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.
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).
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
Marcks and Marcksl1 are abundant proteins that shuttle between the cytoplasm and membrane to modulate multiple cellular processes, including cytoskeletal dynamics, proliferation, and secretion. Here, we performed loss- and gain-of-function experiments in Xenopus laevis to reveal the novel roles of these proteins in spinal cord development and regeneration. We show that Marcks and Marcksl1 have partly redundant functions and are required for normal neurite formation and proliferation of neuro-glial progenitors during embryonic spinal cord development and for its regeneration during tadpole stages. Rescue experiments in Marcks and Marcksl1 loss-of-function animals further suggested that some of the functions of Marcks and Marcksl1 in the spinal cord are mediated by phospholipid signaling. Taken together, these findings identify Marcks and Marcksl1 as critical new players in spinal cord development and regeneration and suggest new pathways to be targeted for therapeutic stimulation of spinal cord regeneration in human patients.
