Phylogenetic relationships of the ANKH amino acid sequence in vertebrate organisms. (a) Results from neighbor-joining tree analysis using the out-group taxon (Eptatretus burgeri) demonstrate the relationships between vertebrate organisms. Bootstrap analysis was performed using 10,000 replicates, and these bootstrap values are included. Monophyletic clustering of teleost fish for Ankha and Ankhb are represented by red and blue branches, respectively. (b) The neighbor-joining tree analysis includes human (Homo sapiens), mouse (Mus musculus), and zebrafish (Danio rerio) Ankha and Ankhb, and the out-group taxon (Eptatretus burgeri) includes values from 10,000-replicate bootstrap analysis. (c) Matrix of pairwise amino acid sequence identities for Ankh/Ankha and Ankhb across various vertebrate species determined using percentage identity matrix analysis. Boxes with a black background that have no values represent self-comparisons. High sequence identity is represented by red, and white shades highlight comparisons with lower sequence identity. Squares that have 90 to 100% identity are shown with a dark pink background, sequence identity values from 80–90% are represented in light pink, sequence identity values from 50–80% in white, and sequence identity values below 50% are represented with a white background. Each identity value is rounded to the nearest whole number.

Temporal expression of ankha and ankhb during zebrafish development. (a) Expression of ankha and ankhb relative to the lsm12b control in this representative-endpoint PCR gel, N = 3 replicates. Thirty embryos or fry from each developmental stage, ranging from the 2-cell stage (0.75 hpf) to the 7 dpf stage, were sampled. (b) RT-qPCR normalized expression of ankha and ankhb genes at 2, 3, 4 and 7 days post-fertilization (dpf) relative to gapdh expression, showing the mean +/− the standard error. Each sample represents 30 pooled larvae, where N = 3, and statistical significance was determined using an unpaired t-test (p < 0.05).

Whole-mount in situ hybridization analysis of ankha and ankhb spatial expression patterns in zebrafish development. Lateral views of zebrafish larval stages at 2, 3 and 4 days post-fertilization (dpf) reveal spatial gene expression patterns for ankha and ankhb. At 2 and 3 dpf, both ankha and ankhb show predominant staining in the craniofacial region, somites and notochord. At 4 dpf, ankha extends below the eye (star) and appears in the somites and notochord (arrows). ankhb expression at 3 dpf and 4 dpf is mainly localized to the craniofacial region and notochord area. For ankhb, staining below the eye is reduced and is more localized in regions below the hindbrain. Noto: notochord, Som: somites. The yolk was removed from the embryo; the scale bar indicates 100 μm.