Fig. 2

CRM activities of the second intron of Xenopus chrd. a Sequence alignment of X. tropicalis and X. laevis chrd-D1 core regions (243 bp). Orange boxes, conserved Lim1 motifs; purple box, conserved FoxH1 motif (a major partner of Smad2/3 in Nodal signaling). Siamois may bind to the Lim1 site [50]. b–f Luciferase reporter assays. Xt_chrd-D1 (b, c), intron 2 sequences of Xl_chrd.L and .S (d), Xt_chrd-D1_104 bp (between light green brackets in a) (e), and Xt_chrd-D1_mt (all four conserved Lim1 motifs are mutated) were analyzed. Xt_chrd-D1 showed synergistic activation by Lim1, Ldb1, Ssbp3, and Otx2 (b). Strong activation through Xt_chrd-D1 was observed in Lim1/Ldb1/Ssbp3, Siamois and activin, but not in Wnt8 (c). Xl_chrd.L and .S intron2 showed conserved enhancer activity, which was activated by Lim1/Ldb1/Ssbp3 (d). No responsiveness of Xt_chrd –D1_104 bp to activin (e) suggests that Nodal signaling activates chrd-D1 through the conserved FoxH1 site. Reporter activation by Lim1/Ldb1/Ssbp3 was abolished by mutating four Lim1 motifs in Xt_chrd-D1 (f), indicating that Lim1 directly activates chrd through the intron2 enhancer. Bars represent mean ± s.e.m. *, P < 0.05; **, P < 0.01 (t-test, two tailed). Dosages of injected mRNAs are as follows: lim1, 100 pg/embryo; ldb1, 100 pg/embryo; ssbp3, 100 pg/embryo; otx2, 40 pg/embryo; simois, 100 pg/embryo; wnt8a, 25 pg/embryo; and activin A, 20 pg/embryo. g Transgenic reporter analysis of Xt_chrd-D1. Panels represent whole mount in situ hybridization of the reporter gene mVenus or endogenous chrd for transgenic embryos with dorso-ventral hemisections. Expression patterns were examined at the early (st. 10), middle (st. 11) and late (st. 12.5) gastrula stages as indicated. In total, 8 of 30 transgenic embryos showed reporter expression in the organizer. Embryos are shown with the animal pole at the top and dorsal to the right. Arrow heads, blastopore