Building the vertebrate codex using the gene breaking protein trap library
- Mayo Clinic, United States
- Oklahoma Medical Research Foundation, United States
- Iowa State University, United States
- Li Ka Shing Knowledge Institute, St. Michael's Hospital, Unity Health Toronto & University of Toronto, Canada
- Carnegie Institution for Science, United States
- CSIR-Institute of Genomics and Integrative Biology, India
- Temple University, United States
- University of Cologne, Germany
- National Human Genome Research Institute, National Institutes of Health, United States
Abstract
One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1,200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 3, Figure 4, Figure 4-Figure Supplement 1, Figure 5 and Figure 6.
Article and author information
Author details
Anthony J Treichel
Matthias Hammerschmidt
Steven Arthur Farber
Funding
National Institutes of Health (GM63904)
- Stephen C Ekker
Council of Scientific and Industrial Research (MLP1801)
- Sridhar Sivasubbu
National Institutes of Health (DA14546)
- Stephen C Ekker
National Institutes of Health (DK093399)
- Stephen C Ekker
National Institutes of Health (HG006431)
- Stephen C Ekker
The Mayo Foundation (Internal)
- Stephen C Ekker
Natural Sciences and Engineering Research Council of Canada (RGPIN 05389-14)
- Xiao-Yan Wen
The intramural Reserch Program of the National Human Genome Research Institute, National Institutes of Health (1ZIAHG000183)
- Shawn M Burgess
The Roy J. Carver Charitable Trust (07-2991)
- Maura McGrail
The Roy J. Carver Charitable Trust (07-2991)
- Jeffrey J Essner
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Wenbiao Chen, Vanderbilt University, United States
Ethics
Animal experimentation: All zebrafish were maintained according to the guidelines and the standard procedures approved by the Mayo Clinic Institutional Animal Care and Use Committee (Mayo IACUC). The Mayo IACUC approved all protocols involving live vertebrate animals (A23107, A21710 and A34513).
Version history
- Received: December 19, 2019
- Accepted: August 7, 2020
- Accepted Manuscript published: August 11, 2020 (version 1)
- Version of Record published: September 11, 2020 (version 2)
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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Building the vertebrate codex using the gene breaking protein trap library
eLife 9:e54572.
https://doi.org/10.7554/eLife.54572
Further reading
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- Genetics and Genomics
Previously we showed the generation of a protein trap library made with the gene-break transposon (GBT) in zebrafish (Danio rerio) that could be used to facilitate novel functional genome annotation towards understanding molecular underpinnings of human diseases (Ichino et al, 2020). Here, we report a significant application of this library for discovering essential genes for heart rhythm disorders such as sick sinus syndrome (SSS). SSS is a group of heart rhythm disorders caused by malfunction of the sinus node, the heart’s primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. From 609 GBT lines screened, we generated a collection of 35 zebrafish insertional cardiac (ZIC) mutants in which each mutant traps a gene with cardiac expression. We further employed electrocardiographic measurements to screen these 35 ZIC lines and identified three GBT mutants with SSS-like phenotypes. More detailed functional studies on one of the arrhythmogenic mutants, GBT411, in both zebrafish and mouse models unveiled Dnajb6 as a novel SSS causative gene with a unique expression pattern within the subpopulation of sinus node pacemaker cells that partially overlaps with the expression of hyperpolarization activated cyclic nucleotide gated channel 4 (HCN4), supporting heterogeneity of the cardiac pacemaker cells.
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- Developmental Biology
Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3, and GNAO proteins, but may also induce unrelated defects. Here, we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner G protein signaling modulator 2 (GPSM2), whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the sub-cellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: (1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and (2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.
