A protein-trap mutagenesis system efficiently disrupts expression and reports endogenous expression patterns of genes in the zebrafish. The authors used it to generate an initial collection of 350 freely available fish lines. We describe a conditional in vivo protein-trap mutagenesis system that reveals spatiotemporal protein expression dynamics and can be used to assess gene function in the vertebrate Danio rerio. Integration of pGBT-RP2.1 (RP2), a gene-breaking transposon containing a protein trap, efficiently disrupts gene expression with >97% knockdown of normal transcript amounts and simultaneously reports protein expression for each locus. The mutant alleles are revertible in somatic tissues via Cre recombinase or splice-site-blocking morpholinos and are thus to our knowledge the first systematic conditional mutant alleles outside the mouse model. We report a collection of 350 zebrafish lines that include diverse molecular loci. RP2 integrations reveal the complexity of genomic architecture and gene function in a living organism and can provide information on protein subcellular localization. The RP2 mutagenesis system is a step toward a unified 'codex' of protein expression and direct functional annotation of the vertebrate genome.

Precise control of gene expression is critical for embryo development in both animals and plants. We report that Arabidopsis thaliana GLUTAMINE-RICH PROTEIN23 (GRP23) is a pentatricopeptide repeat (PPR) protein that functions as a potential regulator of gene expression during early embryogenesis in Arabidopsis. Loss-of-function mutations of GRP23 caused the arrest of early embryo development. The vast majority of the mutant embryos arrested before the 16-cell dermatogen stage, and none of the grp23 embryos reached the heart stage. In addition, 19% of the mutant embryos displayed aberrant cell division patterns. GRP23 encodes a polypeptide with a Leu zipper domain, nine PPRs at the N terminus, and a Gln-rich C-terminal domain with an unusual WQQ repeat. GRP23 is a nuclear protein that physically interacts with RNA polymerase II subunit III in both yeast and plant cells. GRP23 is expressed in developing embryos up to the heart stage, as revealed by β-glucuronidase reporter gene expression and RNA in situ hybridization. Together, our data suggest that GRP23, by interaction with RNA polymerase II, likely functions as a transcriptional regulator essential for early embryogenesis in Arabidopsis.

Background—: Genetic studies of cardiomyopathy and heart failure have limited throughput in mammalian models. Adult zebrafish have been recently pursued as a vertebrate model with higher throughput, but genetic conservation must be tested. Methods and Results—: We conducted transcriptome analysis of zebrafish heart and searched for fish homologues of 51 known human dilated cardiomyopathy–associated genes. We also identified genes with high cardiac expression and genes with differential expression between embryonic and adult stages. Among tested genes, 30 had a single zebrafish orthologue, 14 had 2 homologues, and 5 had ≥3 homologues. By analyzing the expression data on the basis of cardiac abundance and enrichment hypotheses, we identified a single zebrafish gene for 14 of 19 multiple-homologue genes and 2 zebrafish homologues of high priority for ACTC1 . Of note, our data suggested vmhc and vmhcl as functional zebrafish orthologues for human genes MYH6 and MYH7 , respectively, which are established molecular markers for cardiac remodeling. Conclusions—: Most known genes for human dilated cardiomyopathy have a corresponding zebrafish orthologue, which supports the use of zebrafish as a conserved vertebrate model. Definition of the cardiac transcriptome and fetal gene program will facilitate systems biology studies of dilated cardiomyopathy in zebrafish.

Zebrafish embryos have been extensively used to study heart development and cardiac function, mainly due to the unique embryology and genetics of this model organism. Since most human heart disease occurs during adulthood, adult zebrafish models of heart disease are being created to dissect mechanisms of the disease and discover novel therapies. However, due to its small heart size, the use of cardiac functional assays in the adult zebrafish has been limited. To address this bottleneck, the transparent fish line casper;Tg(cmlc2:nuDsRed) that has a red fluorescent heart can be used to document beating hearts in vivo and to quantify cardiac functions in adult zebrafish. Here, we describe our methods for quantifying shortening fraction and heart rate in embryonic zebrafish, as well as in the juvenile and adult casper;Tg(cmlc2:nuDsRed) fish. In addition, we describe the red blood cell flow rate assay that can be used to reflect cardiac function indirectly in zebrafish at any stage.

An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart. Hearts of the tr265/tr265 Danio rerio mutant become larger than those of the sibling by week 4 post fertilization and gradually exhibit characteristics of human cardiomyopathy, such as muscular disarray, re-activated fetal gene expression, and severe arrhythmia. At the cellular level, we found both increased individual cardiomyocyte size and increased myocyte proliferation can be detected in week 4 to week 12 tr265/tr265 fish. Interestingly, all tr265/tr265 fish that survive after week-12 have many more cardiomyocytes of smaller size than those in the sibling, suggesting that myocyte hyperplasia allows the long-term survival of these fish. We also show the cardiac hypertrophy process can be recapitulated in wild-type fish using the anemia-inducing drug phenylhydrazine (PHZ). The anemia-induced cardiac hypertrophy models reported here are the first adult zebrafish cardiac hypertrophy models characterized. Unlike mammalian models, both cardiomyocyte hypertrophy and hyperplasia contribute to the cardiac remodeling process in these models, thus allowing the effects of cardiomyocyte hyperplasia on cardiac remodeling to be studied. However, since anemia can induce effects on the heart other than biomechanical, non-anemic zebrafish cardiac hypertrophy models shall be generated and characterized.

Animals respond to adverse environments by slowing down or arresting growth and development. Upon returning to normal conditions, they often show compensatory acceleration in growth and developmental rate. This phenomenon, known as `catch-up' growth, is widely documented in the animal kingdom. The underlying molecular mechanisms, however, are poorly understood. Using the zebrafish embryo as an experimental model system, we tested the hypothesis that changes in IGF signaling activities play an important role in the accelerated growth and temporal development resulting from re-oxygenation following hypoxia. We show that chronic hypoxia reduced, and re-oxygenation accelerated, embryonic growth and developmental rate. Whereas hypoxia repressed the Igf1 receptor and its downstream Erk1/2 and Akt signaling activities, re-oxygenation restored their activities. Specific inhibition of Igf1 receptor signaling during re-oxygenation by genetic and pharmacological approaches attenuated catch-up growth. Further analysis showed that whereas PI3K-Akt is required in both normal and catch-up growth, Mek1/2-Erk1/2 activation induced by elevated IGF signaling during re-oxygenation is particularly crucial for catch-up growth. These results suggest that the evolutionarily conserved IGF signaling pathway coordinates growth and temporal development in zebrafish embryos in response to oxygen availability.