Primary Cilia Are Not Required for Normal Canonical Wnt Signaling in the Mouse Embryo

Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands

Normal spatial pattern of canonical Wnt response in IFT mutants.

<p>The BAT-gal transgene expresses β-galactosidase under the control of seven tandem TCF/LEF sites that mediate responses to canonical Wnt signals. A. Reporter expression patterns are similar in e9.0 wild type and <i>Ift172</i> mutant embryos that carry one copy of the BAT-gal reporter. B. Higher magnification view of the anterior of wild type and mutant embryos shows indistinguishable patterns and levels of reporter expression. BAT-gal reporter expression in <i>Ift88</i> (C), <i>Kif3a</i> (D) and <i>Dync2h1</i> (E) mutant embryos with wild type littermates at e9.5.</p

Quantitation of BAT-gal reporter in whole embryo lysates.

<p>Transcriptional activation in response to canonical Wnt signals was quantitated in e9.5 embryos that carried one copy of the BAT-gal reporter using a spectrophotometric assay for β-galactosidase enzyme activity (data are mean, ±s.d., n≥5; ANOVA, Tukey's post-hoc tests, n.s.).</p

The response of fibroblasts to Wnt3a does not depend on cilia.

<p>A. Wild type, <i>Ift88</i>, <i>Ift172</i> and <i>Dync2h1</i> MEFs stained for cilia (acetylated α-tubulin, green) and basal bodies (centrin, red). MEFs derived from e9.5 wild type and <i>Dync2h1</i> embryos generate cilia within 24 hours of culture, but not <i>Ift172</i> or <i>Ift88</i> mutant MEFs. B. <i>Ift88</i>, <i>Ift172</i> and <i>Dync2h1</i> MEFs fail to respond to Shh. Cells were transfected with a Hh-responsive Gli-luciferase reporter and stimulated with Shh-enriched media. Wild-type cells showed robust activation of the reporter in response to Shh treatment, whereas <i>Ift88</i>, <i>Ift172</i> and <i>Dync2h1</i> MEFs were completely non-responsive to Shh. C. <i>Ift88</i>, <i>Ift172</i> and <i>Dync2h1</i> MEFs respond normally to Wnt3a. SuperTOP-Flash reporter activity was assayed in response to 100 ng/mL recombinant Wnt3a. Reporter activity presented as relative light units (RLU) normalized to Renilla luciferase control. <i>Ift172</i> and <i>Dync2h1</i> mutant MEFs activated the reporter in response to different levels of Wnt3a similar to wild type levels (data are mean ±s.d., n = 4).</p