The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function
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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 small 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 produced no GFP expression, suggesting that these lens responsive elements in the mouse are not used in the zebrafish. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the αBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αa, aBa and αBb were all detected by 1 day post fertilization (dpf). Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, αBa, 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 also 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, although their use in each species may reflect evolutionary changes in developmental roles for α-crystallins. An ontogenetic shift in zebrafish αBa-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.
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2017. The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function. PeerJ Preprints 5:e2889v1 https://doi.org/10.7287/peerj.preprints.2889v1note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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Supplemental Information
Three dimensional render of GFP expression in zebrafish notochord produced by a zebrafish αBb-crystallin promoter fragment
Three dimensional render of GFP expression in zebrafish skeletal muscle produced by a mouse αB-crystallin promoter fragment
Three dimensional render of GFP expression dorsal to the yolk produced by a zebrafish αA-crystallin promoter fragment
Sequence alignments of upstream regions of mouse αA (A) and αB-crystallin (B) showing conservation across vertebrate taxa
Green boxes highlight conserved regions between fishes and mammals. Images include the full extent of zebrafish genomic sequence available in the alignment. The two conserved regions in the αB alignment may be due to retention of two lens specific enhancers in the mouse promoter. However, our data suggest that these lens enhancers are not functional through 7 days post fertilization.
Targeted mass spectrometric detection of αA-crystallin peptide 52-65 (NILDSSNSGVSEVR) in digests of 1-6 day post fertilization (dpf) embryos
Spectral library: MS2 spectrum of αA peptide 52-65 created during a data-dependent analysis of adult zebrafish lens digest. Y10-12 fragment ions were the most abundant in this spectrum, and these were used to detect the peptide in embryo digests, based on their simultaneous elution at approximately 29.5 min during the LC/MS analyses (colored traces marked with an arrow). These fragment ion peaks were integrated for each digest from 1-6 dpf embryos, and results are shown in the Peak Integration Results bar graph, indicating that the αA-crystallin reached its greatest abundance at 4 days dpf. The bar in the graph labeled Library shows the relative proportion of the y10-12 ions in the MS2 spectrum from the lens library, set at the same relative abundance as the fragment ions in 4 dpf digest. The relative intensities of the y10-12 fragment ions detected in the 2-6 dpf samples were very similar to those observed in the MS2 spectrum from the library, as evidenced by their dot product (dotp) values ranging from 0.97 to 0.99 marked above each bar.
Targeted mass spectrometric detection of αA-crystallin peptide 89-99 (VTDDYVEIQGK) in digests of 1-6 day post fertilization (dpf) embryos
Spectral library: MS2 spectrum of αA peptide 89-99 created during a data-dependent analysis of adult zebrafish lens digest. Y 5, 6, and 9 fragment ions were the most abundant in this spectrum, and these were used to detect the peptide in embryo digests, based on their simultaneous elution at approximately 25.8 min during the LC/MS analyses (colored traces). These fragment ion peaks were integrated for each digest from 1-6 dpf embryos, and results are shown in the Peak Integration Results bar graph, indicating that the αA-crystallin reached its greatest abundance at 5 days dpf. The bar in the graph labeled Library shows the relative proportion of the y 5, 6, and 9 ions in the MS2 spectrum from the lens library, set at the same relative abundance as the fragment ions in 5 dpf digest. The relative intensities of the y 5, 6, and 9 fragment ions detected in the 2-6 dpf samples were very similar to those observed in the MS2 spectrum from the library, as evidenced by their dot product (dotp) values ranging from 0.97 to 0.99 marked above each bar.
Targeted mass spectrometric detection of αBa-crystallin peptide 79-88 (HFSPDELTVK) in digests of 1-6 day post fertilization (dpf) embryos
Spectral library: MS2 spectrum of αBa peptide 79-88 created during a data-dependent analysis of adult zebrafish lens digest. b2, y8, and y9 fragment ions were the most abundant in this spectrum, and these were used to detect the peptide in embryo digests, based on their simultaneous elution at approximately 27.5 min during the LC/MS analyses (colored traces). These fragment ion peaks were integrated for each digest from 1-6 dpf embryos, and results are shown in the Peak Integration Results bar graph, indicating that the αBa-crystallin remained in nearly equal abundance during 1-6 dpf. The bar in the graph labeled Library shows the relative proportion of the b2, y8, and y9 ions in the MS2 spectrum from the lens library, set at the same relative abundance as the fragment ions in the 3 dpf digest. The relative intensities of the b2, y8, and y9 fragment ions detected in the 1-6 dpf samples were very similar to those observed in the MS2 spectrum from the library, as evidenced by their dot product (dotp) values ranging from 0.94 to 0.97 marked above each bar.
Targeted mass spectrometric detection of αA-crystallin peptide 52-65 (NILDSSNSGVSEVR) in digests of 4 and 7 day post fertilization (dpf) dissected embryo eyes and trunks
Spectral library: MS2 spectrum of αA peptide 52-65 created during a data-dependent analysis of adult zebrafish lens digest. Y10-12 fragment ions were the most abundant in this spectrum, and these were used to detect the peptide in embryo digests, based on their simultaneous elution at approximately 25.5 min during the LC/MS analyses (colored traces marked with an arrow). These fragment ion peaks were integrated for each digest from 4 and 7 dpf embryo eyes and trunks, and results are shown in the Peak Integration Results bar graph, indicating that the αA-crystallin was only detectable in eyes and not trunks and reached its highest contraction at 7 days dpf. The bar in the graph labeled Library shows the relative proportion of the y10-12 ions in the MS2 spectrum from the lens library, set at the same relative abundance as the fragment ions in 7 dpf eye digest. The relative intensities of the y10-12 fragment ions detected in the eye 4 and 7 dpf samples were very similar to those observed in the MS2 spectrum from the library, as evidenced by their dot product (dotp) values 0.98 marked above each bar.
Targeted mass spectrometric detection of αA-crystallin peptide 71-78 (FTVYLDVK) in digests of 4 and 7 day post fertilization (dpf) dissected embryo eyes and trunks
Spectral library: MS2 spectrum of αA peptide 71-78 created during a data-dependent analysis of adult zebrafish lens digest. Y5-7 fragment ions were the most abundant in this spectrum, and these were used to detect the peptide in embryo digests, based on their simultaneous elution at approximately 37.2 min during the LC/MS analyses (colored traces marked with an arrow). These fragment ion peaks were integrated for each digest from 4 and 7 dpf embryo eyes and trunks, and results are shown in the Peak Integration Results bar graph, indicating that the αA-crystallin was only detectable in eyes and not trunks and reached its highest contraction at 7 days dpf. The bar in the graph labeled Library shows the relative proportion of the y5-7 ions in the MS2 spectrum from the lens library, set at the same relative abundance as the fragment ions in the 7 dpf eye digest. The relative intensities of the y5-7 fragment ions detected in the eye 4 and 7 dpf samples were very similar to those observed in the MS2 spectrum from the library, as evidenced by their dot product (dotp) values 0.99 marked above each bar.
Targeted mass spectrometric detection of αA-crystallin peptide 89-99 (VTDDYVEIQGK) in digests of 4 and 7 day post fertilization (dpf) dissected embryo eyes and trunks
Spectral library: MS2 spectrum of αA peptide 71-78 created during a data-dependent analysis of adult zebrafish lens digest. y5, 7, and 9 fragment ions were the most abundant in this spectrum, and these were used to detect the peptide in embryo digests, based on their simultaneous elution at approximately 21.7 min during the LC/MS analyses (colored traces marked with an arrow). These fragment ion peaks were integrated for each digest from 4 and 7 dpf embryo eyes and trunks, and results are shown in the Peak Integration Results bar graph, indicating that the αA-crystallin was only detectable in eyes and not trunks and reached its highest contraction at 7 days dpf. The bar in the graph labeled Library shows the relative proportion of the y5, 7, and 9 ions in the MS2 spectrum from the lens library, set at the same relative abundance as the fragment ions in the 7 dpf eye digest. The relative intensities of the y5, 7, and 9 fragment ions detected in the eye 4 and 7 dpf samples were very similar to those observed in the MS2 spectrum from the library, as evidenced by their dot product (dotp) values ranging from 0.96 - 0.97 marked above each bar.
Targeted mass spectrometric detection of αBa-crystallin peptide 79-88 (HFSPDELTVK) in digests of 4 and 7 day post fertilization (dpf) dissected embryo eyes and trunks
Spectral library: MS2 spectrum of αBa peptide 79-88 created during a data-dependent analysis of adult zebrafish lens digest. b2, y8, and y9 fragment ions were the most abundant in this spectrum, and these were used to detect the peptide in embryo digests, based on their simultaneous elution at approximately 27.2 min during the LC/MS analyses (colored traces marked with an arrow). These fragment ion peaks were integrated for each digest from 4 and 7 dpf embryo eyes and trunks, and results are shown in the Peak Integration Results bar graph, indicating that the αBa-crystallin was most abundant in eyes and while detectable in 4 dpf trunks, was not observed in 7 dpf trunks. The bar in the graph labeled Library shows the relative proportion of the b2, y8, and y9 ions in the MS2 spectrum from the lens library, set at the same relative abundance as the fragment ions in the 7 dpf eye digest. The relative intensities of the b2, y8, and y9 fragment ions detected in the eye 4 and 7 dpf digests, and trunk 4 dpi digest were very similar to those observed in the MS2 spectrum from the library, as evidenced by their dot product (dotp) values ranging from 0.94 - 0.95 marked above each bar.
Sequences of tryptic peptides used to detect proteins by mass spectrometry
The sequences of 9 tryptic peptides, 3 from each αA-, αBa-, and αBb-crytallin used to detect these proteins in digests from zebrafish embryos are given, along with their charge states, and m/z values.
Additional Information
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Mason Posner conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Kelly Murray performed the experiments, wrote the paper, reviewed drafts of the paper.
Hayden Eighinger performed the experiments, wrote the paper, reviewed drafts of the paper.
Amy Drossman conceived and designed the experiments, performed the experiments, reviewed drafts of the paper.
Zachary Haley performed the experiments, reviewed drafts of the paper.
Justin Nussbaum performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Larry L David conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Kirsten J Lampi performed the experiments, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.
Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):
Work with vertebrate animals was approved by Ashland University's Institutional Animal Use and Care Committee (approval number MP 2015-1)
Data Deposition
The following information was supplied regarding data availability:
The raw data is included is uploaded as supp files.
Funding
This work was supported by an R15 AREA grant from the National Eye Institute of the National Institutes of Health to MP (EY013535) and from grants to support faculty/student research from the Provost Office of Ashland University. A summer student research stipend was provided to KM as part of a Choose Ohio First scholarship grant to Ashland University. The proteomic analysis was also partially supported by National Eye Institute grants (EY027012 and EY10572). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
