Fig. 5

Luciferase reporter analysis of the gsc 5′ region in Xenopus embryos. a Luciferase reporter assays of Bj_gsc 5′ regions for responsiveness to endogenous factors. Reporter constructs were injected into the animal pole (AP), ventral equatorial region (VER), or dorsal equatorial region (DER) at the four-cell stage to examine responsiveness of constructs to endogenous dorsal signals. Results were normalized with activity of embryos injected with reporter constructs into the animal pole. b Luciferase reporter assays of Xl_gsc-U1 and the Bj_gsc 5′ region for responsiveness to exogenous factors. Lim1/Ldb1/Ssbp3a strongly activated reporter gene expression through Xl_gsc-U1 but only slightly through the Bj_gsc 5′ region. While, Wnt and Nodal signaling synergistically activated reporter gene expression through the Bj_gsc 5′ region. c Luciferase reporter assays of Bj_gsc 5′ region with mutations of three FoxH1 motifs for responsiveness to Nodal signaling. The mutation construct greatly reduced responsiveness to activin, suggesting that Nodal/FoxH1 signaling directly regulates Bj_gsc through the 5′ region. See Fig. 4b for details of the FoxH1 motif mutation. Reporter constructs were injected into the animal pole with combinations of mRNAs with dosages as follows: lim1, 100 pg/embryo (Xl_gsc-U1) or 50 pg/embryo (Bj_gsc 5′ regions); ldb1, 100 pg/embryo (Xl_gsc-U1) or 50 pg/embryo (Bj_gsc 5′ regions); ssbp3, 100 pg/embryo (Xl_gsc-U1) or 50 pg/embryo (Bj_gsc 5′ regions); otx2, 50 pg/embryo; wnt8, 25 pg/embryo; and activin A, 40 pg/embryo. Bars represent mean ± s.e.m. *, P < 0.05; **, P < 0.01 (t-test, two tailed)