Journal: bioRxiv

Article Title: Genome-scale functional mapping of the mammalian whole brain with in vivo Perturb-seq

doi: 10.64898/2026.03.16.711480

Figure Lengend Snippet: ( A ) Schematic of mouse whole brain in vivo Perturb-seq using 10x Genomics Flex Apex platform. ( B ) UMAP of whole brain in vivo Perturb-seq dataset encompassing 7.7 million sequenced nuclei, colored by developmental neighborhoods, anatomical region, and neurotransmitter type, inferred using MapMyCells . ( C ) Heatmap of the gene expression levels of 1,947 neurodevelopmental disease-associated risk genes in non-targeting control nuclei across different developmental neighborhoods. ( D ) Histogram of in vitro gRNA activity distribution of 45 selected gRNAs (15 genes, 3 gRNAs per gene) compared to safe-targeting controls by insertion-deletion analysis. ( E ) Immunofluorescence image of sagittal section of a P37 mouse brain retro-orbitally administered with 6e8 total vg per gram of body weight of AAV PHP.eB encoding either GFP or mScarlet (1:1 ratio) (scale bar, 1 mm), accompanied by zoomed in images to show representative MOI in each major brain region (scale bar, 50 μm), and stacked bar plot quantifying GFP and mScarlet viral labeling efficiency as well as double labeling rate. ( F ) UMAPs of whole brain in vivo Perturb-seq dataset separated by neighborhoods, colored by inferred cell subclass using MapMyCells . ( G ) Violin plots of number of genes and RNA UMIs recovered per nucleus from each developmental neighborhood. ( H ) Ranked bar plot showing proportion of sampled nuclei by brain region. ( I ) Stacked bar plot showing percentage of nuclei with no guide, single, double, or multiple guide assignment within each developmental neighborhood. ( J ) Histogram of total nuclei number distribution of nuclei recovered per perturbation. ( K ) Ranked dot plot of nuclei number in each perturbation and cell type pair and the minimum cell number cut off for perturbation and cell type pair for downstream analyses (dashed line). ( L ) Scatter plot of weighted mean log fold-changes of target genes across all cell types against their weighted mean expression levels in non-targeting control nuclei.

Article Snippet: Briefly, HEK293T cells were transfected with above pooled plasmid library or plasmid encoding a PiggyBac transposase , along with AAV-PHP.eB capsid (Addgene #103005) and pHelper plasmids using PEI (Polysciences, #24765-1) at 80-90% confluency.

Techniques: In Vivo, Gene Expression, Control, In Vitro, Activity Assay, Immunofluorescence, Labeling, Expressing

Journal: bioRxiv

Article Title: Genome-scale functional mapping of the mammalian whole brain with in vivo Perturb-seq

doi: 10.64898/2026.03.16.711480

Figure Lengend Snippet: (A) Immunofluorescence image of sagittal section of a P37 mouse brain retro-orbitally injected at P16 with high (1e9), mid (6e8), or low (1.5e8) total vg per gram of body weight of AAV PHP.eB encoding either GFP or mScarlet (1:1 ratio) (scale bar = 1 mm). (B) Quantification of GFP and mScarlet viral labeling efficiency as well as double labeling rate in (A). (C) Representative FACS gating strategy to enrich transduced neuronal nuclei. (D) Bar plot of sex and weight at harvest of animals used in this study. (E) Animal tracking information showing the litter, age at harvest for each animal, as well as AAV-labeling rate by FACS and total nuclei number per hemisphere used for Flex hybridization. (F) Schematic of snRNA-seq data processing and quality control workflow.

Article Snippet: Briefly, HEK293T cells were transfected with above pooled plasmid library or plasmid encoding a PiggyBac transposase , along with AAV-PHP.eB capsid (Addgene #103005) and pHelper plasmids using PEI (Polysciences, #24765-1) at 80-90% confluency.

Techniques: Immunofluorescence, Injection, Labeling, Hybridization, Control

Journal: Nature Communications

Article Title: Molecular signatures of resilience to Alzheimer’s disease in neocortical layer 4 neurons

doi: 10.1038/s41467-026-68920-4

Figure Lengend Snippet: a Violin plots showing KCNIP4 gene expression across major cell types (left) and excitatory neuronal subtypes from BA9 and BA17 (right). b Violin plots showing KCNIP4 expression across AD disease groups in Ex2 and Ex5 neurons from BA9 and BA17. Log-normalized expression levels of KCNIP4 are shown. c Immunostaining for KCNIP4, EYA4, and NeuN in cryosections from low, intermediate, and high pathology stages illustrating increased expression of KCNIP4 in L4 EYA4 + neurons in BA17. d Quantification of KCNIP4 protein expression levels in L4 EYA4 + neurons, L4 EYA4 − neurons, and L2/3 neurons from BA17 across disease stages ( n = 6 donors per disease group). Data are shown as median ± IQR; whiskers represent minimum and maximum values. One-way ANOVA with two-sided Tukey’s test was used for multiple comparisons (* p -value < 0.05; *** p -value < 0.001; **** p -value < 0.0001; exact p -values are available in the Source Data file). Scale bars: 200 µm for low magnification images; 30 µm for high magnification images. Source data are provided as a Source Data file.

Article Snippet: Neurons were transduced on day in vitro 7 (DIV7) with AAV-PHP.eB-CaMKIIa- Kcnip4 -P2A-EGFP or control AAV-PHP.eB-CaMKIIa-EGFP (Addgene #50469-PHPeB) at a multiplicity of infection (MOI) of 5000 viral genomes (vg)/cell.

Techniques: Gene Expression, Expressing, Immunostaining

Journal: Nature Communications

Article Title: Molecular signatures of resilience to Alzheimer’s disease in neocortical layer 4 neurons

doi: 10.1038/s41467-026-68920-4

Figure Lengend Snippet: a In vitro approach to evaluate AAV-mediated Kcnip4 overexpression on neural activity in primary excitatory cortical neurons using calcium imaging. b Representative neuronal Ca 2+ transients quantified as ΔF/F₀ at DIV 14 for each condition. c , Quantification of Ca 2+ transient frequency for each condition. Event frequency (events per minute) was averaged at the well level, with each well considered a biological replicate (4 wells per condition, 2 fields per well, 3 GFP-positive neurons per field). d In vivo approach to evaluate AAV-mediated Kcnip4 overexpression in App SAA and WT mice, and representative coronal section (50-µm thick) of a treated mouse illustrating transduction of cortical neurons. e Western blot representative image and quantification of KCNIP4 levels in cerebral cortex lysates following two different doses of Kcnip4 AAV ( n = 3 per group). f Representative images of cerebral cortex and hippocampus from Kcnip4 AAV-treated mice and quantification of transduction efficiency of the different AAVs in SSC in WT and App SAA mice. g − i Representative images and quantification of cortical amyloid beta, GFAP, and IBA1 immunostaining in App SAA mice treated with Kcnip4 AAV or control AAV (6−7 mice per group). j Representative immunofluorescence image through the SSC co-stained with GFP and c-Fos. k Percentage of c-Fos-positive cells in all cortical neurons across study groups. l − o Quantification of c-Fos in GFP + compared to GFP − neurons from App SAA and WT mice treated with Kcnip4 AAV or control AAV (5−7 mice per group). p Representative immunofluorescence image through the SSC co-stained with GFP and Arc. q Mean Arc staining intensity in all cortical neurons across groups; r−u , Quantification of Arc staining intensity in GFP + compared to GFP − neurons from App SAA and WT mice treated with Kcnip4 AAV or control AAV (5−7 mice per group). Data are shown as median ± IQR. A two-sided t-test was used for pairwise comparisons, and one-way ANOVA with two-sided Tukey’s test was used for multiple comparisons (* p -value < 0.05; ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001; exact p -values are available in the Source Data file). Scale bars: 200 µm ( f ); 50 µm ( j , p ). Source data are provided as a Source Data file.

Article Snippet: Neurons were transduced on day in vitro 7 (DIV7) with AAV-PHP.eB-CaMKIIa- Kcnip4 -P2A-EGFP or control AAV-PHP.eB-CaMKIIa-EGFP (Addgene #50469-PHPeB) at a multiplicity of infection (MOI) of 5000 viral genomes (vg)/cell.

Techniques: In Vitro, Over Expression, Activity Assay, Imaging, In Vivo, Transduction, Western Blot, Immunostaining, Control, Immunofluorescence, Staining