PUBLICATION

N-Ethylmaleimide-Sensitive Factor b (nsfb) Is Required for Normal Pigmentation of the Zebrafish Retinal Pigment Epithelium

Authors
Hanovice, N.J., Daly, C.M., Gross, J.M.
ID
ZDB-PUB-151201-3
Date
2015
Source
Investigative ophthalmology & visual science   56: 7535-7544 (Journal)
Registered Authors
Gross, Jeffrey, Hanovice, Nick
Keywords
none
MeSH Terms
  • DNA Mutational Analysis
  • Melanosomes/metabolism
  • Albinism/embryology
  • Albinism/genetics*
  • Albinism/metabolism
  • Microscopy, Electron, Transmission
  • DNA/genetics*
  • Pigmentation/genetics*
  • Zebrafish/embryology*
  • Retinal Pigment Epithelium/embryology
  • Retinal Pigment Epithelium/metabolism*
  • Retinal Pigment Epithelium/ultrastructure
  • N-Ethylmaleimide-Sensitive Proteins/genetics*
  • N-Ethylmaleimide-Sensitive Proteins/metabolism
  • Disease Models, Animal
  • Animals
  • In Situ Hybridization
  • Mutation*
(all 18)
PubMed
26618645 Full text @ Invest. Ophthalmol. Vis. Sci.
Abstract
Despite the number of albinism-causing mutations identified in human patients and animal models, there remain a significant number of cases for which no mutation has been identified, suggesting that our understanding of melanogenesis is incomplete. Previously, we identified two oculocutaneous albinism mutations in zebrafish, au13 and au18. Here, we sought to identify the mutated loci and determine how the affected proteins contribute to normal pigmentation of the retinal pigment epithelium (RPE).
Complementation analyses revealed that au13 and au18 belonged to a single complementation group, suggesting that they affected the same locus. Whole-genome sequencing and single nucleotide polymorphism (SNP) analysis was performed to identify putative mutations, which were confirmed by cDNA sequencing and mRNA rescue. Transmission electron microscopy (TEM) and image quantification were used to identify the cellular basis of hypopigmentation.
Whole-genome sequencing and SNP mapping identified a nonsense mutation in the N-ethylmaleimide-sensitive factor b (nsfb) gene in au18 mutants. Complementary DNA sequencing confirmed the presence of the mutation (C893T), which truncates the nsfb protein by roughly two-thirds (Y297X). No coding sequence mutations were identified in au13, but quantitative PCR revealed a significant decrease in nsfb expression, and nsfb mRNA injection rescued the hypopigmentation phenotype, suggesting a regulatory mutation. In situ hybridization revealed that nsfb is broadly expressed during embryonic development, including in the RPE. Transmission electron microscopy analyses indicated that average melanosome density and maturity were significantly decreased in nsfb mutants.
au18 and au13 contain mutations in nsfb, which encodes a protein that is required for the maturation of melanosomes in zebrafish RPE.
Genes / Markers
Figures
Figure Gallery (6 images)
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Expression
Phenotype
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
au13
    Unknown
    au18
      Point Mutation
      1 - 2 of 2
      Show
      Human Disease / Model
      Sequence Targeting Reagents
      No data available
      Fish
      1 - 3 of 3
      Show
      Antibodies
      No data available
      Orthology
      Gene Orthology
      nsfa
      nsfb
      1 - 2 of 2
      Show
      Engineered Foreign Genes
      No data available
      Mapping
      No data available