Zebrafish snow white as a model for Hermansky-Pudlak Syndrome

Abstract

Purpose: : Defects in the formation or function of lysosome-related organelles (LROs) are observed in several human albinism disorders, including Chediak-Higashi Syndrome, Hermansky-Pudlak Syndrome and Griscelli Syndrome. While much has been learned about the phenotypic characteristics and the genetic loci involved in these disorders, the molecular and cellular mechanisms underlying many of them remain to be elucidated. Melanosomes are a commonly studied LRO, as they are easy to identify and their biogenesis has been well described. The purpose of this study was to establish the zebrafish snow white (snw) mutant as a model to study the molecular and cellular underpinnings of HPS.

Methods: : Histology and transmission electron microscopy were utilized to examine the morphological and ultrastructural defects in the snw eye. Total melanin levels in whole snw and sibling embryos were biochemically quantified. The density and size of melanosomes in the RPE was measured using ImageJ software. Linkage mapping and candidate gene sequencing identified the possible mutated locus in snw as Hermansky-Pudlak Syndrome 5 (hps5). A splice-blocking morpholino oligonucleotide (MO) against Hps5 was injected into 1 cell stage embryos to investigate gene knockdown effect. Phenotypic rescue by injection of either hps5^+/+ or hps5^mut mRNA into 1 cell stage embryos was performed. Formation of the Bloc2 protein complex (containing Hps5, Hps3, and Hps6) was investigated via expression of hps5^+/+ and hps5^mut constructs in COS7 cells followed by co-immunoprecipitation analyses.

Results: : The snw eye is microphthalmic but morphologically normal, with oculocutaneous hypopigmentation in both melanophores and iridophores by 2 days post fertilization (dpf), continuing through 7 dpf. snw embryos show decreased total melanin in both the eye and whole body, ranging from 60% to 37% of the levels found in wild-type siblings. Further, both the number and size of melanosomes is substantially decreased in snw embryos. The mutation was mapped to chromosome 25 and candidate genes in the region were sequenced to identify the mutated loci. A candidate mutation was identified in hps5, and MO knockdown phenocopies the snw mutant. Injection of wildtype, but not mutant, hps5 mRNA rescues pigmentation in snw mutants.

Conclusions: : The snw mutant posseses ocular defects similar to those observed in human albinism patients displaying Hermansky-Pudlak Syndrome. We demonstrate the usefulness of hps5 as a model to study melanosome biogenesis in the retinal pigmented epithelium and more generally as model for the formation, trafficking and function of lysosome-related organelles.

Keywords: retinal pigment epithelium • melanocytes • visual impairment: neuro-ophthalmological disease