Figure 4

Schematic depiction of chimeras of mature ligand domains, Finger 1 (F1), Heel (H), and Finger 2 (F2), between Xenopus ActivinβB and zebrafish Sqt. HA indicates an hemagglutinin epitope tag. Schematic is not drawn to scale. The letters in these three-lettered (XXX) chimeras represent the Finger 1, Heel, and Finger 2, respectively. S denotes Squint; A denotes ActivinβB. Synthetic mRNAs (200 pg) encoding chimeras were injected into wild-type and MZoep embryos. gsc and ntl mRNA expression is shown at shield stage; animal pole views are dorsal to the right. gsc is expressed in the dorsal organizer (shield) in wild-type embryos, but is absent in MZoep mutants. ntl is expressed around the entire margin in wild-type embryos, but the dorsal margin expression is lost in MZoep mutants. The presence of the ActivinβB prodomain and epitope tag does not alter the specificity or functionality of wild-type ActivinβB (AAA) or Sqt (SSS). AAA can induce ectopic gsc and ntl expression in both wild-type and MZoep embryos. In contrast, SSS can induce ectopic gsc and ntl expression in only wild-type embryos. Similar to ActivinβB, chimeras SSA, SAS, ASA, and SAA can induce ectopic gsc and ntl expression in both wild-type and MZoep embryos. Chimeras ASS and AAS are inactive in both wild-type and MZoep embryos. Western blot analysis indicated that all chimeric constructs produce stable ligands (data not shown).