Fig 1.
Hyd binds the Hedgehog pathway’s key transcriptional effector Ci155 and the Ci-regulatory kinase Sgg.
Co-immunoprecipitation (A,D) and affinity-purification (B,C) studies with the indicated affinity reagents were examined by SDS-PAGE and Western blotting with the indicated antibodies. (A) Drosophila CL8 cells were lysed and incubated with either Hyd or control IgG antibodies and affinity purified by Protein G beads. An arrow indicates the position of the expected size band and an asterisk indicates the presence of an uncharacterised faster migrating Hyd species. (B) Mammalian HEK293 cells were transfected with the indicated constructs and lysates underwent Streptactin-mediated purification (Strp) to purify Haemagglutinin-Streptactin-EDD (HS-EDD) and detect co-purified Myc-GLI2. (C) Drosophila S2 cells were transfected with either HS-hyd or HS-vector control, lysed and then incubated with Streptactin-affinity resin. Control and Hyd-coated beads were then incubated with Sgg-FLAG expressing S2 lysate and, following washing, analysed for bound Sgg-FLAG. Only the HS-Hyd beads purified FLAG-Sgg. (D) Drosophila S2 cells were co-transfected with the indicated hyd mutant and sgg-FLAG constructs and FLAG-affinity purified complexes were analysed with the indicated antibodies.
Fig 2.
hydK7.19 is defective in HECT E3 function and causes abnormal head development.
(A) Schematic representation of the full length Hyd protein containing the Ubiquitin Association Domain (UBA), Regulator of Chromatin Condensation-like (RCC), Ubiquitin-Protein Ligase E3 Component N-Recognin (UBR) domain, Poly(A)-Binding Protein C-Terminal (PABC) and Homologous to the E6AP Carboxyl Terminus (HECT) domains and the potential protein products encoded by hydk7.19 and hyd15. In comparison to control heads (B-D), hydk7.19 flies (E-G) exhibited disruption of the adult eye and increased head-capsule area. Co-expression of the hydWT (K-M), but not hydC>A (H-J), transgene suppressed the hydk7.19 phenotype. Scale bar = 200μm. (O) hydK3.5 flies exhibit eye tissue outgrowths that are not present in hydk7.19 heads. (P) Quantification of the head capsule area of the indicated genotype. % values are normalised to control. n = >10 of each genotype. s.e.m and indicated p value determined by Student’s t-test.
Fig 3.
hydK7.19 EA discs exhibit aberrant Ci155 expression patterns and morphogenetic furrow-associated features.
(A-D) Deconvolved widefield and confocal image (F-Q) sections of control FRT82B (A,B, F-K)) and hydk7.19 (C,D, L-Q) EA discs imaged for direct GFP fluorescence (A,C,G,K), Ci155 immunofluorescence (B,D,H,I,K) and DAPI (A,B,C,E,F,G). (A-D) hydk7.19 EA discs exhibit abnormal Ci155 expression patterns. A “Union Jack” lookup table was applied to Ci155 images to visualise low (blue), medium (white) and high (red) intensity levels and arrows marks the presumed Ci155 DVS / morphogenetic furrow and an asterisk indicates increased Ci155 staining as a result of the tissue folding over in itself (B). (D) hydk7.19 EA discs exhibited ectopic Ci155 expression in the posterior compartment (E, marked by a dashed yellow line, which is also overlaid onto C). (E) Quantification of the area of medium-to-high Ci155 signal in control and hydk7.19 EA discs. n = 5, s.e.m and indicated p value determined by Student’s t-test. (F-Q) hydk7.19 EA discs exhibit abnormal markers of the morphogenetic furrow. Control FRT82B EA discs exhibited normal nuclei distribution (F) and DVS Ci155 expression (H), while hydk7.19 discs exhibited irregular patterns (L,N). (F-H) Dashed lines indicated the DVS’s associated high anterior and low posterior Ci155 expression margins (H), which is overlaid onto (F,G). (L-N) A region of low Ci155 expression flanked by two DVS-like regions of high Ci155 expression is marked by a dashed outline (N), which is overlaid onto (L,M). Arrows mark high Ci155 DVS (H), or DVS-like (N), signals. Scales bars (A-D) 50μm and (F-Q) 10μm.
Fig 4.
hydk7.19 clones exhibit distinct patterns of Ci155 expression.
Confocal image sections of hydk7.19 EA discs imaged for direct GFP fluorescence (B,E,F,G,I,J,M,O) Ci155 immunofluorescence (C,D,F,H,I,L,N,O) and DAPI (A,D,E,K,M,N). (A-F) hydk7.19 discs exhibited curved arrays of nuclei (A, dashed line) that were reflected in the Ci155 DVS (C, dashed line). (G-I) Posterior hydk7.19 clones near the DVS exhibited increased Ci155 expression (H, dashed outline). (J-O) Anterior hydk7.19 clones near the DVS exhibited decreased Ci155 expression (L, low Ci155 marked by dashed lines, which are overlaid onto J,K). Scale bars = 10μm.
Fig 5.
hydk7.19 EA discs exhibit abnormal Ptc expression.
(A-D) Deconvolved widefield and (F-K) confocal image sections of control FRT82B (A,B) and hydk7.19 (C,D and F-K) EA discs imaged for GFP fluorescence and the indicated antigens for IF. “Union Jack” lookup table applied to Ptc images (A,C) to visualise low (blue), medium (white) and high (red) intensity levels. (E) Quantification of the area of medium and high Ptc signal. n = 3, s.e.m and indicated p value determined by Student’s t-test. (F-K) Overlapping expression of Ci155 and Ptc immunofluorescence within a hydk7.19 GFP-positive clone anterior to the Ci155 DVS (F yellow dotted outline, which is overlaid onto G,H). The yellow dashed line indicates the Ci155 DVS, which is overlaid onto H). Scale bars = 50μm (A-D) and 10μm (F-K).
Fig 6.
hydk7.19 EA discs exhibit increased hh-lacZ-associated β-Gal expression within the posterior compartment and DVS-region.
Confocal image sections of FRT82B control (A-E, L-N)) and hydk7.19 (F-J, O-T) EA discs imaged for direct GFP fluorescence (A,F,N,Q,R), β-Gal (B,C,G,H,L-Q,S) and Ci155 immunofluorescence (D,E,I,J,M,P,T). (A-K) hydk7.19 EA discs exhibited increased β-Gal expression (H) relative to FRT82B controls (C). Non-clonal regions (GFP—ve regions) were ‘masked off’ to help visualise β-Gal and Ci155 expression only within GFP-positive clones (C,H and E,J, respectively). Yellow dotted lines indicate the division between anterior and posterior compartment (B—E and G-J). Dashed yellow lines indicate regions of high hh expression (H) and corresponding low Ci155 expression (E). (K) Quantification of the β-Gal average pixel intensity of the masked off images. n = 5, s.e.m and indicated p value determined by Student’s t-test. Scale bars = 50μm. (L-Q) hydk7.19 DVS regions exhibited abnormal β-Gal (O) and Ci155 (P) expression. Dashed lines indicate high Ci155 DVS expression (M,P), which are overlaid onto the other panels. The dotted line marks the anterior front of high β-Gal expression (L), which is overlaid on (M,N). (R-T) Two GFP positive hydk7.19 clones (R), located in the posterior compartment clearly overexpressed β-Gal (S). Of those clones, only one (R yellow dashed line, which is overlaid onto S,T) also harboured increased Ci155 expression (T). A specific clonal subregion (T, dotted line) with the clone coincided with low β-Gal expression (S, dotted line). Scale bars = 10μm.
Fig 7.
Sgg regulates hh-lacZ expression in both the posterior and anterior compartments.
(A-U) Confocal images of EA disc of the indicated genotypes imaged for GFP (A,D,G,J,M,P,S) fluorescence and β-Gal (B,E,H,K,N,Q,T) and Ci155 (C,F,I,L,O,R,U) immunofluorescence. Dashed lines indicate the division between the anterior and posterior compartments, dotted lines indicate regions of high β-Gal and Ci155 expression within and anterior to the DVS region (N,O, respectively). The boxed regions (P-R) indicate a region harbouring three clones overexpressing β-Gal and Ci155 in the anterior compartment, which are enlarged in (S-U). (V) Boxplots of quantification of the average β-Gal pixel intensity of non-GFP masked off images (not shown). n = >5 for each genotype, s.e.m indicated. Statistical analysis by one-way ANOVA and Tukey’s multiple comparison tests, which revealed all comparisons to be statistically significant, except those indicated as non-significant (ns). (W) Potential model to explain the effects observed in the posterior EA disc. The double-headed arrow indicates a physical interaction, the single-headed arrow a positive regulatory action and the round-headed arrow a negative regulatory action. Scale bar = 50μm.
Fig 8.
Sgg and Hyd genetically interact to govern animal viability and head and wing development.
(A-J) Sgg perturbation modifies the hydk7.19 head phenotype. (A-D) Brightfield images of adult Drosophila heads of the indicated genotypes shown either ‘head on’ (upper panels) or ‘side on’ (lower panels). Both gain (C) and loss (D) of sgg function appeared to rescue the hydk7.19 phenotype. Boxplots indicating head width (E, n = ≥8 for each genotype) and counts of eye scars (F, n = ≥8 for each genotype) of the indicated genotypes, with statistical analysis by one-way ANOVA (E) and Fishers exact test (F) revealed statistical significance (asterisks). (G-J) Representative GFP fluorescent signals in adult Drosophila heads of the indicated genotypes revealed only hydk7.19+sggS9A animals lack a GFP signal (n = ≥4 for each genotype). Scale bars = 175μm. (K-P) hydWT overexpression promotes the sggS9A-mediated wing phenotype. (K-N) Brightfield images of adult Drosophila wings showing (K) normal, (L) mildly deformed, (M) severely deformed and (N) wing-to-notum phenotypes. (O) Percentage of adult wing phenotypes of vg-GAL4 flies expressing the indicated transgenes, revealing that the hydWT transgene enhanced, and the hydC>A transgene suppressed the severity of the sggS9A wing defects (n ≥12 for each genotype). (P) Model showing the genetic interaction between sggS9A and hyd UAS-transgenes with respect to the wing-to-notum phenotype. Arrows indicate promotion and blockhead arrows inhibition. (Q-R) hydWT overexpression rescues sggRNAi-mediated embryonic lethality. Percentage viability of sca-GAL4 flies expressing the indicated transgenes revealed a >95% rescue of embryonic lethality upon co-expression with the UAS-hydWT, but not UAS-hydC>A, transgene (16 individual crosses per genotype). (R) Model showing the genetic interaction between sgg and hyd UAS-transgenes. Arrows indicate promotion, blockhead arrows inhibition and dotted blockhead arrow weak inhibition. Scale bar = 250μm.