Targeting notch signaling to restore neural development and behavior in mouse models of ASD

Domain: biology
Published: 2026-03-30
Editorial outcome: major_revision

Abstract
Autism spectrum disorder (ASD) is a neurodevelopmental disorder with diverse genetic and environmental origins, yet whether these factors converge on common molecular pathways remains unclear. This study identifies dysregulation of the Notch signaling pathway as a shared mechanism in both hereditary and nonhereditary ASD models. Aberrant histone deacetylase 3-mediated epigenetic regulation of Notch signaling during embryonic forebrain development disrupts the specification of vasoactive intestinal peptide (VIP+) GABAergic interneuron subtypes (VIP-INs), which originate in the caudal ganglionic eminence (CGE). CGE-specific ablation of Notch1/2 genes in ASD models restores the loss of VIP-INs, normalizes maladaptive excitatory and inhibitory balance, and selectively improves social behaviors. A single antenatal dose of a γ-secretase inhibitor ameliorates multiple ASD-associated neuronal, behavioral, and transcriptomic changes in adult models. The study indicates a strong convergence of ASD-related factors on Notch signaling dysregulation and establishes this pathway as a promising therapeutic target for developmental and behavioral deficits in ASD.

Introduction
Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by deficits in social communication and language, and repetitive behaviors. While approximately 60% of ASD cases are considered to have a genetic basis1,2, the remaining cases arise from a complex interplay between genetic susceptibility and environmental influences, such as prenatal exposure to anticonvulsants, infections, or maternal nutritional deficiencies, which can significantly increase the incidence of ASD in offspring3–6. Despite extensive genetic studies identifying de novo and inherited variants in approximately 15% of ASD cases7,8, the underlying molecular mechanisms remain poorly understood.

Epigenetic modifications, such as histone acetylation and DNA methylation, are emerging as key regulators linking genetic and environmental risk factors of ASD during neurodevelopment9–11. Mutations in chromatin regulators, such as chromodomain-helicase-DNA-binding protein 8 (CHD8) and methyl-CpG binding protein 2 (MeCP2), have been strongly linked to ASD12–14, while environmental risk factors of ASD, such as prenatal exposure to valproic acid (VPA), affect chromatin structure, leading to widespread neurodevelopmental defects15. These findings underscore the role of epigenetic dysregulation in ASD; however, the specific downstream genes that are affected and how they contribute to ASD pathology remain unclear. Elucidating these pathways is a key to improving our understanding of ASD mechanisms.

A core neuropathological feature of ASD is an imbalance between excitatory and inhibitory (E/I) neural signaling16, which disrupts cortical circuit function and social behaviors. Many ASD models exhibit E/I imbalance17–22. In humans, many individuals with ASD also have epilepsy, further confirming the link between ASD and hyperexcitation of neural circuits23,24. A direct causal relationship between E/I imbalance and social deficits has been demonstrated in multiple ASD models25,26. Studies have shown that restoring GABAergic function can alleviate some behavioral abnormalities in ASD models18,19,27. Given the diverse genetic and environmental factors implicated in ASD, we hypothesize that these ASD-related factors would share molecular pathways that influence neuronal development and E/I balance. Elucidating such a common mechanism could provide therapeutic insights into ASD pathophysiology.

In this study, we uncover a convergent mechanism that links ASD heterogeneity, epigenetic dysregulation, and E/I imbalance. Our findings reveal a key pathogenic process involving dysregulation of the Notch signaling pathway—a key regulator of cell fate decisions during brain development28–30. Enhanced Notch signaling, primarily driven by epigenetic dysregulation, disrupts the differentiation of caudal ganglionic eminence (CGE)-derived GABAergic neurons, including cortical vasoactive intestinal peptide (VIP+) interneurons (VIP-INs), which play a crucial role in maintaining E/I balance31,32. Given the recent evidence that VIP-IN dysfunction is implicated in an ASD-related disorder, Dravet syndrome33, our results suggest that notch-mediated VIP-IN deficits are a key driver of ASD pathophysiology. We demonstrate that genetic or pharmacological inhibition of Notch signaling rescues neural deficits and alleviates ASD-like behaviors, highlighting its potential as a therapeutic target across diverse ASD etiologies.

Results
Enhanced notch signaling pathway is a shared molecular signature across developing ASD models
To investigate the molecular pathogenesis underlying ASD, we conducted transcriptome analysis of the fetal brain from two non-genetic rodent models of ASD: in utero exposure to the environmental risk factors, VPA at E13 or polyinosinic:polycytidylic acid [poly(I:C)] at E12. Ribonucleic acid sequencing (RNA-seq) analysis identified significant gene expression changes, with 2734 altered genes (1520 upregulated, 1214 downregulated) in VPA-exposed brains and 1634 altered genes (906 upregulated, 728 downregulated) in poly(I:C)-exposed brains. Among these, 442 genes were commonly upregulated across both models, representing approximately 50% of the total upregulated genes in poly(I:C) mice (Fig. 1a).

Gene ontology (GO) analysis of the upregulated genes in both VPA and poly(I:C) models highlighted significant enrichment in terms of epigenetic regulation and neural development. Chromatin-related terms such as “Chromatin organization,” “Chromatin remodeling,” and “Chromatin binding” emerged as the frequent common GO terms with high signal (Fig. 1b and Supplementary Fig. 1a, b), supporting the hypothesis that epigenetic dysregulation plays a central role in ASD pathogenesis. The extensive gene upregulation by environmental ASD-related factors may primarily disrupt epigenetic regulation, affecting downstream pathways critical for neural development. Notably, the term “Notch signaling pathway” was enriched in both ASD models (Fig. 1b and Supplementary Fig. 1a, b). As a conserved mechanism for cell fate decisions during development28,29, Notch signaling appeared to be specifically activated in these ASD models. Indeed, upregulation of key components, including Notch1, Notch2, and the downstream transcription factor Hes5, was confirmed in both models (Fig. 1c and Supplementary Fig. 1c). Heatmap analysis exhibited a hyperactivation of multiple Notch pathway genes across both ASD models compared with controls (Fig. 1d). Notch signaling pathway had strong functional associations with chromatin regulation in VPA-exposed brains (Supplementary Fig. 2a).

To extend these findings to human ASD models, we examined the expression of the Notch pathway gene in transcriptomic data from neuronal cells derived from human embryonic stem cells (hESCs) carrying ASD-associated copy number variants (CNVs). Interestingly, expression of multiple Notch pathway genes was enhanced in the 16p11.2 deletion model34, a most frequent CNV in hereditary ASD, but not in 1q21.1 deletion/duplication models35 (Fig. 1e). Additionally, meta-analysis of ASD-associated human ESCs/iPSCs revealed that Notch pathway-associated genes were activated in MeCP2 R168X mutant36 and tuberous sclerosis complex knockout (TSCKO)37 cell lines (Supplementary Fig. 2b and Supplementary Data 1). These results suggest the possibility that Notch pathway dysregulation is a molecular hallmark in some cases of hereditary ASD. Our findings reveal that aberrant Notch signaling is a shared molecular signature in developing brains in both non-hereditary and certain hereditary ASD models, providing important insights into the common mechanisms underlying ASD pathogenesis.

Cortical VIP-IN deficits are a common pathology in non-hereditary ASD models
To investigate the postnatal consequences of prenatal Notch signaling dysregulation, we focused on its relationship with E/I imbalance—a hallmark phenotype in ASD. Despite its significance, the molecular basis of E/I imbalance remains poorly understood.

Using an established in vitro ASD model38, where temporal VPA exposure at the initial stage of cortical culture induces E/I imbalance, we performed transcriptomic analysis to identify VPA-susceptible genes (Supplementary Fig. 3a). These genes were significantly enriched in ASD-associated genes listed in the Simons Foundation Autism Research Initiative (SFARI) Gene database (Supplementary Fig. 3b). Exon array analysis revealed significant changes in 126 genes (64 upregulated, 62 downregulated) (Supplementary Data 2). The transcript with the highest fold-change among downregulated genes was Vip, expressed in a cortical GABAergic interneuron subtype. Additionally, the top 10 downregulated genes included transcripts related to GABAergic interneuron subtypes, such as Neuron-derived neurotrophic factor (Ndnf), Reelin (Reln), and Somatostatin (Sst) (Fig. 2a and Supplementary Fig. 3c). Similarly, cell-type proportion analysis indicated the largest reduction of GABAergic interneurons (Fig. 2b). These drastic and selective effects on VIP-INs were confirmed by quantitative analysis of transcripts and immunostaining for markers of cortical interneuron subtypes (Supplementary Fig. 4a, b). Together with previously reported morphological and physiological evidence38, these findings emphasize the significant impact of VPA exposure on E/I balance in vitro.

To validate these findings in vivo, we analyzed major interneuron subtypes in the mature cerebral cortex of VPA- and poly(I:C)-exposed mice. VIP-INs strikingly decreased in both ASD models by 38% and 49% relative to controls, respectively; meanwhile, other subtypes, such as Parvalbumin (PV)-, SST-, and RELN-INs, showed no significant changes, except for a modest reduction in PV-INs in poly(I:C)-exposed mice (Fig. 2c, d). VIP-INs decreased even after a single VPA exposure on E13 or E17 (Supplementary Fig. 5a). These results support the robust reduction in VIP-INs as a shared morphological pathology across these two nonhereditary ASD models.

Absolut reduction of cortical VIP-INs in ASD models is linked to impaired precursor-to-neuron specification in CGE
Cortical VIP-INs are major late-born GABAergic interneurons derived exclusively from the CGE, while PV-INs and SST-INs originate from the medial ganglionic eminence (MGE) during earlier stages39–41. Therefore, we inquired whether other minor CGE-derived interneuron subtypes, not only VIP-INs, were preferentially impaired in the ASD models. We found that subsets of VIP-INs co-expressing calretinin (CR) and NDNF+ neurons modestly decreased to 60% and 70% of control levels in the VPA-exposed mice, respectively (Supplementary Fig. 5b, c). Collectively, these findings indicate that prenatal insults broadly disrupt the development of CGE-derived interneurons, not only the VIP+ subtype but also the NDNF+ subtype.

To explore the mechanism underlying the selective reduction of CGE-derived cell population in the ASD models, we focused on the developmental origins of cortical interneurons, as prenatal exposure to these nonhereditary ASD-risk factors is predicted to target interneuron progenitors. Transcriptional markers Coup-TFII (CGE-derived) and Sox6/Lhx6 (MGE-derived) were used for lineage tracing (Fig. 2e). In VPA-exposed embryonic brains, the COUP-TFII-expressing area in the CGE significantly expanded, whereas SOX6 and LHX6 expression areas in the MGE remained unchanged (Fig. 2f, g, and Supplementary Fig. 6a). These results suggest an inversely increased number of CGE-derived progenitors despite the reduced number of VIP-INs in postnatal ASD brains (Fig. 2c, d). This finding suggests that ASD-risk factors may disrupt the process of specification from mitotic progenitors to postmitotic neuronal progenitors in the CGE. To confirm this supposition, we labeled proliferating cells in the CGE with 5-ethynyl-2’-deoxyuridine (EdU) (Fig. 2h). EdU incorporation was observed in CGE progenitors co-expressing Ki-67 (Supplementary Fig. 6b). Both VPA and poly(I:C) exposures significantly increased the number of EdU+ cells in the CGE (Fig. 2h, i), indicating enhanced proliferation of VIP-IN progenitors in both ASD models.

During development, the CGE-derived cells tangentially migrate to the neocortex and are mainly integrated into layers II/III (Fig. 2e). Next, we chased the fate of CGE-derived cells in the neonatal cortex 6 days after EdU injection. EdU-labeled postnatal CGE markers (PROX1+/EdU+ and COUP-TFII+/EdU+) significantly reduced by VPA exposure (Fig. 2j, k and Supplementary Fig. 6c, d), consistent with the selective reduction of VIP-INs in mature ASD brains. By contrast, SOX6+/EdU+ cells and total EdU+ cells were unaffected (Fig. 2j, k and Supplementary Fig. 6e). These findings suggest that prenatal exposure to ASD-risk factors selectively disrupts the transition from proliferative progenitors to postmitotic VIP-IN precursors in the CGE, leading to a subsequent reduction in cortical VIP-INs.

Aberrant notch activity in the ganglionic eminence region disrupts cortical VIP-IN specification in ASD models
Given the crucial role of Notch signaling in cell fate determination during development28–30, aberrant Notch activity is hypothesized to impair the transition from progenitors to neurons, leading to VIP-IN reduction. To determine whether aberrant Notch activation upon VPA and poly(I:C) exposure (Fig. 1) contributes to the altered VIP-IN progenitor pool and reduced VIP-INs observed in ASD models (Fig. 2), we manipulated Notch signaling in the in vitro ASD model using gain-of-function, loss-of-function, and rescue approaches.

We first confirmed that Notch expression was substantially higher in the ganglionic eminence (GE) than in the cortex (Fig. 3c and Supplementary Fig. 7a), indicating an abundance of Notch-positive neural progenitors in the GE during the critical developmental stages. These Notch-positive progenitors increased approximately twofold in the in vitro ASD model (Fig. 3b and Supplementary Fig. 7b). We also verified activation of Notch signaling at the protein level: the active (cleaved) Notch form was elevated in the in vitro ASD model, and this effect was attenuated by DAPT, a γ-secretase inhibitor that blocks Notch activation (Fig. 3c).

To examine the impact of Notch hyperactivation on neural progenitors, we introduced the intracellular domain of Notch (ICN), its active form, into forebrain neuronal cultures using a retroviral vector that specifically targets proliferative cells. ICN expression markedly reduced both the number of VIP-INs and the expression of Vip transcripts (Fig. 3d, e, h).

To evaluate the loss-of-function effect, we used CRISPR/Cas9-mediated removal of the Notch1 gene or pharmacological inhibition with DAPT. Both approaches significantly increased Vip transcripts, whereas Sst transcripts remained unaffected under all conditions (Fig. 3d, f). VIP-INs strongly reduced in the in vitro VPA model used for transcriptomic screening (Supplementary Fig. 4). Under this condition, both Notch1 gene removal and DAPT treatment significantly rescued the reduction in Vip transcripts and the number of VIP-INs in the VPA model (Fig. 3d, g, i). These findings reveal an inverse correlation between Notch activity and the differentiation of cortical VIP-INs, highlighting the pivotal role of precise regulation of the Notch signaling level during the late embryonic period. Therefore, the hyperactivation of Notch in the GE region at this developmental stage likely disrupts VIP-IN differentiation.

Aberrant histone deacetylase 3-mediated epigenetic regulation enhances notch signaling and impairs VIP-IN specification
We thus demonstrated that hyperactive Notch signaling disrupts the differentiation of CGE-derived VIP-INs (Fig. 3a–e), directly contributing to the observed reduction of VIP-INs in ASD models. However, the upstream mechanisms driving this aberrant Notch activation remain unclear.

Given the widespread epigenetic dysregulation observed in the ASD model (Fig. 1a) and the established role of class I histone deacetylase (HDAC) inhibitors such as VPA in altering gene expression during brain development42–46, we examined the role of class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) in regulating Notch signaling and VIP-IN specification. First, we screened the effects of selective inhibitors for these HDACs on cortical VIP-IN specification (Fig. 4a). Prenatal exposure to MS275 (a class I HDAC inhibitor), and RGFP966 (HDAC3 inhibitor), similar to VPA, significantly increased the number of COUPTFII+/EdU+ proliferating cells in the CGE (Fig. 4b–d) and decreased PROX1+ cells in the neonatal cortex (Supplementary Fig. 8a). Both inhibitors also reduced the number of cortical VIP-INs and Vip transcripts in both animals and forebrain cultures (Fig. 4e, f and Supplementary Fig. 8b, c), indicating disrupted VIP-IN specification. However, no such effects were observed with CAY10683 (HDAC2 inhibitor), highlighting HDAC3 as a key regulator (Fig. 4b–f). Further, RGFP966 exposure increased the active Notch proteins, suppressed by DAPT treatment (Supplementary Fig. 8d), and gene disruption of Notch1 restored RGFP966-induced reduction in Vip transcripts (Supplementary Fig. 8e), similar to VPA exposure (Fig. 3b, f). These findings suggest that HDAC3 plays a critical role in VIP-IN development. Given that HDAC3 is enriched in CGE and associates with the PROX1 (Supplementary Fig. 9a–c), we propose a model in which HDAC3 inhibition leads to aberrant Notch activation, thereby impairing the differentiation of CGE progenitors into VIP-INs (Supplementary Fig. 9d).

Specific HDAC3 inhibition results in repetitive behaviors and social deficits linked to ASD
Based on the morphological abnormalities caused by HDAC3-specific inhibition (Fig. 4a–f), we next investigated whether prenatal inhibition of HDAC3 replicates the behavioral phenotypes characteristic of ASD, as seen in VPA- and poly(I:C)-exposed ASD models. Mice prenatally exposed to RGFP966 exhibited normal development and body weight comparable to control mice (data not shown). Nevertheless, these mice displayed significant increases in self-grooming behavior—a hallmark of repetitive behaviors often observed in ASD (Fig. 4g). Additionally, RGFP966-exposed mice showed social abnormalities, including reduced ultrasonic vocalization (USV) calls (Fig. 4h) and impaired preference for social novelty, but not sociability, in the three-chamber test (Fig. 4i–n), although general sociability remained partially intact (Supplementary Fig. 10). These findings indicate that HDAC3 inhibition alone is sufficient to recapitulate core behavioral phenotypes associated with ASD. Taken together, these data highlight the crucial role of HDAC3-mediated epigenetic regulation in normal social and behavioral development and its disruption as a potential mechanism underlying ASD pathology.

CGE region-targeted disruption of notch signaling rescues VIP-IN loss and social abnormalities in ASD models
To directly test whether the local hyperactivation of Notch signaling in developing brain underlies the morphological and behavioral abnormalities observed in ASD models, we generated CGE-specific Notch1/2 conditional knockout (cKO) mice (Fig. 5a). The deletion of Notch genes targets CGE progenitors around E18, based on the expression pattern of Htr3a47. Both Notch1/2 cKO and control (WT) mice were prenatally exposed to VPA to evaluate the roles of Notch signaling on neural and behavioral ASD-like phenotypes (Fig. 5a).

In the absence of VPA exposure, there is no difference in both the number of cortical VIP-INs and PV-INs, and ASD-like behaviors between WT mice and Notch1/2 cKO mice (Fig. 5b–g and Supplementary Fig. 11a, b). However, while VPA-exposed WT mice exhibited a significant loss of cortical VIP-INs and pronounced social abnormalities in the same way as shown in Fig. 4g–n and Supplementary Fig. 10, these impairments were largely prevented in the Notch1/2 cKO mice (Fig. 5b, d–g, and Supplementary Fig. 11c, d). This conditional deletion of Notch1/2 did not affect repetitive self-grooming behavior observed in VPA-exposed mice (Fig. 5c). These results demonstrate that CGE-specific Notch1/2 deletion reversed VIP-IN loss and social deficits in VPA-exposed mice, highlighting Notch dysregulation as a key driver of VIP-IN circuit abnormalities and consequent social impairments.

Developmental loss of CGE-derived interneuron circuits accompanies cortical E/I imbalance
Next, we investigated the molecular and circuit-level consequences of this prenatal genetic intervention by bulk RNA-seq analysis on the adult forebrain. The transcriptomic differences between WT and Notch1/2 cKO mice were modest, indicated by the small number of differentially expressed genes (DEGs) (Supplementary Data 3) and the close clustering profiles in principal component analysis (PCA) plots (WT vs. cKO, Fig. 5h, i). This is reasonable given that the Htr3a-Cre driver induces conditional Notch loss only in a small subset of CGE-derived interneurons. However, under VPA exposure, transcriptomic profiles diverged significantly between genotypes. VPA-exposed WT mice exhibited over 2101 DEGs (873 upregulated, 1228 downregulated) (WT vs. WT-VPA, Fig. 5h), reflecting widespread dysregulation of gene expression. Notably, gene expression in 49% of these DEGs was reversed in Notch1/2 cKO mice under VPA exposure (WT-VPA vs. cKO-VPA, Fig. 5h), and the aberrant transcriptomic profile observed in WT-VPA mice was partially normalized in the cKO-VPA condition (Fig. 5i).

Enriched GO terms of upregulated genes by VPA exposure (WT vs. WT-VPA) were “Synapse”-related categories with “Glutamatergic synapse” showing the highest enrichment (Fig. 5j and Supplementary Fig. 12). Importantly, the same GO terms were enriched for downregulated genes of Notch1/2 cKO mice under VPA exposure (WT-VPA vs. cKO-VPA, cKO vs. cKO-VPA or WT vs. cKO-VPA) (Supplementary Figs. 12, 13). Consistently, genes associated with glutamatergic synapse transmission including Grin2a, Grin2b, Scn2a, Nrxn1, and CamK2d were upregulated by VPA exposure, while these were normalized in Notch1/2 cKO-VPA mice (Fig. 5k–m and Supplementary Data 4). Transcriptomic changes in Grin2b and Grin2a, NMDA receptor subunits, were further supported by quantitative analysis (Supplementary Fig. 12c).

In the embryonic brain exposed to VPA or poly(I:C), RNA-seq analysis revealed the transcript level of Neurogenin2 (Neurog2)—a transcriptional activator promoting the differentiation of glutamatergic neuron—to be upregulated. However, the upregulation was restored in Notch1/2 cKO-VPA mice (Supplementary Fig. 12d), suggesting that Neurog2 elevation represents a secondary event of impaired inhibitory networks. Together, these results suggest that the developmental loss of CGE-derived interneurons may trigger maladaptive excitatory gene expression programs.

By contrast, the genes localized on medium spiny neurons (MSNs) in the striatum, such as Ppp1r1b, Pde10a/1b, Drd1/2, Penk, and Tac1, account for 11 out of the top 20 downregulated genes following VPA exposure, indicating a pronounced reduction of striatal MSNs, GABAergic neurons derived from the lateral ganglionic eminence (LGE) (Supplementary Fig. 13c, d). However, unlike glutamatergic regulators (Fig. 5m), these downregulated expressions were less normalized in the CGE-specific KO-VPA mice (Supplementary Fig. 13e).

These data suggest that the developmental loss of VIP-INs broadly impacts not only inhibitory circuits but also surrounding excitatory circuits, leading to maladaptive E/I balance and subsequent social deficits in ASD. Thus, Notch1/2 deletion demonstrates potential to restore both morphological and functional disruptions caused by exposure to ASD-related environmental factors.

A single dose of potent notch inhibitor prevents cortical and behavioral deficits in the ASD model
To explore the therapeutic potential of inhibiting Notch signaling against ASD, we examined whether a pharmacological approach could ameliorate the morphological and behavioral abnormalities in the ASD model. We used Ro4929097, a γ-secretase inhibitor that potently inhibits Notch activity and has previously been used for clinical purposes. This compound was originally developed as a candidate drug for Alzheimer’s disease and then advanced to Phase II clinical trials in 2011 for anticancer applications in melanoma, T-cell acute lymphoblastic leukemia, and other indications48–50. A single dose of Ro4929097 was prenatally administrated to VPA-exposed mice (Fig. 6a). The potent inhibition effect of Notch activity by Ro4929097 was confirmed in forebrain neuronal cultures (Fig. 6b). Ro4929097 potently restored the reduction of cortical VIP-INs caused by VPA exposure, almost to normal levels (Fig. 6c, d). This result aligns with an earlier finding that hyperactive Notch signaling underlies the VIP-IN loss (Figs. 3, 5). Similarly, behavioral abnormalities in VPA-exposed ASD mice were restored by Ro4929097 (Fig. 6e–i). We noted hair loss—which appeared to be due to long-term self-grooming behavior (>13 weeks-old)—in 27% of VPA-exposed mice, but this was nearly absent in Ro4929097-treated mice (Fig. 6e). Ro4929097 also prevented repetitive self-grooming behavior as well as deficits in social behaviors (Fig. 6g–i), compared with the outcome achieved by CGE-specific Notch deletion, which did not prevent repetitive behavior (Fig. 5).

Restoration of neural lineages in neonatal ASD models by pharmacological notch inhibition via single-cell transcriptomics
To elucidate the cellular mechanisms underlying robust rescue of ASD-like behaviors by the Notch inhibitor Ro4929097, we performed single-cell RNA seq (scRNA-seq) on neonatal whole brain and revealed 20 distinct cell clusters in the uniform manifold approximation and projection (UMAP) representation (Fig. 6j and Supplementary Fig. 14), with significant shifts in cluster composition in the ASD model compared with controls (Fig. 6k). Remarkably, the single dose of Ro4929097 almost restored this composition to control levels. To statistically validate these observations, we performed chi-square tests followed by residual analyses to compare cell-type proportions across the three groups. The resulting heatmap showed residual patterns consistent with the stacked barplot in Fig. 6k (Fig. 6l). Pairwise chi-square tests revealed significant nonindependence between groups. Additionally, Pearson correlation coefficients indicated that administration of Ro4929097 shifted the ASD model profile toward that of controls (Fig. 6l).

Focusing on Notch1 and Notch2-positive populations, their expression was observed in neural progenitors (NSC/NPs) marked by Nestin (Supplementary Fig. 15). Their enrichment in proliferating clusters was also confirmed by the labeling of S-phase or G2/M-phase (Fig. 6m). We further re-clustered the NSC/NP population into four clusters and identified two clusters (clusters 0 and 3) that express the GABA precursor markers Dlx1 and Dlx5 (Fig. 6m and Supplementary Fig. 16). In ASD models, there was an expansion of a re-clustered progenitor subtype (cluster 3) enriched for CGE-selective markers (Dlx1/5, Htr3a, Prox1), consistent with the increased CGE populations seen in embryonic ASD brains (Fig. 2). Treatment with Ro4929097 reversed this expansion, restoring the cellular composition of CGE to control levels (Fig. 6m).

We extended this analysis to other Notch1/Notch2-positive cell types, revealing abnormalities in non-neuronal populations, including glial fibrillary acidic protein (GFAP)-positive astrocytes and Olig2-positive oligodendrocytes. Notably, while Ro4929097 significantly corrected abnormalities in oligodendrocyte-related populations (cluster numbers 5, 8, 12, 14, and 18) (Fig. 6n), it had limited effects on astrocytic populations (Fig. 6o). These findings underscore that the transient blockade of Notch activity is sufficient to restore ASD-like behavioral phenotypes and the disruption of developmental trajectories, including multiple neural and glial lineages in the ASD model.

Methods
Generation of the mouse ASD models
C57BL6/J mice (Japan SLC, Inc., Shizuoka, Japan) and ICR (CD-1) mice (Charles River Laboratories, Inc., Wilmington, MA) were used in this study. Pregnancy was confirmed by the presence of a vaginal plug, designated as embryonic day 0 (E0). The dosages of drugs were adjusted according to the body weight of the dam on the day of injection.

The drugs were administered by intraperitoneal injections on the determined gestational days as follows: VPA (Sigma–Aldrich, St. Louis, MO) or histone deacetylase (HDAC) inhibitors were administered at E13, E14, E15, and/or E18, and poly(I:C) (Sigma–Aldrich) was administered at E12. ICR (CD-1) mice were used, except for the experiment using Htr3a-Cre; Notch1/2 conditional knockout mice. VPA, poly(I:C), and CAY10683 (Cayman Chemicals, Ann Arbor, MI) were suspended in 0.9% saline. RGFP966 and MS275 (Cayman Chemicals) were dissolved in dimethyl sulfoxide and further diluted in saline. The administered doses were as follows: VPA, 290–330 mg/kg; poly(I:C), 20 mg/kg; Ro4929097 (Cayman Chemicals), 1 mg/kg. For HDAC inhibitors, MS275 was 5–9.25 mg/kg for cortical analysis and 10–12.5 mg/kg for embryonic CGE/MGE analysis. RGFP966 was 6.5–10 mg/kg for cortical analysis and 7.5–10 mg/kg for embryonic analysis. CAY10683 was 35–40 µg/kg for cortical analysis and 85 µg/kg for embryonic analysis. Control dams received saline injections. After delivery, the dams were housed individually and allowed to raise their own litters. Both female and male offspring were used for the experiments up to 15 weeks of age.

All mice were housed at an animal facility of the Tokai University with constant temperature and air humidity, and a 12-h light/12-h dark cycle. All animal procedures complied with institutional and governmental regulations on animal experimentation, reviewed and approved by the Institutional Animal Care and Use Committee and Genetic Modification Safety Committee of Tokai University (nos. 244018 and 23-010-28). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize animal suffering. Sex was not considered in the study design and analysis, since there were no significant sex-related differences in the behavioral, histological and biochemical analysis of this study.

Generation of Htr3a-Cre; Notch1/2 conditional knockout mice
Htr3a-Cre mice (MMRRC, ID: 37089, C57BL6/J background)64 were crossed with Notch1 flox/flox (C57BL6/J background)65 and Notch2 flox/flox (C57BL6/J background)66 mice to generate Htr3a-Cre; Notch1 flox/flox; and Notch2 flox/flox conditional knockout (cKO) mice. Genotyping was performed using polymerase chain reaction (PCR) for the Cre transgene and floxed alleles as previously described65,66. All genetically modified mice were maintained on a C57BL/6J background and were bred under specific pathogen-free (SPF) conditions.

Neuronal cell culture and in vitro ASD model
Primary neuronal cultures were prepared from the forebrains of embryonic day (E) 14.5-15.5 mouse embryos of ICR strain or Cas9 knockin mice (Jackson, Stock#026179)67. The dissociation was performed using 0.05% trypsin in the presence of DNase I (Roche Applied Science) for 10 min at 37°C. Trypsin digestion was inactivated using soybean trypsin inhibitor (Gibco; Thermo Fisher Scientific). Dissociated cells were plated onto six-well or 24-well culture dishes at a density of (1–2×10⁵ cells/cm²) and maintained in Neurobasal medium (Gibco) supplemented with 2% B27 (Gibco), 2 mM Glutamax (Gibco), and penicillin/streptomycin (Gibco) for up to 26 days.

For the in vitro ASD model, the cell line involved in E/I imbalance, neuronal cultures were treated with (1–2 mM) valproic acid (VPA) from DIV0 to 6, following a protocol established previously38. Cells were harvested at DIV15 for biochemical analysis.

For the in vitro treatment with HDAC inhibitors shown in Fig. 4, neurons were exposed to 750 nM MS275, 1–2 µM RGFP966, or 1–10 nM CAY10683 for 4–6 days from the beginning of the culture, then maintained in a normal growth medium until the designated experimental time points.

To specifically modulate Notch1 signaling in proliferating cells, retroviral infection was performed at DIV3–4. For Notch1 intracellular domain (ICN) overexpression, a retrovirus carrying Notch1 ICN was introduced into neuronal cultures. For Notch1 gene disruption, neuronal cultures derived from Cas9-expressing transgenic mice were infected with retroviruses carrying sgRNAs targeting Notch1, following a previously described protocol68.

Antibodies
The following commercially available primary antibodies were used:
• Rabbit anti-VIP (1:1000, 20077, ImmunoStar)
• Mouse anti-Reelin (1:500, D223-3 (CR-50), MBL)
• Rat anti-Somatostatin (1:300, MAB354 (YC7), Merck Millipore)
• Rabbit anti-Parvalbumin (1:1000, MSFR105210, Nittobo Medical)
• Rabbit anti-Calretinin (1:1000, MSFR100440, Nittobo Medical)
• Mouse anti-Calretinin (1:1000, MAB1568 (6B8.2), Merck Millipore)
• Mouse anti-GAD67 (1:1000, MAB5406 (1G10.2), Merck Millipore)
• Rabbit anti-PROX1 (1:1000, ab199359, Abcam)
• Mouse anti-COUP-TFII (1:1000, PP-H7147-00 (H7147), Perseus Proteomics)
• Rabbit anti-SOX6 (1:1000, ab30455, Abcam)
• Mouse anti-LHX6 (1:1000, H00026468-M02 (1B11), Abnova)
• Mouse anti-HDAC3 (1:1000, #3949 (7G6C5), Cell Signaling)
• Rabbit anti-cleaved NOTCH1 (1:1000, #4147 (Val1744) (D3D8), Cell Signaling,)
• Mouse anti-GAPDH (1:1000, Santa Cruz Biotechnology)

For immunostaining, Alexa Fluor 488- or 546-conjugated secondary antibodies (Molecular Probes) were used. For western blot analysis, horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit immunoglobulin G (IgG) secondary antibodies (Thermo Fisher Scientific) were used.

RNA isolation and analysis
Total RNA was isolated using RNAiso Plus (Takara, Kyoto, Japan), followed by DNase treatment with Turbo DNase (Ambion, Austin, TX, USA) to remove contaminating DNA. Two micrograms of total RNA were reverse transcribed using random hexamers and PrimeScript II Reverse Transcriptase (Takara).

Quantitative PCR (qPCR) was performed on a StepOnePlus qPCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Green Master Mix (Applied Biosystems) and the comparative CT method. Custom primer sets (Supplementary Data 5) were designed with Primer3web 4.1.0, and mRNA levels were normalized to Gapdh mRNA.

Bulk RNA-sequence analysis
Total RNA was extracted from brain tissues and subjected to quality control procedures. mRNA was enriched using oligo(dT) beads, followed by rRNA depletion with the Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA, USA). The enriched mRNA was randomly fragmented using fragmentation buffer, and cDNA synthesis was carried out using the NEBNext Ultra RNA Library Kit (Illumina) according to the manufacturer’s protocol.

After library preparation, sequencing was performed on a NovaSeq 6000 platform (Illumina), with pooling optimized based on effective concentration and anticipated data volume. The sequencing data underwent a filtering process to remove reads containing adapters, reads with undetermined bases (N > 10%), and reads with low-quality bases (Q score ≤ 5) that accounted for more than half of the total bases. Clean reads were then aligned to the mouse reference genome (mm10) using the HISAT2 program 47. Data analysis was performed using Metascape (https://metascape.org/gp/index.html) and STRING for gene ontology (GO) and protein interaction analysis. Using Metascape, the enriched GO terms were listed by the percentage of the genes on the hit list to the genes annotated with the GO term. Using STRING, the enriched GO terms were scored by the signal intensity defined as a weighted harmonic mean between the ratio of the annotated gene with the observed/expected annotated with a term and -log(false discovery rate; FDR). The enriched GO terms were sub-categorized into Biological Process, Cellular Component, Molecular Function, and/or Pathways, compared between experimental groups.

Single-cell RNA-sequence analysis
For analyzing VPA-exposed brains, single-cell RNA sequencing (scRNA-seq) was performed using the BD Rhapsody™ Whole Transcriptome Analysis (WTA) Amplification Kit (BD Biosciences). Briefly, viable cells were isolated from neural tissue using the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec), following the manufacturer’s protocol. The resulting single-cell suspension was sorted using the BD Rhapsody™ Single-Cell Analysis System. Cell Sorter to enrich for viable single cells. A total of approximately 25,000 cells were sorted per animal group before proceeding to single-cell capture and cDNA synthesis. Library preparation was conducted according to the BD Rhapsody™ WTA Amplification Kit protocol. Sequencing was performed on an appropriate platform, generating approximately 5,000 high-quality single-cell transcriptomes per sample across three experimental groups, with an average sequencing depth of 19,164 reads per cell.

Data analysis was performed using Seurat (v5.0.3) in R 4.3.0. After quality control, cells with >12% mitochondrial gene expression, fewer than 1,500 detected genes, or more than 5,000 genes were removed. Normalization was performed using Seurat’s “NormalizeData” function with the “LogNormalize” method, followed by feature selection and scaling. Three datasets were then integrated using Seurat’s integration anchors method. Dimensionality reduction was conducted using principal component analysis. Clustering was performed using the Louvain algorithm with a resolution parameter of 0.5, and visualization was performed using uniform manifold approximation and projection (UMAP). Differentially expressed genes were identified using the Wilcoxon rank-sum test with Bonferroni correction to characterize cluster-specific markers.

For the analysis of human ESC-derived neurons after differentiation, previously published scRNA-seq datasets were used (DDBJ accession IDs: DRX256644, DRX256645, DRA011369, and DRA010907)34,35. Single-cell expression data were converted into pseudo-bulk profiles for downstream analysis. Pseudo-bulk analysis was performed using 10X Genomics Loupe Browser v8.1.2. To investigate Notch signaling dysregulation in ASD models, gene expression patterns were analyzed and visualized using heatmaps. The Log2 fold change values shown in the heatmaps represent the ratio of the normalized mean gene unique molecular identifier counts between conditions.

Mouse exon array and cell-type proportion analysis
Total RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). One hundred nanograms of total RNA were labeled using the Low Input Quick Amp WT Labeling Kit, One-Color*3, and hybridized to the SurePrint G3 Mouse Exon Microarray 2×400K (Agilent Technologies, Folsom, CA, USA) following the manufacturer’s protocol.

Hybridization signals were detected using a DNA Microarray Scanner G2600D (Agilent Technologies, Santa Clara, CA, USA). All scanned images were analyzed with Agilent Feature Extraction Software (v11.5.1.1), and raw data were processed using GeneSpring GX software (v13.0, Agilent Technologies). After 75th percentile shift normalization, differentially expressed genes in the VPA-treated groups were identified by comparing raw probe signal intensities, averaging across genotypes, and filtering out probes with signal intensities below 100. Scatterplots with Pearson’s correlation coefficients were generated, and a volcano plot was constructed using a moderated t-test with Benjamini–Hochberg multiple testing correction. The altered genes were aligned with the cell-types database of the Allen Brain Map (https://portal.brain-map.org/atlases-and-data/rnaseq).

For cell-type proportion analysis, significantly altered 126 genes (64 upregulated, 62 downregulated) in the mouse exon array were analyzed using Bisque69, a computational tool for estimating cell-type composition from bulk RNA-seq data by leveraging single-cell RNA-seq references.

Flow cytometry analysis by fluorescence activated cell sorting
Cells were enzymatically dissociated using trypsin and resuspended in the neuronal culture medium described above. To block nonspecific Fc receptor binding, mouse Fc Blocker (BioLegend) was added to the cell suspension and incubated at 4°C for 10 min. Cells were then incubated with a biotin-conjugated anti-NOTCH1 primary antibody (1:1000, HMN1-12, BioLegend) or the corresponding isotype control (Armenian Hamster IgG, 0.5 mg/mL, BioLegend) at 4°C for 30 min. After washing with phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA), cells were stained with allophycocyanin (APC)-conjugated streptavidin (Thermo Fisher) for secondary detection. Propidium iodide (PI) was used for live/dead cell discrimination.

Flow cytometric data were acquired using a BD LSR Fortessa™ cell analyzer (BD Biosciences) and analyzed with BD FACSDiva software v9.0 and FlowJo v10 (BD Biosciences). Dead cells were excluded based on PI staining, and debris was gated out using forward scatter (FSC) and side scatter (SSC) parameters. Doublets were excluded using FSC-H vs. FSC-W and SSC-H vs. SSC-W. HMN1-12–positive cells were gated using the isotype control as a reference.

Protein analysis
Brain tissues or cultured cells were homogenized in lysis buffer containing PBS, 10 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.2% sodium dodecyl sulfate (SDS), and a protease inhibitor cocktail (Roche Applied Science). For quantitative western blot analysis, 40 µg of total protein was separated by polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Merck Millipore). Visualization was performed using an HRP-conjugated secondary antibody and enhanced chemiluminescent detection (Pierce; Thermo Fisher).

Signal acquisition was conducted with an image analyzer (LAS500, GE Healthcare, Chicago, IL), and quantification was performed using ImageJ (National Institutes of Health (NIH), Bethesda, MD). Signal intensities were normalized to GAPDH as an internal control. The uncropped blots are provided in the Source Data file.

Immunocytochemistry, image acquisition, and analysis
Cultured neurons were fixed in ice-cold 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 20 min. After fixation, neurons were permeabilized with blocking solution containing 0.15% Triton X-100 and 5% normal donkey serum in PBS for at least 30 min at room temperature. Primary antibody incubation was performed overnight at 4°C, followed by visualization using Alexa Fluor 546- or 488-conjugated secondary antibodies.

Images were acquired using a Zeiss LSM700 confocal microscope (Zeiss, Oberkochen, Germany) and processed with Adobe Photoshop and Illustrator (Adobe, San Jose, CA). For quantification, the positive cell of each GABAergic-INs was defined by the double stained cell with cell type-specific antibody and GAD67 antibody. The total number of the positive cells on coverslip was counted and expressed as a proportion of the total number of cells. Representative images were captured from randomly selected fields in each experimental condition.

Immunohistochemistry, image acquisition, and analysis
Postnatal week 3−14 mice were transcardially perfused with 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4). Brains were dissected and post-fixed for 60 min. Embryonic and neonatal brains (E14–P0) were collected and fixed in the fixative for 24 h. After washing with PBS, brains were cryoprotected in 25% sucrose/PBS until they sank. Sections were cut at 50 µm (P21-adult) or 14 µm (E14, P0) using a cryostat (Leica Microsystems, Wetzlar, Germany). For HDAC3 and PROX1 double staining, fixed E15 brains were embedded into paraffin blocks and sectioned at 3 μm. Sections were permeabilized with blocking solution containing 0.3% Triton X-100 and 10% normal donkey serum in PBS for 30 min at room temperature. Primary antibody incubation was performed overnight at 4°C, followed by visualization using Alexa Fluor 546- or 488-conjugated secondary antibodies.

Images were acquired from the CGE/MGE of embryonic brains and the cerebral cortex (layers I–IV) of postnatal brains using a Zeiss LSM700 confocal microscope. Image analysis was performed using ImageJ (NIH). For quantification in postnatal brains, the number or density of marker-positive cells was measured per unit area (/mm²). A single threshold was set for each staining condition to ensure clear cell identification. For quantification in embryonic brains, the proportion of marker-positive and/or EdU-positive areas relative to the total CGE or MGE area was measured.

In utero EdU labeling
Pregnant female mice were exposed to VPA, poly(I:C), or HDAC inhibitors, simultaneously or prior to a single intraperitoneal injection of 8 mg/kg EdU (Click-iT EdU Imaging Kit, Thermo Fisher Scientific) on E13 or E14. The mice were sacrificed 1 day (Figs. 2h-i, 4b-d) or 6 days (Fig. 2j-k) after EdU injection. Embyonic and newborn brains were dissected and fixed in 4% paraformaldehyde for 24 h. EdU-labeled cells were detected according to the manufacturer’s protocol.

Behavioral analysis
Pregnant female mice were housed individually and raised their litters until weaning (P28). After weaning, offspring were housed in same-sex groups (2−5 mice/cage). Behavioral tests were performed in adult mice (8−13-weeks-old), except for the ultrasonic vocalization (USV) test (P7), as described previously70. Both male and female mice were tested. Before each test, mice were habituated in the test room for at least 30 min. Between tests, equipment was cleaned with 70% ethanol and dried. Behavior was recorded using a digital camera placed on the side of (self-grooming) or above (three-chamber test, reciprocal social interaction) equipment and analyzed with Smart 3.0 software.

USV (P7). Newborn pups emit USVs in response to maternal separation. USV calls were recorded for 5 min using an ultrasound microphone (Avisoft Bioacoustics, Berlin, Germany) placed 15 cm above the pup inside a soundproof chamber, and analyzed with Avisoft-SASLab Pro software. Body weight and axillary temperature were measured afterward.

Self-grooming (8−10 w). To assess repetitive behaviors, each test mouse was placed in a clean cage under white light (160 lux). The total duration of self-grooming was recorded over 25 min.

Three-chamber test (9−11 w). The test apparatus consisted of three chambers separated by Plexiglas walls with small openings (5×3 cm) for free movement. A small wire cage was placed in each side chamber. The test consisted of three 10-min sessions under white light (20 lux):
1. Habituation: The test mouse was placed in the centre chamber and allowed to explore all three chambers for 10 min.
2. Sociability: A novel mouse (stranger 1) was placed in one of the wire cages, and the test mouse was returned to the centre chamber to explore freely. Time spent in the stranger’s chamber was compared with that in the empty chamber.
3. Social Novelty: A second unfamiliar mouse (stranger 2) was placed in the opposite wire cage, whereas stranger 1 remained in the same location. The test mouse was again allowed to explore freely, and the time spent in the chamber with stranger 2 was compared with the time spent with stranger 1.

The sociability index (SI) and social novelty index (SNI) were calculated as follows:
• SI = (time in stranger chamber − time in empty chamber) / (time in stranger chamber + time in empty chamber)
• SNI = (time in stranger chamber − time in familiar chamber) / (time in stranger chamber + time in familiar chamber)

Reciprocal social interaction (11−13 w). Pairs of mice (matched for genotype, drug exposure, sex, age, and weight, with no prior contact) were placed in an open-field arena (45×45 cm) under white light (100 lux). Social interactions were recorded for 10 min, and the number of contacts, total contact duration, and resting time were measured.

Statistical analysis
GraphPad Prism 10 was used for statistical analyses. Pairwise comparisons were performed using the unpaired t-test or Mann–Whitney test. For multiple-group comparisons, analysis of variance (ANOVA) was followed by Bonferroni’s or Dunnett’s post-hoc tests. Data are presented as mean ± standard error of the mean (SEM). Statistical significance is indicated as follows: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In their manuscript, Hanno et al. study the role of Notch signaling in mouse models of Valporic acid (VPA) and Poly-IC induced (PIC) Autism like phenotypes. The authors treat pregnant mice at mid-late gestation with VPA and PIC which induces behavioral traits resembling autism in the offspring. They report a reduction in VIP neurons in the cerebral cortex. VIP neurons are generated from the caudal ganglionic eminence (CGE) during late embryogenesis, and the authors claim that delayed neurogenesis reduces the number of VIP neurons in the postnatal cerebral cortex. They propose that the effect is due to maintained/increased Notch signaling in the CGE following treatment, which leads to a reduction in VIP neuron production. They show that conditional ablation of Notch1/Notch2 in the LGE rescues the VIP production. Hence, they claim that in sporadic cases of Autism, an aberrant increase in Notch signaling could play a role in regulating inhibitory neuron formation, leading to an unbalance in excitatory and inhibitory neuron signaling in the cortex. The authors present extensive data and a combination of in vitro and in vivo experiments to support their hypothesis. The authors have some interesting findings but the link to Autism is, in my opinion, not substantiated, and the animal model is not ideal without considerable further characterization.\n\nMain concerns\n1. The authors need to describe the brain defects of the VPA and PIC treatment in greater detail. mRNA-seq analysis is suggestive but not definitive and miss-regulated gene expression is likely not only linked to a slight reduction in VIP neuron production.\n2. The effects on VIP neuron production is relatively modest. The authors need to validate that the effects are really caused by the reduction in VIP neurons. Currently, the conclusions are based on correlations rather than substantiated evidence.\n3. The conditional loss of Notch signaling needs to be described in greater detail. Changes in proliferation and neuronal composition of the brains of the treated and animals compared to Notch-ablated controls need to be expanded.\n4. The authors focus on the effects of VPA on the CGE, but a more detailed analysis of other brain regions and subtypes should be added.\n5. The authors need to show or at least discuss why, in their model and potentially in Autism, the effects they show and claim are selective for CGE progenitors and their progeny. Are other late-born neuron populations affected by the VPA and PIC treatment?\n6. The human ESC experiment are very superficial and would require a more detailed analysis of cell-fate and differentiation in the different lines. Otherwise, the authors may be comparing cell-line specific differentiation effects that are nothing to do with the Notch signaling.\n7. Some of the images, particularly for VIP immunostainings in the brain and in the cell cultures, are not convincing. The number of VIP neurons generated in the in vitro assays (10-30 VIP+ cells / 10000 cells plated) is very small and the differences with VPA treatment modest in absolute terms. The Notch rescue experiment in Figure 3h are also very small and the number of VIP+ cells generated in the Notch1 knockout cells is lower or at best, the same as in the NIC results presented in Figure 3g.\n8. The authors need to discuss the relevance of the FACS plots in Figure 3a. It would have been interesting to compare the number of Notch-positive cells in the different brain regions of control and VPA and PIC-treated embryos. Figure 3a does not show an increase in Notch signaling per se but an increase in the number of Notch-expressing cells in the different brain regions. The differences likely reflect the difference in the proportion of neural progenitors in the dissected brain regions over the total cells isolated.\n9. The link to HDAC3 is interesting but the mechanistic link to the VPA and Notch regulation remains unclear.\n10. Figure 5b: The immunostaining is very difficult to judge.\n11. Without further evidence for the link to Autism, the authors should tone down their claims for a role of aberrant Notch signaling in the CGE in autism.\n\nMinor comments\nThe manuscript lacks formal proof of a mechanism of VPA and PIC regulation of Notch signaling. So currently, the data are interesting but correlative. The link to Autism is tenuous and should, at this point, be toned down.\nIn places, the manuscript is difficult to read and would benefit from proofreading and editing by a native English speaker.", "reviewer_2": "The paper is well-written overall with strong experimental design. Below are specific recommendations to strengthen the manuscript:\n1. Throughout the manuscript, please specify details of the models used (e.g., on page 5, line 27, the \"in vitro ASD model\" should include the cell lines involved in E/I imbalance comparisons).\n2. In the introduction (page 3, line 6), please include a reference to Mordaunt et al. (DOI: 10.1186/s13229-019-0287-z), which provides relevant context.\n3. In the introduction (page 3, line 24), a citation to Sharifi et al. (https://doi.org/10.1038/s42003-024-06990-0) would strengthen the background.\n4. Figure 1d requires clarification regarding which models the abbreviations correspond to.\n5. Regarding Figure 1e (page 5, lines 9-19): The authors should address the opposing directions of Notch1 dysregulation between human ASD models (downregulation) and mouse models (upregulation). This cross-species difference warrants explanation.\n6. On page 6 (lines 1-18), there is an inconsistency in gene notation: human gene notation is used in the text while mouse notation appears in the Figure 2a heatmap. Please standardize.\n7. On page 9, the discussion of HDAC3 inhibition as a mechanism for ASD-like behavior would benefit from distinguishing between MeCP2 and HDAC3 contributions to the phenotype (consider referencing DOI: 10.1038/nn.4347).\n8. The GO analysis (page 10, lines 34-35) requires additional methodological details, specifically how these particular GO terms were selected and the number of input genes used in the analysis.\n9. Regarding single-cell clustering (page 12, lines 12-18; Fig. 6j; Supplementary Fig. S10a,b): The cell type labeling methodology requires validation against the Allen Brain Atlas or published literature, as the selected gene markers currently show expression across multiple clusters.\n10. Figure 6k would benefit from statistical analysis to substantiate claims about cell proportion restoration, assuming accurate cell type labeling.\n11. The statistical methodology for differential gene expression (page 20) relies on the Wilcoxon rank-sum test with Bonferroni correction. Consider that alternative methods may offer lower false discovery rates for single-cell data analysis (see https://doi.org/10.1038/s41467-021-25960-2).", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank Reviewer #1 for the constructive comments. Considering all the comments below, we note that the major concerns raised by Reviewer #1 are largely related to the following: (i) whether the reduction of VIP interneurons is causally linked to autism-like social phenotypes and (ii) whether our transcriptomic analyses experimentally provide more than correlative evidence.\n\nMain concerns\n1. The authors need to describe the brain defects of the VPA and PIC treatment in greater detail. mRNA-seq analysis is suggestive but not definitive and miss-regulated gene expression is likely not only linked to a slight reduction in VIP neuron production.\nResponse: We assume that the reviewer is referring to the transcriptomic alterations observed in differentiated ASD-model cultures and brains shown in Fig. 2. Our qPCR and exon array analyses revealed a reduction in VIP and in other neuronal subtype markers, in particular, a robust decrease in NDNF transcript levels (Fig. 2a, Supplementary Fig. 4a). NDNF-positive interneurons are CGE-derived interneurons enriched in cortical layer I.\nNo reliable antibody was available for NDNF immunohistochemistry; therefore, we recently generated a NDNF‒HA knockin mouse line, enabling us to directly quantify NDNF-positive neurons (Supplementary Fig. 5b,c). We found a small but significant reduction (~30% decrease compared with controls) in the number of NDNF-positive cells in VPA-exposed offspring. Thus, the decreased expression of VIP and NDNF transcripts (Fig. 2a, Supplementary Fig. 4) were consistent with the observed reductions in corresponding cell numbers (Fig. 2c,d, Supplementary Fig. 5c).\nWe further detected significant reductions in a VIP subtype, calretinin (CR)-co-expressing VIP neurons located in deeper cortical layers (Supplementary Fig. 5c).\nTogether, these findings indicate that VPA exposure broadly disrupts CGE-derived neuronal populations, including both VIP- and NDNF-positive neurons, rather than only VIP neurons. This supports the view that behavioral abnormalities in these ASD models may result from a broad impairment of late-phase CGE interneuron development. Importantly, although these results expand the range of affected interneurons, our main conclusion remains unchanged: disruption of VIP neuron development likely represents a major and central contributor to ASD-like phenotypes in these models.\n2. The effects on VIP neuron production is relatively modest. The authors need to validate that the effects are really caused by the reduction in VIP neurons. Currently, the conclusions are based on correlations rather than substantiated evidence.\nResponse: We agree with the reviewer that inhibitory neurons are not the only cell type affected in the VPA and PIC models. Indeed, our scRNA-seq analysis revealed broad changes in cellular composition within the VPA model (Fig. 6k).\nOur qPCR and RNA-seq analyses further revealed that both VPA and PIC models exhibit a common alteration in the excitation/inhibition (E/I) balance, which appears to be partially mediated by upregulation of neurogenin2 (Neurog2) transcript levels, which strongly induces the differentiation of glutamatergic neurons (Supplementary Fig. 12d). This alteration may underlie the increased expression of excitatory synaptic molecules, such as Grin2b and Grin2a in transcript (Supplementary Fig. 12c) and protein levels (Iijima et al., 2016; PMID: 27264355), observed in the mature VPA-exposed mice (Fig. 5j-m).\nHowever, the elevated Neurog2 expression following VPA exposure was fully restored in Notch1/2 conditional knockout (cKO) brains, suggesting that upregulation of Neurog2 occurs as a secondary event after activation of Notch signaling in CGE-interneuron. These results support the notion that disruption of inhibitory neuronal networks indirectly drive maladaptive excitatory gene expression programs.\nWe have incorporated these points into the revised Results (pages 11‒12) and Discussion (page 16).\n3. The conditional loss of Notch signaling needs to be described in greater detail. Changes in proliferation and neuronal composition of the brains of the treated and animals compared to Notch-ablated controls need to be expanded.\nResponse: We appreciate the reviewerʼs request for additional details regarding the conditional loss of Notch signaling. To address this, we examined cortical PV⁺ neurons̶a major class of GABAergic interneurons̶in control and Notch1/2 cKO brains, and found no differences in either their number or distribution. The new data are provided in Supplementary Fig. 11a.\nDifferential gene expression analysis revealed that only a small number of genes were altered in the cKO (Fig. 5h), and principal component analysis (PCA) further showed that cKO samples were largely comparable to controls (Fig. 5i). These results indicate that conditional loss of Notch signaling does not cause major transcriptomic changes in the forebrain. This is consistent with the fact that Htr3a-Cre drives recombination only in a small subset of interneurons, and thus the overall impact of conditional knockout on brain development is expected to be quite small under baseline conditions.\nWe then conducted GO analyses comparing cKO versus cKO + VPA (Supplementary Fig. 13a). Interestingly, the term “glutamatergic synapse” appeared among the downregulated GO categories, whereas the same term was found among the upregulated categories in the WT versus WT + VPA comparison (Supplementary Fig. 12a). The supplementary figures have been revised to present these findings clearly (page 11).\nTogether, these analyses provide a more complete view by the impact of conditional Notch loss on the VPA response and complement our discussion of alterations in proliferation and neuronal composition.\n4. The authors focus on the effects of VPA on the CGE, but a more detailed analysis of other brain regions and subtypes should be added.\nResponse: We expected the reviewer to raise the question of whether MGE- or LGE-derived lineages are also affected. We already examined established MGE markers (SOX6, LHX6) at E14 and P0 and MGE-derived neurons (PV, SST) at the adult cortex, and found no significant differences between control and VPA-exposed brains (Fig.2f‒k, Supplementary Fig. 6). This suggests that MGE lineages are largely preserved.\nRegarding LGE-derived neurons, we note the limitation of the study: there are no specific markers exclusively restricted to the LGE, especially during embryonic stages. Nevertheless, our RNA-seq analysis revealed a trend of reduced expression of medium spiny neuron (MSN)-related genes (e.g., Drd1, Drd2, Ppp1r1b), which are known to arise from the LGE (Supplementary Fig. S13). While these genes are not purely LGE-specific, the reduction supports the possibility that VPA exposure may also impact LGE-derived neuronal populations, not only CGE.\nWe have now clarified these points in the Results (pages 7 and 12) and Discussion (page 15).\n5-1. The authors need to show or at least discuss why, in their model and potentially in Autism, the effects they show and claim are selective for CGE progenitors and their progeny.\nResponse: We appreciate the reviewerʼs insightful comment. Regarding why CGE-derived interneurons appear to be strongly affected in the VPA and PIC models, we speculate that this selectivity reflects the timing of drug exposure. In the ASD models, both VPA and PIC were exposed around E13, a developmental stage when CGE-derived interneurons are actively generated. However, majority of MGE-derived interneurons are differentiated. Therefore, at this stage, CGE progenitors are potently regulated by Notch signaling to maintain their proliferative state or differentiate into interneurons. Our data indicate that VPA and PIC exposure disrupt Notch-related transcriptional programs at this critical period, which may preferentially impair CGE interneuron production and maturation.\nImportantly, our experimental data support this idea. FACS-sorted Notch-positive proliferating cells are more enriched in the ganglionic eminence (GE) regions than the cortical regions (Fig. 3a). Considering that MGE-derived interneuron production largely occurs before E13, it is likely that the majority of these Notch-positive cells in the GE are CGE-derived progenitors. Consistent with this perspective, our previous study showed that GE cultures prepared at E13 are highly enriched in VIP-positive neurons relative to other interneuron subtypes (Sato et al., Neurochem. Res., 2022). These findings strongly support the notion that late-phase CGE progenitors are particularly dependent on Notch signaling and therefore more vulnerable to its disruption by VPA and PIC.\nFurthermore, as described in our response to reviewer #1ʼs comment 9, we have provided additional data showing that HDAC3 is strongly expressed in the CGE region and colocalizes with the transcription factor Prox1, which is essential for differentiation of interneuron. This spatially enriched expression and molecular interaction likely contribute to the preferential impact of HDAC3-mediated Notch regulation on CGE-derived neuronal lineages.\nTaken together, the combination of (1) the late onset of CGE interneuron generation, (2) the high Notch dependency of these progenitors at E13, and (3) the spatiotemporal interaction between HDAC3 and Prox1 within the CGE likely makes them more vulnerable to VPA- and PIC-induced disruptions. Therefore, this developmental window effect may explain the apparent selective impairment of CGE-derived interneurons in our models.\nWe have now clarified these points in the Discussion section (pages 15‒16).\n5-2. Are other late-born neuron populations affected by the VPA and PIC treatment?\nResponse: We agree with the reviewer that other late-born neuronal populations are also likely to be affected by VPA and PIC exposure. Indeed, both our bulk RNA-seq (Fig. 5) and scRNA-seq (Fig. 6) analyses indicate that VPA treatment induces broad transcriptional alterations beyond CGE-derived interneurons, including changes in other genes associated with neurogenesis.\nHowever, for other late-born cell types that do not originate from the CGE̶such as layer 2/3 pyramidal neurons or chandelier cells̶the lack of reliable and developmentally restricted genetic markers currently prevents their selective labeling and quantification during embryonic or early postnatal stages.\nMoreover, as several abnormalities in excitatory neurons and glial cells have already been reported in many previous studies, we do not believe that a more detailed analysis of these additional populations would add substantial novelty within the scope of the present work. Rather, we are concerned that inclusion of too many additional phenotypic datasets from the VPA and PIC models could obscure our main focus on cortical interneuron development.\n6. The human ESC experiment are very superficial and would require a more detailed analysis of cell-fate and differentiation in the different lines. Otherwise, the authors may be comparing cell-line specific differentiation effects that are nothing to do with the Notch signaling.\nResponse: The previous manuscript lacked sufficient evidence linking our findings to human neurodevelopmental disorders. To address the issue, we further added a meta-analysis of publicly available transcriptomic datasets (10 lines) derived from human ES/iPS cell‒based neuronal differentiation models of neurodevelopmental disorders. Our analysis revealed that several of these datasets (Mecp2 mutant, Chd8, and Tsc KO) indicate activation of the Notch pathway (Supplementary Fig. 2b and Supplementary Table 1).\nAlthough neurodevelopmental disorders could not be explained simply by aberrant Notch signaling, new results indicate that developmental hyperactivation of Notch signaling may contribute to the pathophysiology of a subset of human neurodevelopmental disorders. We have incorporated these points into the revised Discussion (page 14).\n7-1. Some of the images, particularly for VIP immunostainings in the brain and in the cell cultures, are not convincing. The number of VIP neurons generated in the in vitro assays (10-30 VIP+ cells / 10000 cells plated) is very small and the differences with VPA treatment modest in absolute terms.\nResponse: We apologize for the errors and appreciate the reviewerʼs careful evaluation. We have corrected the y-axis units and numeric values and now explicitly state the normalization procedure in the Methods (page 25).\nRegarding the staining images, there is a technical limitation for VIP immunostaining. To improve clarity, we have now indicated representative VIP-positive cells with arrows in the revised figures. Please see our response to reviewer #1ʼs comment 10 for additional details.\n7-2. The Notch rescue experiment in Figure 3h are also very small and the number of VIP+ cells generated in the Notch1 knockout cells is lower or at best, the same as in the NIC results presented in Figure 3g.\nResponse: As for the Notch rescue experiment illustrated in Fig. 3h (Fig.3i in the revised manuscript), Notch1 disruption was achieved by retroviral delivery of genome-editing constructs, which yields mosaic knockout. This underestimates the true effect size relative to complete genetic ablation, which may cause the small rescue effect especially at DIV22‒26 (Fig. 3i). Despite this technical limitation of the experiment, the Notch1 loss-of-function still produced a statistically significant change in VIP+ neuron yield relative to controls (Fig. 3f). Importantly, these results are directionally consistent with Notch gain-of-function (NICD) experiments and pharmacological Notch inhibition, both of which independently reproduce the expected bidirectional effects on VIP differentiation. Taken together, these orthogonal manipulations strengthen the causal link between Notch activity and VIP-IN differentiation.\n8-1. The authors need to discuss the relevance of the FACS plots in Figure 3a.\nResponse: In our original submission, the description of Fig. 3a was not sufficiently clear. The purpose of this analysis was to demonstrate that Notch1-positive progenitors are highly enriched in the ganglionic eminence (GE) compared with the cortical primordium (Cx) in the embryonic brain. This regional enrichment provides an essential context for interpreting the subsequent effects of VPA treatment on interneuron development. Considering the timing of interneuron generation, we believe that the majority of these Notch1-positive cells in the GE at this stage are possibly derived from the caudal ganglionic eminence (CGE). We have revised the Discussion section to explicitly state this point (page 15).\n8-2. It would have been interesting to compare the number of Notch-positive cells in the different brain regions of control and VPA and PIC-treated embryos. Figure 3a does not show an increase in Notch signaling per se but an increase in the number of Notch-expressing cells in the different brain regions. The differences likely reflect the difference in the proportion of neural progenitors in the dissected brain regions over the total cells isolated.\nResponse: We thank the reviewer for this valuable recommendation. As suggested, we performed new in vitro experiments using embryonic progenitors exposed to VPA and analyzed them by FACS. These experiments directly demonstrated a robust increase in the fraction of Notch1-positive cells in VPA-treated cultures compared with controls (Fig. 3b). We also confirmed that VPA treatment significantly upregulated Notch1 and its downstream target Hes5 expression (Supplementary Fig. 1c).\nTogether, these results demonstrate the mechanistic relevance of our findings: VPA indeed promotes the expansion of Notch signaling-active progenitors, consistent with our transcriptomic data and supporting the notion that VPA subsequently impairs their differentiation trajectory.\n9. The link to HDAC3 is interesting but the mechanistic link to the VPA and Notch regulation remains unclear.\nResponse: To address the mechanistic involvement of HDAC3 in Notch regulation, we performed additional analyses and experiments, as follows:\n1. Coimmunoprecipitation assays demonstrated a physical interaction between HDAC3 and Prox1, a transcription factor highly expressed in CGE-derived neurons (Supplementary Fig. 9d).\n2. Immunohistochemistry showed strong expression of HDAC3 in the CGE (Supplementary Fig. 9b).\n3. Biochemical fractionation of embryonic brain tissue followed by Western blotting confirmed that HDAC3 and Prox1 colocalize in the soluble nuclear fraction (Supplementary Fig. 9c).\nAlthough we currently lack direct ChIP-seq evidence of HDAC3 binding to the Notch1 promoter, several open database ChIP-seq analyses reveal that the Notch1 promoter region undergoes deacetylation by HDACs during neuronal differentiation (data not shown). These findings collectively support a model in which HDAC3 is highly expressed in the CGE and forms complexes with Prox1 to regulate transcriptional activity through epigenetic deacetylation.\n10. Figure 5b: The immunostaining is very difficult to judge.\nResponse: Although we agree that the immunostaining of VIP-positive cells may appear less distinct compared with other neuronal markers, this is an inherent limitation of VIP immunostaining, as VIP is a secreted neuropeptide that diffuses into the extracellular space, and many neighboring cells could express VIP receptors. Therefore, it is technically difficult to visualize the sharp and cell-restricted labeling of VIP-expressing neurons.\nIn the revised manuscript, we have replaced the original panel with high-magnification images (to ensure transparency, unprocessed source images and full fields of view have been provided in Supplementary Fig. 11b)\n11. Without further evidence for the link to Autism, the authors should tone down their claims for a role of aberrant Notch signaling in the CGE in autism.\nResponse: We agree with the reviewerʼs suggestion that our previous wording might have overstated the direct link between Notch signaling and autism. We have therefore carefully revised the manuscript to avoid overgeneralization. In particular, we now limit our conclusions to autism models, and we have also modified the title as follows:\n“Targeting Notch Signaling to Restore Neural Development and Behavior in Autism Models.”\nNevertheless, our additional analyses of human ES/iPS cell-derived neurons indicate that Notch signaling abnormalities may indeed contribute to early developmental processes relevant to at least a subset of human neurodevelopmental disorders. While we recognize that this is not currently definitive evidence, we plan to further investigate this possibility in our future studies.\n\nMinor comments\nThe manuscript lacks formal proof of a mechanism of VPA and PIC regulation of Notch signaling. So currently, the data are interesting but correlative. The link to Autism is tenuous and should, at this point, be toned down.\nResponse: We completely agree with the reviewer that the present study does not provide a full mechanistic proof of how VPA and poly(I:C) exposure dysregulates Notch signaling. Indeed, elucidating the molecular intermediates that link not only diverse environmental but also genetic risk factors to Notch pathway activation remains an open and challenging question in the field.\nIn the current work, we have provided evidence that both VPA and poly(I:C) exposure converge on abnormal Notch signaling during embryonic development, leading to specific alterations in late-born interneuron populations. While these findings are still correlative, we believe they are important in highlighting Notch dysregulation as a convergent pathway in multiple ASD models.\nWe fully recognize the importance of dissecting the upstream mechanisms that couple VPA, poly(I:C), or genetic mutations to Notch activity. This will be the focus of our future work, as we anticipate that understanding such mechanisms may ultimately reveal novel and potentially preventive strategies against ASD onset.\nIn places, the manuscript is difficult to read and would benefit from proofreading and editing by a native English speaker.\nResponse: We apologize for the parts of the manuscript that were difficult to read. We have thoroughly revised the text to improve clarity and flow. In addition, the revised manuscript has been professionally proofread and edited by a native English editing service (Editage) to ensure that the language meets publication standards.\n\nReviewer #2 (Remarks to the Author):\nThe paper is well-written overall with strong experimental design. Below are specific recommendations to strengthen the manuscript:\nWe sincerely thank Reviewer #2 for the positive and helpful comments. We have revised the manuscript accordingly, as detailed below.\n1. Throughout the manuscript, please specify details of the models used (e.g., on page 5, line 27, the \"in vitro ASD model\" should include the cell lines involved in E/I imbalance comparisons).\nResponse: In our study, the “in vitro ASD model” did not use immortalized cell lines but rather primary cultured cortical neurons, in which ASD-like conditions were pharmacologically induced as described previously (Iijima et al., 2016; PMID: 27264355). We have now clarified this point in the revised Methods (page 19) and figure legends, including detailed descriptions of the culture origin, treatment conditions, and parameters used for the E/I imbalance comparisons.\n2. In the introduction (page 3, line 6), please include a reference to Mordaunt et al. (DOI: 10.1186/s13229-019-0287-z), which provides relevant context.\nResponse: We have added the recommended reference in the Introduction (page 3, line 6) to provide additional context on epigenetic and transcriptomic alterations in ASD.\n3. In the introduction (page 3, line 24), a citation to Sharifi et al. (https://doi.org/10.1038/s42003-024-06990-0) would strengthen the background.\nWe have added the suggested citation in page 3, line 24 (line 27 in the revised manuscript) in the Introduction to strengthen the background on Notch signaling and ASD-related mechanisms.\n4. Figure 1d requires clarification regarding which models the abbreviations correspond to.\nResponse: We have revised the legend of Figure 1d to clearly explain which abbreviations correspond to which models.\n5. Regarding Figure 1e (page 5, lines 9-19): The authors should address the opposing directions of Notch1 dysregulation between human ASD models (downregulation) and mouse models (upregulation). This cross-species difference warrants explanation.\nResponse: Increased Notch signaling has also been suggested in human neurodevelopmental disorders. As noted in the original Discussion, the example is 1p21 duplication syndrome, which presents with macrocephaly, is one such case. We used an ES cell line with 1p21 duplication (Fig. 1e), but this line does not carry a Notch gene copy at 1p21 and therefore could not be suitable for direct testing. However, in the newly performed screen of human ES/iPS cell‒derived differentiated neurons, we observed a tendency toward Notch pathway upregulation in the 16q11 deletion line (Fig. 1e) and in Mecp2 mutant, Chd8, and Tsc KO lines (Supplementary Fig. 2b). These results importantly suggest that aberrant elevation of Notch signaling during early development may represent a shared pathological feature in at least a subset of ASD cases.\nAlthough several mutations in Notch-related genes have been reported, it has not been clearly demonstrated whether they cause downregulation or upregulation of the pathway. Future studies will be necessary to clarify how these variants affect Notch signaling.\n6. On page 6 (lines 1-18), there is an inconsistency in gene notation: human gene notation is used in the text while mouse notation appears in the Figure 2a heatmap. Please standardize.\nResponse: We have carefully corrected the manuscript to ensure consistency in gene nomenclature: human gene symbols are in uppercase italics (e.g., VIP), whereas mouse gene symbols are presented with only the first letter capitalized and in italics (e.g., Vip), both in the text (page 6) and the Fig. 2a.\n7. On page 9, the discussion of HDAC3 inhibition as a mechanism for ASD-like behavior would benefit from distinguishing between MeCP2 and HDAC3 contributions to the phenotype (consider referencing DOI: 10.1038/nn.4347).\nResponse: We sincerely appreciate the reviewerʼs insightful suggestion regarding the discussion of HDAC3 inhibition and the reference to the study (DOI: 10.1038/nn.4347). We carefully checked this paper and found that the study investigated social behaviors in Camk2a-Cre; Hdac3 conditional knockout mice, which represent HDAC3 loss in “glutamatergic neurons.” By contrast, our study focuses on the pharmacological inhibition of HDAC3 in “GABAergic neuron progenitors,” which results in distinct behavioral phenotypes from those reported in Camk2a-Cre models.\nGiven the confusion by these mechanistic differences, we wondered whether we should cite this reference directly in the revised manuscript. However, we are grateful to the reviewer for bringing this important study to our attention.\n8. The GO analysis (page 10, lines 34-35) requires additional methodological details, specifically how these particular GO terms were selected and the number of input genes used in the analysis.\nResponse: We have expanded the Methods section (page 21) to clarify how the GO terms were selected, the criteria for enrichment, and the number of input genes used in the analysis. This additional detail has helped improve transparency and reproducibility.\n9. Regarding single-cell clustering (page 12, lines 12-18; Fig. 6j; Supplementary Fig. S10a,b): The cell type labeling methodology requires validation against the Allen Brain Atlas or published literature, as the selected gene markers currently show expression across multiple clusters.\nResponse: We appreciate the reviewerʼs important comment regarding the validation of our single-cell clustering. As the reviewer correctly pointed out, most publicly available references such as the Allen Brain Atlas are based on mature brains. By contrast, our single-cell dataset was obtained at postnatal day 2, a developmental stage in which many canonical differentiation markers are still weakly expressed. Indeed, when we attempted validation using the Allen Brain Atlas reference, the results were not informative (see the figure for the annotation summary below).\nWe acknowledge that some degree of subjectivity is unavoidable in annotating developing cell types. Nevertheless, we believe that our clustering is largely accurate, as it is supported by the expression patterns of well-established neuronal and glial marker genes. To further substantiate this point, we have now added a supplemental figure summarizing feature plots of representative developmental marker genes (Supplementary Fig. 15). These analyses demonstrate consistent and biologically plausible marker expression patterns across clusters.\n10. Figure 6k would benefit from statistical analysis to substantiate claims about cell proportion restoration, assuming accurate cell type labeling.\nResponse: We thank the reviewer for raising this important point regarding the statistical evaluation of cell-type composition across groups. In the revised manuscript, we have now conducted chi-square tests to assess differences in cell-type proportions across the three groups, followed by residual analyses. The resulting heatmap (Fig. 6l) demonstrates patterns broadly consistent with the stacked bar plot shown in Fig. 6k.\nIn addition, pairwise chi-square tests indicated significant nonindependence in most group comparisons. To further validate these results, we calculated Pearsonʼs correlation coefficients of cell-type distributions (Fig. 6l). These complementary analyses consistently suggested that administration of the Notch inhibitor Ro4929097 shifted the ASD model profile toward that of wild-type mice.\nTogether, these additional analyses provide strengthened statistical support for the conclusion that pharmacological inhibition of Notch signaling partially rescues cell-type composition abnormalities in the ASD model.\n11. The statistical methodology for differential gene expression (page 20) relies on the Wilcoxon rank-sum test with Bonferroni correction. Consider that alternative methods may offer lower false discovery rates for single-cell data analysis (see https://doi.org/10.1038/s41467-021-25960-2).\nResponse: We appreciate the reviewerʼs suggestion. We used the Wilcoxon rank-sum test with Bonferroni correction, as implemented in Seurat, which is a commonly adopted and conservative approach for single-cell differential gene expression analyses. In response to the reviewerʼs comment, we carefully re-evaluated our analysis description and noticed that the original text also mentioned pathway enrichment analysis using the Gene Ontology (GO) database. However, this analysis was not included in the final version of the manuscript. We apologize for the confusion.\nThe GO analysis was initially attempted to annotate clustered neuronal populations, but as our dataset was derived from postnatal day 2 (P2) mouse brains, differentiation markers were not sufficiently enriched to enable reliable functional annotation. Therefore, we decided not to include the GO analysis results in the current study. We have now deleted the corresponding sentence from the Methods section (pages 21‒22) to avoid confusion and ensure consistency with the presented data.\n\nValidation of cell-type annotation using differentiation markers from the Allen Brain Atlas.\nFeature plots showing representative developmental cell-type markers used to validate cluster identities in the postnatal day 2 dataset. Each plot displays the normalized expression levels of selected marker genes across the UMAP embedding. The accompanying table summarizes the corresponding cluster annotations based on the expression of neuronal and glial lineage markers. Marker gene references were derived from the Allen Brain Atlas.\n\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe sincerely thank Reviewer #3 for their contribution to the peer-review process. We appreciate the time and effort they devoted, and we welcome the Nature Communications initiative to support Early Career Researchers in peer review."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In their revised manuscript, Hanno et al. have addressed most of my initial concerns. They have added additional data and reworked the manuscript, which has improved significantly. The data remain somewhat correlational rather than definitive. However, the data will be of interest for the field.\n\nMajor concerns\n1. It is not clear to this reviewer why the figures are duplicated at the end of the merged document.\n2. Clarification to my original point 1. A more detailed analysis of cortical layering and other neural cell populations in the brains of the VPA- and PIC-treated animals would have been good to clarify the global effects of the treatments on brain architecture.\n3. The authors have tried to improve the immunostaining panels, particularly for VIP. However, they are still very difficult to interpret, and it is hard to imagine how these images can be interpreted.\n\nMinor concerns\nFigure 3 legend: A description of panel b is missing.", "reviewer_2": "The authors have appropriately responded to prior reviewer critiques and the manuscript is improved by the revisions.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We are grateful for the positive feedback and for the recognition that the revised manuscript has improved. We have addressed all remaining concerns through making corrections clarifications on the figure. We believe these adjustments further strengthen the manuscript.\n\nMajor concerns\n1. It is not clear to this reviewer why the figures are duplicated at the end of the merged document.\nResponse: We sincerely apologize for the confusion caused by the duplicated figures at the end of the document. We have carefully reconstructed the merged PDF document. The re-revised submission files contain only one complete set of figures.\n2. Clarification to my original point 1. A more detailed analysis of cortical layering and other neural cell populations in the brains of the VPA- and PIC-treated animals would have been good to clarify the global effects of the treatments on brain architecture.\nResponse: We agree that a more detailed analysis of cortical layering and other neural cell populations may provide additional insights, given that some previous reports have shown significant increases in the thickness of cortical layers II‒IV (Fujimura et al., 2016) and neuronal cell loss in layers II‒V (Kataoka et al., 2013) in prenatal VPA-exposed mice.\nTo address the reviewerʼs concern, we have performed additional immunostaining using a cortical layer-specific marker (Blue: SATB2) and a non-neuronal marker (Red: GFAP), and did not detect robust differences in the length of layers II‒IV or in cell density, at least in the adult somatosensory area of our ASD model mice (as shown in the supplementary rebuttal figures).\nTo avoid potential confusion̶particularly because some previous studies have reported cortical abnormalities that we did not observe in our samples̶we have chosen to include these additional data in the rebuttal for transparency rather than in the main manuscript.\n3. The authors have tried to improve the immunostaining panels, particularly for VIP. However, they are still very difficult to interpret, and it is hard to imagine how these images can be interpreted.\nResponse: We appreciate the reviewerʼs concern regarding the immunostaining panels. To address this and improve figure quality, we replaced the previous grayscale images with the original color images showing single VIP staining and double staining for VIP/GAD67 (Magenta: VIP; Green: GAD67), as shown in Figures 3h and 3j. These color images provide improved contrast and reduced background, facilitating clearer interpretation.\nInterpretation of VIP-positive cells was supported by co-staining with GAD67, a pan-GABAergic neuronal marker, and only neurons positive for both VIP and GAD67 were counted in the experiments. This point has been clarified in the re-revised Methods (page 24).\n\nMinor concerns\nFigure 3 legend: A description of panel b is missing.\nResponse: We apologize for missing the part of description in Fig.3 legend. We have added the description of panel b.\n\nReviewer #2 (Remarks to the Author):\nThe authors have appropriately responded to prior reviewer critiques and the manuscript is improved by the revisions.\nWe thank the reviewer for finding the improvements in the revised manuscript and appreciate the positive feedback.\n\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe appreciate the reviewerʼs contribution and the initiative by Nature Communications to support training and recognition for Early Career Researchers. Thank you again for your involvement in the review process."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed all of my remaining concerns."}, "author_response": "We thank the reviewer for their positive assessment and greatly appreciate their helpful feedback during the revision process."}]

Decision letter