Figure 4

Sclerotome culture. (A) Sclerotome was isolated from E11.5 day embyos and grown in micromass culture. (B) RT-PCR to show purity of cultures. RNA was isolated from neural tube (NT), notochord (NC), and sclerotome (Sc) immediately after dissection. cDNA was made from the RNA and expression of Pax1, a marker for sclerotome, Pax3, a marker for myotome and neural tube, and brachyury (T), a marker for the notochord, were determined using PCR. 18S was used as a loading control. Pax1 was expressed in the sclerotome but T and Pax3 were not detected indicating that there was no contamination with myotome, neural tube or notochord in the sclerotome preparation. (C) Alcian blue stained micromass cultures 72 hours after treatment with BMP or TGF-β. Untreated (un) cultures are shown as a control. Very little alcian blue stain was seen in untreated controls. Treatment with BMP resulted in a lot of darkly stained nodules with cartilage morphology. Treatment with TGF-β resulted in an increase in alcian blue stain but discreet nodules with cartilage morphology were not detected. (D) RT-PCR was used to determine expression of Fmod in untreated (UN) and BMP or TGF-β treated cultures. Fmod was induced after treatment with TGF-β.