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.

No abstract

14The clinical and largely unpredictable heterogeneity of phenotypes in patients with 15 mitochondrial disorders demonstrates the ongoing challenges in the understanding of this semi-16 autonomous organelle in biology and disease. Here we present a new animal model that 17 recapitulates key components of Leigh Syndrome, French Canadian Type (LSFC), a 18 mitochondrial disorder that includes diagnostic liver dysfunction. LSFC is caused by allelic 19 variations in the Leucine Rich Pentatricopeptide repeat-containing motif (LRPPRC) gene. 20 LRPPRC has native functions related to mitochondrial mRNA polyadenylation and translation as 21 well as a role in gluconeogenesis. We used the Gene-Breaking Transposon (GBT) cassette to 22 create a revertible, insertional mutant zebrafish line in the LRPPRC gene. lrpprc zebrafish 23 homozygous mutants displayed impaired muscle development, liver function and lowered levels 24 of mtDNA transcripts and are lethal by 12dpf, all outcomes similar to clinical phenotypes 25 observed in patients. Investigations using an in vivo lipidomics approach demonstrated 26 accumulation of non-polar lipids in these animals. Transcript profiling of the mutants revealed 27 dysregulation of clinically important nuclearly encoded and mitochondrial transcripts. Using 28 engineered liver-specific rescue as a genetic model therapy, we demonstrate survival past the 29 initial larval lethality, as well as restored normal gut development, mitochondrial morphology 30 2 and triglyceride levels functionally demonstrating a critical role for the liver in the 31 pathophysiology of this model of mitochondrial disease. Understanding the molecular 32mechanism of the liver-mediated genetic rescue underscores the potential to improve the clinical 33 diagnostic and therapeutic developments for patients suffering from these devastating disorders. 35

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

Background: Retinoblastoma binding protein 4 (Rbbp4) is a component of transcription regulatory complexes that control cell cycle gene expression. Previous work indicated that Rbbp4 cooperates with the Rb tumor suppressor to block cell cycle entry. Here, we use genetic analysis to examine the interactions of Rbbp4, Rb, and Tp53 in zebrafish neural progenitor cell cycle regulation and survival. Results: Rbbp4 is upregulated across the spectrum of human embryonal and glial brain cancers. Transgenic rescue of rbbp4 mutant embryos shows Rbbp4 is essential for zebrafish neurogenesis. Rbbp4 loss leads to apoptosis and γ-H2AX in the developing brain that is suppressed by tp53 knockdown or maternal zygotic deletion. Mutant retinal neural precursors accumulate in M phase and fail to initiate G0 gene expression. rbbp4; rb1 mutants show an [Correction added on May 16, 2022, after first online publication: Iowa State funding statement has been added.]