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A. Experimental timeline. Pregnant heterozygous mice were injected with LPS or saline at E16, E17, and E18. 4 hours after the last injection, the fetal forebrain was dissected for RNA-seq analysis. B, C. A volcano plot illustrating DEGs in the forebrain of E18. The red dots represent significantly upregulated genes, the blue dots represent significantly downregulated genes (|log2 FC| ≥ 1 and FDR < 0.05), and the black dots represent insignificant differentially expressed genes. (B) DEGs in the forebrain of Lcn2 KO animals at E18 (C) DEGs in the forebrain of WT MIA at E18, 4 hours after the last LPS injection. D, E. Gene ontology enrichment analysis using Metascape based on the genes deregulated (both up- and downregulated). A bar graph showing the top 5 clusters with the highest p-value. E. Heatmap of enriched terms across input differentially expressed gene lists. F. A Venn diagram of unique and overlapping gene expression between Lcn2 KO and MIA groups. G. The heat map of overlapping genes between Lcn2 KO and MIA. H, I. RT-qPCR confirmation of the results obtained with RNAseq analysis for <t>Pax2</t> (H) and En2 (I) genes. mRNA expression levels in the forebrain were measured 4 hours after the final injection of LPS or saline in pregnant dams. (H) Two-way ANOVA: procedure: F (1, 33) = 4.616; p = 0.0391, genotype: F (1, 33) = 8.232; p = 0.0071, and interaction: F (1, 33) = 1.933; p = 0.1737, followed by Fisher’s LSD test. WT ctrl n = 7, WT MIA n = 11; Lcn2 KO n = 8, Lcn2 KO MIA n = 11. (I) Two-way ANOVA: procedure: F (1, 34) = 11.60; p = 0.0017, genotype: F (1, 34) = 4.019; p = 0.0530, and interaction: F (1, 34) = 7.669, p = 0.0090, followed by Fisher’s LSD post hoc test. WT ctrl n = 8, WT MIA n = 10; Lcn2 KO n = 9, Lcn2 KO MIA n = 11. Data are presented as mean ± SEM.
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A. Experimental timeline. Pregnant heterozygous mice were injected with LPS or saline at E16, E17, and E18. 4 hours after the last injection, the fetal forebrain was dissected for RNA-seq analysis. B, C. A volcano plot illustrating DEGs in the forebrain of E18. The red dots represent significantly upregulated genes, the blue dots represent significantly downregulated genes (|log2 FC| ≥ 1 and FDR < 0.05), and the black dots represent insignificant differentially expressed genes. (B) DEGs in the forebrain of Lcn2 KO animals at E18 (C) DEGs in the forebrain of WT MIA at E18, 4 hours after the last LPS injection. D, E. Gene ontology enrichment analysis using Metascape based on the genes deregulated (both up- and downregulated). A bar graph showing the top 5 clusters with the highest p-value. E. Heatmap of enriched terms across input differentially expressed gene lists. F. A Venn diagram of unique and overlapping gene expression between Lcn2 KO and MIA groups. G. The heat map of overlapping genes between Lcn2 KO and MIA. H, I. RT-qPCR confirmation of the results obtained with RNAseq analysis for <t>Pax2</t> (H) and En2 (I) genes. mRNA expression levels in the forebrain were measured 4 hours after the final injection of LPS or saline in pregnant dams. (H) Two-way ANOVA: procedure: F (1, 33) = 4.616; p = 0.0391, genotype: F (1, 33) = 8.232; p = 0.0071, and interaction: F (1, 33) = 1.933; p = 0.1737, followed by Fisher’s LSD test. WT ctrl n = 7, WT MIA n = 11; Lcn2 KO n = 8, Lcn2 KO MIA n = 11. (I) Two-way ANOVA: procedure: F (1, 34) = 11.60; p = 0.0017, genotype: F (1, 34) = 4.019; p = 0.0530, and interaction: F (1, 34) = 7.669, p = 0.0090, followed by Fisher’s LSD post hoc test. WT ctrl n = 8, WT MIA n = 10; Lcn2 KO n = 9, Lcn2 KO MIA n = 11. Data are presented as mean ± SEM.
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A. Experimental timeline. Pregnant heterozygous mice were injected with LPS or saline at E16, E17, and E18. 4 hours after the last injection, the fetal forebrain was dissected for RNA-seq analysis. B, C. A volcano plot illustrating DEGs in the forebrain of E18. The red dots represent significantly upregulated genes, the blue dots represent significantly downregulated genes (|log2 FC| ≥ 1 and FDR < 0.05), and the black dots represent insignificant differentially expressed genes. (B) DEGs in the forebrain of Lcn2 KO animals at E18 (C) DEGs in the forebrain of WT MIA at E18, 4 hours after the last LPS injection. D, E. Gene ontology enrichment analysis using Metascape based on the genes deregulated (both up- and downregulated). A bar graph showing the top 5 clusters with the highest p-value. E. Heatmap of enriched terms across input differentially expressed gene lists. F. A Venn diagram of unique and overlapping gene expression between Lcn2 KO and MIA groups. G. The heat map of overlapping genes between Lcn2 KO and MIA. H, I. RT-qPCR confirmation of the results obtained with RNAseq analysis for <t>Pax2</t> (H) and En2 (I) genes. mRNA expression levels in the forebrain were measured 4 hours after the final injection of LPS or saline in pregnant dams. (H) Two-way ANOVA: procedure: F (1, 33) = 4.616; p = 0.0391, genotype: F (1, 33) = 8.232; p = 0.0071, and interaction: F (1, 33) = 1.933; p = 0.1737, followed by Fisher’s LSD test. WT ctrl n = 7, WT MIA n = 11; Lcn2 KO n = 8, Lcn2 KO MIA n = 11. (I) Two-way ANOVA: procedure: F (1, 34) = 11.60; p = 0.0017, genotype: F (1, 34) = 4.019; p = 0.0530, and interaction: F (1, 34) = 7.669, p = 0.0090, followed by Fisher’s LSD post hoc test. WT ctrl n = 8, WT MIA n = 10; Lcn2 KO n = 9, Lcn2 KO MIA n = 11. Data are presented as mean ± SEM.
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A. Experimental timeline. Pregnant heterozygous mice were injected with LPS or saline at E16, E17, and E18. 4 hours after the last injection, the fetal forebrain was dissected for RNA-seq analysis. B, C. A volcano plot illustrating DEGs in the forebrain of E18. The red dots represent significantly upregulated genes, the blue dots represent significantly downregulated genes (|log2 FC| ≥ 1 and FDR < 0.05), and the black dots represent insignificant differentially expressed genes. (B) DEGs in the forebrain of Lcn2 KO animals at E18 (C) DEGs in the forebrain of WT MIA at E18, 4 hours after the last LPS injection. D, E. Gene ontology enrichment analysis using Metascape based on the genes deregulated (both up- and downregulated). A bar graph showing the top 5 clusters with the highest p-value. E. Heatmap of enriched terms across input differentially expressed gene lists. F. A Venn diagram of unique and overlapping gene expression between Lcn2 KO and MIA groups. G. The heat map of overlapping genes between Lcn2 KO and MIA. H, I. RT-qPCR confirmation of the results obtained with RNAseq analysis for <t>Pax2</t> (H) and En2 (I) genes. mRNA expression levels in the forebrain were measured 4 hours after the final injection of LPS or saline in pregnant dams. (H) Two-way ANOVA: procedure: F (1, 33) = 4.616; p = 0.0391, genotype: F (1, 33) = 8.232; p = 0.0071, and interaction: F (1, 33) = 1.933; p = 0.1737, followed by Fisher’s LSD test. WT ctrl n = 7, WT MIA n = 11; Lcn2 KO n = 8, Lcn2 KO MIA n = 11. (I) Two-way ANOVA: procedure: F (1, 34) = 11.60; p = 0.0017, genotype: F (1, 34) = 4.019; p = 0.0530, and interaction: F (1, 34) = 7.669, p = 0.0090, followed by Fisher’s LSD post hoc test. WT ctrl n = 8, WT MIA n = 10; Lcn2 KO n = 9, Lcn2 KO MIA n = 11. Data are presented as mean ± SEM.
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A. Experimental timeline. Pregnant heterozygous mice were injected with LPS or saline at E16, E17, and E18. 4 hours after the last injection, the fetal forebrain was dissected for RNA-seq analysis. B, C. A volcano plot illustrating DEGs in the forebrain of E18. The red dots represent significantly upregulated genes, the blue dots represent significantly downregulated genes (|log2 FC| ≥ 1 and FDR < 0.05), and the black dots represent insignificant differentially expressed genes. (B) DEGs in the forebrain of Lcn2 KO animals at E18 (C) DEGs in the forebrain of WT MIA at E18, 4 hours after the last LPS injection. D, E. Gene ontology enrichment analysis using Metascape based on the genes deregulated (both up- and downregulated). A bar graph showing the top 5 clusters with the highest p-value. E. Heatmap of enriched terms across input differentially expressed gene lists. F. A Venn diagram of unique and overlapping gene expression between Lcn2 KO and MIA groups. G. The heat map of overlapping genes between Lcn2 KO and MIA. H, I. RT-qPCR confirmation of the results obtained with RNAseq analysis for <t>Pax2</t> (H) and En2 (I) genes. mRNA expression levels in the forebrain were measured 4 hours after the final injection of LPS or saline in pregnant dams. (H) Two-way ANOVA: procedure: F (1, 33) = 4.616; p = 0.0391, genotype: F (1, 33) = 8.232; p = 0.0071, and interaction: F (1, 33) = 1.933; p = 0.1737, followed by Fisher’s LSD test. WT ctrl n = 7, WT MIA n = 11; Lcn2 KO n = 8, Lcn2 KO MIA n = 11. (I) Two-way ANOVA: procedure: F (1, 34) = 11.60; p = 0.0017, genotype: F (1, 34) = 4.019; p = 0.0530, and interaction: F (1, 34) = 7.669, p = 0.0090, followed by Fisher’s LSD post hoc test. WT ctrl n = 8, WT MIA n = 10; Lcn2 KO n = 9, Lcn2 KO MIA n = 11. Data are presented as mean ± SEM.
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A. Experimental timeline. Pregnant heterozygous mice were injected with LPS or saline at E16, E17, and E18. 4 hours after the last injection, the fetal forebrain was dissected for RNA-seq analysis. B, C. A volcano plot illustrating DEGs in the forebrain of E18. The red dots represent significantly upregulated genes, the blue dots represent significantly downregulated genes (|log2 FC| ≥ 1 and FDR < 0.05), and the black dots represent insignificant differentially expressed genes. (B) DEGs in the forebrain of Lcn2 KO animals at E18 (C) DEGs in the forebrain of WT MIA at E18, 4 hours after the last LPS injection. D, E. Gene ontology enrichment analysis using Metascape based on the genes deregulated (both up- and downregulated). A bar graph showing the top 5 clusters with the highest p-value. E. Heatmap of enriched terms across input differentially expressed gene lists. F. A Venn diagram of unique and overlapping gene expression between Lcn2 KO and MIA groups. G. The heat map of overlapping genes between Lcn2 KO and MIA. H, I. RT-qPCR confirmation of the results obtained with RNAseq analysis for Pax2 (H) and En2 (I) genes. mRNA expression levels in the forebrain were measured 4 hours after the final injection of LPS or saline in pregnant dams. (H) Two-way ANOVA: procedure: F (1, 33) = 4.616; p = 0.0391, genotype: F (1, 33) = 8.232; p = 0.0071, and interaction: F (1, 33) = 1.933; p = 0.1737, followed by Fisher’s LSD test. WT ctrl n = 7, WT MIA n = 11; Lcn2 KO n = 8, Lcn2 KO MIA n = 11. (I) Two-way ANOVA: procedure: F (1, 34) = 11.60; p = 0.0017, genotype: F (1, 34) = 4.019; p = 0.0530, and interaction: F (1, 34) = 7.669, p = 0.0090, followed by Fisher’s LSD post hoc test. WT ctrl n = 8, WT MIA n = 10; Lcn2 KO n = 9, Lcn2 KO MIA n = 11. Data are presented as mean ± SEM.

Journal: bioRxiv

Article Title: Lcn2 deficiency leads to social impairments independent of maternal immune activation

doi: 10.1101/2025.06.27.661499

Figure Lengend Snippet: A. Experimental timeline. Pregnant heterozygous mice were injected with LPS or saline at E16, E17, and E18. 4 hours after the last injection, the fetal forebrain was dissected for RNA-seq analysis. B, C. A volcano plot illustrating DEGs in the forebrain of E18. The red dots represent significantly upregulated genes, the blue dots represent significantly downregulated genes (|log2 FC| ≥ 1 and FDR < 0.05), and the black dots represent insignificant differentially expressed genes. (B) DEGs in the forebrain of Lcn2 KO animals at E18 (C) DEGs in the forebrain of WT MIA at E18, 4 hours after the last LPS injection. D, E. Gene ontology enrichment analysis using Metascape based on the genes deregulated (both up- and downregulated). A bar graph showing the top 5 clusters with the highest p-value. E. Heatmap of enriched terms across input differentially expressed gene lists. F. A Venn diagram of unique and overlapping gene expression between Lcn2 KO and MIA groups. G. The heat map of overlapping genes between Lcn2 KO and MIA. H, I. RT-qPCR confirmation of the results obtained with RNAseq analysis for Pax2 (H) and En2 (I) genes. mRNA expression levels in the forebrain were measured 4 hours after the final injection of LPS or saline in pregnant dams. (H) Two-way ANOVA: procedure: F (1, 33) = 4.616; p = 0.0391, genotype: F (1, 33) = 8.232; p = 0.0071, and interaction: F (1, 33) = 1.933; p = 0.1737, followed by Fisher’s LSD test. WT ctrl n = 7, WT MIA n = 11; Lcn2 KO n = 8, Lcn2 KO MIA n = 11. (I) Two-way ANOVA: procedure: F (1, 34) = 11.60; p = 0.0017, genotype: F (1, 34) = 4.019; p = 0.0530, and interaction: F (1, 34) = 7.669, p = 0.0090, followed by Fisher’s LSD post hoc test. WT ctrl n = 8, WT MIA n = 10; Lcn2 KO n = 9, Lcn2 KO MIA n = 11. Data are presented as mean ± SEM.

Article Snippet: RNA was reverse transcribed with SuperScript™ IV VILO (# 11766050, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. cDNA was amplified with TaqMan probes (Lcn2 Mm01324470_m1, Gapdh Mm99999915_g1, En2 Mm00438710_m1, Pax2: Mm01217939_m1, ThermoFisher, Waltham, MA, USA) that were specific for the mouse.

Techniques: Injection, Saline, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Expressing