Fig. 2. GDF3 acts as a BMP inhibitor in frog embryos and mouse pluripotent cells. (A) Stage 41 embryos injected with 2 ng GDF3 RNA in the VMZ at the 4-cell stage (upper embryo) or uninjected sibling embryo (lower embryo). (B) Luciferase units of total embryo lysate from frog embryos injected with BRE-Lux and GDF3, BMP4 or both RNAs. (C) Relative luciferase units (luciferase:renilla) from total cell lysate of P19 cells transfected with BRE-Lux and the BMP signaling components Smad1, Smad4 and OAZ and Gdf3, Bmp4 or both. (D) Diagram of frog embryo showing ectodermal explant (animal cap), and VMZ and DMZ. (E,F) RT-PCR of GDF3-injected and uninjected animal caps matured to sibling stage 12.5 or stage 21 with whole embryo as positive control and whole embryo with no RT (-RT) as a negative control. ODC is shown as a loading control. (G) GDF3 protein produced in oocytes injected with either water or GDF3 RNA (50 ng). Oocyte CM was collected and oocytes were lysed at the end of culture (lysate) and western blot was performed with pGDF3. The prepro form (^**) of GDF3 is 45 kD and the mature form (^*) is 18 kD. (H) VMZ and DMZ explants cultured in 0.5x MMR (-), in the presence of activin protein secreted from oocytes (diluted 1:50 or 1:500), or in the presence of GDF3 protein secreted from oocytes (diluted 1:100 or 1:1000).