The zebrafish as a model system for analyzing mammalian and native [alpha]-crystallin promoter function

• Published in: PeerJ (San Francisco, CA) CA), 2017-11, Vol.5, p.e4093
• Main Authors: Posner, Mason, Murray, Kelly L, McDonald, Matthew S, Eighinger, Hayden, Andrew, Brandon, Drossman, Amy, Haley, Zachary, Nussbaum, Justin, David, Larry L, Lampi, Kirsten J
• Format: Article
• Language: English
• Subjects: Animal development; Animal genetics; Crystalline lens; Physiological aspects; Promoters (Genetics); Zebra fish
• ISSN: 2167-8359; 2167-8359
• DOI: 10.7717/peerj.4093

Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish [alpha]-crystallin promoter function, how it compares to that of mammals, or whether mammalian [alpha]-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of [alpha]-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish [alpha]-crystallins. Our data showed that mouse and zebrafish [alpha]A-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse [alpha]B-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish [alpha]B-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the [alpha]Bb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish [alpha]A increased between 1 and 2 days post fertilization (dpf), [alpha]Ba increased between 4 and 5 dpf, but [alpha]Bb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect [alpha]A, aBa, and [alpha]Bb peptides in digests of zebrafish embryos. In whole embryos, [alpha]A-crystallin was first detected by 2 dpf, peaked in abundance by 4-5 dpf, and was localized to the eye. [alpha]Ba was detected in whole embryo at nearly constant levels from 1-6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4-7 dpf. In contrast, due to its low abundance, no [alpha]Bb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mamma