mention that FCD type IIb was confirmed in the patient included in this study. The authors should mention in the text how diagnosis was performed. Since FCD type IIb is defined by the presence of cortical dyslamination, as well as cellular hallmarks such as dysmorphic neurons and balloon cells, the authors should provide, if available, histological images supporting diagnosis. Also, did histopathology reveal mTOR hyperactivity in the patient brain tissue such as by pS6 staining?\nResponse: We confirm that the diagnosis of FCD type II was established by histological examination. We have now included the corresponding histological images in Figure 1C. Since pS6 immunostaining is not routinely performed in clinical diagnostic practice, such data were not available from the patient tissue. However, we demonstrated that pS6 levels were significantly increased in Foxj3-knockdown neurons (Supplementary Figure 12; p20, lines 476-483), supporting hyperactivation of the mTOR pathway.\n2. The authors mention that three individuals were sequenced whole-genome to identify the causative variant. They should mention in main text what type of DNA was sent for sequencing (blood?) and the depth of the sequencing. Also, they should mention the Sanger sequencing reported in Fig.1 since it is unclear from the main text how the variant was identified in the mother (she did not undergo WGS).\nResponse: We appreciate this suggestion and have revised the text for clarity. The pedigree in Figure 1A has been corrected, and the table in Figure 1D has been revised to clearly indicate the individuals who underwent WGS and Sanger sequencing. Information about the sequencing was also updated (p29, lines 683-684, 697).\n3. Since FCD type II is recognized as a mosaic brain disorder and an mTORopathy, the authors should discuss the possibility that the variable penetrance of the FCD and epilepsy phenotypes may involve somatic mosaic variants. Do the authors think that second hit variants in FOXJ3 may be present in the brain of the patient? Could this be assessed by deep targeted sequencing of DNA extracted from the surgically resected tissue? This is particularly relevant in view of the fact that PTEN is an mTOR pathway inhibitor, and it has been shown that double-hit somatic variants in other mTOR inhibitors lead to FCD type II.\nResponse: We agree with the reviewer that this is an important consideration. To address this, we performed paired high-depth whole-exome sequencing (WES) on DNA extracted from both the resected FCD tissue and peripheral blood of the index patient. No significant somatic mutations were detected in FOXJ3 or genes in the PTEN-mTOR pathway. We repeated the analysis with re-examined data and confirmed the absence of relevant somatic variants. These findings have been included in the Discussion (p26, lines 626-631). Corresponding methodological details have been added under “DNA extraction from fresh frozen brain tissues” (p 29, lines 701-714) and “Identifying somatic mutations by whole exome sequencing” (p30, lines 716-730).\n4. The authors propose FOXJ3 as a new gene potentially implicated in FCD type II, and show that FOXJ3 interacts with PTEN, an mTOR pathway regulator. However, there is no FCD and mTOR hallmark assessment performed in the mouse model. The authors should check for presence of mTOR hyperactivity for example by pS6 staining. Furthermore, they could perform immunostaining to assess whether their model recapitulates the generation of dysmorphic neurons or balloon cells in the electroporated area.\nResponse: We thank the reviewer for the suggestion. We examined the neuronal morphology following expression of the FCD-associated FOXJ3 mutant and observed enlarged soma size, reminiscent of dysmorphic neurons, in electroporated regions (Supplementary Figure 14; p22, lines 515-528). Furthermore, we also showed that pS6 was indeed increased in Foxj3-knockdown neurons (Supplementary Figure 12; p20, lines 476-483). These findings further support that FOXJ3 dysfunction contributes to FCD pathogenesis through dysregulation of the PTEN-mTOR pathway.\n5. It would be interesting to know whether the mice develop seizures postnatally following FOXJ3 knockdown?\nResponse: We thank the reviewer for the comment. We did not observe seizures in the IUE-mediated FOXJ3 knockdown mouse model. The is likely due to the relatively small proportion of cortical neurons transfected during electroporation. We have added this limitation into the Discussion section (p25, lines 601-605).\n6. The authors nicely show that FOXJ3 knockdown in progenitors producing lower/middle-layer neurons causes a depletion of these lower/middle-layer neurons in favor of upper-layer neurons. They also show a delay in neuronal migration that may or may not contribute to this fate change. They then claim that knocking-down FOXJ3 in postmitotic cells does not cause a fate change and suggest that prevention of cell cycle exit may be at the origin of the fate change. It would be interesting for the authors to perform EOMES staining to assess the effect of FOXJ3 knockdown on intermediate progenitors and whether there is any change in direct versus indirect neurogenesis.\nResponse: We appreciate the reviewer’s helpful suggestion. We performed EOMES/TBR2 immunostaining in embryonic brains electroporated at E12.5 and found that Foxj3 knockdown increased the proportion of EOMES/TBR2-positive intermediate progenitors (Supplementary Figure 5). This suggests that FOXJ3 regulates cell cycle exit in both radial glial cells and intermediate cells, providing further mechanistic insight into the observed fate switch. These new data have been added to the Results section (p15, lines 357-362).\n7. The schematic in Figure 8 is not super interesting as it is. The authors should rather show the two scenarios with and without functional FOXJ3.\nResponse: We thank the reviewer for this helpful suggestion. Figure 8 has been revised to depict two scenarios: cortical development with normal FOXJ3 function versus FOXJ3 dysfunction, highlighting the impact on neuronal specification and migration.\n8. There are few writing errors: line 244 (in both controls and Foxj3 knockdown brains); line 276 (samples were collected at P7); line 508 (AKT instead of ATK).\nResponse: We thank the reviewer for these points. They have been corrected in the revised manuscript.\nReviewer #3 (Remarks to the Author):\nKey Points\n• Pten is a major negative regulator of mTOR signaling. Assessment of mTOR signaling would strengthen the evidence for Pten involvement. One would predict increased mTOR signaling (i.e. phosphorylated S6 and others) in the shFoxJ3 treated tissues, and reduced signaling with Pten overexpression. Hyperactivating mTOR pathway mutations are proposed to be the key mechanism of FCDII, so these studies are important to better link FOXJ3 to FCDII.\nResponse: We thank the reviewer for this excellent suggestion. To address this, we examined PTEN expression and S6 phosphorylation following knockdown of Foxj3 in cultured neurons. As expected, PTEN expression was reduced, and importantly, phosphorylation of S6 was significantly increased (Supplementary Figure 12), indicating hyperactivity of the mTOR pathway. This finding corroborates the pathological hallmarks in FCDII and have now been added to the Results section (p20, lines 476-483).\n• Although not viewed as a required experiment, downregulation of Pten should replicate the shFoxJ3 phenotype if the effect is mediated by Pten.\nResponse: We appreciate the reviewer for the suggestion. We performed Pten knockdown using IUE at E13.75 and observed cell distribution defects similar to those in FoxJ3 knockdown brains (Supplementary Figure 13A). These findings strengthen the conclusion that PTEN is a critical downstream effector of FOXJ3 in cortical development (p21, lines 506-509).\n• Inclusion of data to assess the impact of the N351S mutation on protein function would strengthen the study. Is this a loss of function mutation, as the study assumes with the KD model?\nResponse: We thank the reviewer for the helpful suggestion. We tested the ability of the N351S variant to induce expression of FOXJ3 downstream targets . The variant failed to increase FOXJ3 downstream gene expression, including Pten and Tsc1 (Figure 6F), supporting the interpretation that N351S is a loss-of-function mutation. These results have been incorporated into the Results section (p20, lines 470-471).\n• Have the authors considered a second-hit mechanism to account for the focality of brain lesions in FOXJ3 patients? It is interesting that some patients with heterozygous FOXJ3 mutations present with focal brain malformations, while other carriers do not have epilepsy or a focal lesion at all. The pattern is suggestive of a second hit mechanism, as has been proposed for tuber and SEGA formation in tuberous sclerosis complex (TSC). In TSC, patients have germline heterozygous mutations in TSC1/TSC2, while some tubers and most SEGAs show somatic second hits. By a similar mechanism, FOXJ3 heterozygosity combined with a second hit in a neural progenitor would explain the appearance of focal malformations. Notably, the shFoxJ3 produces 70% KD, so the model is closer to a two-hit/KO rather than a 50% reduction that might occur in patients. This would explain why the mice consistently show a phenotype while some family carriers are unaffected.\nResponse: We agree that this is an important consideration. As noted in our response to Reviewer #2 (Question 3), we performed high-depth paired WES of both resected FCD tissue and peripheral blood from the index patient. No somatic second-hit mutations were detected in FOXJ3 or PTEN-mTOR pathway genes. While we cannot entirely rule out low-level somatic mosaicism, our data argue against a two-hit mechanism in this family. This has been added to the Discussion (p26, lines 626-631).\n• Fig.5: The panels are too small/low resolution to discern whether GFP+ cells are co-labeled or not. Higher resolution micrographs are needed.\nResponse: We thank the reviewer for noting this. We have uploaded high-resolution images for Figure 5.\n• Fig.5: The arrangement and description of figure 5 is confusing. This figure should be extensively reworked and checked for accuracy. Specifically, the figure lettering and arrangement of panels is confusing. A,C,E,G describe the first experiment and are discussed in lines 334-346, and B,D,F,H describe a second experiment and are discussed in lines 347-366. Line 359 appears to refer to the wrong panels, and the description of the findings in the text uses different group names than on the y-axis of panels D and H. As presented, the result is hard to interpret.\nResponse: We thank the reviewer for the comment. We have extensively revised the figure layout, panel labeling, and corresponding text to ensure clarity and consistency.\n• Animals studies, for the most part, use n’s of 3. The data appear convincing, in that data is generally well clustered for a group, but use of small groups sizes reduces experimental rigor.\nResponse: We acknowledge the reviewer’s concern. In our studies, each animal included multiple electroporated embryos, providing several biological replicates and reducing variability. Thus, even with n = 3 animals, the dataset provides sufficient statistical power, while minimizing animal use is consistent with ethical guidelines. We have clarified this rationale in the revised manuscript (p32, lines 767-769).\nMinor points\nWe thank the reviewer for these helpful comments. All points have been addressed and corrected in the revised manuscript as detailed below:\n• Line 113: revised terminology for mTOR is “mechanistic” target of rapamycin.\nResponse: Corrected (p6, line 125)\n• Line 117; the implication that FCD is “often” caused by Pten mutations misrepresents their frequency. Mutations in mTOR itself are most common, while Pten mutations tend to be relatively rare. A better characterization would be “can be”.\nResponse: Corrected (p6, line 130)\n• Family member #56 is described as “affected”, but the phenotype is not described. The pedigree also indicates another family member had epilepsy, but no details are provided. Was this also FCDII?\nResponse: The pedigree has been revised (Figure 1A) and the legend now includes a description of the patient #56 (p49, lines 1285-1286).\n• Fig.1A; a line is missing between the 3rd and 4th gen in the pedigree\nResponse: The pedigree has been revised.\n• Figure 2: The text describes FOXJ3 levels as “peaking” at E14.5, but levels are similar at E12.5 and E13.5, so this is not really a peak and should be rephrased.\nResponse: We have rephrased the sentence (p9, lines 216-217)\n• Fig.4E. The y-axis label in the figure does not match the text (E12.5 – E17.5)\nResponse: Corrected (Figure 4C).\n• Line 323: text refers to panel S2E, but no E panel is labeled in the figure. Figure S2 labels appear to be incorrect.\nResponse: Corrected (p14, line 335).\n• Figure 8 does not appear to be referenced in the text\nResponse: Corrected (p22, line 534)\n• Figure 8 could be further developed to better convey the details of the model. Colors are used to denote different layers, but are not referenced in the figure text to help explain the model. The impact of FOXJ3 dysfunction is described in the figure legend, but is not shown in the model. Pten, shown in the figure, is not described in the figure legend and this part of the figure is not well annotated (e.g. is pten increased, decreased? Is mTOR signaling increased?). In its current form the figure is not particularly helpful, and should either be eliminated or substantially reworked.\nResponse: The figure and legend have been substantially revised (p54, lines 1415-1433).\n• Suppl Figure 5: Microscope images in panel A for FOXJ3 data do not appear to be representative of the data in B. Large numbers of GFP+,SOX2+ cells appear in the image.\nResponse: We have replaced the panel with a more representative image (Supplementary Figure 7A, lower panels)\n• Suppl Figure 7A: key is misaligned in the figure; Neurod6 is misspelled.\nResponse: Corrected (Supplementary Figure 9)\n• Suppl Figure 10: Addition of an experimental timeline, as in other figures, would help with interpretation\nResponse: We have added an experimental timeline in the figure (Supplementary Figure 13).\n• Mouse source is not reported\nResponse: The mouse source is now added (p32, lines 756-757)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I think my concerns have been addressed in the revisions of the paper.", "reviewer_2": "This is a very nice and rigorous work that contributes significantly to the field of nurodevelopmental disorders by providing new genetic mechanisms of FCD, including functional work. I appreciate the fact that the authors addressed all my comments and concerns by performing new experiments and integrating convincing data into the new version of the manuscript. I recommend the manuscript for publication.", "reviewer_3": "The authors have addressed my main concerns. Correction of a few additional minor items will improve the final manuscript\nMinor points\n• Lines 130-131: grammar errors\n• Figure 5D: The authors have indicated that they have uploaded the original images at higher-resolution to improve the figure quality. Perhaps this is so, but images still appear pixelated, and it is not possible to discern whether cells are co-labeled or not. Addressing this likely requires new images collected at higher resolution rather than increasing the pixel density of the originals.\n• Figure 5D: There is an “H” in the middle of the upper right panel\n• Figure 5: Legend refers to panel E instead of D\n• Supplemental figure 12: The text indicates (line 480 and figure legend) that FoxJ3 and Pten expression are reduced, however, the data and statistics are not provided. Addition of graphs to suppl fig 12 showing reduced FoxJ3 and Pten and statistical analyses are needed to support the claims."}, "author_response": "Response: We thank the reviewer for the constructive comments and suggestions, which have helped to further improve the quality and clarity of the manuscript. We have addressed all minor points as detailed below.\nMinor points\n• Lines 130-131: grammar errors\nResponse: We have corrected the grammatical errors.\n• Figure 5D: The authors have indicated that they have uploaded the original images at higher-resolution to improve the figure quality. Perhaps this is so, but images still appear pixelated, and it is not possible to discern whether cells are co-labeled or not. Addressing this likely requires new images collected at higher resolution rather than increasing the pixel density of the originals.\nResponse: We have replaced the panels in Figure 5D with higher-resolution images.\n• Figure 5D: There is an “H” in the middle of the upper right panel\nResponse: The extraneous letter has been removed.\n• Figure 5: Legend refers to panel E instead of D\nResponse: This error has been corrected in the figure legend.\n• Supplemental figure 12: The text indicates (line 480 and figure legend) that FoxJ3 and Pten expression are reduced, however, the data and statistics are not provided. Addition of graphs to suppl fig 12 showing reduced FoxJ3 and Pten and statistical analyses are needed to support the claims.\nResponse: We have now added quantitative graphs and statistical analyses to Supplementary Figure 12B."}]

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