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. 2008 Aug;237(8):2030-8.
doi: 10.1002/dvdy.21551.

Intraflagellar transport, cilia, and mammalian Hedgehog signaling: analysis in mouse embryonic fibroblasts

Polloneal Jymmiel R Ocbina  1 , Kathryn V Anderson
Affiliations

Intraflagellar transport, cilia, and mammalian Hedgehog signaling: analysis in mouse embryonic fibroblasts

Polloneal Jymmiel R Ocbina et al. Dev Dyn. 2008 Aug.

Abstract

Genetic studies in the mouse have shown that Intraflagellar Transport (IFT) is essential for mammalian Hedgehog (Hh) signal transduction. In this study, we take advantage of wild type and IFT mutant mouse embryonic fibroblasts (MEFs) to characterize additional aspects of the relationship between IFT and Hh signaling. Exposure to Sonic hedgehog (Shh) ligand or expression of an activated allele of Smo, SmoA1, activates an Hh reporter in wild-type MEFs, but not in MEFs derived from embryos that lack IFT172 or the Dync2h1 subunit of the retrograde IFT motor. Similarly, decreased activity of either Sufu or PKA, two negative regulators of Hh signal transduction, activates the pathway in wild-type, but not IFT mutant, MEFs. In contrast to wild-type MEFs, Smo is constitutively present in the cilia of Dync2h1 mutant MEFs. This finding suggests that IFT-dependent trafficking of Hh pathway components through the cilium is essential for their function.

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Figures

Figure 1
Figure 1. The presence of cilia correlates with Hedgehog responsiveness in wild-type (A) and Ptch1 (B) mouse embryonic fibroblasts (MEFs)
A. (Left panel) Most wild-type MEFs grown at low density lacked cilia, whereas confluent MEFs almost always grew primary cilia. Wild-type MEFs were initially plated at 1.0, 0.2 and 0.1 × 105 cells/cm2 in 24-well plates. (Right panel) Hedgehog responsiveness was assayed by transfecting wild type MEFs with Renilla luciferase transfection control and a Hh-responsive Gli-luciferase reporter, then stimulating with Shh-conditioned media (Materials and Methods). The activation of the reporter in response to Shh treatment correlates with cell density, and therefore with the presence of primary cilia on the cells. B. Ptch1 MEFs grow cilia and activate the reporter in a similar cell-density dependent manner in the absence of Shh treatment. Data are mean ± s.d.
Figure 2
Figure 2. Characterization of cilia phenotypes in Ift172wim and Dync2h1ttn mutant MEFs
A-D. Immunofluorescent staining of wild type and mutant MEFs for acetylated α-tubulin (red) and DAPI (blue) show that although Ift172wim cells do not possess cilia (C), mutants that lack the heavy chain of the retrograde IFT motor, Dync2h1, are able to grow cilia (D). Scale bar = 5μm. E-H. HIgher magnification views of MEFs using scanning electron microscopy. Ift172wim MEFs lack cilia (G). Dync2h1ttn MEFs are able to grow primary cilia (H). but their morphology is abnormal compared to WT (E) and Ptch1 (F) primary cilia. From 18 Dync2h1 mutant cilia examined, the mutant cilia were shorter (565 ± 20nm) than wild type (800 ± 30nm). Each mutant cilia had a bulb-like structure along the length of the axoneme. Scale bars, 5μm and 2μm (inset). I. Activation of the Gli-luciferase reporter in wild type and mutant MEFs. Wild type cells show robust activation of the reporter in response to Shh treatment. As expected, Ptch1 mutant MEFs mutant activate the reporter even in the absence of ligand. In contrast, Ift172wim and Dync2h1ttn MEFs show no activation of the reporter in response to Shh treatment. Data are mean ± s.d.
Figure 3
Figure 3. Smo, Sufu and PKA depend on IFT proteins for activity
A. Expression of an activated form of Smo, SmoA1, activates the Gli-luciferase reporter in wild-type, but not Ift172wim or Dync2h1ttn mutant cells. B. Expression of a dnPKA construct activates the Hh pathway in wild type MEFs, whereas Ift172wim and Dync2h1ttn MEFs are unresponsive to this treatment. C. Expression of shRNAs against Sufu in wild type MEFs causes strong activation of the Gli-luciferase promoter. Knockdown of Sufu in MEFs that have a mutation in Ift172 or Dync2h1 had no effect on reporter activity. Data are mean ± s.d.
Figure 4
Figure 4. Smoothened localization to cilia is not sufficient to activate the Hh pathway A-B
Localization of Smo (green) in cilia of wild type, Ptch1, Ift172wim, Dync2h1ttn and Dync2h1GT MEFs in the absence (A) or presence (B) of Shh ligand. Acetylated α-tubulin (red) was used to identify cilia and DAPI (blue) was used to label nuclei. In the absence of Shh (A), Smo is found in the cytoplasm of wild-type MEFs and is localized to the cilium following 48 hours treatment with Shh (B). In MEFs that lack the Hh receptor Ptch1, Smo is enriched in cilia regardless of Shh presence. Ift172wim MEFs, which do not grow cilia, show cytoplasmic staining of Smo in the presence or absence of Shh. Constitutive enrichment of Smo in the primary cilium was observed in Dync2h1ttn and Dync2h1GT MEFs, independent of Shh treatment.
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