Fig. 4

Reporter analyses of the lim1 intron 1 and chrd intron 2 in the Xenopus embryo. a, b Luciferase reporter assays of lim1 intron 1. Responsiveness of reporter constructs to Nodal signaling was tested with or without activin A mRNA (40 pg/embryo). A reporter construct mutated in two FoxH1 motifs (Fm), but not that in a Smad motif (Sm) exhibited no response to Nodal signaling (b), suggesting that Nodal signaling directly regulates Bj_lim1 through FoxH1 binding to the intron1 enhancer. c, d Luciferase reporter assays of Bj_chrd intron 2. Responsiveness of Bj_chrd intron 2 (region g) to Lim1 and Otx2 was tested with or without lim1, ldb1, ssbp3, and otx2 mRNAs (100, 100, 100 and 40 pg/embryo, respectively). Lim1/Ldb1/Ssbp3 significantly activated the reporter gene through Bj_chrd intron 2 (C), but the activation level was significantly reduced by mutating three Lim1 motifs (D). See Fig. 2f for details of the Lim1 motif mutation. Bars represent mean ± s.e.m. *, P < 0.05, **, P < 0.01; †, P < 0.1 (t-test, two tailed)