Screen for pathways corresponding to developmental HSC maturation reveals Jak-Stat1. (A) WGCNA. The horizontal bar represents all genes in the sample. Identified co-expressed genes (modules) are assigned individual colors in the first row. In subsequent rows, red indicates upregulation and blue indicates downregulation in each sample. The module of interest, which shows low expression in the AGM and higher expression in the FL and BM is indicated by an arrow. (B) Expression levels of genes in this module identified in (A) during embryonic development. (C) Top 5 gene ontology biological process terms identified from genes in this module suggesting inflammatory pathway activation. (D) Top 5 KEGG pathway terms identified from genes in this module. Jak-Stat pathways are shown in red. (E) GSEA of samples displayed in columns compared against AGM HSCs. Red indicates that the gene set is more highly enriched in the AGM compared with tissues indicated, whereas blue indicates that the gene set is less enriched. The color scale is determined by the absolute enrichment score from the GSEA. (F) Quantitative RT-PCR for IFN-α and IFN-γ transcripts in whole E11.5 AGM, and E11.5 and E13.5 FL; n = 4. IFNs are detected in the AGM at Ct values <30, confirming their presence. (G) Percent of E11.5 AGM (VE-cadherin⁺CD45⁺) and E13.5 FL HSCs (Lineage⁻Sca1⁺c-Kit⁺), which are positive for intracellular staining of phospho-Stat1 (pacific orange). Absolute MFI for phospho-Stat1 is quantified. Relative MFI was determined by dividing MFI of sample by that of the IgG isotype control; n = 5-7. Statistical significance: *P < .05; **P < .01; ***P < .001. Ct, cycle threshold; IgA, immunoglobulin A; mRNA, messenger RNA; YS, yolk sac.