Zhao Y et al. (2011), The expression of αA- and βB1-crystallin during...

XB-ART-43153

Mol Vis 2011 Mar 23;17:768-78.

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The expression of αA- and βB1-crystallin during normal development and regeneration, and proteomic analysis for the regenerating lens in Xenopus laevis.

Zhao Y , Ju F , Zhao Y , Wang L , Sun Z , Liu M , Gao L .

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To 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. Real 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. αA-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. The 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.

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Species referenced: Xenopus laevis
Genes referenced: dnai1

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	Figure 1. SDS–PAGE for induced expression of recombinant proteins (αA-crystallin-pet28, βB1-crystallin-pet28) and western blotting analysis for the two specific antibodies. Recombinant proteins were expressed in E. coli Rosetta (DE3). Their respective molecular weigh are 19 kDa and 23 kDa. In western blotting analysis, sample 1, 2, and 3 are total lens proteins; samples 4 and 5 are purified fusion proteins. By results, good specificity is shown.
	Figure 2. Real-time PCR analysis for transcripts of αA-crystallin and βB1-crystallin during the lens development. Stages are according to the normal table of Nieuwkoop & Faber. The red curve illustrates the relative expression of transcripts of αA-crystallin. From the beginning of expression at stage 26 to stage 38, it displayed an increasing trend. After stage 38, the expression began to decrease. Finally, the expression quantity maintained at a relative stable level. The same went for βB1-crystallin, which is shown by the black curve.
	Figure 3. Real time PCR analysis for transcripts of αA-crystallin and βB1-crystallin during lens regeneration. The eyeballs of regenerated lenses were dissected at pre-operation, 0 day, 3 days, 5 days, 7 days, 9 days, 14 days, 21 days, and 30 days after lentectomy. The transcripts of two crystallins showed the same variation trends.
	Figure 4. Immunofluorescence for αA-crystallin and βB1-crystallin during embryonic lens development. Sections double-stained with αA-crystallin and βB1-crystallin antisera at different developmental stages, analyzed by confocal microscopy. Some cells express predominantly βB1-crystallin (red) and some αA-crystallin (green). Overall, there is a strong co-localization of these two crystallin proteins throughout the lens cells. First positive immunofluorescence was detected at stage 29/30 (A). At stage 32 (B), a number of cells in the area of the lens rudiment where lens fibers will form. With further differentiation, the lens primary fibers and secondary primary fibers are formed during stage 34–46 (C-H). Negative controls: I (without antibodies); J (only secondary antibodies); K (only primary antibodies); L: differential interference contrast (DIC). Abbreviations: Le, lens; PLF, primary lens fiber; SLF, secondary lens fiber.
	Figure 5. Immunofluorescence for αA-crystallin and βB1-crystallin during the lens regeneration. The images show the distribution of αA-crystallin and βB1-crystallin in the process of the regeneration, analyzed by confocal microscopy. The regenerated lenses were dissected at operation: 3 days, 5 days, 7 days, 9 days, 11 days, 15 days, and 21 days, as is shown in A-G, respectively. Negative controls: H (only primary antibodies); I (only secondary antibodies); J: differential interference contrast (DIC). Abbreviations: Le, lens; PLF, primary lens fiber; SLF, secondary lens fiber.
	Figure 6. Two-dimensional electrophoresis photography of regenerated lens. A-F: The patterns of protein spots in regenerated lens (5 days, 7 days, 9 days, 11 days, 15 days, 0 day, respectively). The protein patterns are significantly different among all stages of regeneration. Although differences existed, some protein spots just showed the variation of expression quantity.

References [+] :

Altmann, Lens induction by Pax-6 in Xenopus laevis. 1997, Pubmed, Xenbase