Fig. 6.

RSF1 collaborates with Ring1 to regulate mesodermal specification during early Xenopus embryogenesis. (A) Both RSF1 and Ring1 regulate Xenopus gastrulation. Injection of RSF1-MO (20 ng) and Ring1-MO (50 ng) induced similar gastrulation defects in Xenopus tadpoles, with embryos displaying short axis and open blastopore. In contrast, RNF2-MO induced a milder phenotype of bent axis and head defects. (B) RSF1-MO and Ring1-MO act cooperatively to induce gastrulation defects. A combination of low doses of Ring1-MO and RSF1-MO resulted in an embryonic phenotype that mimicked or surpassed that with the high doses of individual MOs. (C) At late gastrula stages, RSF1-MO and Ring1-MO cause delay in blastopore closure. Combination of low doses of RSF1-MO and Ring1-MO induced similar gastrulation defects as that when higher doses of individual MOs were used. RNF2-MO did not have obvious effects on blastopore closure. (D) In situ hybridization demonstrated that KD of RSF1 or Ring1 reduced mesodermal markers in a similar fashion. The pan-mesodermal gene Brachyury (Bra), the dorsal mesodermal marker Chordin (Chd), and the ventrolateral mesodermal marker Wnt8 were all reduced upon RSF1 or Ring1 KD. KD of RNF2 did not alter expression of these mesodermal markers. The embryos were injected with the MOs and the lineage tracer encoding nuclear β-Gal into the marginal zone of one cell at the two-cell stage, and the injected region was revealed by staining with the β-Gal substrate Red-Gal.