Clark, Karl J

Rationale: Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ, such as the heart, especially those exhibiting adult phenotypes on depletion. Objective: We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes. Methods and Results: The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of 1 embryonic lethal, 1 larval lethal, and 1 adult recessive mutant exhibiting cardiac hypertrophy at 1 year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, α a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy. Conclusions: We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a zebrafish insertional cardiac mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens.

AbstractVEGF induces vascular permeability (VP) in ischemic diseases and cancer, leading to many pathophysiological consequences. The molecular mechanisms by which VEGF acts to induce hyperpermeability are poorly understood and in vivo models that easily facilitate real-time, genetic studies of VP do not exist. In the present study, we report a heat-inducible VEGF transgenic zebrafish (Danio rerio) model through which VP can be monitored in real time. Using this approach with morpholino-mediated gene knock-down and knockout mice, we describe a novel role of phospholipase Cβ3 as a negative regulator of VEGF-mediated VP by regulating intracellular Ca2+ release. Our results suggest an important effect of PLCβ3 on VP and provide a new model with which to identify genetic regulators of VP crucial to several disease processes.

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.

AbstractThe stress response is a normal reaction to a real or perceived threat. However, stress response systems that are overwhelmed or out of balance can increase both the incidence and severity of diseases including addiction and mood and anxiety disorders. Using an animal model with both genetic diversity and large family size can help discover the specific genetic and environmental contributions to these behavioral diseases. The stress response has been studied extensively in teleosts because of their importance in food production. The zebrafish (Danio rerio) is a major model organism with a strong record for use in developmental biology, genetic screening, and genomic studies. More recently, the stress response of larval and adult zebrafish has been documented. High-throughput automated tracking systems make possible behavioral readouts of the stress response in zebrafish. This non-invasive measure of the stress response can be combined with mutagenesis methods to dissect the genes involved in complex stress response behaviors in vertebrates. Understanding the genetic and epigenetic basis for the stress response in vertebrates will help to develop advanced screening and therapies for stress-aggravated diseases such as addiction and mood and anxiety disorders.

Endothelial tubulogenesis is a crucial step in the formation of functional blood vessels during angiogenesis and vasculogenesis. Here, we use in vivo imaging of living zebrafish embryos expressing fluorescent fusion proteins of β-Actin, α-Catenin, and the ERM family member Moesin1 (Moesin a), to define a novel cord hollowing process that occurs during the initial stages of tubulogenesis in intersegmental vessels (ISVs) in the embryo. We show that the primary lumen elongates along cell junctions between at least two endothelial cells during embryonic angiogenesis. Moesin1-EGFP is enriched around structures that resemble intracellular vacuoles, which fuse with the luminal membrane during expansion of the primary lumen. Analysis of silent heart mutant embryos shows that initial lumen formation in the ISVs is not dependent on blood flow; however, stabilization of a newly formed lumen is dependent upon blood flow. Zebrafish moesin1 knockdown and cell transplantation experiments demonstrate that Moesin1 is required in the endothelial cells of the ISVs for in vivo lumen formation. Our analyses suggest that Moesin1 contributes to the maintenance of apical/basal cell polarity of the ISVs as defined by adherens junctions. Knockdown of the adherens junction protein Ve-cadherin disrupts formation of the apical membrane and lumen in a cell-autonomous manner. We suggest that Ve-cadherin and Moesin1 function to establish and maintain apical/basal polarity during multicellular lumen formation in the ISVs.

Abstract Visual data collection is paramount for the majority of scientific research. The added transparency of the zebrafish ( Danio rerio ) allows for a greater detail of complex biological research that accompanies seemingly simple observational tools. We developed a visual data analysis and collection approach that takes advantage of the cylindrical nature of the zebrafish allowing for an efficient and effective method for image capture that we call Specimen in a Corrected Optical Rotational Enclosure imaging. To achieve a nondistorted image, zebrafish were placed in a fluorinated ethylene propylene tube with a surrounding optically corrected imaging solution (water). By similarly matching the refractive index of the housing (fluorinated ethylene propylene tubing) to that of the inner liquid and outer liquid (water), distortion was markedly reduced, producing a crisp imagable specimen that is able to be fully rotated 360°. A similar procedure was established for fixed zebrafish embryos using convenient, readily available borosilicate capillaries surrounded by 75% glycerol. The method described here could be applied to chemical genetic screening and other related high-throughput methods within the fish community and among other scientific fields.

Tobacco use is predicted to result in over 1 billion deaths worldwide by the end of the 21 st century. How genetic variation contributes to the observed differential predisposition in the human population to drug dependence is unknown. The zebrafish ( Danio rerio ) is an emerging vertebrate model system for understanding the genetics of behavior. We developed a nicotine behavioral assay in zebrafish and applied it in a forward genetic screen using gene-breaking transposon mutagenesis. We used this method to molecularly characterize bdav /cct8 and hbog /gabbr1.2 as mutations with altered nicotine response. Each have a single human ortholog, identifying two points for potential scientific, diagnostic, and drug development for nicotine biology and cessation therapeutics. We show this insertional method generates mutant alleles that are reversible through Cre-mediated recombination, representing a conditional mutation system for the zebrafish. The combination of this reporter-tagged insertional mutagen approach and zebrafish provides a powerful platform for a rich array of questions amenable to genetic-based scientific inquiry, including the basis of behavior, epigenetics, plasticity, stress, memory, and learning.

Abstract Morpholino oligonucleotides are the most common anti-sense “knockdown” technique used in zebrafish ( Danio rerio ). This review discusses common practices for the design, preparation, and deployment of morpholinos in this vertebrate model system. Off-targeting effects of morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting morpholino. Finally, new uses of morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.