The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function

Mason Posner, Kelly L. Murray, Matthew S. McDonald, Hayden Eighinger, Brandon Andrew, Amy Drossman, Zachary Haley, Justin Nussbaum, Larry David, Kirsten Lampi

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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 α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-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 α-crystallins. Our data showed that mouse and zebrafish αA-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 αB-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 αB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αA increased between 1 and 2 days post fertilization (dpf), αBa increased between 4 and 5 dpf, but αBb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, aBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4-5 dpf, and was localized to the eye. αBa 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 αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.

Original languageEnglish (US)
Article number4093
JournalPeerJ
Volume2017
Issue number11
DOIs
StatePublished - Jan 1 2017

Fingerprint

crystallins
Crystallins
Zebrafish
Danio rerio
promoter regions
embryo (animal)
Embryonic Structures
Lens
Lenses
Fertilization
Notochord
Muscle
Green Fluorescent Proteins
green fluorescent protein
skeletal muscle
Skeletal Muscle
protein synthesis
eyes
Tissue
mice

Keywords

  • Crystallins
  • Gene expression
  • GFP
  • Lens
  • Mass spectrometry
  • Promoters
  • Proteomics
  • Vision
  • Zebrafish

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Posner, M., Murray, K. L., McDonald, M. S., Eighinger, H., Andrew, B., Drossman, A., ... Lampi, K. (2017). The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function. PeerJ, 2017(11), [4093]. https://doi.org/10.7717/peerj.4093

The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function. / Posner, Mason; Murray, Kelly L.; McDonald, Matthew S.; Eighinger, Hayden; Andrew, Brandon; Drossman, Amy; Haley, Zachary; Nussbaum, Justin; David, Larry; Lampi, Kirsten.

In: PeerJ, Vol. 2017, No. 11, 4093, 01.01.2017.

Research output: Contribution to journalArticle

Posner, M, Murray, KL, McDonald, MS, Eighinger, H, Andrew, B, Drossman, A, Haley, Z, Nussbaum, J, David, L & Lampi, K 2017, 'The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function', PeerJ, vol. 2017, no. 11, 4093. https://doi.org/10.7717/peerj.4093
Posner M, Murray KL, McDonald MS, Eighinger H, Andrew B, Drossman A et al. The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function. PeerJ. 2017 Jan 1;2017(11). 4093. https://doi.org/10.7717/peerj.4093
Posner, Mason ; Murray, Kelly L. ; McDonald, Matthew S. ; Eighinger, Hayden ; Andrew, Brandon ; Drossman, Amy ; Haley, Zachary ; Nussbaum, Justin ; David, Larry ; Lampi, Kirsten. / The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function. In: PeerJ. 2017 ; Vol. 2017, No. 11.
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KW - Proteomics

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