The creation of molecular tools able to unravel in vivo spatiotemporal activation of specific cell signaling events during cell migration, differentiation and morphogenesis is of great relevance to ...developmental cell biology. Here, we describe the generation, validation and applications of two transgenic reporter lines for Wnt/β-catenin signaling, named TCFsiam, and show that they are reliable and sensitive Wnt biosensors for in vivo studies. We demonstrate that these lines sensitively detect Wnt/β-catenin pathway activity in several cellular contexts, from sensory organs to cardiac valve patterning. We provide evidence that Wnt/β-catenin activity is involved in the formation and maintenance of the zebrafish CNS blood vessel network, on which sox10 neural crest-derived cells migrate and proliferate. We finally show that these transgenic lines allow for screening of Wnt signaling modifying compounds, tissue regeneration assessment as well as evaluation of potential Wnt/β-catenin genetic modulators.
► TCF responsive elements linked to a fluorescent reporter reveal Wnt/b-catenin activity in a whole zebrafish. ► Wnt reporter (7xTCFsiam) fish unveils novel tissues of wnt/b-catenin dynamic expression. ► Wnt activity can be followed in vivo in embryonic, larval and adult sensory organs and brain endothelia. ► Wnt signaling is revealed in vivo in regenerating tissues. ► Wnt reporter fish can be used both for drug screenings and wnt mutants phenotyping.
Cardiovascular disease (CVD) is one of the leading causes of death worldwide. The most significant risk factors associated with the development of heart diseases include genetic and environmental ...factors such as hypertension, high blood cholesterol levels, diabetes, smoking, and obesity. Coronary artery disease accounts for the highest percentage of CVD deaths and stroke, cardiomyopathies, congenital heart diseases, heart valve defects and arrhythmias follow. The causes, prevention, and treatment of all forms of cardiovascular disease remain active fields of biomedical research, with hundreds of scientific studies published on a weekly basis. Generating animal models of cardiovascular diseases is the main approach used to understand the mechanism of pathogenesis and also design and test novel therapies. Here, we will focus on recent advances to finding the genetic cause and the molecular mechanisms of CVDs as well as novel drugs to treat them, using a small tropical freshwater fish native to Southeast Asia: the zebrafish (
Danio rerio
). Zebrafish emerged as a high-throughput but low-cost model organism that combines the advantages of forward and reverse genetics with phenotype-driven drug screenings. Noninvasive imaging allows in vivo analyses of cardiovascular phenotypes. Functional verification of candidate genes from genome-wide association studies has verified the role of several genes in the pathophysiology of CVDs. Also, zebrafish hearts maintain their ability to regenerate throughout their lifetime, providing novel insights to understand human cardiac regeneration.
Protein kinase D2 belongs to a family of evolutionarily conserved enzymes regulating several biological processes. In a forward genetic screen for zebrafish cardiovascular mutants, we identified a ...mutation in the
gene. Homozygous mutant embryos develop as wild type up to 36 h post-fertilization and initiate blood flow, but fail to maintain it, resulting in a complete outflow tract stenosis. We identified a mutation in the
gene that results in a T757A substitution at a conserved residue in the kinase domain activation loop (T714A in human PRKD2) that disrupts catalytic activity and drives this phenotype. Homozygous mutants survive without circulation for several days, allowing us to study the extreme phenotype of no intracardiac flow, in the background of a functional heart. We show dysregulation of atrioventricular and outflow tract markers in the mutants and higher sensitivity to the Calcineurin inhibitor, Cyclosporin A. Finally we identify TBX5 as a potential regulator of PRKD2. Our results implicate PRKD2 catalytic activity in outflow tract development in zebrafish.This article has an associated First Person interview with the first author of the paper.
ABSTRACT
First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. ...Panagiota Giardoglou and Despina Bournele are co-first authors on ‘A zebrafish forward genetic screen identifies an indispensable threonine residue in the kinase domain of PRKD2’, published in BiO. Panagiota is a PhD student in the lab of Dr Dimitris Beis at the Biomedical Research Foundation Academy of Athens, Greece, investigating modelling human cardiovascular diseases and underlying the mechanisms involved in their pathophysiology. Despina is a toxicologist in the lab of Dr Kyriaki Machera at the Benaki Phytopathological Institute, Kifissia, Athens, Greece, investigating molecular and developmental biology, and zebrafish toxicology.
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