Analysis of Hh pathway member expression in different hematopoietic developmental stages. (A) Affymetrix U133A arrays were normalized across samples and hierarchical clustering of Hh pathway genes using the Pearson correlation was performed, demonstrating an increased expression in earlier developmental stages. (B) Schematic of Hh signaling indicating genes identified by expression array analysis and small molecules used to agonize (puromorphamine) or antagonize (cyclopamine) the pathway. Green coloring is used for stimulatory and red is used for repressive proteins and processes. Gli3 is shown as a composite because it functions in both stimulating and repressing the pathway. (C) PCA was used to generate unbiased groupings of arrays, which demonstrated clustering based on developmental stage. (D) GSEA shows statistical significance (P < .05) for augmented expression of Hh pathway members in embryonic versus fetal, neonatal, and adult samples. (E) PCR (30-40 cycles) confirming higher expression of the Hh pathway members Gli1/2/3 and SMO at early developmental stages. LIN28 serves as a positive control for early ontogeny, RUNX1 for hematopoietic progenitors, CD45 for later hematopoietic differentiation, and 18S rRNA as a loading control. hFL indicates human fetal liver; hFB, fetal blood; MPB, human mobilized peripheral blood, and CB, human cord blood. All somatic sources except FL were lineage depleted (Lin⁻).