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THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia

Abstract

Characterization of previously described intraflagellar transport (IFT) mouse mutants has led to the proposition that normal primary cilia are required for mammalian cells to respond to the sonic hedgehog (SHH) signal. Here we describe an N-ethyl-N-nitrosourea–induced mutant mouse, alien (aln), which has abnormal primary cilia and shows overactivation of the SHH pathway. The aln locus encodes a novel protein, THM1 (tetratricopeptide repeat–containing hedgehog modulator-1), which localizes to cilia. aln-mutant cilia have bulb-like structures at their tips in which IFT proteins (such as IFT88) are sequestered, characteristic of Chlamydomonas reinhardtii and Caenorhabditis elegans retrograde IFT mutants. RNA-interference knockdown of Ttc21b (which we call Thm1 and which encodes THM1) in mouse inner medullary collecting duct cells expressing an IFT88–enhanced yellow fluorescent protein fusion recapitulated the aln-mutant cilial phenotype, and live imaging of these cells revealed impaired retrograde IFT. In contrast to previously described IFT mutants, Smoothened and full-length glioblastoma (GLI) proteins localize to aln-mutant cilia. We hypothesize that the aln retrograde IFT defect causes sequestration of IFT proteins in aln-mutant cilia and leads to the overactivated SHH signaling phenotype. Specifically, the aln mutation uncouples the roles of anterograde and retrograde transport in SHH signaling, suggesting that anterograde IFT is required for GLI activation and that retrograde IFT modulates this event.

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Figure 1: Ttc21b is mutated in aln-mutant mice.
Figure 2: SHH signaling targets are misexpressed in aln mutants.
Figure 3: Genetic analyses of epistasis between mice mutant for aln and mice mutant for Shh, Smo or Gli2.
Figure 4: GLI3 protein and transcript levels are elevated in aln mutants.
Figure 5: IFT88 is sequestered in aln-mutant primary cilia.
Figure 6: SMO and GLI proteins translocate to aln-mutant cilia.
Figure 7: Retrograde IFT is impaired in cilia of THM1-deficient IMCD cells.

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Acknowledgements

We thank B. Wang (Cornell University) for the antibody to GLI3, A. McMahon (Harvard University) for the Shh-null and Smo-null mutant mice, J. Eggenschwiler (Princeton University) and A. Joyner (Memorial Sloan-Kettering Cancer Center) for the Gli2-null mutant mice, K. Parker and C. Rao for technical assistance, C. Yang for mouse husbandry and R. Stearns for assistance with scanning electron microscopy at the electron microscopy facility at the Harvard School of Public Health. This research was supported by National Institutes of Health grants R01HD36404 (to D.R.B.) and R01HD056030 (to B.K.Y.) and the Harvard PKD Center (P50DK074030 to J.V.S.).

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Authors and Affiliations

Authors

Contributions

P.V.T. carried out most experiments. C.J.H. carried out immunostaining of IFT88 on limb cryosections and GLI localization to aln cilia. T.Y.B. generated the m368-2 cell line and performed live-cell imaging experiments with P.V.T. A.T.-D. carried out immunofluorescence analysis of neural tube sections and whole-mount in situ hybridization experiments. B.J.H. generated the aln mutant and mapped the aln locus to chromosome 2. A.L.C. generated the antibody to THM1, and H.Q. conducted resequencing analysis of candidate loci for aln. P.J.S. did the initial GLI3 western blot on aln tissue. D.R.B., B.K.Y., J.V.S., C.J.H. and P.V.T. designed the experiments. R.W.S. provided thoughtful discussions and was instrumental throughout the study. P.V.T., R.W.S. and D.R.B. wrote the manuscript. J.V.S., B.K.Y., C.J.H. and T.Y.B. critically commented on the manuscript.

Corresponding author

Correspondence to David R Beier.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1 (PDF 2924 kb)

Supplementary Video 1

Movement of IFT88-EYFP in live EV4 cells. 50, 0.4 sec images taken at 0.75-second intervals were assembled into a movie and played forward. (AVI 1762 kb)

Supplementary Video 2

Movement of IFT88-EYFP in live R1-4 cells. 50, 0.4 sec images taken at 0.75-second intervals were assembled into a movie and played forward. (AVI 1723 kb)

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Tran, P., Haycraft, C., Besschetnova, T. et al. THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia. Nat Genet 40, 403–410 (2008). https://doi.org/10.1038/ng.105

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