Dataset Information

Analysis of a zebrafish dync1h1 mutant reveals multiple functions for cytoplasmic dynein 1 during retinal photoreceptor development.

ABSTRACT:

Background

Photoreceptors of the retina are highly compartmentalized cells that function as the primary sensory neurons for receiving and initiating transmission of visual information. Proper morphogenesis of photoreceptor neurons is essential for their normal function and survival. We have characterized a zebrafish mutation, cannonball, that completely disrupts photoreceptor morphogenesis.

Results

Analysis revealed a non-sense mutation in cytoplasmic dynein heavy chain 1 (dync1h1), a critical subunit in Dynein1, to underlie the cannonball phenotypes. Dynein1 is a large minus-end directed, microtubule motor protein complex that has been implicated in multiple, essential cellular processes. In photoreceptors, Dynein1 is thought to mediate post-Golgi vesicle trafficking, while Dynein2 is thought to be responsible for outer segment maintenance. Surprisingly, cannonball embryos survive until larval stages, owing to wild-type maternal protein stores. Retinal photoreceptor neurons, however, are significantly affected by loss of Dync1h1, as transmission electron microscopy and marker analyses demonstrated defects in organelle positioning and outer segment morphogenesis and suggested defects in post-Golgi vesicle trafficking. Furthermore, dosage-dependent antisense oligonucleotide knock-down of dync1h1 revealed outer segment abnormalities in the absence of overt inner segment polarity and trafficking defects. Consistent with a specific function of Dync1h1 within the outer segment, immunolocalization showed that this protein and other subunits of Dynein1 and Dynactin localized to the ciliary axoneme of the outer segment, in addition to their predicted inner segment localization. However, knock-down of Dynactin subunits suggested that this protein complex, which is known to augment many Dynein1 activities, is only essential for inner segment processes as outer segment morphogenesis was normal.

Conclusions

Our results indicate that Dynein1 is required for multiple cellular processes in photoreceptor neurons, including organelle positioning, proper outer segment morphogenesis, and potentially post-Golgi vesicle trafficking. Titrated knock-down of dync1h1 indicated that outer segment morphogenesis was affected in photoreceptors that showed normal inner segments. These observations, combined with protein localization studies, suggest that Dynein1 may have direct and essential functions in photoreceptor outer segments, in addition to inner segment functions.

SUBMITTER: Insinna C

PROVIDER: S-EPMC2880287 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Analysis of a zebrafish dync1h1 mutant reveals multiple functions for cytoplasmic dynein 1 during retinal photoreceptor development.

Insinna Christine C Baye Lisa M LM Amsterdam Adam A Besharse Joseph C JC Link Brian A BA

Neural development 20100422

<h4>Background</h4>Photoreceptors of the retina are highly compartmentalized cells that function as the primary sensory neurons for receiving and initiating transmission of visual information. Proper morphogenesis of photoreceptor neurons is essential for their normal function and survival. We have characterized a zebrafish mutation, cannonball, that completely disrupts photoreceptor morphogenesis.<h4>Results</h4>Analysis revealed a non-sense mutation in cytoplasmic dynein heavy chain 1 (dync1h1 ...[more]

PMID: 20412557

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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

Analysis of a zebrafish dync1h1 mutant reveals multiple functions for cytoplasmic dynein 1 during retinal photoreceptor development.

Background

Results

Conclusions

Publications

Analysis of a zebrafish dync1h1 mutant reveals multiple functions for cytoplasmic dynein 1 during retinal photoreceptor development.

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