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Nicholas Hanovice, Christina M.S. Daly, Jeffrey Gross; N ethyl maleimide-sensitive factor b (nsfb) is required for melanogenesis in the zebrafish retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2318.
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© ARVO (1962-2015); The Authors (2016-present)
Melanogenesis in the retinal pigment epithelium (RPE) involves endocytic and secretory pathways, and is critical for proper retinal development and visual system function in vertebrates. To better understand how melanogenesis occurs in vivo, we isolated a recessive zebrafish oculocutaneous albino mutant (au18), and identified a nonsense mutation in the N-ethyl maleimide sensitive factor b (nsfb) gene, which encodes a protein responsible for SNAP/SNARE complex recycling. Virtually nothing is known about the function of nsfb, or its role in in melanogenesis. We hypothesized that loss of nsfb activity disrupts melanosome maturation in au18 RPE.
To identify the causative mutation in au18, heterozygous carriers were crossed to a polymorphic strain of zebrafish to generate a mapping line. From incrosses of this line, 200 homozygous mutant embryos were collected, genomic DNA was isolated and utilized in whole-genome Illumina sequencing. Sequence data were analyzed via the MegaMapper single nucleotide polymorphism (SNP)-mapping pipeline. cDNA sequencing from au18 mutants was performed to confirm the putative nsfb mutation.<br /> To identify the cellular basis of the hypopigmentation phenotype in au18, transmission electron microscopy (TEM) was utilized. Since au18 hypopigmentation varies in severity, mildly- and severely-hypopigmented embryos (n=3) were analyzed at 48, 58 and 72 hours post fertilization. TEM images at 11,500X were taken from dorsal, central and ventral RPE and the number, size, and maturity of melanosomes were quantified using Image J software.
Whole genome sequencing and SNP mapping identified a nonsense mutation in nsfb in au18 mutants. cDNA sequencing confirmed the presence of the mutation (C893T), which truncates the nsfb protein by roughly two-thirds (Y297X).<br /> TEM analyses indicate that while the average perimeter of melanosomes in au18 RPE was not decreased compared to sibling RPE at 48hpf, average melanosome number and maturity were significantly decreased (p<0.05). 58hpf au18 RPE contained fewer and less mature melanosomes compared to sibling, but melanosome perimeter was not significantly changed, a trend that continued in 72hpf embryos.
These results support a model in which nsfb activity is required for the maturation of melanosomes in the vertebrate RPE, and are the first to identify a role for nsfb during RPE development.
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