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Ectopic proliferation contributes to retinal dysplasia in the juvenile zebrafish patched2 mutant eye.

ABSTRACT: PURPOSE:Patched is a well-studied tumor suppressor and negative regulator of the Hedgehog (Hh) pathway. Earlier work in this laboratory has shown that embryonic zebrafish patched2 (ptc2) mutant retinas possess an expanded ciliary marginal zone (CMZ) and phenotypes similar to those in human patients with basal cell naevus syndrome (BCNS), a congenital disorder linked to mutations in the human PTCH gene. This study extends the analysis of retinal structure and homeostasis in ptc2-/- mutants to juvenile stages, to determine whether Patched 2 function is essential in the postembryonic eye. METHODS:Histologic, immunohistochemical, and molecular analyses were used to characterize retinal defects in the 6-week-old juvenile ptc2-/- retina. RESULTS:Juvenile ptc2-/- mutants exhibited peripheral retinal dysplasias that included the presence of ectopic neuronal clusters in the inner nuclear layer (INL) and regions of disrupted retinal lamination. Retinal dysplasias were locally associated with ectopic proliferation. BrdU/EdU labeling and immunohistochemistry assays demonstrated that a population of ectopically proliferating cells gave rise to the ectopic neuronal clusters in the INL of ptc2-/- mutants and that this contributed to retinal dysplasia in the mutant eye. CONCLUSIONS:These results demonstrate a direct link between overproliferation and retinal dysplasia in the ptc2-/- juvenile retina and establish ectopic proliferation as the likely cellular underpinning of retinal dysplasia in juvenile ptc2-/- mutants.

SUBMITTER: Bibliowicz J

PROVIDER: S-EPMC3231842 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Ectopic proliferation contributes to retinal dysplasia in the juvenile zebrafish patched2 mutant eye.

Bibliowicz Jonathan J Gross Jeffrey M JM

Investigative ophthalmology & visual science 20111117 12

<h4>Purpose</h4>Patched is a well-studied tumor suppressor and negative regulator of the Hedgehog (Hh) pathway. Earlier work in this laboratory has shown that embryonic zebrafish patched2 (ptc2) mutant retinas possess an expanded ciliary marginal zone (CMZ) and phenotypes similar to those in human patients with basal cell naevus syndrome (BCNS), a congenital disorder linked to mutations in the human PTCH gene. This study extends the analysis of retinal structure and homeostasis in ptc2-/- mutant ...[more]

PMID: 22003118

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Project description:Introduction: Pathogenic mutations in RPGR ORF15, one of two major human RPGR isoforms, were responsible for most X-linked retinitis pigmentosa cases. Previous studies have shown that RPGR plays a critical role in ciliary protein transport. However, the precise mechanisms of disease triggered by RPGR ORF15 mutations have yet to be clearly defined. There are two homologous genes in zebrafish, rpgra and rpgrb. Zebrafish rpgra has a single transcript homologous to human RPGR ORF15; rpgrb has two major transcripts: rpgrb ex1-17 and rpgrb ORF15, similar to human RPGR ex1-19 and RPGR ORF15, respectively. rpgrb knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. However, the impact of knocking down rpgra in zebrafish remains undetermined. Here, we constructed a rpgra mutant zebrafish model to investigate the retina defect and related molecular mechanism. Methods: we utilized transcription activator-like effector nuclease (TALEN) to generate a rpgra mutant zebrafish. Western blot was used to determine protein expression. RT-PCR was used to quantify gene transcription levels. The visual function of embryonic zebrafish was detected by electroretinography. Immunohistochemistry was used to observe the pathological changes in the retina of mutant zebrafish and transmission electron microscope was employed to view subcellular structure of photoreceptor cells. Results: A homozygous rpgra mutant zebrafish with c.1675_1678delins21 mutation was successfully constructed. Despite the normal morphological development of the retina at 5 days post-fertilization, visual dysfunction was observed in the mutant zebrafish. Further histological and immunofluorescence assays indicated that rpgra mutant zebrafish retina photoreceptors progressively began to degenerate at 3-6 months. Additionally, the mislocalization of cone outer segment proteins (Opn1lw and Gnb3) and the accumulation of vacuole-like structures around the connecting cilium below the OSs were observed in mutant zebrafish. Furthermore, Rab8a, a key regulator of opsin-carrier vesicle trafficking, exhibited decreased expression and evident mislocalization in mutant zebrafish. Discussion: This study generated a novel rpgra mutant zebrafish model, which showed retinal degeneration. our data suggested Rpgra is necessary for the ciliary transport of cone-associated proteins, and further investigation is required to determine its function in rods. The rpgra mutant zebrafish constructed in this study may help us gain a better understanding of the molecular mechanism of retinal degeneration caused by RPGR ORF15 mutation and find some useful treatment in the future.

Dataset Information

Ectopic proliferation contributes to retinal dysplasia in the juvenile zebrafish patched2 mutant eye.

Publications

Ectopic proliferation contributes to retinal dysplasia in the juvenile zebrafish patched2 mutant eye.

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