Figure 5. sac1 phosphatase and stt4 PI4 Kinase cause elevated membrane levels of Smoothened protein.

The wing pouch is marked by a dotted line. Red channel only shown in gray scale in C’, D’, E’, F’ and G’ for clarity.

(A) A schematic representation of the third instar wing disc. The wing pouch is marked by white dotted line. The A/P compartment boundary (A/P) demarcates the anterior and posterior compartments. The D/V compartment (D/V) boundary demarcates the dorsal and the ventral compartments. ap-Gal4 is expressed throughout the dorsal compartment (shown in blue). Smo is expressed in the posterior compartment of the pouch (red dots)

(B) In wild-type, Smo (red) is expressed in the posterior compartment while the Hh target reporter dpp-lacZ (green) is activated along the A/P compartment boundary.

(C-C’) Knockdown of sac1 in the dorsal compartment of the wing disc using the combination ap-Gal4, UAS-GFP; UAS-sac1^RNAi (green cells), causes Smo (red) membrane localization to expand to the dorsal-anterior compartment of the wing (arrow) (compare with B). Red channel only shown in gray scale for clarity (C’).

(D-D’) Knockdown of stt4 kinase in the dorsal compartment of the wing disc using the combination ap-Gal4, UAS-stt4^RNAi UAS-GFP (green). Smo (red) membrane localization is reduced in the dorsal posterior compartment (arrow). Red channel only shown in gray scale for clarity (D’)

(E-E’) Double knockdown of sac1 and stt4 in the dorsal compartment of the wing disc using ap-Gal4, UAS-GFP; UAS-stt4^RNAi; UAS-sac1^RNAi (green) genetic combinations. Smo (red) accumulation in the dorsal anterior compartment (arrow) and dorsal posterior compartment (arrowhead) is significantly reduced. Red channel only shown in gray scale for clarity (E’).

(F-F’) As a control, wild-type Ptc (red) is expressed along the A/P boundary at similar levels in cells of the dorsal (green) and ventral (non-green) regions of the wing pouch.

(G-G’) Knockdown of stt4 kinase in the dorsal (green) compartment of the wing disc using the genetic combination ap-Gal4, UAS-stt4^RNAiUAS-GFP causes significant reduction in Ptc (red) expression.

(H-H’) Over-expression of Ptc in the dorsal compartment of the wing disc using the combination ap-gal4, UAS-ptc-YFP (green). Smo (red) membrane accumulation is significantly reduced in the dorsal posterior compartment (arrow). Note that the fully functional Ptc-YFP fusion can be directly visualized in the green channel. Red channel only shown in gray scale for clarity (H’)

(I-I’) Over expression of Ptc in a sac1 knockdown background in the dorsal compartment of the wing disc using ap-gal4 (green). Smo (red) membrane localization in the posterior dorsal compartment is reduced (compare with C and C’).

(J-J’) sac1 knockdown in the dorsal compartment of the wing disc using ap-Gal4, UAS-GFP; UAS-sac1^RNAi (green) in the background of dpp-lacZ. Ectopic activation of dpp-lacZ (red) is seen the dorsal anterior compartment (compare with 5B). It is currently unclear why the expression of dpp-lacZ is highest near the A/P boundary and lower near the distal edges of the pouch.

(K-K’) Double knockdown of sac1 and stt4 in the dorsal compartment of the wing disc using the combination ap-Gal4, UAS-GFP/ UAS-stt4^RNAi; UAS-sac1^RNAi (green). Ectopic dpp-lacZ as a read out for activation of the Hh pathway seen upon loss of sacI alone is suppressed (compare with J-J’).