Fig. 8. Wnt7b as a potential endogenous signal regulating osteogenesis. (A,B) Induction of Wnt genes in C3H10T1/2 cells stimulated with NShh for 24 hours (A) or 48 hours (B), as determined by real-time PCR. Results are presented as fold changes over unstimulated cells. Wnt proteins not detectable in either stimulated or unstimulated cells are not included in the graphs. ^*P 0.05. (C-E) Effects of cycloheximide on Hh induction of Wnt5a (C), Wnt7b (D) or Wnt9a (E) expression. mRNA levels were determined by real-time PCR and normalized to Gapd for cells stimulated for 24 hours with N-Shh in the presence of either DMSO (C) or 10 µg/ml cyclohexamine (CHX). (F-H') In situ hybridization using ³⁵S-labeled riboprobes against Wnt5a (F,F'), Wnt7b (G,G') or Wnt9a (H,H') on sections of E14.5 humeri (F,F',H,H') or ulna (G,G') from wild type (F-H) or Ihh^-/- (F'-H') embryos. Red arrows indicate the perichondrium. Yellow arrows indicate the joint regions. PH, prehypertrophic chondrocytes; H, hypertrophic chondrocytes. (I) Expression of Wnt7b in cartilage isolated from the hindlimbs of E14.5 wild-type or Ihh^-/- embryos, assayed by real-time PCR. The average is presented for three pairs of wild type versus Ihh^-/- littermates. The levels of mRNA were normalized to Gapd and presented in arbitrary units with the level in the wild-type embryo set at 1. (J) AP assay for C3H10T1/2 cells transfected with either the empty vector pCIG or constructs expressing Wnt5a, Wnt7b or Wnt9a. Average is shown for triplicate samples from a representative experiment.