Dataset Information

A conserved role of ?A-crystallin in the development of the zebrafish embryonic lens.

ABSTRACT: ?A- and ?B-crystallins are small heat shock proteins that bind thermodynamically destabilized proteins thereby inhibiting their aggregation. Highly expressed in the mammalian lens, the ?-crystallins have been postulated to play a critical role in the maintenance of lens optical properties by sequestering age-damaged proteins prone to aggregation as well as through a multitude of roles in lens epithelial cells. Here, we have examined the role of ?-crystallins in the development of the vertebrate zebrafish lens. For this purpose, we have carried out morpholino-mediated knockdown of ?A-, ?Ba- and ?Bb-crystallin and characterized the gross morphology of the lens. We observed lens abnormalities, including increased reflectance intensity, as a consequence of the interference with expression of these proteins. These abnormalities were less frequent in transgenic zebrafish embryos expressing rat ?A-crystallin suggesting a specific role of ?-crystallins in embryonic lens development. To extend and confirm these findings, we generated an ?A-crystallin knockout zebrafish line. A more consistent and severe lens phenotype was evident in maternal/zygotic ?A-crystallin mutants compared to those observed by morpholino knockdown. The penetrance of the lens phenotype was reduced by transgenic expression of rat ?A-crystallin and its severity was attenuated by maternal ?A-crystallin expression. These findings demonstrate that the role of ?-crystallins in lens development is conserved from mammals to zebrafish and set the stage for using the embryonic lens as a model system to test mechanistic aspects of ?-crystallin chaperone activity and to develop strategies to fine-tune protein-protein interactions in aging and cataracts.

SUBMITTER: Zou P

PROVIDER: S-EPMC4638411 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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A conserved role of αA-crystallin in the development of the zebrafish embryonic lens.

Zou Ping P Wu Shu-Yu SY Koteiche Hanane A HA Mishra Sanjay S Levic Daniel S DS Knapik Ela E Chen Wenbiao W Mchaourab Hassane S HS

Experimental eye research 20150704

αA- and αB-crystallins are small heat shock proteins that bind thermodynamically destabilized proteins thereby inhibiting their aggregation. Highly expressed in the mammalian lens, the α-crystallins have been postulated to play a critical role in the maintenance of lens optical properties by sequestering age-damaged proteins prone to aggregation as well as through a multitude of roles in lens epithelial cells. Here, we have examined the role of α-crystallins in the development of the vertebrate ...[more]

PMID: 26149094

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Project description:BackgroundThe teleost Astyanax mexicanus is a single species consisting of eyed surface-dwelling (surface fish) and blind cave-dwelling (cavefish) morphs. Cavefish eyes are lost through apoptosis of the lens, which in turn promotes the degeneration of other optic tissues. The αA-crystallin (αA-crys) gene is strongly downregulated in the cavefish lens and is located in a genomic region (QTL) responsible for eye loss. Therefore, αA-crys has been proposed as a candidate for regulating cavefish eye degeneration. The purpose of this study was to determine the mechanism of αA-crys downregulation and its role in cavefish eye degeneration.ResultsThe involvement of αA-crys in eye degeneration was confirmed by knocking down its expression in surface fish, which led to apoptosis of the lens. The underlying reason for αA-crys downregulation in cavefish was investigated by comparing genomic αA-crys DNA sequences in surface fish and cavefish, however, no obvious cis-regulatory factors were discovered. Furthermore, the cavefish αA-crys allele is expressed in surface fish x cavefish F1 hybrids, indicating that evolutionary changes in upstream genes are most likely responsible for αA-crys downregulation. In other species, Sox2 is one of the transcription factors that regulate lens crystallin genes during eye development. Determination of sox2 expression patterns during surface fish and cavefish development showed that sox2 is specifically downregulated in the cavefish lens. The upstream regulatory function of Sox2 was demonstrated by knockdown in surface fish, which abolished αA-crys expression and induced lens apoptosis.ConclusionsThe results suggest that αA-crys is required for normal eye development in cavefish via suppression of lens apoptosis. The regulatory changes involved in αA-crys downregulation in cavefish are in trans-acting factors rather than cis-acting mutations in the αA-crys gene. Therefore, αA-crys is unlikely to be the mutated gene(s) associated with an Astyanax eye QTL. The results reveal a genetic pathway leading from sox2 to αA-crys that is required for survival of the lens in Astyanax surface fish. Defects in this pathway may be involved in lens apoptosis and thus a cause of cavefish eye degeneration.

Project description:PurposeTo explore the expression of the lens crystallins (αA- and βB1-crystallin) in Xenopus laevis embryonic lens development and regeneration and to analyze the order of different crystallins generated in the regenerating lens.MethodsReal Time-PCR, Immunofluorescence, and 2D-PAGE were used to analyze the expressions of αA-crystallin and βB1-crystallin, and related factors during embryonic lens development and regeneration in Xenopus laevis.ResultsαA-crystallin and βB1-crystallin were first detected at stage 29/30 during normal development, and the two crystallins were simultaneously detected in regeneration. During embryonic lens development, the relative expression level of the βB1-crystallin gene was higher than that of the αA-crystallin gene. In the process of the lens regeneration, however, the relative expression level of the βB1-crystallin gene was lower than that of the αA-crystallin gene. Throughout embryonic lens development, the two crystallin transcripts showed the same variation trends, and similar occurrence did in the regeneration process. Crystallins showed different localization and distribution during the ontogeny and regeneration, especially in the lens fiber region. 2D-electrophores revealed the patterns of the sequential synthesis of crystallins, with regard to the different classes and apparent variations of some auxiliary regulatory factors.ConclusionsThe ontogeny and localization of the crystallins during embryonic lens development and regeneration indicated a different development program, although they have identical origins, the ectoderm. The expression level of crystallin transcripts displayed a consistent variation tendency, but the presence of appreciable differences was still exposed. In addition to stably producing the crystallins of different classes in accordance with established procedure, these auxiliary factors may perform the function, to some extent, because of significant changes in their expression throughout the process of lens regeneration.

Dataset Information

A conserved role of ?A-crystallin in the development of the zebrafish embryonic lens.

Publications

A conserved role of αA-crystallin in the development of the zebrafish embryonic lens.

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