Zebrafish pinhead was cloned into pCS2(+) vectors containing a C-terminal HA or FLAG tag for eukaryotic expression. For Pinhead-GFP, the sequence encoding GFP protein was inserted downstream from the pinhead coding sequence with an “EFLQDIIDGSPGLE” linker separating the fluorescent protein and the Pinhead protein. C-terminal epitope-tagged Admp, Chd, Noggin1, and Bmp1a were cloned in pCS2-Flag, pCS2-HA, or pCS2-Myc vectors. All the resulting constructs were confirmed by sequence analysis.

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