Yanyan Zhang et al. npg 1447 Figure 7 In vivo activities of dimerized Fu variants. (A-H) Wing discs expressing the indicated dimerized Fu variants by act>CD2>Gal4. The immunostainings show the expression of Fu variants, dpp-lacZ (dppZ) (A-D) and endogenous Ptc (E-H). In the Div-Fu, Div-FuK and Div-FuK* clones, which were marked by green, dppZ or Ptc (blue) expression was turned on in A-compartment (B, F, Div-Fu; C and G, Div-FuK; D, H, Div-FuK*). However, as the control, DivIVA protein failed to upregulate dppZ or Ptc expression (A, E). 7D). Conversely, Div-FuKG13V, Div-FuK*G13V or wild-type Fu failed to activate dpp expression (Supple- mentary information, Figure S10B-S10D″). However, the ptc expression levels are not very strong, suggesting that the dimerization of Fu alone could not mimic the high- est levels of Hh signaling. Div-Fu, Div-FuK and Div- FuK*, but not Div, also activated ptc expression in the A-compartment and Div-FuK* appeared to have a higher activity than Div-FuK (Figure 7E-7H). Fu, Div-FuG13V, Div-FuKG13V or Div-FuK*G13V failed to induce ptc expression in the A-compartment (data not shown). Alto- gether, although dimerization of Fu alone does not seem to fully activate the Hh pathway, these data still suggest that Fu dimerization promotes Hh pathway activity in vivo. Discussion The Hh family of morphogens controls distinct cellu- lar outcomes through graded signal activities [1, 17, 36, 37]. As one of the main morphogens, Hh forms a gradi- ent when it diffuses from Hh secreting cells to receiving cells. In Drosophila wing imaginal discs, a Hh gradient is required for the patterning of the adult wing. As a long- www.cell-research.com | Cell Research range morphogen, low concentrations of Hh activate dpp and collier (col) expression, whereas as a short-range morphogen, high Hh concentrations activate ptc and en expression [38-40]. However, how the Hh gradient regu- lates differential gene expression remains elusive. The seven-transmembrane protein Smo is thought to relay different levels of Hh activity through differential phos- phorylation and the graded conformational change of its carboxyl-terminal cytoplasmic tail [9, 17, 41]. Previous studies on the Cos2/Fu complex suggested that Smo re- cruits the Cos2/Fu complex to membranes by physically interacting with the Cos2/Fu complex through its C-tail [11, 22]. In the present study, we found that Smo recruits the Cos2/Fu complex to the membrane through the in- teraction between Smo and Cos2, which is regulated by graded Hh or Smo C-tail phosphorylation levels. More- over, the motor domain of Cos2 is important for this recruitment. Upon Hh binding with Ptc, the inhibition of Ptc on Smo is terminated. Under this condition, the Smo C-tail interacts with Cos2 at amino acids 818 to 974, and Fu fails to co-localize with the Smo C-tail in the absence of Cos2. Cos2 appears to be essential for the interaction between Cos2/Fu and Smo in the presence of Hh. Fu is important for the interaction between Cos2/Fu and Smo