Fig. 1

necab2, conserved in the vertebrate lineage, is expressed in the developing zebrafish brain. (A) Protein Diagrams of NECAB2 orthologs in H. sapiens, M. musculus, and D. rerio. Diagrams represented the longest isoform. The diagrams were denoted by the species name and chromosome number (Chr.) All major functional domains are conserved. Scale bar = 50 amino acids (aa). (B) Protein Diagrams of the Necab family in D. rerio. Diagrams represented the longest isoforms. Diagrams are denoted by the gene name. All the major functional domains were conserved in Necab1, Necab2, and Necab3. Scale bar = 50 amino acids (aa). (C) The phylogenetic tree of the evolutionary relationship of NECAB2. Diverse animal phyla were identified by a best reciprocal BLAST search with human NECAB2 and were mapped onto a phylogenetic tree. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances. All of the sequences were available from the NCBI protein database. Scale bar = 0.10. (D) Whole-mount in situ hybridization of necab2 in wild-type embryos at 24, 48, and 96 hpf. necab2 was expressed in selected brain regions reminiscent of the locations of nascent neurons. Scale bar = 500 μm. (E) Whole-mount immunofluorescence of Necab2 in wild-type larvae at 7 dpf. Necab2 was abundant in the telencephalon, habenula, and cerebellum. Scale bar = 100 μm. Necab2 was detected in structures reminiscent of synapse boutons (arrowheads) and the cell cytoplasm/membrane avoiding the nucleus (asterisks). The region in the dashed white box was shown at a higher magnification on the right. Scale bar = 5 μm. NECAB2 protein domains: EF, EF-Hand; NHR, NECAB Homologous Region; ABM, Antibiotic Biosynthesis Monooxygenase. hpf, hour post-fertilization; dpf, day post-fertilization; Tel, telencephalon; Di, diencephalon; Ce, cerebellum; Re, retina; Ha, habenula; OT, optic tectum; MHB, midbrain-hindbrain boundary; HB, hindbrain.