Expression analyses of purified osteoblasts and HSCs during normal steady state and after systemic stress. (A) The autocrine production of Wnt3a by Wif1 transgenic HSCs stimulates Wnt signaling that is normalized on exposure to exogenous Wnt3a. Wif1 and control OB mice were crossed to TOPgal mice and injected with control L-CM or Wnt3a-expressing L-CM (Wnt3a-CM). LSK48⁻ cells were flow sorted 18 hours after treatment, mRNA isolated, and assessed for TOPgal reporter activity, Axin2, and Wnt3a mRNA levels by quantitative real-time RT-PCR. β-actin normalized data are expressed as the ratio of expression in Wif1 versus OB mice after injection of either control L-CM (black bars) or Wnt3a-CM (white bars). (B) Known niche regulators are up-regulated in Wif1-expressing osteoblasts during steady state but are down-regulated after 5-FU. GFP⁺ osteoblasts from Wif1 and control OB mice were sorted from collagenased bone isolated during normal steady state (black bars) or 9 days after 5-FU injection (white bars). Gene expression was assayed by quantitative RT-PCR for the indicated genes and normalized to β-actin or GAPDH. Data are expressed as the ratio of relative gene expression in Wif1 versus control OB mice ± SD. (C) HSC gene expression in a dysregulated niche is altered at steady state and after systemic stress. HSCs were isolated from Wif1 and OB BM during normal, steady state, LSKCD34^−/lo, or LSKCD48⁻ for Wnt 3a and Axin2 (black bars), and 9 days after 5-FU injection, LSKCD48⁻ (white bars). Expression of the indicated genes was interrogated by quantitative RT-PCR and normalized to β-actin or GAPDH. Data are expressed as the ratio of relative gene expression in Wif1 versus control OB mice ± SD. ND indicates not detected.