title: Monitoring Wnt Signaling in Zebrafish Using Fluorescent Biosensors.
abstract: In this chapter, we are presenting methods to monitor and quantify in vivo canonical Wnt signaling activities at single-cell resolution in zebrafish. Our technology is based on artificial enhancers, obtained by polymerization of TCF binding elements, cloned upstream to ubiquitous or tissue-specific promoters. The different promoter/enhancer combinations are used to drive fluorescent protein reporter constructs integrated in the zebrafish germline by microinjection of fertilized zebrafish eggs. Fish with a single integration site are selected by Mendelian analysis of fluorescent carriers, and heterozygous offspring are used to monitor and quantify canonical Wnt activities. Open source public domain software such as ImageJ/Fiji is used to calculate the integrated densities in the region of interest and compare the effect of experimental conditions on control and treated animals.
authors: Dipartimento di Biologia, Università degli Studi di Padova, Via Ugo Bassi 58/B, Padova, 35131, Italy.
authors: Nicola
authors: N
authors: Facchinello
authors: Dipartimento di Biologia, Università degli Studi di Padova, Via Ugo Bassi 58/B, Padova, 35131, Italy.
authors: Marco
authors: M
authors: Schiavone
authors: Dipartimento di Biologia, Università degli Studi di Padova, Via Ugo Bassi 58/B, Padova, 35131, Italy.
authors: Andrea
authors: A
authors: Vettori
authors: Dipartimento di Biologia, Università degli Studi di Padova, Via Ugo Bassi 58/B, Padova, 35131, Italy. francesco.argenton@unipd.it.
authors: Francesco
authors: F
authors: Argenton
authors: Dipartimento di Biologia, Università degli Studi di Padova, Via Ugo Bassi 58/B, Padova, 35131, Italy.
authors: Natascia
authors: N
authors: Tiso
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015374:Biosensing Techniques / Q000379:methods*; D004742:Enhancer Elements, Genetic; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D008845:Microinjections / Q000379:methods; D011401:Promoter Regions, Genetic; D051153:Wnt Proteins / Q000235:genetics* / Q000302:isolation & purification; D060449:Wnt Signaling Pathway / Q000235:genetics; D015027:Zebrafish / Q000235:genetics
chemical_list: D051153:Wnt Proteins; D049452:Green Fluorescent Proteins
keywords: Chemical biology; Transgenic reporter; Transposase; Zebrafish