Bioinformatic and expression analysis of the zebrafish orthologues of Cacna2d4. (A) Maximum likelihood phylogenetic tree of the Cacna2d family. Eight members were identified in the zebrafish genome, two of which are clear orthologues of mammalian Cacna2d4 (dashed box). (B) Schematic representation of predicted protein structure applying to both members of the Cacna2d4 subclade (dashed box in A). Cacna2d4 proteins contain Van-Willebrand factor (VWA) and VWA N-terminal (VWA-N) domains, as well as one Cache domain, an N-terminal signal peptide and a ω site adjacent to a short C-terminal hydrophobic region, predicted to mediate proteolytic cleavage and GPI anchoring. Proteolytic cleavage (dashed line) of the Cacna2d4 pro-form gives rise to α2 and δ peptides, which remain attached to each other through disulfide bonds in mature α2δ proteins. (C) In situ hybridization of zebrafish cacna2d4a and cacna2d4b. In 3 dpf zebrafish, cacna2d4a is expressed throughout the retinal INL and in photoreceptors (Phs) of the retinal periphery (arrowheads). In the adult retina, cacna2d4a is expressed in the INL and the distal half of the Ph layer. In larval zebrafish, cacna2d4b shows strong expression in Phs, weak expression in the INL and additional signal in the cerebellum. In the adult retina, cacna2d4b is expressed across the Ph layer and in the INL. Arrows in (B) indicate proteolytic cleavage. aa, amino acids; CB, cerebellum; Dr, Danio rerio; GCL, ganglion cell layer; Hs, Homo sapiens; Mm, Mus musculus. Scale bars correspond to 100 μm in images of 3 dpf zebrafish and 50 μm in adult sections.
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