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Fig. 1. The HhGFP chimera behaves as the wild-type Hh protein. (A) Scheme of the
predicted HhGFP fusion protein processing. HhGFP-F, full-length protein;
HhGFP-U, the unprocessed protein without the signal peptide; HhC, the
C-terminal region of the processed protein; HhGFP-Np, the N-terminal region of
the processed protein with the GFP fragment and the palmitic acid and
cholesterol modifications. (B) Western blot of protein extracts from
AB1-Gal4/UAS-Hh and AB1-Gal4/UAS-HhGFP salivary glands
stained with anti-Hh or anti-GFP. U, unprocessed protein (HhU); N, processed
protein (HhNp); C, catalytic fragment (HhC). (C) Ectopic clones of HhGFP
(green) in A cells (labeled by ß-Gal staining, blue) of a wing imaginal
disc stained with anti-En (red). Ectopic expression of HhGFP in A wing pouch
cells is able to induce En expression inside the clone and non-autonomously in
a region of three to four cell diameters around the clone (arrowheads in red
panel) as wild-type Hh. Note that HhGFP vesicles are detected in cells far
from the source (arrows in green panel). (D) hh-Gal4/UAS-HhGFP wing
imaginal disc stained with anti-Ptc (red). (d) Magnification of the boxed area
in D. HhGFP signal is observed in A cells co-localizing with Ptc protein in
vesicles (arrowheads). (E) disp- clones (labeled by the
lack of ß-gal staining, red) in a en-Gal4/UASHhGFP (green) wing
imaginal disc. There is an accumulation of Hh-GFP in the
disp- mutant cells (arrowheads) with respect to the
wild-type region (asterisk). (F) en-Gal4/UASHhGFP; hhts2
pharate raised at restrictive temperature during larvae development. All the
structures were rescued except for some head structures (arrow) where the
expression of en depends on Hh expression. In this and all other
figures, the A compartment of the wing disc is towards the left and the P is
towards the right. A broken line indicates the AP compartment border. Scale
bars: 15 µm in C,d; 50 µm in D,E.
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