Wnt3a plays a nonredundant role in the regulation of FL HSC function. (A) Fresh wild-type or Wnt3a^−/− FLs were isolated and Ter119⁺ erythrocytes were depleted before mRNA isolation. Gene expression was analyzed by quantitative polymerase chain reaction for the indicated Wnt genes and expression levels normalized for GAPDH using established primer/probe sets. Results correspond to mean ± SEM of 2 independent experiments. In each experiment a pool of 4 to 8 fetal livers from each genotype was used. Measurements were performed in duplicate for each sample. ND indicates not detectable. Fetal thymic lobes and fetal brain were used as positive controls for all genes analyzed. (B) Canonical Wnt signaling is completely abolished in HSCs from Wnt3a^−/− embryos. Wnt3a mice were crossed with Bat-Gal Wnt reporter mice in which a LacZ cassette is under control of Tcf/Lef-binding motifs, resulting in LacZ expression when the pathway is activated. Activation of the pathway was determined by LacZ staining on individual FLs from Bat-Gal^Tg/+ wild-type or Wnt3a^−/− littermate embryos. LacZ expression was analyzed by flow cytometry⁴  on electronically gated LSK cells. (C) Frequency of LacZ⁺ cells inside LSK population. Results correspond to mean ± SEM of 2 independent experiments with 2 wild-type and 1 Wnt3a^−/− littermate embryos each. *P = .03. (D) Littermate wild-type embryos not carrying the reporter transgene were used as negative controls to determine the background staining of LacZ.