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. 2009 Jun 15;330(2):452-60.
doi: 10.1016/j.ydbio.2009.04.009. Epub 2009 Apr 14.

Suppressor of Fused inhibits mammalian Hedgehog signaling in the absence of cilia

Jinping Jia  1 , Asa Kolterud, Huiqing Zeng, Amber Hoover, Stephan Teglund, Rune Toftgård, Aimin Liu
Affiliations

Suppressor of Fused inhibits mammalian Hedgehog signaling in the absence of cilia

Jinping Jia et al. Dev Biol. .

Abstract

The Hedgehog (Hh) family of secreted proteins regulates mammalian development and cancer formation through Gli transcription factors, which exist in both activator and repressor forms. In vertebrates, the primary cilia play an essential role in Hh signal transduction and are required for both the activator and repressor activities of Gli proteins. In the current study, we demonstrate that mouse Suppressor of Fused (Sufu) interacts with Gli proteins and inhibits Gli activator activity in the absence of cilia. Removal of Sufu in both Smoothened (Smo) and Ift88 mutants, respectively, leads to full activation of Hh signaling, suggesting that Smo-mediated repression of Sufu, but not the inhibitory function of Sufu, requires cilia. Finally, we show that Sufu is important for proper activator/repressor ratio of Gli3 protein in mice, both in the presence and absence of cilia.

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Figures

Figure 1
Figure 1
Cilia do not form in Ift88 mutant MEFs. (A) Genomic PCRs using primers specific to wild type or Ift88 mutant allele are performed on MEF cell lysates. No wild type product is detected in the Ift88 mutant cells. DNA prepared from the tail of a heterozygous Ift88 mutant mouse is used as control. (B) Western blot is performed on MEF cell lysates using an IFT88-specific antibody. No IFT88 protein is detected in the Ift88 mutant MEFs. Tubulin is used as a loading control. (C) Acetylated α-tubulin is present in the basal body (arrowhead) and axoneme (arrow) of the cilia in wild type cells. (D) Acetylated α-tubulin is present in punctuate doublets (arrowheads) in Ift88 mutant cells. (E, F) γ-tubulin is present in punctuate doublets (arrowheads) in both wild type (E) and Ift88 mutant (F) cells. DNA is stained blue in C–F.
Figure 2
Figure 2
Sufu physically interacts with all Gli proteins in the absence of cilia. (A–C) Sufu co-immunoprecipitates with Gli1 (A), Gli2 (B) and Gli3 (C) when over-expressed in wild type and Ift88 mutant cells. (D) Endogenous Gli3 co-immunoprecipitates with Sufu, but not by unrelated antibodies (anti-Myc) in wild type and Ift88 mutant cells. Panels shown in D are assembled from one single experiment in which same experimental procedure was applied to both experimental and control samples. For each panel, the input lanes are loaded with 10% of protein lysate used for immunoprecipitation.
Figure 3
Figure 3
Sufu inhibits Gli activities in the absence of cilia. Wild type and Ift88 mutant MEFs are co-transfected with a Gli-responsive luciferase reporter and various effectors. Activities of the firefly luciferase are plotted to reflect the relative expression level of the reporter. (A) Sufu inhibits Gli1-mediated activation of the reporter in wild type and Ift88 mutant cells. (B) Sufu inhibits Gli2-mediated activation of the reporter in wild type and Ift88 mutant cells. (C) Reducing the level of Sufu protein by RNA interference activates a Gli-responsive reporter expression in both wild type and Ift88 mutant cells. Immunoblots below each chart shows the relevant proteins present in the same volume of lysate used for luciferase activity measurement. In C, the relative amount of Sufu protein is calculated and shown below the immunoblots. The amount in cells treated with scrambled shRNA is arbitrarily set to 1.
Figure 4
Figure 4
Sufu is epistatic to Ift88. (A–D) lateral views of E9.5 mouse embryos. Arrowheads in B point to elevated neural ridges in the brain of an Ift88 mutant embryo. Note that there is no neural ridge elevation in the brains of the Sufu (C) and Sufu/Ift88 double (D) mutant embryos. (E–H) Ptch1 expression in E9.5 whole embryos detected by wholemount RNA in situ hybridization. Ptch1 expression is reduced in an Ift88 mutant embryo (F), but upregulated in the Sufu (G) and Sufu/Ift88 double (H) mutant embryos. (I–J) Gli1 expression in E9.5 whole embryos detected by wholemount RNA in situ hybridization. Gli1 expression is reduced in an Ift88 mutant embryo (J), but upregulated in the Sufu (K) and Sufu/Ift88 double (L) mutant embryos
Figure 5
Figure 5
Sufu regulates spinal cord patterning in the absence of cilia. (A) Foxa2 labels the floor plate in wild type spinal cord. (B) Nkx2.2 labels the precursors for V3 interneurons. (C) Pax6 is present in the neural progenitor cells of the dorsal two-thirds of the spinal cord. (D) Foxa2 and (E) Nkx2.2 are absent in Ift88 mutants. (F) Pax6 is present throughout the spinal cord in Ift88 mutants. (G) Foxa2 and (H) Nkx2.2 are present throughout the spinal cord of the Sufu mutants. (I) Pax6 is absent in the Sufu mutant spinal cord. (J) Foxa2 and (K) Nkx2.2 are present throughout the spinal cord of Sufu/Ift88 double mutants. (L) Pax6 is absent in the Sufu/Ift88 double mutant spinal cord. Shown are immunofluorescent images of transverse sections of E9.5 embryos. Dashed lines outline the spinal cords.
Figure 6
Figure 6
Sufu regulates spinal cord patterning downstream of Smo. (A) Nkx2.2 labels the precursors for V3 interneurons. (B) Pax6 is present in the neural progenitor cells of the dorsal two-thirds of the spinal cord. (C) Pax7 is present in the dorsal progenitor cells of the wild type spinal cord. (D) Nkx2.2 is absent in Smo mutants. (E) Pax6 is present throughout the spinal cord in Smo mutants. (F) Pax7 is present throughout the spinal cord in Smo mutants. (G) Nkx2.2 is present throughout the spinal cord of Sufu mutant. (H) Pax6 is absent in the Sufu mutant spinal cord. (I) Pax7 is absent in the Sufu mutant spinal cord. (J) Nkx2.2 is present throughout the spinal cord of Sufu/Smo double mutant. (K) Pax6 is absent in the Sufu/Smo double mutant spinal cord. (L) Pax7 is absent in the Sufu/Smo double mutant spinal cords. Shown are immunofluorescent images of transverse sections of E9.5 embryos. Dashed lines outline the spinal cord.
Figure 7
Figure 7
Sufu regulates the ratio between the activator and repressor forms of Gli3 protein independent of cilia. (A) Immunoblot showing the level of Gli3-190 and Gli3-83 in E9.5 wild type and different combinations of Sufu and Ift88 mutants. Each lane was loaded with protein extracted from a single embryo. (B) Immunoblot showing the levels of full length Gli3 (Gli3-190) and processed product (Gli3-83) in wild type E12.5 limb bud, and MEFs derived from E9.5 wild type, Sufu and Ptch1 mutant embryos. Tubulin was used as loading control in both A and B. (C) Graphical representation of the Gli3-190 versus Gli3-83 ratio in E9.5 wild type and different combinations of Sufu and Ift88 mutants. (D) Graphical representation of the relative level of total Gli3 protein (Gli3-190 and Gli3-83). The relative level of Gli3 protein in wild type was set to one. Note that the ratios in C and D may be under-estimated due to saturation of band density in A and B. Graphics in C and D summarize data from at least 3 embryos of each genotype.
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References

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