In the experiments presented here, we find that four quite different mechanisms that can activate the mammalian Hedgehog pathway all depend on IFT. The Hh pathway reporter is activated by addition of Shh ligand, expression of activated Smoothened, or knockdown of Sufu or PKA activity in wild-type embryonic fibroblasts, but all of these treatments fail to activate the Hh target in fibroblasts that lack either IFT172 or the Dync2h1. It is striking that the phenotypes of Ift172wim and Dync2h1ttn mutant MEFs are identical in all our assays, even though the Dync2h1ttn cells have cilia of nearly normal length, as assayed by both SEM and staining with acetylated α-tubulin. These results support the view that cilia and retrograde IFT trafficking are essential to activate the Hh pathway.
The mechanisms of action of Sufu and PKA in mammalian Hh signaling are not yet clear. Mammalian Sufu interacts directly with Gli proteins, and may tether Gli proteins outside the nucleus, just as Drosophila
Sufu tethers Ci in the cytoplasm in the absence of ligand (Kogerman et al., 1999
; Dunaeva et al., 2003
). However, mammalian Sufu has a much stronger phenotype than the Drosophila
gene and is thought to have roles within the nucleus (Paces-Fessy et al., 2004
; Barnfield et al., 2005
; Svard et al., 2006
). Sufu protein is enriched both in the cilium and in the nucleus (Haycraft et al., 2005
), and our data are consistent with the possibility that Sufu tethers Gli proteins in cilia to prevent their release to the nucleus in the absence of ligand. However, our findings do not rule out that Sufu has an additional later function within the nucleus.
Decreased PKA activity strongly activates the mammalian Hh pathway (Huang et al., 2002
). PKA acts, at least in part, by priming Gli3 for processing to allow generation of the repressor form of the protein by proteasome-dependent proteolysis (Pan et al., 2006
; Wang and Li, 2006
). The site where biochemical events that promote Gli3 processing take place is not known. Proteasomes are enriched at the basal body, but none have been found within the cilium (Wigley et al., 1999
), suggesting that proteolysis does not take place within the cilium. Our findings indicate that PKA activity in the mammalian Hh pathway depends on cilia, which would suggest that PKA may act within cilia.
Genetic experiments to define the relationships between IFT components, Sufu and PKA would require several generations of mouse breeding, but could be carried out much more quickly in cell culture. Nevertheless, the cell culture experiments have a number of limitations. For example, previous experiments indicated that simple treatment with shRNAs did not fully inactivate Sufu or fully activate the Hh pathway, presumably due to the stability of the protein (Varjosalo et al., 2006
). Therefore our results indicate that a partial loss of Sufu activity that is sufficient to activate the pathway in wild-type cells does not activate the pathway in the absence of cilia or retrograde IFT. Similarly, the MEF experiments allow us to conclude that a decrease in the activity of PKA that is sufficient to activate the pathway in wild-type cells has no effect in IFT mutant MEFs. Analysis of the phenotypes of double mutants with null alleles of Sufu, PKA and IFT mutants will provide definitive information on pathway relationships in vivo. In addition, there are clear cell-type specific differences in the requirement for some cilia proteins in the embryo. For example, Shh signaling is disrupted to different extents along the rostrocaudal axis of Dync2h1
mutant embryos (Huangfu and Anderson, 2005
). Such cell-type specific differences in the relationship between retrograde IFT and Shh signaling can only be studied in vivo
We find that Smo accumulates in the cilia of the Dync2h1
mutant cells in the absence of Shh. This suggests that Smo continually traffics through cilia in the absence of Hh ligand, and accumulates to high levels within the cilium when retrograde IFT is disrupted. Our findings contrast with the report of May et al., who did not detect either Smo or acetylated α-tubulin in the cilia of the embryonic node in Dync2h1
mutant embryos (May et al., 2005
). Because we find that Smo accumulates in the cilia of all three Dync2h1
mutants examined, we suggest that the difference between the experiments may reflect the greater ease of detection of ciliary Smo in cultured cells than in embryonic tissues.
Our experiments in MEFs suggest that Shh may act by modulating the kinetics of Smo transit through the cilium, rather than by regulating an on/off switch of Smo localization. Shh may increase the rate of delivery of Smo-containing vesicles to the base of the cilium, where Smo is transferred to an anterograde IFT-dependent trafficking mechanism. Once at the tip, Smo may be released, like other IFT cargo, and there it can interact with other Hh pathway components localized at the cilia tip. In either the presence or absence of Shh ligand, if retrograde transport is blocked, both Smo and modified Gli proteins fail to move out of the cilium. The consequence of this is that Smo and Gli proteins both remain at the cilia tip and the nuclear pathway is not activated. More complex models are also possible: for example, Shh could act by decreasing the rate of retrograde IFT, thereby increasing the amount of time Smo can associate with other pathway components at the cilia tip and promoting downstream signaling events. Direct measurements of the rates of Smo trafficking and of anterograde and retrograde IFT in the presence or absence of Shh would, in principle, distinguish between these hypotheses.