Divergent Wnt8a gene expression in teleosts

Abstract

The analysis of genes in evolutionarily distant but morphologically similar species is of major importance to unravel the changes in genomes over millions of years, which led to gene silencing and functional diversification. We report the analysis of Wnt8a gene expression in the medakafish and provide a detailed comparison to other vertebrates. In all teleosts analyzed there are two paralogous Wnt8a copies. These show largely overlapping expression in the early developing zebrafish embryo, an evolutionarily distant relative of medaka. In contrast to zebrafish, we find that both maternal and zygotic expression of particularly one Wnt8a paralog has diverged in medaka. While Wnt8a1 expression is mostly conserved at early embryonic stages, the expression of Wnt8a2 differs markedly. In addition, both genes are distinctly expressed during organogenesis unlike the zebrafish homologs, which may hint at the emergence of functional diversification of Wnt8a ligands during evolution.

Figure 1. Phylogenetic analysis of Wnt8a genes.

The medaka genome contains two Wnt8a genes (bold). The Wnt8a cDNA sequence alignment of various species shows that the medaka paralogous copies cluster well with those of evolutionarily related teleosts. acul., aculeatus; nigro., nigroviridis.

Figure 2. Differential early Wnt8a1 and Wnt8a2 gene expression.

(A–D) 8-cell stage embryos are shown with the animal pole to the top. Sense RNA probes were used to validate the maternal Wnt8a1 expression. (E,F) RT-PCR for Wnt8a1 and Wnt8a2 transcripts on extracted RNA at stages indicated to the left. (G,H) Pictures focused on the dorsal margin of the dissected blastoderm of embryos at 40% epiboly with animal pole to the top. While Wnt8a1 (G) is expressed in cells of the blastoderm margin but not in cells of the shield (framed by black lines), Wnt8a2 (H) is weakly expressed in cells of the shield and the margin. Open rectangle indicates the broad Wnt8a2 expression domain at the margin. (I,J) Embryos at 80% epiboly with anterior to the left; (I) dotted lines frame the developing embryonic axis; inset and (J) show embryos dissected from the yolk for clarity. (J) Dotted line marks the blastoderm margin. 40%, 40% epiboly; c, cells; ma, margin; ea, embryonic axis; n.c., negative control; pam, paraxial mesoderm; st., stage; vma, ventral margin.

Figure 3. Wnt8a gene paralog expression pattern differ during somitogenesis and organogenesis.

(A,B,E,G–I) Dorsal and (C,D,F) lateral views with anterior to the left, (A–F) focused on the tail region. (A,B) At 38 hours post fertilization (hpf), Wnt8a1 transcripts are found in the axial part of the tailbud including the posterior end of the notochord (arrow), while Wnt8a2 is widely expressed in the tailbud, presomitic mesoderm and developing somites. (C–F) Wnt8a1 expressing cells are found in the tailbud at 2,5 days post fertilization (dpf) and 3,5 dpf and Wnt8a2 is expressed in caudal hematopoietic tissue (arrows in D). Line in (F) marks the position of the transversal section shown in the inset. (G) After 4 dpf, Wnt8a1 is expressed in the entire gut, oesophagus (not before 4 dpf (I)), swim- and gall bladder. The anatomy of these structures has been well described previously . (I) The dissected gut system is shown for clarity. The inset shows Wnt8a1 expression in the outflow tract of the dissected heart. (H) Wnt8a2 transcripts are present in the otic vesicles. a, atrium; ca, caudal artery; cv, caudal vein; g, gall bladder; l, liver; no, notochord; nt, neural tube; oe, oesophagus; oft, outflow tract; ov, otic vesicle; psm, presomitic mesoderm; sb, swim bladder; som, somites; tb, tailbud; v, ventricle.