Several recent studies in a number of model systems including zebrafish, Arabidopsis, and mouse have revealed phenotypic differences between knockouts (i.e., mutants) and knockdowns (e.g., ...antisense-treated animals). These differences have been attributed to a number of reasons including off-target effects of the antisense reagents. An alternative explanation was recently proposed based on a zebrafish study reporting that genetic compensation was observed in egfl7 mutant but not knockdown animals. Dosage compensation was first reported in Drosophila in 1932, and genetic compensation in response to a gene knockout was first reported in yeast in 1969. Since then, genetic compensation has been documented many times in a number of model organisms; however, our understanding of the underlying molecular mechanisms remains limited. In this review, we revisit studies reporting genetic compensation in higher eukaryotes and outline possible molecular mechanisms, which may include both transcriptional and posttranscriptional processes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Optogenetic Control of Cardiac Function Arrenberg, Aristides B; Stainier, Didier Y.R; Baier, Herwig ...
Science (American Association for the Advancement of Science),
11/2010, Letnik:
330, Številka:
6006
Journal Article
Recenzirano
The cardiac pacemaker controls the rhythmicity of heart contractions and can be substituted by a battery-operated device as a last resort. We created a genetically encoded, optically controlled ...pacemaker by expressing halorhodopsin and channelrhodopsin in zebrafish cardiomyocytes. Using patterned illumination in a selective plane illumination microscope, we located the pacemaker and simulated tachycardia, bradycardia, atrioventricular blocks, and cardiac arrest. The pacemaker converges to the sinoatrial region during development and comprises fewer than a dozen cells by the time the heart loops. Perturbation of the activity of these cells was entirely reversible, demonstrating the resilience of the endogenous pacemaker. Our studies combine optogenetics and light-sheet microscopy to reveal the emergence of organ function during development.
The vertebrate vasculature forms an extensive branched network of blood vessels that supplies tissues with nutrients and oxygen. During vascular development, coordinated control of endothelial cell ...behaviour at the levels of cell migration, proliferation, polarity, differentiation and cell-cell communication is critical for functional blood vessel morphogenesis. Recent data uncover elaborate transcriptional, post-transcriptional and post-translational mechanisms that fine-tune key signalling pathways (such as the vascular endothelial growth factor and Notch pathways) to control endothelial cell behaviour during blood vessel sprouting (angiogenesis). These emerging frameworks controlling angiogenesis provide unique insights into fundamental biological processes common to other systems, such as tissue branching morphogenesis, mechanotransduction and tubulogenesis.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
A major goal of regenerative medicine is to instruct formation of multipotent, tissue-specific stem cells from induced pluripotent stem cells (iPSCs) for cell replacement therapies. Generation of ...haematopoietic stem cells (HSCs) from iPSCs or embryonic stem cells (ESCs) is not currently possible, however, necessitating a better understanding of how HSCs normally arise during embryonic development. We previously showed that haematopoiesis occurs through four distinct waves during zebrafish development, with HSCs arising in the final wave in close association with the dorsal aorta. Recent reports have suggested that murine HSCs derive from haemogenic endothelial cells (ECs) lining the aortic floor. Additional in vitro studies have similarly indicated that the haematopoietic progeny of ESCs arise through intermediates with endothelial potential. Here we have used the unique strengths of the zebrafish embryo to image directly the generation of HSCs from the ventral wall of the dorsal aorta. Using combinations of fluorescent reporter transgenes, confocal time-lapse microscopy and flow cytometry, we have identified and isolated the stepwise intermediates as aortic haemogenic endothelium transitions to nascent HSCs. Finally, using a permanent lineage tracing strategy, we demonstrate that the HSCs generated from haemogenic endothelium are the lineal founders of the adult haematopoietic system.
Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate ...gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.
About the Authors: Didier Y. R. Stainier * E-mail: didier.stainier@mpi-bn.mpg.de (DYRS); cmoens@fredhutch.org (CBM) Affiliation: Department of Developmental Genetics, Max Planck Institute for Heart ...and Lung Research, Bad Nauheim, Germany ORCID http://orcid.org/0000-0002-0382-0026 Erez Raz Affiliation: Institute of Cell Biology, ZBME, University of Münster, Münster, Germany ORCID http://orcid.org/0000-0002-6347-3302 Nathan D. Lawson Affiliation: Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America Stephen C. Ekker Affiliation: Mayo Clinic, Rochester, Minnesota, United States of America Rebecca D. Burdine Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America Judith S. Eisen Affiliation: Institute of Neuroscience, University of Oregon, Eugene, Oregon, United States of America ORCID http://orcid.org/0000-0003-1229-1696 Philip W. Ingham Affiliations Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, The Living Systems Institute, University of Exeter, Exeter, United Kingdom Stefan Schulte-Merker Affiliation: Institute of Cardiovascular Organogenesis and Regeneration, WWU Münster, Faculty of Medicine, Münster, Germany Deborah Yelon Affiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, California, United States of America Brant M. Weinstein Affiliation: Division of Developmental Biology, NICHD, NIH, Bethesda, Maryland, United States of America Mary C. Mullins Affiliation: Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America ORCID http://orcid.org/0000-0002-9979-1564 Stephen W. Wilson Affiliation: Department of Cell and Developmental Biology, University College London, London, United Kingdom ORCID http://orcid.org/0000-0002-8557-5940 Lalita Ramakrishnan Affiliation: Molecular Immunity Unit, Department of Medicine, University of Cambridge, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom Sharon L. Amacher Affiliation: Departments of Molecular Genetics and Biological Chemistry and Pharmacology, Ohio State University, Columbus, Ohio, United States of America Stephan C. F. Neuhauss Affiliation: Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland ORCID http://orcid.org/0000-0002-9615-480X Anming Meng Affiliation: School of Life Sciences, Tsinghua University, Beijing, China Naoki Mochizuki Affiliation: National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan ORCID http://orcid.org/0000-0002-3938-9602 Pertti Panula Affiliation: Department of Anatomy and Neuroscience Center, University of Helsinki, Helsinki, Finland Cecilia B. Moens * E-mail: didier.stainier@mpi-bn.mpg.de (DYRS); cmoens@fredhutch.org (CBM) Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of AmericaCitation: Stainier DYR, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, et al. Additionally, mutant alleles for many genes are now readily available through zebrafish community resource centers. ...MOs should be used alongside mutant(s) for the corresponding gene. ...a word of caution that previous publication of MOs is not a guarantee of their fidelity, particularly if a new phenotype is being described. ...we hope that these brief and mostly conceptual guidelines will assist scientists working with zebrafish as well as those assessing manuscripts and grant proposals based on experiments using zebrafish.
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A pandemic of metabolic diseases (atherosclerosis, diabetes mellitus, and obesity), unleashed by multiple social and economic factors beyond the control of most individuals, threatens to diminish ...human life span for the first time in the modern era. Given the redundancy and inherent complexity of processes regulating the uptake, transport, catabolism, and synthesis of nutrients, magic bullets to target these diseases will be hard to find. Recent studies using the worm Caenorhabditis elegans, the fly Drosophila melanogaster, and the zebrafish Danio rerio indicate that these "lower" metazoans possess unique attributes that should help in identifying, investigating, and even validating new pharmaceutical targets for these diseases. We summarize findings in these organisms that shed light on highly conserved pathways of energy homeostasis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The burden of cardiovascular and metabolic diseases worldwide is staggering. The emergence of systems approaches in biology promises new therapies, faster and cheaper diagnostics, and personalized ...medicine. However, a profound understanding of pathogenic mechanisms at the cellular and molecular levels remains a fundamental requirement for discovery and therapeutics. Animal models of human disease are cornerstones of drug discovery as they allow identification of novel pharmacological targets by linking gene function with pathogenesis. The zebrafish model has been used for decades to study development and pathophysiology. More than ever, the specific strengths of the zebrafish model make it a prime partner in an age of discovery transformed by big-data approaches to genomics and disease. Zebrafish share a largely conserved physiology and anatomy with mammals. They allow a wide range of genetic manipulations, including the latest genome engineering approaches. They can be bred and studied with remarkable speed, enabling a range of large-scale phenotypic screens. Finally, zebrafish demonstrate an impressive regenerative capacity scientists hope to unlock in humans. Here, we provide a comprehensive guide on applications of zebrafish to investigate cardiovascular and metabolic diseases. We delineate advantages and limitations of zebrafish models of human disease and summarize their most significant contributions to understanding disease progression to date.
Genetic robustness, or the ability of an organism to maintain fitness in the presence of harmful mutations, can be achieved via protein feedback loops. Previous work has suggested that organisms may ...also respond to mutations by transcriptional adaptation, a process by which related gene(s) are upregulated independently of protein feedback loops. However, the prevalence of transcriptional adaptation and its underlying molecular mechanisms are unknown. Here, by analysing several models of transcriptional adaptation in zebrafish and mouse, we uncover a requirement for mutant mRNA degradation. Alleles that fail to transcribe the mutated gene do not exhibit transcriptional adaptation, and these alleles give rise to more severe phenotypes than alleles displaying mutant mRNA decay. Transcriptome analysis in alleles displaying mutant mRNA decay reveals the upregulation of a substantial proportion of the genes that exhibit sequence similarity with the mutated gene's mRNA, suggesting a sequence-dependent mechanism. These findings have implications for our understanding of disease-causing mutations, and will help in the design of mutant alleles with minimal transcriptional adaptation-derived compensation.
Genetic manipulations with a robust and predictable outcome are critical to investigate gene function, as well as for therapeutic genome engineering. For many years, knockdown approaches and reagents ...including RNA interference and antisense oligonucleotides dominated functional studies; however, with the advent of precise genome editing technologies, CRISPR-based knockout systems have become the state-of-the-art tools for such studies. These technologies have helped decipher the role of thousands of genes in development and disease. Their use has also revealed how limited our understanding of genotype-phenotype relationships is. The recent discovery that certain mutations can trigger the transcriptional modulation of other genes, a phenomenon called transcriptional adaptation, has provided an additional explanation for the contradicting phenotypes observed in knockdown versus knockout models and increased awareness about the use of each of these approaches. In this review, we first cover the strengths and limitations of different gene perturbation strategies. Then we highlight the diverse ways in which the genotype-phenotype relationship can be discordant between these different strategies. Finally, we review the genetic robustness mechanisms that can lead to such discrepancies, paying special attention to the recently discovered phenomenon of transcriptional adaptation.
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