Fig. 1. A nonsense mutation in the retinal anterior homeobox gene (rax) results in an eyeless phenotype. (A) The Rax protein structure includes an octapeptide domain (OP, blue), homeodomain (HD, green) and OAR domain (orange). Nuclear localization signals (NLSs, red) are found in the homeodomain. A nonsense mutation results in a truncation prior to the homeodomain and NLSs. (B and C) Tadpoles homozygous for the rax mutant allele fail to form eyes (B, right; C, top). A small amount of retinal pigmented epithelium (RPE) is sometimes seen in mutant animals (B and C, orange arrows) whereas some mutant embryos have no sign of RPE (B, blue arrow); this variation is observed within the same clutch of embryos. The eyeless phenotype can be rescued with the injection of 5 pg of rax mRNA (C, bottom, 10 out of 24 injected). Sections through the mutant, wildtype, and mRNA-rescued mutant tadpole eye regions are shown in the middle panel, illustrating the loss of eye tissue, except small residual RPE in some mutant embryos, and rescue of the retinal and lens structures in mRNA-injected mutant. Far right of (C), insets of Sanger-sequencing showing point mutation in exon 2, which results in a premature stop codon. (D) A Xenopus tropicalis BAC clone containing a rax–gfp fusion gene can rescue eye formation when transiently expressed in mutant embryos. Note that BAC injection tends to be effective dominantly (or only) in one side as described before ( Fish et al. 2012). E1–E3 indicate rax exons; the gfp3 open reading frame (ORF) has been fused to the 3′ end of the final rax exon (E3) to produce a rax–gfp3 fusion gene. Quality of rescue is highly dose-sensitive, with the best rescue observed with 5 pg mRNA (C, data not shown) and 10 pg of BAC DNA injected (D).