1
Manipulating amounts of the lens protein alpha A-crystallin can alter the development of lens cataract Cassie Craig, Dept. of Biology & Environmental Science Program, Ashland University, OH Mentor: Mason Posner, Ph.D. Examination of lens crystallin promoters Adult zebrafish were placed in breeding tanks to collect eggs for injection experiments. All injections were done with a Harvard Apparatus picoinjector. Linearized plasmids containing either a promoter for aBa-, aBb- or bB1-crystallin attached to the GFP gene were injected into single-celled zygotes. Seventy picograms of each plasmid with 0.2% phenol red as a tracer dye was injected in a volume of 1 nanoliter. Twenty-four hours post fertilization (hpf) the chemical PTU was added to the water holding the injected embryos to block melanin production so that GFP expression could be visualized by fluorescent microscopy. Morpholino injections to block aA-crystallin production Using a similar injection technique as above, 4.5 ng of an MO that blocks aA-crystallin translation was injected into zebrafish embryos, which were then examined by Nomarski microscopy to identify cataracts and embryos were sectioned and stained with hematoxylin and eosin to identify any abnormalities in lens development. The purpose of this study was to show that altering aA-crystallin levels can impact the development of cataracts and that the cloche zebrafish mutant can be a valuable model for studying this costly visual disease. BACKGROUND PURPOSE AND HYPOTHESIS MATERIALS AND METHODS DISCUSSION REFERENCES Cataracts are the leading cause of human blindness. Zebrafish have become a valuable model for human diseases because of their similar genetics, ability to produce many offspring for study, and the ease with which they can be genetically modified. Alpha A and alpha B-crystallin are small heat shock proteins that are components of the vertebrate eye lens. Mutations in a-crystallins are known to cause lens diseases such as cataract. A previous study showed that low levels of A-crystallin are associated with lens cataract in the cloche zebrafish mutant (Goishi 2006). In this study we examined how altering a- crystallin levels affect cataract development. Morpholino anti-sense RNAs (MO) were injected into wildtype and cloche embryos to block the production of aA-crystallin protein. We previously showed that prevention of aA-crystallin production had no effect on the wildtype lens (Posner 2013). Here we tested whether the cloche lens is more susceptible to cataract development. We also examined the effectiveness of various lens protein promoters to introduce crystallins into the cloche zebrafish so that we can study their ability to prevent cataract. This was done by visualizing the ability of various crystallin promoters to drive the expression of green fluorescent protein (GFP) in zebrafish embryos. Our results will lead to future studies that use the cloche zebrafish as a model to test the ability of native and modified a-crystallins to prevent lens cataract. ACKNOWLEDGMENTS Human bB1-crystallin promoter can be used to add proteins to the cloche zebrafish lens: Because the cloche zebrafish mutant develops cataract if it has two copies of the mutant allelle, this strain has the potential to be used as a model system for studying cataract development and prevention. We show here that the human bB1-crystallin promoter can efficiently drive expression of a foreign protein into the zebrafish lens. We can now use this promoter to introduce various native and modified a-crystallins to determine whether they can prevent the development of cataract. The work reported here provides a foundation for these future studies. Zebrafish aBa- and aBa-crystallin promoters are not active in lens: Adult zebrafish produce both aB- crystallin proteins in their lens. Interestingly, the promoters for these genes were not active in the embryonic lens. This suggests that the expression and function of these two proteins changes during zebrafish development. Our results also show that neither of these promoters will be useful for introducing proteins into the zebrafish lens. Interestingly, aBa-crystallin is only found in the lens of adult zebrafish, but here we show it expressed in skeletal muscle and notochord of embryos. Future studies will need to address why and how this protein becomes restricted to the lens later in development. This topic is relevant to a broader question about how the location of protein expression can evolve. MO injection causes cataracts in the heterozygous cloche mutant allele: We previously showed that the lens of wildtype zebrafish develop normally if aA-crystallin production is blocked by morpholino injection (MO). The results of this present study, however, show that embryos heterozygous for the cloche mutant allele will develop cataract after MO injection. This result suggests that the cloche lens is more susceptible to cataract than wildtype zebrafish, perhaps due to greater physiological stress related to the cloche mutation. In the heterozygous cloche embryo one allele is mutant and the other is not. The lens research community has been interested in determining the role that aA-crystallin may play in preventing cataract. Our results here suggest that the impact of aA-crystallin may depend on the genetic background of the individual being examined. • Goishi, K., Shimizu, A., Najarro, G., Watanabe, S., Rogers, R., Zon, L. I., & Klagsbrun, M. (2006). αA-crystallin expression prevents γ-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens. Development (09501991), 133(13), 13. • Posner, M., Skiba, J., Brown, M., Liang, J. O., Nussbaum, J., & Prior, H. (2013). Loss of the small heat shock protein αA- crystallin does not lead to detectable defects in early zebrafish lens development. Experimental Eye Research, 116227-233. doi:10.1016/j.exer.2013.09.007 • Smith, A. A., Wyatt, K., Vacha, J., Vihtelic, T. S., Zigler Jr, J. S., Wistow, G. J., & Posner, M. (2006). Gene duplication and separation of functions in αB-crystallin from zebrafish (Danio rerio). FEBS Journal, 273(3), 481-490. doi:10.1111/j.1742-4658.2005.05080.x Thank you Mason Posner and Kelly Murray for the help and mentoring of the project. Funding for this project was provided by the Department of Biology. For additional information, please contact [email protected] or [email protected] Figure 2: Embryos showed GFP expression in the notochord, skeletal muscle and geometric shapes (insert). The proportion of embryos with GFP expression in skeletal muscle and notochord differed between the two promoters. The aBa promoter was more active in notochord while the aBb promoter was more active in skeletal muscle. Geometric shapes expressing GFP were identified in the skin of the embryos, but reasons for this expression and the identity of this tissue are unclear. Lens expression was very rare or absent with these promoters. Error bars indicate standard error (n = 3 and 2 respectively). Figure 1: The human bb1 promoter drove GFP expression in the zebrafish lens (insert). Two batches of 40 eggs each were injected with the bb1/GFP plasmid. Over a course of five days the embryos were checked for expression. GFP Expression occurred after 48 hours in the lens, increased up until 72 hours and then stabilized after 72 hours. It was found that 38% of the embryos showed GFP expression. There was no expression anywhere else outside of the lens. Figure 3: Sections through zebrafish embryos. Wildtype embryos without lens cataracts showed normal lens development (A). A heterozygous cloche mutant embryo injected with an MO to block aA-crystallin production shows abnormal lens development (B). The lens contains “dents” and the central fiber cells have not lost their nuclei, as normally happens during lens development. Fifty percent of injected cloche embryos had cataract. 0 48 72 120 0% 5% 10% 15% 20% 25% 30% 35% 40% Hours post fertilization Percent of Embryos With GFP Expression The human bB1-crystallin promoter can drive GFP expression in the zebrafish lens Skeletal Muscle Notochord Geometric Shapes Lens 0 10 20 30 40 50 60 70 80 90 Alpha-Ba Promotor Injection Alpha-Bb Promotor Injection Location on Fish Average Percent of Zebrafish with GFP Expression The aBa and aBb promoters differ in expression and show rare expression in lens MO injections resulted in cataract for cloche embryos but not in wildtype embryos Picoinjector Zygo te Yolk Embryo A B RESULTS
