ue58 mutant zebrafish have a severe, peripheral nerve myelin pathology. (A) Confocal images of the spinal cord of Tg(mbp:EGFP-CAAX) control (left) and ue58 mutant (right) at 7 dpf showing disruption to CNS myelin (region within brackets). Scale bar, 10 µm. (B) Confocal images of the pLLn in Tg(mbp:EGFP-CAAX) control (top) and ue58 mutant (bottom) animals at 5 dpf showing major disruption to myelin. Scale bar, 10 µm. (C) Higher magnification images of areas demarcated in B showing myelin in control (left) and ue58 mutant (right) animals. Scale bar, 10 µm. (D) DIC images of Tg(mbp:EGFP-CAAX) control (left) and ue58 mutants (right) at 6 dpf showing appearance of tissue edema. Scale bar, 10 µm. (E) Brightfield images of control (left) and ue58 mutants (right) at 6 dpf showing generally normal morphological development. Scale bar, 0.5 mm. (F) Genomic structure of the zebrafish slc12a2b gene, showing exons (boxes) and introns (lines). White boxes denote untranslated regions. Exons in black were annotated in partial genomic sequences LOC100537771 and LOC100329477 and matched homologous exons in the orthologue slc12a2a. Exons in blue did not align with any annotated genomic sequence, and their limits were inferred by homology with slc12a2a genomic structure. Exons are drawn to scale relative to each other; introns in pink contain unknown bases (N) and are of unknown size. The start (ATG) and stop (TGA) codons are indicated in green and red, respectively. The ue58 allele has a T>A mutation in exon 26 leading to a premature stop codon. (G) Alignment of the 40 most C-terminal amino acids of NKCC1b shows high similarity between species in this domain. Arrowhead indicates the position of the premature stop codon introduced by ue58. (H) Protein structural prediction algorithms, using CCTOP, indicate that NKCC1b in zebrafish is likely to have intracellular N and C termini and 12 transmembrane domains. (I) Sequence similarities of the protein products of zebrafish NKCC1a, NKCC1b, and murine and human NKCC1 homologues.
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