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 fl uorescent 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 fl uorescent 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.

Monitoring Wnt signaling in zebrafish using fluorescent biosensors

FACCHINELLO, NICOLA;SCHIAVONE, MARCO;VETTORI, ANDREA;ARGENTON, FRANCESCO;TISO, NATASCIA
2016

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 fl uorescent 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 fl uorescent 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.
Methods in Molecular Biology
978-1-4939-6391-1
978-1-4939-6393-5
978-1-4939-6391-1
978-1-4939-6393-5
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3208546
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