Sections of retina (A–D) and cleared tissue (E). DCX positive cells (green arrows) are detected in A. burtoni retina (A) but not in zebrafish (C). Magnification of (A,C) are presented in (B,D). The proliferating cell nuclear antigen (PCNA; red arrows) binding antibodies detect cells in the peripheral germinal zone (PGZ; of both species. In addition, DCX (green arrows) and PCNA (red arrows) are detected in the ONL corresponding to cones and rod precursors, respectively. Nuclei are stained with DRAQ5 (blue). No double positive cells for DCX (green arrows) and PCNA (red arrows) are found. By analyzing the whole transparent eyeball, it is possible to follow DCX positive processes in the neural fiber layer (green arrow) into the optic nerve (E; white arrow). Scale bar 20 µm (A,C); 10 µm (B,D). ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer; NFL: neural fiber layer.

Western blot analysis of protein extracts derived from zebrafish, A. burtoni, and mice brains. Increasing concentrations of proteins were loaded in each well (40, 60 and 80 μg per well). Analysis shows the expression of DCX in A. burtoni and mice, but not in zebrafish extracts. β-actin was used as the loading control after the membrane stripping.

Alignments of DCX and DCLK amino acid sequences. Conserved residues between mice and A. burtoni DCX proteins (A); Conservation of the DCLK2 immunogen sequence use to detecting DCLK protein among the studied species (B).