Cargando…
The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function
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...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2017
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708185/ https://www.ncbi.nlm.nih.gov/pubmed/29201567 http://dx.doi.org/10.7717/peerj.4093 |
_version_ | 1783282595908288512 |
---|---|
author | Posner, Mason Murray, Kelly L. McDonald, Matthew S. Eighinger, Hayden Andrew, Brandon Drossman, Amy Haley, Zachary Nussbaum, Justin David, Larry L. Lampi, Kirsten J. |
author_facet | Posner, Mason Murray, Kelly L. McDonald, Matthew S. Eighinger, Hayden Andrew, Brandon Drossman, Amy Haley, Zachary Nussbaum, Justin David, Larry L. Lampi, Kirsten J. |
author_sort | Posner, Mason |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5708185 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-57081852017-12-03 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 L. Lampi, Kirsten J. PeerJ Developmental Biology 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. PeerJ Inc. 2017-11-27 /pmc/articles/PMC5708185/ /pubmed/29201567 http://dx.doi.org/10.7717/peerj.4093 Text en ©2017 Posner et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
spellingShingle | Developmental Biology Posner, Mason Murray, Kelly L. McDonald, Matthew S. Eighinger, Hayden Andrew, Brandon Drossman, Amy Haley, Zachary Nussbaum, Justin David, Larry L. Lampi, Kirsten J. The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function |
title | The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function |
title_full | The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function |
title_fullStr | The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function |
title_full_unstemmed | The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function |
title_short | The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function |
title_sort | zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function |
topic | Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708185/ https://www.ncbi.nlm.nih.gov/pubmed/29201567 http://dx.doi.org/10.7717/peerj.4093 |
work_keys_str_mv | AT posnermason thezebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT murraykellyl thezebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT mcdonaldmatthews thezebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT eighingerhayden thezebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT andrewbrandon thezebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT drossmanamy thezebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT haleyzachary thezebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT nussbaumjustin thezebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT davidlarryl thezebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT lampikirstenj thezebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT posnermason zebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT murraykellyl zebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT mcdonaldmatthews zebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT eighingerhayden zebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT andrewbrandon zebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT drossmanamy zebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT haleyzachary zebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT nussbaumjustin zebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT davidlarryl zebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction AT lampikirstenj zebrafishasamodelsystemforanalyzingmammalianandnativeacrystallinpromoterfunction |