Fig. 3: IκBα KO LT-HSCs maintain an AGM-specific HSC gene expression program.
From: IκBα controls dormancy in hematopoietic stem cells via retinoic acid during embryonic development
A Heatmap of 1476 differentially expressed genes (DEGs) from the comparison of IκBα KO against WT/HET samples. DEGs were called with absolute shrunken logFC > 1 and adjusted p-values (Benjamini–Hochberg procedure, FDR < 0.05). A Wald-test was applied for DEG analysis. B GSEA comparing IκBα WT/HET and KO LT-HSCs bulk RNA-seq data against the top 300 genes defining (top panel) fetal liver LT-HSCs signature (Manesia et al.42) or (bottom panel) AGM HSCs (Thambyrajah et al. 2023)74. Indicated p-value is a nominal p-value obtained from GSEA (C), Bar plot depicting significantly enriched MSigDB HALLMARK pathways in IκBα KO compared to WT/HET with negative Normalized Enrichment Score (NES) and identified by GSEA from the bulk RNA-seq data (Benjamini–Hochberg procedure (FDR) adjusted p-value < 0.05). D IHC on E11.5 (43–45 s) sagittal AGM section for endothelial marker (DLL4, green) and IκBα (yellow) and DAPI (blue) on Gfi1:tomato (red) embryos. Representative image from n = 4 WT and 5 KO AGM 12 um sections derived from 2 embryos for each genotype. Scale bar: 20 μm. Imaged using an SPE (Leica) with a 20× oil lens. E IHC on E11.5 (42–45 somites) cross-section of AGM for pSer32/36 IκBα (yellow), cKIT (red), and DAPI (blue). Scale bar: 10 μm. Images were taken using an SPE (Leica) with a 20× oil lens and processed using Imaris. F Bar chart depicting the number of p-IκBα Ser32/36 positive cells within cKIT+ IAHC at E10.5 (12 embryos of 33–36 somites). Each bar represents one IAHC. Counts were performed manually. Imaged using an SPE (Leica) with a 20x oil lens. G Bar chart depicting individual values for the frequency of (i) cKIT+(IAHC) in CD31+ cells and (ii) SCA1+EPCR+ positive cells within IAHC of E11.5 (42–46 somites) AGMs of IκBα WT (n = 7) and KO (n = 6) in 3 independent experiments. Statistical test: Multiple linear regression model with Experiment and Genotype covariates, significance obtained from applying t-test to corresponding model coefficient estimate (*p-value < 0.05, ns p-value > 0.05). Bars indicate mean values and error bars refer to ± standard deviation. Source data are provided as a Source Data file.
