Fig. 5

The feelgood line carries a missense mutation in creb3l2. (A) The feelgood mutation was mapped to chromosome 4 between SSLP markers z20533 and z7104. Novel SSLP markers with the indicated number of recombinants reduced the critical interval to a <50 kb region that contained two genes. The physical map of the critical region was based on the Zv7 assembly. (B) Schematic diagram of the Creb3l2 primary structure illustrating a missense N301K mutation in the DNA-binding basic motif. Basic, basic motif; Lzip, leucine zipper motif; TM, transmembrane domain; S1P, Site-1 protease recognition sequence. (C) Electropherograms of wild-type (+/+), heterozygous (+/-) and feelgood (-/-) genomic DNA. The arrow points to the C′G transversion that results in lysine for asparagine substitution at position 301 (N301K). (D) Comparison of the DNA-binding basic motif of creb3l2 across several species. The amino acid changed in feelgood mutants is underlined in red. (E) Schematic diagram of the Tol2kit-based construct containing mCherry-tagged creb3l2 under the ubiquitous β-actin (bactin2) promoter. (F–I) Alcian blue staining of cartilage elements at 5 dpf of non-injected (NIC) wild-type embryo (F); non-injected feelgood embryo (G); creb3l2-mCherry rescued feelgood mutant embryo (H); and mutant embryo injected with feelgood (N301K) creb3l2-Cherry that failed to rescue the phenotype (I). The chromatograms of sequences surrounding the feelgood lesion for the embryos shown in F–I have been included in the corresponding adjacent images. Arrows indicate an increased length of lower jaw in rescued mutant (H) compared with NIC feelgood control (G) or embryos injected with creb3l2 N301K (I). (J) Quantification of the number of mutant, wild-type and rescued phenotypes in the β-actin2 creb3l2-mCherry injection experiments. The feelgood genotype of these fish was confirmed by sequencing.