Adenylyl cyclases require ciliary localization to regulate the Hh pathway. (A) CGNP cultures were transfected for 24 h with HA-tagged constructs of AC1, AC3, AC5 or AC6 cloned in pCIG, a nuclear-GFP-expressing bicistronic vector. The cells were stained with anti-acetylated-tubulin (AcTubulin, cilium marker, red), anti-HA (green) and anti-GFP (transfection, blue) antibodies. AC3, AC5 and AC6 but not AC1 accumulated in the cilium. (B) Endogenous expression of AC5 and/or AC6 (green) was detected in the cilium of CGNPs and NIH3T3 cells with an antibody that detects both molecules with similar affinity (see supplementary material Fig. S1B). Acetylated Tubulin (red) and DAPI (blue) were used as markers of the cilium and nucleus, respectively. (C) The chart shows inter-species sequence conservation and intramolecular location of a putative AC5 ciliary location motif. The motif was predicted through multiple alignment of AC5 with other similar ciliary proteins. (D) Mutation of amino acids 76–77 of AC5 to AA (AC5^WR76-77AA–HA) impaired its ciliary location in NIH-3T3 cells. Cultures were stained with anti-AcTubulin (red), anti-HA (green) anti-GFP (purple) antibodies and DAPI (blue). The boxed areas are shown magnified with only the HA staining on the right. (E,F) Shh-induced proliferation of CGNPs was not inhibited by transfection with the AC5^WR76-77AA mutant as assessed by [³H]thymidine or BrdU incorporation. All the bar graphs in this figure show the mean±s.d. of at least three independent experiments. The total number of cells counted for each data point in panels F is indicated inside each bar. *P<0.05, ***P<0.001 (one-way ANOVA followed by the Tukey's test). Scale bars: 10 µm.

Expression of AC5 and AC6 represses the Hh pathway in the developing chicken neural tube. (A–D) HH-12 chicken neural tubes were electroporated with different constructs cloned in pCIG (a diagram of the vector is shown above B) and they were left to develop for 16 h. (A) Scheme of the working protocol and expression levels of AC1, AC3, AC5 and AC6 in the neural tubes studied by q-PCR. (B) BrdU (∼1 µl at 0.5 µg/µl) was injected into the neural tube lumen 4 h prior to fixation and transverse sections of the neural tube were then stained with antibodies against BrdU (proliferation, red) and GFP (transfection, green), and DAPI (nucleus, blue). (C,D) The percentage of BrdU-positive cells among the transfected population was calculated separately for the dorsal and ventral neural tube. PKACα^QR was used as a positive control. (E) In an experiment similar to that for A–D, the cell cycle phase distribution was studied in the population of transfected cells (GFP-positive) by FACS analysis, and the Gli3R and Bcl2 combination was used as a positive control. (F,G) The expression of Olig2 (a marker of motoneuron precursors, red) was studied in HH-12 chicken neural tubes expressing AC3, AC5 or AC6 at 16 hpe (green). The number of Olig2-positive cells in the electroporated side (EP) was compared in each case to the non-electroporated side (NEP). (H,I) HH-12 chicken neural tubes were transfected for 16 h with AC3, AC5 or AC6 cloned in pCIG (green) together with pGBSRED, a RFP-expressing reporter of Hh pathway activity (red). Diagrams of the vectors are shown above the panels. The percentage of RFP-positive cells among the transfected population is shown in the bar graph. All the bar graphs in this figure show the mean±s.d. of at least three independent experiments. The total number of cells counted in each data point in panels C, D, F and I is indicated inside each bar. *P<0.05, ***P<0.001 (one-way ANOVA followed by the Tukey's test). Scale bars: 50 µm.

Adenylyl cyclase knockdown activates the Hh pathway. (A) Mouse CGNP cultures grown in the presence or absence of low doses of Shh (0.3 µg/ml), were transfected for 24 h with pSHIN expressing different short hairpin molecules directed against mouse AC3, AC5 or AC6. [³H]thymidine incorporation in the last 4 h of cultures was used to study cell proliferation. (B) HH-12 chicken neural tubes were transfected for 16 h with pSHIN expressing selected short hairpin molecules directed against AC3, AC5 or AC6, or a combination of these, together with the pGBSLUC luciferase reporter of Hh pathway activity. The efficiency of the different shRNAs is shown in supplementary material Fig. S1F,G. (C,D) As in B but using pGBSRED as the reporter. The dominant-negative (dn)PKA construct was used as a positive control of Hh pathway activation. The percentage of transfected cells that are RFP-positive is shown in the bar graph. Diagrams of the expression vector are shown above the panels. All the bar graphs in this figure show the mean±s.d. of at least three independent experiments. The total number of cells counted in each data point in panel D is indicated inside each bar. *P<0.05, ***P<0.001 (one-way ANOVA followed by the Tukey's test). Scale bar: 50 µm.