Morphant

Abstract: Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.

Abstract: 1 Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany, 2 Institute of Cell Biology, ZBME, University of Münster, Münster, Germany, 3 Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 4 Mayo Clinic, Rochester, Minnesota, United States of America, 5 Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America, 6 Institute of Neuroscience, University of Oregon, Eugene, Oregon, United States of America, 7 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, 8 The Living Systems Institute, University of Exeter, Exeter, United Kingdom, 9 Institute of Cardiovascular Organogenesis and Regeneration, WWU Münster, Faculty of Medicine, Münster, Germany, 10 Division of Biological Sciences, University of California, San Diego, La Jolla, California, United States of America, 11 Division of Developmental Biology, NICHD, NIH, Bethesda, Maryland, United States of America, 12 Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America, 13 Department of Cell and Developmental Biology, University College London, London, United Kingdom, 14 Molecular Immunity Unit, Department of Medicine, University of Cambridge, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, 15 Departments of Molecular Genetics and Biological Chemistry and Pharmacology, Ohio State University, Columbus, Ohio, United States of America, 16 Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland, 17 School of Life Sciences, Tsinghua University, Beijing, China, 18 National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan, 19 Department of Anatomy and Neuroscience Center, University of Helsinki, Helsinki, Finland, 20 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America

Abstract: Angiogenesis is a fundamental vertebrate developmental process that requires signalling by the secreted protein vascular endothelial growth factor-A (VEGF-A). VEGF-A functions in the development of embryonic structures, during tissue remodelling and for the growth of tumour-induced vasculature. The study of the role of VEGF-A during normal development has been significantly complicated by the dominant, haplo-insufficient nature of VEGF-A-targeted mutations in mice. We have used morpholino-based targeted gene knock-down technology to generate a zebrafish VEGF-A morphant loss of function model. Zebrafish VEGF-A morphant embryos develop with an enlarged pericardium and with major blood vessel deficiencies. Morphological assessment at 2 days of development indicates a nearly complete absence of both axial and intersegmental vasculature, with no or reduced numbers of circulating red blood cells. Molecular analysis using the endothelial markers fli-1 and flk-1 at 1 day of development demonstrates a fundamental distinction between VEGF-A requirements for axial and intersegmental vascular structure specification. VEGF-A is not required for the initial establishment of axial vasculature patterning, whereas all development of intersegmental vasculature is dependent on VEGF-A signalling. The zebrafish thus serves as a quality model for the study of conserved vertebrate angiogenesis processes during embryonic development.