Zili prevents the formation of complex Smad1/5/9/4 and inhibits BMP signaling. A–D were performed in human HEK293 cells too. A, interactions of Zili with different zebrafish Smads. B–D, binding of Myc-Smad4 to HA-Smad1/5/9 was inhibited by coexpression of an increasing amount of FLAG-Zili (1 and 4 μg). E, bmp2b and vent expression in embryos injected with zili-MO or zili-cMO or zili mRNA. The embryos at the shield stage are shown in animal pole views with the dorsal oriented toward the right. Injection with zili-MO induced bmp2b and vent expression and zili mRNA abolished these factors in embryos. WB, Western blot; IP, immunoprecipitation; TCL, total cell lysate.