The extracellular domain of Smoothened regulates ciliary localization and is required for high-level Hh signaling

Abstract

Members of the Hedgehog (Hh) family of secreted proteins function as morphogens to pattern developing tissues and control cell proliferation. The seven-transmembrane domain (7TM) protein Smoothened (Smo) is essential for the activation of all levels of Hh signaling. However, the mechanisms by which Smo differentially activates low- or high-level Hh signaling are not known. Here we show that a newly identified mutation in the extracellular domain (ECD) of zebrafish Smo attenuates Smo signaling. The Smo agonist purmorphamine induces the stabilization, ciliary translocation, and high-level signaling of wild-type Smo. In contrast, purmorphamine induces the stabilization but not the ciliary translocation or high-level signaling of the Smo ECD mutant protein. Surprisingly, a truncated form of Smo that lacks the cysteine-rich domain of the ECD localizes to the cilium but is unable to activate high-level Hh signaling. We also present evidence that cilia may be required for Hh signaling in early zebrafish embryos. These data indicate that the ECD, previously thought to be dispensable for vertebrate Smo function, both regulates Smo ciliary localization and is essential for high-level Hh signaling.

Figure 1. s294 is a hypomorphic allele of smo with a C125Y substitution in the extracellular domain

(A–C) Comparison of wild-type (A), smo^s294 mutant (B) and smo^hi1640 mutant (C) embryos at 24 hpf shows that s294 mutants have similar, although weaker, phenotypes as the smo null allele hi1640. (D–F) Immunostaining for Prox1 (green) and sMHC (red) assesses medium-to-low levels Hh signalling in wild-type (D), smo^s294 mutant (E) and smo^hi1640 mutant (F) embryos at 24 hpf. (G–I) Immunostaining for Engrailed (red) assesses high levels Hh signalling in wild-type (G), smo^s294 mutant (H) and smo^hi1640 mutant (I) embryos at 24 hpf. (J) Graphic representation of Smo. Each amino acid is represented by a circle, conserved cysteine residues in the ECD are shown in green. The s294 mutation is shown in red; similar Drosophila cysteine substitution alleles are shown in purple.

Figure 2. The extracellular domain of Smo is required for high level signalling

Purmorphamine treatment of wild-type and smo^s294 mutant embryos. DMSO-treated wild-type (A, E, I), DMSO-treated smo^s294 mutant (C, G, K), purmorphamine-treated wild-type (B, F, J) and purmorphamine-treated smo^s294 mutant (D, H, L) embryos at 24 hpf. Purmorphamine treatment did not significantly affect morphology (B), and caused only a modest increase in Prox1 (green) and sMHC staining (red) (F), but strongly induced ectopic Eng positive MPs in wild-type embryos (J). Purmorphamine-treatment of smo^s294 mutants gave significant rescue of the morphological phenotype (D), and restored Prox1 (green) and sMHC (red) expression (H), but not Eng expression (L). (M–T) Injection of SmoΔCRD mRNA in wt and smo^hi1640 mutant embryos. (M–P) Prox1 (green) and sMHC (red) staining, (Q–T) Eng staining. Injection of 250 pg SmoΔCRD mRNA caused a complete rescue of Prox1 and sMHC expression in smo^hi1640 mutants (P) compared to uninjected mutants (O), and a slight increase in Prox1 and sMHC expression in wild-type (N) compared to control (M). In contrast, injection of SmoΔCRD mRNA had no effect on Eng expression in wild-type (R) compared to control (Q), and did not rescue Eng expression in smo^hi1640 mutants (T). For quantification see Suppl. Tables 1–2.

Figure 3. SmoC151Y does not localise to the cilium

Expression of the ciliary marker acetylated Tubulin (green) and either Myc-tagged wild-type Smo or Myc-tagged SmoC151Y (red) in NIH-3T3 cells (A–D) and zebrafish embryos at 10 hpf (E–J). Nuclei of NIH-3T3 cells were visualised with DAPI (blue). (A–B) In NIH-3T3 cells, wild-type Smo localised to the cilium in response to Hh (A), whereas SmoC151Y (B) did not. (C–D) Treatment with the Smo agonist purmorphamine induced ciliary localisation of wild-type Smo (C), but did not induce detectable ciliary localisation of SmoC151Y (D). Myc-tagged Smo mRNA was injected into Tg(-1.8gsc:GFP)^ml1 zebrafish embryos, which express GFP in the dorsal midline. Wild-type Smo localised to the cilia of cells surrounding the dorsal midline in embryos treated with DMSO (E), whereas SmoC151Y was never detected on the cilium (F). Purmorphamine treatment increased the ciliary localisation of Smo (G), but did not induce the ciliary localisation of SmoC151Y (H). In contrast, SmoΔCRD localised to the cilia in untreated embryos (I). (J) The ciliary localisation of Smo was quantified in locations away from the dorsal midline in sections (insert J), and the mean percentage of cilia that exhibited co-localisation with the Myc-tagged Smo constructs are shown. Error bars show s.e.m. Total number of sections counted are given below. Scale bar in (E) is 10 μm.

Figure 4. Inhibition of ciliogenesis causes loss of Hh target gene expression in zebrafish embryos

(A–E) Expression of the ciliary marker acetylated Tubulin (green) and the basal body marker γ-Tubulin (red) in Kupffer’s vesicle of 16 hpf zebrafish embryos. Injection of either dnKif3b mRNA (B), qilin MO1 (C) or qilin MO2 (D) caused a reduction of KV cilia, which were restored in embryos co-injected with qilin MO2 and qilin mRNA (E). Scale bar in (A) is 10 μm. (F–O) in situ hybridisation of 10 hpf control and injected embryos. In wild-type embryos, ptc1 (F) and myod (K) are expressed in two stripes flanking the dorsal midline. Injection of dnKif3b (G), qilin MO1 (H), or qilin MO2 (I, L) caused a reduction or loss of ptc1 and myod expression, which could be restored by co-injection of qilin MO2 with qilin mRNA (J, M). (N–O) shh expression in qilin MO2 injected embryos (O) was comparable to controls (N), with a broadened expression reflecting convergence-extension defects. For quantification of these experiments, see Supplementary Fig. 10.