Fig. 5. Infection of embryonic metatarsal bone rudiments with adenovirus. Metatarsal rudiments infected with adenovirus that express the ß-gal reporter gene (A,C) or a dominant-negative mutation of the TGFß type II receptor (B,D) in the perichondrium were untreated (A,C), treated with TGFß (1 or 10 ng/ml; B) or Shh (2 µg/ml; D) for 5 days. Treatment with TGFß1 in rudiments infected with ß-gal virus resulted in a decrease in the overall length of the metatarsal bones and in a decrease in the length of the hypertrophic zone (black lines) when compared with untreated cultures. (A) The effects of TGFß1 were dose dependent. (B) TGFß1 treatment did not affect the overall length of the metatarsal or the length of the hypertrophic zone (black line) in rudiments previously infected with DNIIR virus. (C) Shh treatment of rudiments infected with the ß-gal virus resulted in a decrease in the length of the hypertrophic zone (black line). (D) Bone rudiments infected with DNIIR virus and treated with Shh did not show any decrease of the length of the hypertrophic area when compared with untreated rudiments. Metatarsal bones infected with an adenovirus that express the ß-gal reporter gene were used to determine the efficiency and location of infected cells. (E) Whole bone rudiments were stained for ß-galactosidase activity with X-gal. Infected cells appear blue. (F) Adenovirus infection was also visualized by whole-mount immunocytochemistry with a fluorescence-conjugated antibody directed to the adenovirus coat protein. Infected cells appear green. Staining only in the plane of focus is visible resulting in dark areas at either end of the bone. Scale bars: 250 µm in A-D; 180 µm in E,F.
