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Melanosomes in pigmented epithelia maintain eye lens transparency during zebrafish embryonic development.

ABSTRACT: Altered levels of trace elements are associated with increased oxidative stress that is eventually responsible for pathologic conditions. Oxidative stress has been proposed to be involved in eye diseases, including cataract formation. We visualized the distribution of metals and other trace elements in the eye of zebrafish embryos by micro X-ray fluorescence (?-XRF) imaging. Many elements showed highest accumulation in the retinal pigment epithelium (RPE) of the zebrafish embryo. Knockdown of the zebrafish brown locus homologues tyrp1a/b eliminated accumulation of these elements in the RPE, indicating that they are bound by mature melanosomes. Furthermore, albino (slc45a2) mutants, which completely lack melanosomes, developed abnormal lens reflections similar to the congenital cataract caused by mutation of the myosin chaperon Unc45b, and an in situ spin trapping assay revealed increased oxidative stress in the lens of albino mutants. Finally transplanting a wildtype lens into an albino mutant background resulted in cataract formation. These data suggest that melanosomes in pigment epithelial cells protect the lens from oxidative stress during embryonic development, likely by buffering trace elements.

SUBMITTER: Takamiya M

PROVIDER: S-EPMC4855227 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Melanosomes in pigmented epithelia maintain eye lens transparency during zebrafish embryonic development.

Takamiya Masanari M Xu Feng F Suhonen Heikki H Gourain Victor V Yang Lixin L Ho Nga Yu NY Helfen Lukas L Schröck Anne A Etard Christelle C Grabher Clemens C Rastegar Sepand S Schlunck Günther G Reinhard Thomas T Baumbach Tilo T Strähle Uwe U

Scientific reports 20160504

Altered levels of trace elements are associated with increased oxidative stress that is eventually responsible for pathologic conditions. Oxidative stress has been proposed to be involved in eye diseases, including cataract formation. We visualized the distribution of metals and other trace elements in the eye of zebrafish embryos by micro X-ray fluorescence (μ-XRF) imaging. Many elements showed highest accumulation in the retinal pigment epithelium (RPE) of the zebrafish embryo. Knockdown of th ...[more]

PMID: 27141993

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Project description:Heat shock proteins (Hsps) were originally identified as proteins expressed after exposure of cells to environmental stress. Several Hsps were subsequently shown to play roles as molecular chaperones in normal intracellular protein folding and targeting events and to be expressed during discrete periods in the development of several embryonic tissues. However, only recently have studies begun to address the specific developmental consequences of inhibiting Hsp expression to determine whether these molecular chaperones are required for specific developmental events. We have previously shown that the heat-inducible zebrafish hsp70 gene is expressed during a distinct temporal window of embryonic lens formation at normal growth temperatures. In addition, a 1.5-kb fragment of the zebrafish hsp70 gene promoter is sufficient to direct expression of a gfp reporter gene to the lens, suggesting that the hsp70 gene is expressed as part of the normal lens development program. Here, we used microinjection of morpholino-modified antisense oligonucleotides (MOs) to reduce Hsp70 levels during zebrafish development and to show that Hsp70 is required for normal lens formation. Hsp70-MO-injected embryos exhibited a small-eye phenotype relative to wild-type and control-injected animals, with the phenotype discernable during the second day of development. Histological and immunological analysis revealed a small, underdeveloped lens. Numerous terminal deoxynucleotidyl transferase-mediated dUTP-fluoroscein nick-end labeling (TUNEL)-positive nuclei appeared in the lens of small-eye embryos after 48 hours postfertilization (hpf), whereas they were no longer apparent in untreated embryos by this age. Lenses transplanted from hsp70-MO-injected embryos into wild-type hosts failed to recover and retained the immature morphology characteristic of the small-eye phenotype, indicating that the lens phenotype is lens autonomous. Our data suggest that the lens defect in hsp70-MO-injected embryos is predominantly at the level of postmitotic lens fiber differentiation, a result supported by the appearance of mature lens organization in these embryos by 5 days postfertilization, once morpholino degradation or dilution has occurred.

Project description:BackgroundWe highlight an unrecognized physiological role for the Greek key motif, an evolutionarily conserved super-secondary structural topology of the βγ-crystallins. These proteins constitute the bulk of the human eye lens, packed at very high concentrations in a compact, globular, short-range order, generating transparency. Congenital cataract (affecting 400,000 newborns yearly worldwide), associated with 54 mutations in βγ-crystallins, occurs in two major phenotypes nuclear cataract, which blocks the central visual axis, hampering the development of the growing eye and demanding earliest intervention, and the milder peripheral progressive cataract where surgery can wait. In order to understand this phenotypic dichotomy at the molecular level, we have studied the structural and aggregation features of representative mutations.MethodsWild type and several representative mutant proteins were cloned, expressed and purified and their secondary and tertiary structural details, as well as structural stability, were compared in solution, using spectroscopy. Their tendencies to aggregate in vitro and in cellulo were also compared. In addition, we analyzed their structural differences by molecular modeling in silico.ResultsBased on their properties, mutants are seen to fall into two classes. Mutants A36P, L45PL54P, R140X, and G165fs display lowered solubility and structural stability, expose several buried residues to the surface, aggregate in vitro and in cellulo, and disturb/distort the Greek key motif. And they are associated with nuclear cataract. In contrast, mutants P24T and R77S, associated with peripheral cataract, behave quite similar to the wild type molecule, and do not affect the Greek key topology.ConclusionWhen a mutation distorts even one of the four Greek key motifs, the protein readily self-aggregates and precipitates, consistent with the phenotype of nuclear cataract, while mutations not affecting the motif display 'native state aggregation', leading to peripheral cataract, thus offering a protein structural rationale for the cataract phenotypic dichotomy "distort motif, lose central vision".

Dataset Information

Melanosomes in pigmented epithelia maintain eye lens transparency during zebrafish embryonic development.

Publications

Melanosomes in pigmented epithelia maintain eye lens transparency during zebrafish embryonic development.

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