Syngap1 and the development of murine neocortical progenitor cells

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: minor_revision

Abstract
SYNGAP1 regulates synaptic plasticity through interactions with scaffold proteins and modulation of Ras and Rap GTPase signaling. Human SYNGAP1 mutations are linked to intellectual disability, epilepsy, and autism. In mice, Syngap1 haploinsufficiency causes impaired LTP, premature maturation of dendritic spines, learning disabilities, and seizures, reflecting the human phenotypes of SYNGAP1 syndrome. Recently, SYNGAP1 was shown to influence cortical neurogenesis and progenitor proliferation in human organoids. Here, we show that Syngap1 absence or haploinsufficiency does not alter neocortical progenitors and their cellular output in mice. Despite careful analysis of cortical progenitor properties, we fail to replicate the main findings from human organoids. This discrepancy suggests species-specific or methodological differences and raises questions about the broader relevance of SYNGAP1’s role in neurogenesis. The absence of cortical progenitor deficits in haploinsufficient mice, which exhibit cognitive deficits and seizures, indicates these arise from SYNGAP1’s regulation of synapse function rather than its role on neurogenesis.

Introduction
Synaptic Ras GTPase-activating protein 1 (SYNGAP1) is a critical postsynaptic protein predominantly expressed in the brain, where it plays a crucial role in synaptic plasticity, learning, and memory1,2. Since its discovery in the late 1990s3,4, SYNGAP1 has been identified as a major regulator of synaptic strength through its interaction with synaptic scaffold proteins and modulation of Ras and Rap GTPase signaling pathways. It is one of the most abundant proteins in the postsynaptic density (PSD)5–7, where it interacts with PSD-95 and other critical scaffolding proteins, positioning it as a pivotal component in regulating excitatory synapses. SYNGAP1’s GAP activity modulates Ras-ERK and PI3K-Akt signaling pathways, which can regulate synaptic plasticity mechanisms, including long-term potentiation (LTP)8.

Syngap1 heterozygous loss-of-function (LoF) mice present strongly impaired LTP, premature maturation of dendritic spines, and learning disabilities9–11. Later studies identified spontaneous seizure activity and seizure susceptibility in these mice12,13. Homozygous knockout mice died within days of birth9–11 coincident with the time frame when the expression of Syngap1 dramatically increases in the brain14–16. These structural and functional abnormalities underline the importance of SYNGAP1 in postnatal neurodevelopment and synapse regulation, supporting its role as a critical molecular hub in synaptic function. Following the identification of SYNGAP1’s role in synaptic plasticity, heterozygous LoF mutations in human SYNGAP1 were linked to a highly penetrant and severe neurodevelopmental disorder (SYNGAP1 syndrome; MRD5; SYNGAP1-related intellectual disability; SYNGAP1 encephalopathy)17–19. Patients with SYNGAP1 mutations often present with intellectual disability, epilepsy, and autism spectrum disorder. With more than 1400 genetically diagnosed patients worldwide and an estimated prevalence at around 6 out of 100,000 individuals20, SYNGAP1 has become a key target for understanding the molecular mechanisms that contribute to neurodevelopmental diseases and cognitive impairment.

The majority of these studies have focused on developmental stages after neuronal migration is complete and when excitatory synapses have started to emerge. More recently, studies have begun exploring the potential involvement of SYNGAP1 in earlier stages of neurodevelopment, including cortical development21,22. SYNGAP1 depletion leads to an increased ratio of neural progenitor cells to neurons and a reduction of ventricle and telencephalon size in Xenopus21. In human cortical organoids, SYNGAP1 haploinsufficiency accelerated neuronal maturation, which decreased the ratio of progenitor cells to neurons and resulted in a reduced ventricular area22. These findings suggest that SYNGAP1 regulates developmental processes during brain development at much earlier stages than previously thought.

In this study, we sought to determine the extent to which these results extend to cortical neurogenesis in mice. Using histological analysis of genetically modified mice carrying heterozygous and homozygous knock-out alleles for Syngap1 (Syngap1 KO), we did not observe significant alterations in the properties of cortical progenitors and the generation of neocortical cell layers. A comprehensive analysis of progenitor numbers, proliferative capacity, and division mode, along with quantifications of overall neocortical thickness and neuronal and glial cell counts at different developmental ages, did not replicate the key findings observed in human organoids. We thus conclude that Syngap1 is not essential for neocortical development in mice that otherwise recapitulate most aspects of SYNGAP1-related intellectual disability9–13. Our findings highlight potential species-specific or methodological differences and raise important questions about the broader applicability of SYNGAP1’s role in neurogenesis.

Results
Syngap1 levels do not affect the number of neocortical progenitor cells in mice
A recent study has proposed that SYNGAP1 plays a role in cortical neurogenesis by affecting the proliferation and differentiation of late progenitor cells in human organoids22. Cortical progenitors have been broadly divided into two classes named apical progenitors (APs = PAX6-positive cells) that undergo mitosis in the ventricular zone (VZ), and intermediate progenitors (IPs = TBR2-positive cells) that undergo mitosis in the subventricular zone (SVZ)23–28. Although SYNGAP1 is mainly expressed in neurons at postnatal ages14,15,29, we show by RNAscope in situ hybridization that some Syngap1 mRNA is detectable in Pax6-positive murine neocortical progenitor cells already at embryonic days 14.5 and 18.5 (E14.5 and E18.5; Supplementary Fig. 1a; Fig. 1a). However, we failed to detect the high expression in ventricle wall cells previously reported22. Analyses of previously published single-cell RNA sequencing datasets have confirmed that Syngap1 is expressed at very low levels during embryonic development in both mouse30 and human31 neocortex (Supplementary Fig. 1b–f’).

Quantitative analysis of histological sections revealed that at E18.5 the number of PAX6+ APs and TBR2+ IPs was not altered in the somatosensory cortex of heterozygous and homozygous Syngap1 KO mice (Fig. 1b, c; rostral→caudal axis – Supplementary Fig. 2a). The number of neocortical progenitor cells was also normal in mice carrying the GAP-AL mutation which eliminates the GAP activity of Syngap1 (Syngap1 GAP*; Supplementary Fig. 2c)8. In addition, the number of PAX6+ APs was not altered in the motor cortex of heterozygous and homozygous Syngap1 KO mice (Supplementary Fig. 2b), and the cortical thickness remained unaltered in Syngap1 KO and Syngap1 GAP* mice at E18.5 (Fig. 1d; Supplementary Fig. 2d).

Syngap1 levels do not affect the properties of progenitor cells in mice
To further investigate the potential role of SYNGAP1 in progenitor function, we injected EdU into pregnant mice at E18.5 and analyzed EdU incorporation into DNA 1 h later in the somatosensory cortex of heterozygous and homozygous Syngap1 KO mice (Fig. 2a, a’). Consistent with an unaltered number of progenitor cells, the number of EdU+ cells was also unaffected in the somatosensory cortex of heterozygous and homozygous Syngap1 KO mice at E18.5 (Fig. 2a). Next, we investigated cell cycle exit by injecting EdU into pregnant mice at E17.5 and analyzing the number of EdU+/Ki67− cells 24 h later (Fig. 2b, b’). The number of EdU+/Ki67− cells was not altered in the somatosensory cortex of heterozygous and homozygous Syngap1 KO mice at E18.5 (Fig. 2b, b’). We conclude that the cell cycle and proliferative capacity of neocortical progenitors are normal in heterozygous and homozygous Syngap1 KO mice.

In human cortical organoids, SYNGAP1 haploinsufficiency affects the division mode of progenitor cells22. In the VZ, depending on their cleavage plane, APs undergo either symmetric proliferative divisions (vertical=60–90° plane) or asymmetric differentiative divisions (oblique=30–60° plane and horizontal=0–30° plane, respectively)32–35. In addition, APs engage in two modes of neurogenesis termed direct and indirect neurogenesis23–28. During direct neurogenesis, APs divide asymmetrically to self-renew and to generate one neuron, while during indirect neurogenesis, APs divide asymmetrically to self-renew and generate an IP that then gives rise to neurons23–27. Although indirect neurogenesis generates neurons for all cortical layers, it is the predominant neurogenic mode that produces upper-layer projection neurons (ULNs)24,25,36–39. Defects in the division mode of neocortical progenitors alter the identity and topography of cortical neurons and can cause several neurological and psychiatric disorders28,40,41. In SYNGAP1 haploinsufficient human organoids, there is an increased proportion of progenitors undergoing differentiative division22. To determine if a similar phenotype was observed in mice, we analyzed the division planes of mitotic ventricular APs (PH3-positive cells) in heterozygous and homozygous Syngap1 KO mice at E18.5. Quantitative analysis of histological sections in the somatosensory cortex revealed a normal ratio of self-renewing and differentiative divisions in neocortical progenitors of heterozygous and homozygous Syngap1 KO mice (Fig. 2c). As a positive control, we analyzed Brn1 and Brn2 conditional knockout (Brn1/2-cKO) mice. Brn1 and Brn2 have previously been shown to regulate the proliferative capacity of cortical progenitor cells and the balance between direct and indirect neurogenesis42. In agreement with earlier findings, the ratio of progenitors to neurons was significantly altered in Brn1/2-cKO mice (Fig. 2c’), demonstrating that our analysis tools are sufficiently sensitive to detect changes in the biology of neocortical progenitor cells.

Neocortical progenitors generate neurons for different cortical layers in sequential order. Deep-layer neurons (DLNs) are generated prior to ULNs, which only begin to emerge in mice around E14.5. Subsequently, these progenitors start to generate glial cells around E16.543. To determine the extent to which Syngap1 affects the properties of progenitor cells during earlier developmental stages at the beginning of ULN generation, we analyzed markers of progenitor cells (PAX6 and TBR2) and proliferation (Ki67) in the somatosensory cortex of homozygous Syngap1 KO mice at E14.5. Immunohistochemical analysis revealed that the number of progenitor cells, as well as their proliferative capacity, was normal in homozygous Syngap1 KO mice at E14.5 (Fig. 2d, d’). Additionally, as a readout of cells actively going through indirect neurogenesis, we analyzed the number of TBR2+ cells that have incorporated EdU 1 h after injection. Levels of indirect neurogenesis were unaltered in the somatosensory cortex of homozygous Syngap1 KO mice at E14.5 (Fig. 2e, e’). Quantitative analysis also revealed a normal ratio of self-renewing and differentiative divisions in neocortical progenitors from the somatosensory cortex of heterozygous and homozygous Syngap1 KO mice at E14.5 (Fig. 2f; Supplementary Fig. 1g). Altogether, these results show that the neocortical properties of progenitor cells are normal in the absence of Syngap1 in mice.

Syngap1 levels do not affect the number of neurons and glial cells generated in mice
To further determine the extent to which Syngap1 may have essential roles during the formation of neocortical cell layers, we analyzed numbers of ULNs, DLNs, and astrocytes in heterozygous and homozygous Syngap1 KO mice at P0 and P13. Immunohistochemical analysis of CTIP2 (highly expressed in DLNs) and BRN2 (highly expressed in ULNs at P0) did not reveal any differences in the numbers of CTIP2+ and BRN2+ neurons in the somatosensory cortex of heterozygous and homozygous Syngap1 KO mice compared to wild-type at P0 (Fig. 3a). Numbers of CTIP2+ and BRN2+ neurons were also normal in Syngap1 GAP* mice at P0 (Supplementary Fig. 2e, e’). In addition, we observed normal numbers of astrocytes (SOX9-positive cells) in the somatosensory cortex of heterozygous and homozygous Syngap1 KO mice at P0 (Fig. 3b, b’). Immunohistochemistry analysis of CTIP2 and CUX1 (highly expressed in ULNs at P13) revealed normal numbers of DLNs and ULNs in the somatosensory cortex of heterozygous Syngap1 KO mice at later postnatal ages (P13; Fig. 3c). Numbers of astrocytes were also unaltered at P13 in these mice (Fig. 3d; Supplementary Fig. 2f).

To further investigate if the late progenitors are generating normal levels of neurons and glial cells, we analyzed cell fate by injecting EdU at E18.5 and quantifying the number of EdU-positive cells expressing the indicated cellular markers in the somatosensory cortex of control and heterozygous Syngap1 KO mice at P13. Syngap1 deficiency did not affect the ratio of EdU-positive ULNs (EdU+ CUX1+ cells) or EdU-positive glial cells (EdU+ SOX9+ cells) in mice (Fig. 3d, d’). In addition, analysis of a previously published whole-brain RNA sequencing dataset16 shows that the expression of neurodevelopmental marker genes and cortical cell layer/type-specific marker genes is unaltered in Syngap1+/− and Syngap1+/c.3583-9G>A haploinsufficient mice (Supplementary Fig. 3).

Overall, these results show that heterozygous and homozygous Syngap1 KO mice have normal numbers of progenitor cells that proliferate and differentiate to generate appropriate numbers of neurons and glia. Interestingly, the migration of ULNs was slightly affected in both homozygous Syngap1 KO and homozygous Syngap1 GAP* mice (arrowheads on Fig. 3a and Supplementary Fig. 2e; Supplementary Fig. 2g, h), suggesting that Syngap1 may play a role in the migration and circuit integration of ULNs and that this effect is GAP activity dependent.

Methods
All the experiments reported in this study adhered to the ethical regulations and guidelines of the National Institute of Health Guide for Care and Use of Laboratory Animals. The experiments involving mice were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University School of Medicine under the study protocol numbers: MO22M217, M023M70, and MO23M52.

Mice
Mice were maintained on a 14 h light/10 h dark cycle. Both male and female mice were used, and no obvious differences between the sexes were noted. All mice were group-housed in pathogen-free facilities with regulated temperature and humidity and given ad libitum access to food and water. All mice used were seemingly free of infection, health abnormalities, or immune system deficiencies, and were employed independently of their gender. None of the mice used had been used for previous experiments. The date of the vaginal plug detection was designated E0.5, and the date of birth P0. Syngap1+/− heterozygous knockout mice10, Syngap1 GAP mutants (+/GAP*, GAP*/GAP*)8, and WT (Control) littermates were maintained on a mixed of C57/B6J and 129S background. To maintain mutants on the mixed genetic background, we chose breeders randomly from within a colony and backcrossed the offspring to F1 hybrids between the two strains that comprise the mixed background (in this case, C57BL/6 and 129 F1 hybrids) about every 10 generations. Emx1-Cre [B6.129S2-Emx1tm1(cre)Krj]51 and Brn1/2 conditional knockout mice have been described42. CD1 time-pregnant wild-type mice were obtained from Charles River Laboratories.

Animal euthanasia
We used the most humane methods for euthanasia following the recommendations of the panel report on Euthanasia of the American Veterinary Medical Association and the guidelines of the National Institute of Health Guide for Care and Use of Laboratory Animals. Different methods were used for mice of different ages, according to the experiments that needed to be performed: neonatal and adult mice — isoflurane inhalation followed by cervical dislocation; newborn mice — chilling and decapitation; embryos 14 and 18 days post coitum — chilling and decapitation; newborn to adult mice — cardiac perfusion under full anesthesia.

RNAscope multiplex in situ hybridization
Brain sections were processed for multiplex fluorescent in situ hybridization RNAscope52 following the manufacturer’s instructions (#323110, Advanced Cell Diagnostics). Gene-specific probes: Syngap1 (417381), Pax6 (412821-C2).

Single-cell RNA sequencing analysis
To analyze the expression of Syngap1 in mice, we reanalyzed the available dataset from mouse cerebral cortex30. The same Uniform Manifold Approximation and Projection (UMAP) coordinates from the publication were used for the feature plot. For the cell type classification, apical progenitors from the original paper were classified as ventricular radial glial cells (vRG), intermediate progenitors were classified as IP, and the neurons were classified as excitatory neurons (EN.) The total Unique Molecular Identifiers (UMI) in each cell was scaled by a factor of 10000, and the count was log-transformed after adding a pseudocount of 1 for the feature plot. The colorbar of the feature plot was scaled by the minimum and maximum expression of a certain gene. To analyze the expression of SYNGAP1 in humans, we reanalyzed the available dataset from human neocortex31. The cell identity was assigned by the original metadata, with all the excitatory neuronal cell types grouped as EN and all other non-neuronal cell types grouped as other. A new UMAP was learned to visualize the feature plot of cell identities (vRG, outer radial glial cells (oRG), IP, EN, and others). The total UMI in each cell was scaled by the median total UMI among all cells, and the count was log-transformed after adding a pseudocount of 1. The normalized counts were scaled to zero mean and unit variance to learn 50 PCAs. Among them, the top 16 were used to learn the UMAP with 30 nearest neighbors and a min_dist of 0.3. The log-transformed data were used to represent each gene in the feature plot, and the color bar of the feature plot was scaled by the minimum and maximum expression of a certain gene. For the dot plots, the size of each circle was scaled to the proportion of cells with positive gene expression. The expression level of each gene was normalized within each cell group and shown by different colors of the circle. To analyze the expression of cellular marker genes in two different Syngap1 haploinsufficiency mouse models, we analyzed the available dataset from Araki et al.16. Differential expression at the gene level was determined using DESeq2 with a false discovery rate of 0.1 (Benjamini–Hochberg correction) and absolute log2 fold change value threshold of 0.1, correcting for rRNA ratio and sex.

Immunohistochemistry
Embryonic brains were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 2–4 h at 4°C. Postnatal mice were transcardially perfused with 20 ml ice-cold 4% PFA using a peristaltic pump at a rate of 2 ml/min. Brains were removed from the skull and post-fixed in 4% PFA overnight at 4°C. Embryonic brains were cryo-preserved with 30% sucrose, embedded in Tissue-Tek O.C.T. compound (#4583, SAKURA), and frozen at −80°C. They were then sectioned coronally at 16 µm with a cryostat (CM3050S, Leica). Postnatal brains were embedded in 3% low-melting-point agarose (#R0801, Thermo Fisher Scientific) in PBS and sectioned coronally at 60 µm with a vibrating microtome (VT1200S, Leica). For immunohistochemistry, brain sections were permeabilized in PBS containing 0.2% Triton X-100 and blocked in 10% goat serum for 1 h at room temperature. Brain slices were then incubated with primary antibodies in blocking solution overnight (or 48 h, depending on the thickness of the section and antibody used) at 4°C and subsequently incubated with the appropriate secondary antibodies diluted in blocking solution for 2 h at room temperature. To label all cell nuclei, the fluorescent nuclear dye DAPI (1 μg/ml, #D9542, Sigma-Aldrich) was included with the secondary antibody solution. The sections were mounted with ProLongTM Gold (#P36930, Thermo Fisher Scientific). Primary antibodies: anti-CTIP2 (1:1000, rat monoclonal, ab18465 Abcam, clone 25B6, current lot: GR322373-4); anti-CUX1 (1:1000, rabbit polyclonal, 11733-1-AP Proteintech, current lot: 00050220); anti-BRN2 (1:1000, rabbit monoclonal, 12137 Cell Signaling, clone D2C1L); anti-Ki67 (1:500, rabbit polyclonal, ab15580 Abcam, current lot: GR3285096-1 or 1:500, rat monoclonal, 14-5698-82 Thermo Fisher Scientific, clone SolA15, current lot: 2496198); anti-PAX6 (1:250, mouse monoclonal, MA1-109 Thermo Fisher Scientific, clone 13B10-1A10, current lot: XD341788 or 1:500, rabbit polyclonal, 901301 Biolegend, clone Poly19013, current lot: B343290); anti-TBR2 (1:500, rat monoclonal, 14-4875-82 Thermo Fisher Scientific, clone Dan11mag, current lot: 2488481 or 1:500, rabbit polyclonal, ab183991 Abcam, clone EPR19012, current lot: GR3237130-1); anti-SOX9 (1:1000, rabbit monoclonal, ab185966 Abcam, clone EPR14335-78 current lot: GR3194558-4); anti-PH3 (1:1000, rat monoclonal, ab10543 Abcam, clone HTA28, current lot: 1052214-1); anti-γ-TUBULIN (1:500, goat polyclonal, A. Holland gift53); anti-Centrin (1:500, rabbit polyclonal, A. Holland gift54). Secondary antibodies: Alexa 555, 647, 488 anti-rat (A21434; A21247, A11006), anti-rabbit (A21430; A21246), and anti-mouse (A21425; A21237) and Alexa 488 anti-rabbit (A11070); all diluted 1:500 and all from Thermo Fisher Scientific. Antigen retrieval55 was performed for all immunohistochemistry at embryonic ages. The method used here was a heat-induced citrate method55. The sections were then mounted with ProLongTM Gold (P36930, Thermo Fisher Scientific).

EdU injections and labeling
EdU (5-ethynyl-2′-deoxyuridine; #A10044, Thermo Fisher Scientific) was diluted at 10 mg/ml in 0.9% NaCl (#114-055-101, Quality Biological) and administered in pregnant females at the specified embryonic pregnancy stage at 50 mg/Kg body weight. For proliferation analysis, a single intraperitoneal EdU injection was administered at E18.5, embryos were fixed after 1 h, and the number of EdU+ cells was quantified. For cell cycle exit calculation, a single intraperitoneal EdU injection was administered at E17.5; embryos were fixed after 24 h, and the percentage of EdU+ cells labeled with Ki67 was calculated. For cell-fate analysis, a single intraperitoneal EdU injection was administered at E18.5, and the number of EdU+ cells expressing the indicated cellular markers were analyzed at P13. EdU was labeled using Click-iT® EdU imaging kits with Alexa 555 (#C10638, Thermo Fisher Scientific). Briefly, brain sections were post-fixed with 3.7% formaldehyde and permeabilized with 0.5% Triton X-100 in PBS. The Click-iT® reaction cocktail was then added, and the sections incubated in the dark for 30 min at room temperature. After the washes, the brain sections were stained for the different proteins of interest using the immunohistochemistry protocol previously described.

Cleavage plane analysis
PH3 immunostaining was used to identify mitotic cells. γ-TUBULIN immunostaining was used to determine the position of both centrosomes (γ-TUBULIN positive) in mitotic cells and to mark the cleavage plane in relation to the ventricular surface. The angle of the cleavage plane was measured using ImageJ.

Imaging
All immunohistochemistry and ISH RNAscope data were acquired in an equivalent latero-medial level of the cortex from at least 3 independent animals per condition or genotype. Images were captured using a Zeiss LSM 800 confocal microscope. Maximum intensity projections were generated in ImageJ or Imaris software. In all fluorescence microscopy figures, different channels of image series were represented in pseudocolor, and contrast and brightness were adjusted manually using ImageJ or Adobe Photoshop software. All markers were counterstained with DAPI to allow visualization of overall cellular density.

Histological analysis
A series of z-stack (depth of 10 μm) confocal tiled images were used for cell quantifications; 150 μm-wide images (embryonic brains) or 300 μm-wide images (postnatal brains) comprising the entire extension of the cortex were analyzed. All quantifications were made in an equivalent latero-medial level of the cortex from at least 3 independent animals per condition or genotype. The number of cells expressing the markers of interest was quantified in radial sections of the same latero-medial level and adjusted per area. Cortical thickness was analyzed by measuring the length from the lower limit of the VZ to the upper limit of the CP. PAX6+ or TBR2+ cortical thickness was analyzed by measuring the length occupied by PAX6+ or TBR2+ cells from the lower limit of the VZ into the CP. To analyze the distribution of BRN2+ cells, we divided the cortex into longitudinal bins, and a rectangle was drawn from the border of the white matter with layer VI to the pial surface. This rectangle was then divided into ten equal bins, and the number of cells within each bin was quantified.

Statistical analysis
Statistical analyses of the immunohistochemistry experiments were done using GraphPad Prism software. Statistically significant differences were assessed by Student’s unpaired t-test or one-way analysis of variance (one-way ANOVA Dunnett’s multiple comparisons test; F DFn, DFd (DFn: degrees of freedom in the numerator; DFd: degrees of freedom in the denominator)), comparing two or more groups, respectively. To compare the distribution of BRN2+ cells in the different bins of the cortex for the different genotypes, we used two-way analysis of variance (two-way ANOVA-Šídák’s multiple comparisons test; F DFn, DFd). P<0.05 was considered a significant difference. All values represent individual animals’ mean ± standard error of the mean (SEM). The statistical test used and the statistical significance are indicated in figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors investigate whether mutations in the neurodevelopmental disorder (NDD) risk gene Syngap1 affect cortical neurogenesis in mice. While SynGAP was originally identified as a major component of the post-synaptic density and a synaptic regulator, recent studies have suggested that it may also regulate early neurodevelopment. With the advent of human brain organoid modeling, many studies have demonstrated alterations in neuronal differentiation and/or maturity in organoids with mutations in NDDs or other psychiatric disorder risk genes. This is somewhat unexpected given that the brains of individuals with these disorders is thought to be grossly normal (i.e. no major cortical developmental abnormalities have been reported). It is therefore important to validate these findings in other systems, including in animal models. Here the authors find no evidence for defects in corticogenesis resulting from Syngap1 loss of function mutations in mice. This important analysis demonstrates that there are either species differences in how these mutations affect brain development and function and/or that brain organoid models may be overly sensitive in detecting abnormalities and that perhaps, in vivo, compensatory systems are engaged to normalize development.\nStrengths:\nThe experiments are well done and are appropriate to address the question. It is appreciated that multiple embryonic and post-natal time points were assessed. It is also a strength that multiple Syngap1 mouse models were investigated. The findings are important and provide a counterpoint to recent studies showing large changes in early neurodevelopment resulting from Syngap1 mutations.\nWeaknesses:\nThe only weakness I see with this study is that it is somewhat limited in scope. While showing that there are not major changes in NPC proliferation/differentiation in mouse models of Syngap1 deficiency is an important conclusion, the scope of the experiments and analyses is limited. This is an editorial call, but if the authors wanted to expand the scope, they could consider following up on the potential migration defect they observed and/or consider performing scRNA-seq as this is a very sensitive method for detecting changes in gene expression during development. If Syngap1 does play any role in early development or cell type proportions, it should be detectable by scRNA-seq. If not, then it strongly rules out changes in development in mice as contributing to their phenotypes.\nSpecific points:\nI appreciate that this study was motivated by two prior studies by Willsey and Birtele and that the conclusions of this study differ from those. The authors have done a good job of presenting their opposing view without being overly confrontational. That said, a minor suggestion would be to not refer to the first authors’ names when referring to these studies. Rather, the authors might consider refer to them as the study in Xenopus or the study in brain organoids, so as not to single out those authors in particular.\nThe authors have focused on somatosensory cortex, but it is possible that other cortical regions could be affected by Syngap1 loss. Examining one other cortical region at one of the time points would strengthen their argument.\nWere both male and female mice used in this study? What was the sex of the cells from which the brain organoids were derived?\nMinor points:\nLine 41: The authors write “Research in knockout mice…”. Perhaps just say “Research in Syngap1 heterozygous loss-of-function mice…”\nIt is unclear why the authors start with E18.5 and then go back to E14.5, it seems more logical to start with the earliest time point and then present later time points. Related to this, it would be helpful if the authors could comment on the correspondence in developmental timing between their mouse models and the human brain organoids in the brain organoid study (Birtele et al).", "reviewer_2": "This is an important follow-up manuscript published by Giorgio Quadrato and colleagues in 2023 describing a potential non-synaptic function for SYNGAP1. As stated by the authors of this manuscript, SYNGAP1 was discovered by the senior author as one of the most abundant synaptic proteins over 20 years ago. Mutations in the SYNGAP1 gene cause a developmental and epileptic encephalopathy in humans. The manuscript by Quadrato used primarily human organdies to suggest that neurogenesis is abnormal with mutations in SYNGAP1 and then reported a similar finding in mice. In this manuscript by Huagnir and colleagues, the authors deeply investigate this finding in mice and come to a differing conclusion that neurogenesis in both Syngap1 haploinsufficient and complete loss of function have no clear deficits in neurogenesis. The experiments are very well done, data well presented and manuscript well written. The authors for the most part conclude that either differences between humans and mice or differences in experimental design lead to the differing results. I have only 2 major point that need to be addressed and a few minor observations.\nMajor points:\n1. In the discussion, the authors state on the bottom of page 9 to beginning of page 10 that \"the observed differences may have resulted from a comparison between different regions of the rostral-caudal axis in control and Syngap1 KO brains\" in reference to the mouse findings of Quadrato and colleagues. This is the only mention of a methodological issue with the Quadrato manuscript and does not seem to be supported by any data that is presented. Is this merely speculation or is there evidence to support this. If only speculation, then it should either be taken out or very clearly stated as mere speculation. If there is evidence of this, it should be provided.\n2. In extended Figure 2, part C, the authors present evidence that there is no difference in cortical thickness between CT, GAP/GAP and GAP/WT. However in closely examining the data and statistical analysis, it seems there may in fact be a difference for which this experiment was underpowered to detect. I would like to see this experiment repeated following a power analysis to determine the number of mice needed to detect the 10-15% difference that appears to be present.\nMinor points:\n1. In the last line of the abstract, \"SYNGAP\" is used. I think the authors want to change this to SYNGAP1.\n2. On page 33, in extended Figure 2C figure legend, the authors say \"T vs GAP*/GAP*...\" I believe the T should be CT.\nThank you for the opportunity to review this manuscript.", "reviewer_3": "In this study, Barão et al. test the hypothesis that non-synaptic mechanisms of SYNGAP1-deficiency affect brain development in mice, as has been suggested by recent studies in other organisms. In three figures, the authors quite convincingly demonstrate that there are no changes in the number of neuroprogenitors, proliferation of neuroprogenitors, and the numbers of neurons and glia generated in the Syngap1 knockout mice. These studies are clearly described and well-controlled.\nOne question is whether there is an alternative transcript of Syngap1 that is not dependent on the deleted exons and continues to be expressed in the knockout animals. Discussion of known alternative transcripts and other possible starts might be helpful. Another possibility would be to examine RNA sequencing data from these animals. The investigators could evaluate the transcripts originating from this locus and determine whether this is a possibility.\nThe other question is whether this is a true species difference or is an unrelated effect of the techniques used in the other studies. The authors might be able to analyze the trajectory of SYNGAP1 in human and mouse neuroprogenitors from available expression data to determine whether there might be a difference that accounts for this discrepancy.\nThird, there appears to be some differences between the mouse background strains used in this manuscript vs those studied in Birtele et al paper. It is possible, and in fact common, that some phenotypes of a pathogenic variant (in this case in Syngap1) can only be detected in a certain mouse strain background but not in others. Adding more details of the mixed background used in this paper to provide a more detailed way to compare the differences between the two papers and adding this possibility to the discussion would be helpful.\nFinally, the literature on the brain/head size of patients with SYNGAP1-related intellectual disability (SYNGAP1-ID) is mixed. There appears to be a subset of patients with microcephaly while this is not a uniform manifestation of this condition. It would be helpful to add some comments related to the significance of these mouse findings in the setting of the clinical picture."}, "author_response": "We thank the reviewers for their insightful comments and the valuable time they invested in their reviews. We are pleased to report that the resulting changes and additions have strengthened the manuscript and have contributed to a better clarification of the role of SYNGAP1 in murine neocortical progenitor cells. Below we provide a detailed response to the reviewer comments.\nSummary of the main changes to the original manuscript:\n- The text was adapted to not include the name of the authors of the previous studies.\n- Analysis of PAX6+ progenitor cells in the motor cortex were included in Supplementary Figure 2b. These results further strengthened our findings showing that the number of PAX6+ progenitor cells also remains unaltered in the motor cortex of these Syngap1 mouse models.\n- Single-cell RNA sequencing analyses were done using previously published datasets from mouse (Di Bella et al. 2021)31 and human neocortex (Polioudakis et al. 2019)32 to better represent the expression patterns of Syngap1 in the two species and support the discussion of the manuscript. These analyses are now part of Supplementary Figure 1. Overall, the expression of Syngap1 is extremely low during embryonic development in both mouse and human neocortex.\n- Whole-brain RNA sequencing analyses to characterize the expression of neurodevelopmental marker genes were done using a previously published dataset from two different Syngap1 haploinsufficient mouse models originally reported in Araki et al., 202316. These analyses are now included in Supplementary Figure 3. In line with the results we reported, this analysis shows that an extensive list of cortical layer and cellular markers are unaltered in Syngap1+/- and Syngap1+/c.3583-9G>A haploinsufficient adult mice.\n- We have confirmed that all analyses passed the normality tests. Two analyses had to be improved: In Figure 2c, the number of cells per animal was set to a minimum of 20 to guarantee the normality of the analysis; In Supplementary Figure 2d (previous Supplementary Figure 2c) and following the remark number 2 from reviewer #2, the number of animals analyzed per group was increased to guarantee the normality of the analysis and make sure we could detect a 10-15% difference if present. Similar to what we have reported in the original manuscript, no statistical differences of cortical thickness were found for Syngap1GAP*/GAP* or Syngap1+/GAP* when compared to the control.\n- In the new version of the manuscript, we have replaced the multiple unpaired t-test by the one-way ANOVA followed by Dunnett's post-hoc test when we analyzed more than two groups to guarantee a statistically rigorous comparison of each mutant to the control group. This approach did not change any of our previous conclusions. The methods, the figure legends, and the DATA source file were updated accordingly.\nResponse to reviewer comments\nReviewer #1 (Remarks to the Author):\n[...]\nWe have taken multiple approaches to address this concern. First, although we fully agree that a single-cell RNA sequencing experiment at different developmental stages in Syngap1 haploinsufficient mice would be a more elegant way to clarify SYNGAP1’s role in neurodevelopment, we re-analyzed the whole mouse brain bulk RNA-sequencing results from Araki et al., 202316 in two different Syngap1 mouse models (Supplementary Figure 3). In line with the results we obtained in this manuscript, this analysis shows that an extensive list of cortical layer and cellular markers are unaltered in Syngap1+/- and Syngap1+/c.3583-9G>A haploinsufficient adult mice, supporting our results that Syngap1 loss-of-function does not affect the specification of the different cell types during early neurodevelopment.\nSecond, to confirm the migration phenotype we observed in Syngap1KO and Syngap1GAP*/GAP* mice, we knocked down Syngap1 by in utero electroporation with Syngap1 shRNA in mice at E16.5 and analyzed the cell fate of the electroporated cells at P13. Syngap1-deficiency did not affect the ratio of RFP-positive ULNs (RFP+CUX1+ cells) or RFP-positive glia cells (RFP+SOX9+ cells) in mice (Revision Figure 1). In these experiments, we did not observe migration defects in the electroporated cells, suggesting SYNGAP1’s role in migration is non-cell-autonomous or developmental stage specific (arrowheads). Nonetheless, more studies are needed to fully understand the role of SYNGAP1 in neuronal migration.\nRevision Figure 2. Syngap1 knocked down by in utero electroporation of progenitor cells did not change their neuron/glia ratio or migration. Cortical cell identities of the control and Syngap1-shRNA condition immunolabeled for RFP (red), CUX1 or SOX9 (grey) at P13 after in utero electroporation (IUE) of wild-type mice at E16.5 (n=4 CT, n=4 Syngap1-shRNA; two-sided unpaired t-test: CUX1 p=0.9289; SOX9 p=0.6387). Arrowheads highlight the correct position of the RFP+ neurons. DL, Deep Layer; UL, Upper Layer; WM, White Matter. Values are mean  SEM; ns, not significant. Scale bars: 100 µm (P13).\nSpecific points:\nI appreciate that this study was motivated by two prior studies by Willsey and Birtele and that the conclusions of this study differ from those. The authors have done a good job of presenting their opposing view without being overly confrontational. That said, a minor suggestion would be to not refer to the first authors’ names when referring to these studies. Rather, the authors might consider refer to them as the study in Xenopus or the study in brain organoids, so as not to single out those authors in particular. The text was changed to incorporate the reviewer’s suggestion along the manuscript.\nThe authors have focused on somatosensory cortex, but it is possible that other cortical regions could be affected by Syngap1 loss. Examining one other cortical region at one of the time points would strengthen their argument. To address this concern, we have quantified the number of PAX6+ cells in the motor cortex of Control (CT), heterozygous (+/-) and homozygous (-/-) Syngap1KO brains at E18.5. These results were incorporated in Supplementary Figure 2 – now panel b. The results further strengthened our findings showing that the number of PAX6+ progenitor cells also remain unaltered in the motor cortex of these Syngap1 mouse models.\nWere both male and female mice used in this study? What was the sex of the cells from which the brain organoids were derived? We now report on gender in the reporting summary. Briefly, we have not determined sex for embryonic or postnatal day 0 samples. For P13 analysis, since no differences have been reported between males and females for the analyzed markers in our previous studies, we have included both sexes in these analysis - Figure 3c and 3d: control = 3 males and 1 female; Syngap1+/- = 2 males and 2 females. Birtele et al.22 used iPSCs derived from mutant and control males to generate the human organoids.\nMinor points:\nLine 41: The authors write “Research in knockout mice…”. Perhaps just say “Research in Syngap1 heterozygous loss-of-function mice…” The text was changed to incorporate the reviewer’s suggestion in the revised manuscript.\nIt is unclear why the authors start with E18.5 and then go back to E14.5, it seems more logical to start with the earliest time point and then present later time points. Related to this, it would be helpful if the authors could comment on the correspondence in developmental timing between their mouse models and the human brain organoids in the brain organoid study (Birtele et al). It is still challenging to establish the exact age equivalent between the time of the organoid cultures and the developmental time of the human brain and even more so to link it to the developmental time of the mouse brain. A 2-month-old brain organoid used by Birtele et al.22 roughly corresponds to the early to mid-fetal brain development stage in humans. Specifically, it may be comparable to prenatal stages in human fetal brain development (10-38 weeks post-conception). This is a vast period for human brain development and therefore also very imprecise when compared to mouse brain development but roughly corresponds to the end stage of embryonic development in mice (E17-E18). Taking this into consideration, we focused the manuscript mainly on the study of E18 brains similar to the age presented by Birtele et al. for the mouse DATA22. We then included E14.5 experiments to understand whether Syngap1 would play a role in the neurogenesis of upper layer neurons and whether in those progenitors its role could be more evident. To address the reviewer concern, we have added modifications in the manuscript to make this reasoning clearer.\nReviewer #2 (Remarks to the Author):\n[...]\nMajor points:\n1. In the discussion, the authors state on the bottom of page 9 to beginning of page 10 that \"the observed differences may have resulted from a comparison between different regions of the rostral-caudal axis in control and Syngap1 KO brains\" in reference to the mouse findings of Quadrato and colleagues. This is the only mention of a methodological issue with the Quadrato manuscript and does not seem to be supported by any data that is presented. Is this merely speculation or is there evidence to support this. If only speculation, then it should either be taken out or very clearly stated as mere speculation. If there is evidence of this, it should be provided. Because the brain sections shown in Figure 5 panels e and f of Birtele et al.22 are clearly taken from different rostral-caudal levels in control and Syngap1KO brains, we can speculate that this discrepancy contributed to the statistical differences reported in panels g and h. Notably, when sections from comparable rostral-caudal levels were analyzed (Extended Data Figure 6, panels h–j), Birtele et al.22 reported no significant differences. Nonetheless, we fully agree with the reviewer’s point, and given that our original statement was speculative, we have removed it from the manuscript.\n2. In extended Figure 2, part C, the authors present evidence that there is no difference in cortical thickness between CT, GAP/GAP and GAP/WT. However, in closely examining the data and statistical analysis, it seems there may in fact be a difference for which this experiment was underpowered to detect. I would like to see this experiment repeated following a power analysis to determine the number of mice needed to detect the 10-15% difference that appears to be present. Taking into consideration this concern, we have confirmed the normality for all the analysis presented in the manuscript and have increased the n in few analyses to guarantee the statistical tests passed the normality test – Figure 2c: number of cells per condition was set to a minimum of 20 to guarantee the normality of the presented analysis; Supplementary Figure 2d (previous Supplementary Figure 2c): number of animals analyzed per group was increased to guarantee the normality of the analysis and make sure we can detect a 10-15% difference if present. In the experiment that the Reviewer 2 mentioned, after power analysis we determined we needed to increase the n for all the groups to be able to confidently detect a 10-15% difference if present (see power analysis reported below – Revision Figure 2). The new analysis is now included in Supplementary Figure 2d (previous Supplementary Figure 2c). Similar to what we have reported before, no statistical differences of cortical thickness were found for Syngap1GAP*/GAP* or Syngap1+/GAP* when compared to the control.\nRevision Figure 2. Power analysis for a two-sample t-test based on the results previously reported in Supplementary Figure 2c\nMinor points:\n1. In the last line of the abstract, \"SYNGAP\" is used. I think the authors want to change this to SYNGAP1. The text is now corrected in the revised manuscript.\n2. On page 33, in extended Figure 2C figure legend, the authors say \"T vs GAP*/GAP*...\" I believe the T should be CT. The text is now corrected in the revised manuscript.\nReviewer #3 (Remarks to the Author):\n[...]\nOne question is whether there is an alternative transcript of Syngap1 that is not dependent on the deleted exons and continues to be expressed in the knockout animals. Discussion of known alternative transcripts and other possible starts might be helpful. Another possibility would be to examine RNA sequencing data from these animals. The investigators could evaluate the transcripts originating from this locus and determine whether this is a possibility.\nWe examined the transcript expression of Syngap1+/- mice extensively previously16 and here to address the reviewer’s question. Overall, there is likely little protein arising from the deleted allele. Syngap1+/− mice were previously generated by deleting the core exons 6 and 7, which shifts the reading frame and leads to reduction of Syngap110. Western blotting of protein extracted from the whole brain of Syngap1+/- mice revealed approximately 50% less Syngap1 protein expression (Revision Figure 3a). Northern blots with a Syngap1 probe targeting exons 8-11 (and a control S26 RNA probe) show a similar reduction of Syngap1 expression and no significant other isoform bands (Revision Figure 3b). Quantitative PCR (qPCR) using exon-exon junction assays on whole-brain mRNA extracts from Syngap1+/- mice revealed a ~40% reduction of mRNA in comparison to wild-type controls (Revision Figure 3c), suggesting that mutant Syngap1 mRNA is subject to nonsense-mediated decay (NMD) as expected due to the premature stop codon. We verified in bulk brain RNA-seq of these mice that exons 6 and 7 are skipped in a large portion of transcripts, which are not completely degraded through NMD (Revision Figure 3c). Perhaps due to this incomplete NMD and significant intron retention, total estimated Syngap1 RNA expression (which includes intron-including pre-mRNA) is not significantly lower in these mice compared to wild-type littermates (Revision Figure 3e). The difference with qPCR and Northern blot quantification could be due to transcripts with intron retention and immature polyA tails, which affect these measures differently. Given the halved Syngap1 protein expression and absence of substantial truncated protein species (Revision Figure 3a), this suggests that the transcripts from the knockout allele may be protected from NMD to an extent but are nevertheless translationally inactive. Substantial intron retention of intron 7 in Syngap1+/− mice (Araki et al. PNAS 2023 SI Appendix, Figure S3) further suggests that nuclear retention of unspliced pre-mRNA may contribute to this resistance to NMD, which occurs in the cytosol only after mRNA maturation. Alternative splicing events, including exon 14 skipping, exon 17 5′ extension (VK), exon 18 5′ extension (β vs. non-β), exon 18 3′ extensions, and exon 19 skipping (α1, α2, γ), did not show significant changes in ratio (Araki et al. PNAS 2023 SI Appendix, Figure S2 B–E). As the reviewer suggested, whereas an N-terminal splice junction unique to full-length Syngap1 (A1/2/4; exon 3-4) was significantly reduced in both Syngap1+/c.3583-9G>A (P = 0.0210, two-way ANOVA and Šídák’s post hoc test) and Syngap1+/− mice (P = 0.0437), other isoforms, including the second-most abundant D isoform, did not show a significant RNA reduction (Revision Figure 3f). This suggests that these N-terminal isoforms may be subject to less efficient NMD. The less pronounced downregulation of these N-terminal isoforms might contribute to the <50% decrease observed in Syngap1 mRNA levels (Revision Figure 3c). These transcripts nevertheless are minor isoforms and likely contribute a small amount of Syngap1 protein (Revision Figure 3a and b).\nRevision Figure 3. mRNA and protein measurements in Syngap1+/- mice. a Western blot of Syngap1+/- and Syngap1+/L813fsX mice brain samples. b Northern blot of Syngap1+/- mouse brains with a Syngap1 probe targeting exons 8-11 and a control S26 RNA probe. c qPCR of Syngap1+/- mice brain samples using exon-exon junction assays. d Exon junction-spanning read abundance in bulk brain RNA-seq of Syngap1+/- mice. Exons 6 and 7 are skipped in exon 5-8 junction reads. e RNA-seq measurement of total Syngap1 expression (which includes intron-including pre-mRNA) is not significantly lower in these mice compared to wild-type littermates. f Exon junction-spanning read abundance of N-terminal splice isoforms of Syngap1 (A1/2/4; A3; B; D; E isoforms).\nThe other question is whether this is a true species difference or is an unrelated effect of the techniques used in the other studies. The authors might be able to analyze the trajectory of SYNGAP1 in human and mouse neuroprogenitors from available expression data to determine whether there might be a difference that accounts for this discrepancy. To address this question, we have analyzed previously published single-cell RNA sequencing datasets from mouse31 and human32 neocortex and included this analysis in Supplementary Figure 1. In support of our findings, the expression levels of Syngap1 are very low in all cell types analyzed during embryonic development in both human and mouse neocortex.\nThird, there appears to be some differences between the mouse background strains used in this manuscript vs those studied in Birtele et al paper. It is possible, and in fact common, that some phenotypes of a pathogenic variant (in this case in Syngap1) can only be detected in a certain mouse strain background but not in others. Adding more details of the mixed background used in this paper to provide a more detailed way to compare the differences between the two papers and adding this possibility to the discussion would be helpful. The Syngap1KO mice used in this manuscript are the same mice and under the same genetic background as the mice used by Birtele et al.22 in their study. We have added details and made this clear in the revised manuscript.\nFinally, the literature on the brain/head size of patients with SYNGAP1-related intellectual disability (SYNGAP1-ID) is mixed. There appears to be a subset of patients with microcephaly while this is not a uniform manifestation of this condition. It would be helpful to add some comments related to the significance of these mouse findings in the setting of the clinical picture. Microcephaly and structural brain anomalies are very rare in SYNGAP1 patients, which aligns with our findings showing no brain size abnormalities in Syngap1 haploinsufficient mice that model key aspects of SYNGAP1-related intellectual disability. Similarly, despite observing changes in the ratio of cell types, Birtele et al.22 reported no overall size differences in the analyzed human organoids. We have revised the manuscript to clarify this point and emphasize that the absence of structural phenotypes in our model is consistent with observations in SYNGAP1 patients."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my main comments and I believe the data are convincing. The inclusion of the sequencing analyses bolsters the conclusions that Syngap1 mRNA is lowly expressed during embryonic development and that there are not major changes in cortical cell type proportions as determined by levels of key cell type marker genes. I just have a few minor comments:\n1) One of the other reviewers mentioned the possibility of another isoform of Syngap1 being expressed in the mouse models that could account for the lack of effect on early development. The authors provided a thorough response in the rebuttal but I think it would be worth discussing this more in the manuscript itself.\n2) There may be a word missing in the sentence in lines 199-200: do the authors mean \"undergoing differentiative division\"?\n3) In line 200 – perhaps remove this part of the statement “…properties of their neocortical progenitor cells” since the properties of the cells were not directly addressed here.\n4) In lines 217-218 the authors state that “only a reduced subset of human SYNGAP1 haploinsufficiency patients are diagnosed with microcephaly or structural brain abnormalities”. It would be helpful to be more specific here, i.e. approximately what percentage of SYNGAP1 patients have microcephaly?\n5) The authors sometimes use “SYNGAP1” when referring to the mouse gene (instead of \"Syngap1\")\n6) In lines 219-220, the authors state that the phenotypes likely arise due to SynGAP's synaptic funcitons. Perhaps this could be broadened to say SynGAP’s roles in neuronal physiology (including synaptic and intrinsic excitability changes)", "reviewer_3": "The authors have addressed all my comments."}, "author_response": "NCOMMS-25-05776_Response to reviewer comments\nWe thank the reviewers for their comments, suggestions and their valuable time. Below we provide a response to the reviewer comments.\nReviewer #1 (Remarks to the Author)\nThe authors have addressed my main comments and I believe the data are convincing. The inclusion of the sequencing analyses bolsters the conclusions that Syngap1 mRNA is lowly expressed during embryonic development and that there are not major changes in cortical cell type proportions as determined by levels of key cell type marker genes. I just have a few minor comments:\n1) One of the other reviewers mentioned the possibility of another isoform of Syngap1 being expressed in the mouse models that could account for the lack of effect on early development. The authors provided a thorough response in the rebuttal but I think it would be worth discussing this more in the manuscript itself.\nThe discussion was changed to incorporate the reviewer’s suggestion.\n2) There may be a word missing in the sentence in lines 199-200: do the authors mean \"undergoing differentiative division\"? The text was changed to correct it.\n3) In line 200 – perhaps remove this part of the statement “…properties of their neocortical progenitor cells” since the properties of the cells were not directly addressed here. The text was changed to incorporate the reviewer’s suggestion.\n4) In lines 217-218 the authors state that “only a reduced subset of human SYNGAP1 haploinsufficiency patients are diagnosed with microcephaly or structural brain abnormalities”. It would be helpful to be more specific here, i.e. approximately what percentage of SYNGAP1 patients have microcephaly? The text was changed to incorporate the reviewer’s suggestion.\n5) The authors sometimes use “SYNGAP1” when referring to the mouse gene (instead of \"Syngap1\")\nThe text was changed to correct this along the manuscript.\n6) In lines 219-220, the authors state that the phenotypes likely arise due to SynGAP's synaptic funcitons. Perhaps this could be broadened to say SynGAP’s roles in neuronal physiology (including synaptic and intrinsic excitability changes) The text was changed to incorporate the reviewer’s suggestion.\nReviewer #3 (Remarks to the Author)\nThe authors have addressed all my comments. N/A."}]

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