Journal: PLOS Biology

Article Title: Hedgehog-stimulated phosphorylation at multiple sites activates Ci by altering Ci–Ci interfaces without full Suppressor of Fused dissociation

doi: 10.1371/journal.pbio.3003105

Figure Lengend Snippet: (A–E) Third instar wing discs with one copy of Ci-WT ( crCi-WT/ ci 94 ), showing a stripe of induction of ptc-lacZ reporter expression (“Ptc-lacZ”), visualized by Beta-galactosidase antibody staining (red), in anterior cells near the center of the disc responding to Hh, released from posterior (right) cells. The posterior edge of Ptc-lacZ defines the AP compartment boundary and is marked by yellow dotted lines. (A’, B’, D, E’’) Full-length Ci (Ci-155) visualized by 2A1 antibody staining (gray-scale), and (C’) en expression visualized by En antibody staining (green). Anterior En induction by Hh is to the left of the AP border (yellow dotted lines) and co-incides with lower Ci-155 levels, which are otherwise elevated in Hh signaling territory. (A–C’) shows wild-type discs using (A–A’) 20× and (B–C’) 63× objective lenses. (D–E’) A Su(fu) LP/LP disc using (D–D’) 20× and (E–E’) 63× objective lenses, showing normal Ptc-lacZ but lower levels of Ci-155 in anterior and AP border cells. (F–M) Third instar wing discs (63× objective) with one copy of the indicated ci CRISPR allele, GFP marking homozygous cos2 mutant clones (green; yellow arrowheads), and yellow dotted lines marking the AP border. (F’–M’) Ptc-lacZ expression (red) and (F’’–M’’) Ci-155 expression (gray-scale) in the same discs. (F’) Ptc-lacZ induction by Ci-WT in cos2 mutant clones was (G’) increased in discs that lack Su(fu) activity, and (H’-M’) was increased to a lesser degree by each of the Ci variants shown. (F”) Ci-155 levels are increased in cos2 mutant clones because proteolytic processing is blocked, but the increase was reduced or absent ( G” ) in the absence of Su(fu) and ( H”–M” ) for most Ci variants, potentially due to reduced Su(fu)-dependent Ci stabilization or En-mediated inhibition of ci transcription; ( K” ) Ci Δ272-346 and ( L” ) Ci Δ270-300 have higher C-155 than other Ci variants. The brightness of G” –J” and M” images was increased relative to others in order to see any changes in Ci levels in clones more easily. Quantitation used 20× images, all obtained under the same conditions. Scale bars are (A, D) 100 μm, (B, C, E) 20 μm, and (all others) 40 μm. (N, O) Bar graphs showing average (N) Ptc-lacZ intensity or (O) Ci-155 signal intensity in cos2 clones relative to the AP border of control discs (with two copies of Ci-WT), together with SEMs ( n -values 20, 10, 16, 22, 53, 29, 27, and 31, respectively, for each graph). Ci-WT ratios align with blue dotted lines. Differences with p < 0.005 (Student t test with Welch correction) are indicated for comparing a Ci variant to (N) Ci-WT (black asterisk) or (O) Ci Δ1,370–1,397 (red asterisk). Please see for details of measurements and expression of all experimental values relative to AP border values of control wild-type wing discs, and for raw data, including all p values. A cartoon of Ci domains and deletions, together with testing for En induction in cos2 clones, are shown in .

Article Snippet: For Ci-155 staining, Rat 2A1 anti-Ci (Developmental Studies Hybridoma Bank) in 1:3 dilution was used.

Techniques: Expressing, Staining, CRISPR, Mutagenesis, Clone Assay, Activity Assay, Inhibition, Quantitation Assay, Control, Variant Assay