Fig. 3. Rspo2 expression is regulated by Sp8 in the limb. Whole-mount in situ hybridization was performed on wild-type (A,B,D-H,J), Sp8^-/- (C) and Rspo2^Tg/Tg (K) embryos of the indicated age [embryonic day (E)]. (A-H) Rspo2 antisense riboprobe corresponding to sequences in EST AK011587. (J,K) Sp8 antisense riboprobe. (A-D) Telencephalon expression (white arrowheads). (A,B) AER precursors (red arrowheads). (B,D) Mature AER (red arrows). (B) Transient domain at base of hindlimb (black arrowhead); inset highlights proximal mesenchymal expression domain. (C) Rspo2 expression is undetectable in limb buds (black arrowheads). (D) Genital ridge expression (black arrowhead). (E) Expression in nasal pits. (F-H) Rspo2 expression in laryngeal regions (arrows) and lung mesenchyme (arrowheads). (I) Real-time RT-PCR products amplified from cDNA prepared from stage 2 hindlimb bud ectoderm from Sp8^-/- (lane 1) or Sp8^+/+ (lane 2), and stage 4 Sp8^+/+ (lane 3) embryos. Rspo2 was dramatically decreased in Sp8^-/- ectoderm and increased during limb outgrowth in control embryos, whereas the expression of other AER markers was changed less than twofold. (J,K) Arrows indicate differences in Sp8 expression in the posterior AER margin; expression is weaker in Rspo2^Tg/Tg forelimb AER (arrowhead).