Detection of Smad Signaling in Zebrafish Embryos.

Nodal and BMPs play critical roles in germ layer induction and patterning in early zebrafish embryos. Smad2/3 and Smad1/5/8 are intracellular effectors of Nodal and BMPs, respectively. These Smads regulate, in cooperation with other factors, transcription of hundreds of target genes in the nucleus. The activity and stability of Smads are regulated by phosphorylation modifications. To better understand the regulatory network of Smads-mediated signaling and its biological implications, it is necessary to monitor the signaling activity in an in vivo model system. In this chapter, we describe the methods used in zebrafish embryos for dissecting Smads signaling, including TGF-β/Nodal- and BMP-responsive luciferase reporter assays, Western blotting for Smads, co-immunoprecipitation for Smads and their interacting proteins, chromatin-immunoprecipitation for identification of Smad2-binding sites, and immunostaining for detection of active Smad1/5/8.