When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response. Rapid changes in heart rate and blood glucose levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic‐pituitary‐adrenal axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent hypothalamic‐pituitary‐adrenal axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires hypothalamic‐pituitary‐interrenal (HPI) axis activation. In teleost fish, interrenal cells are functionally homologous to the adrenocortical layer. We derived eight frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), five in exon 2 or 5 of nr3c1 (glucocorticoid receptor [GR]) and two in exon 2 of nr3c2 (mineralocorticoid receptor [MR]). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show that this locomotor response requires a functioning HPI axis via the action of mc2r and the canonical GR encoded by nr3c1 gene, but not MR ( nr3c2 ). Our rapid behavioral assay paradigm based on HPI axis biology can be used to screen for genetic and environmental modifiers of the hypothalamic‐pituitary‐adrenal axis and to investigate the effects of corticosteroids and their cognate receptor interactions on behavior.
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.
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.
14Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. 15The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37 16 gene set and overall genomic structure ranging from 16,596 base pairs (bp) in the teleost 17 zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most 18 prevalent inherited diseases affecting roughly 1 in every 5000 individuals. Currently, few 19 effective treatments exist for those with mitochondrial ailments, representing a major unmet 20 patient need. Mitochondrial dysfunction is also implicated to be a common component of a wide 21 variety of other human illnesses ranging from neurodegenerative disorders like Huntington's 22 mitochondrial function with the goal of widening our understanding of their role in biology and 37 human disease. 38The vertebrate mitochondrial chromosome is circular and includes 37 genes, 13 encoding for 61 protein subunits of the electron transport chain, 22 coding for transfer RNAs, and 2 encoding 62 ribosomal RNAs (Figure 1) [11,12]. The mitochondrial gene order, strand specific nucleotide 63 bias and codon usage is highly conserved [13]. However, mtDNA encoded genes lack introns 64 and utilize a divergent genetic code than their nuclear counterparts [14,15]. For instance, AUA 65 codon codes for methionine as per mitochondrial genetic code, whereas the same sequence codes 66 for isoleucine in the nuclear genetic code. Similarly, nuclear stop codon UGA is read as the 67 tryptophan amino acid by the mitochondrial codon cypher. 68Mitochondria are unique cellular compartments with different DNA and RNA repair and editing 69 rules, hampering attempts at directly manipulating these nucleic acid components. For example, 70DNA nucleases that introduce double stranded breaks and subsequent repair in nuclear DNA 71 induce the degradation of mtDNA [16][17][18]. Indeed, none of the canonical DNA repair pathways 72 found in the nucleus have been shown to be active in mitochondria [18][19][20]. Finally, no system 73 has demonstrated the ability to deliver exogenous DNA or RNA to mitochondria, restricting the 74 tools available for mtDNA editing [21,22]. All of these factors are distinct from the nuclear 75 genome, making mtDNA a far less accessible genome for traditional gene editing methods and 76 reagents. 77The diverse functions that mitochondria are capable of, including oxidative phosphorylation, 78would not be possible with the small subset of 13 proteins encoded in mtDNA. Discoveries led 79 by high throughput proteomic approaches have enhanced our knowledge of the mitochondrial 80 proteome [23][24][25][26][27]. There are approximately 1158 nuclear-encoded proteins that localize to the 81 mammalian mitochondria, exerting a dual genetic control via its nuclear counterpart. Nuclear-82 encoded mitochondrial proteins on the basis of their mitochondrial function can be broadly 83 many research amenable characteristics of zebrafish are their high genetic orthology to hu...
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