The results described thus far demonstrate that IFT proteins are required for Hh signaling and that some Hh signaling proteins are enriched in cilia. These findings do not exclude the possibility that IFT proteins traffic Hh pathway components outside of cilia and that the enrichment of Smo, Glis and Sufu in cilia is not essential for their activity. However, mutations in several mouse cilia-associated proteins also show altered Hh signaling. Analysis of these mutants argues that cilia, not just the intraflagellar transport machinery, are required for Hh signaling. In addition, some of these mutants suggest that normal Hh signal transduction depends on active participation of ciliary proteins.
Loss of function of mouse Ofd1
, which was first identified as the gene affected in human Oral-Facial-Digital Sydrome 1, causes phenotypes similar to those of IFT mutants. The OFD1 protein has been seen localized to the basal body and centrosome rather than in the cilium (Romio et al., 2004
), but it is required for cilia formation (Ferrante et al., 2006
). Ofd1 is X-linked, although the human Ofd1
gene is not subject to X-inactivation; females heterozygous for Ofd1
mutations have a syndrome that includes craniofacial and digit abnormalities and, occasionally, polycystic kidneys, presumably due to haploinsufficiency of the gene. Ofd1
mutations are apparently lethal to hemizygous human males. In mice, the Ofd1
gene is subject to X-inactivation and, as a result, Ofd1
mutant female mice have a more severe phenotype than the human females, presumably because of mosaic loss of function due to random inactivation of the X-chromosome (Ferrante et al., 2006
null mutant mice fail to form cilia in the embryonic node, and show the associated randomization of left-right patterning. Ventral neural cell fates are not specified in Ofd1
mutant male embryos and the mutants express the direct Hh-target gene Patched1
at low levels, indicating that Hh signaling is defective in these embryos. Thus loss of this centrosome/basal body protein blocks the formation of cilia and that disrupts Hh signaling, which supports the hypothesis that cilia are required for Hh signaling, beyond the requirement for IFT proteins.
Ofd1 mutant limb buds exhibit ectopic expression of Gli3R target genes such as HoxD genes in the anterior limb buds, suggesting that, as in IFT mutants, formation of Gli3R is impaired, which is consistent with the polydactylous phenotype observed in Ofd1/+ female embryos. However, in contrast to Ift88 and Dynch2 mutants, Ofd1-deficient male limb buds exhibit normal levels of Patched1 and Gli1 expression in the posterior limb bud. These data suggest that OFD1 is not required for Gli activator function in the limb bud, although it does appear to be required for Gli activator function in the neural tube. Because it is not known whether cilia are present on Ofd1 mutant limb bud cells, it is possible that these mutant cells retain some aspects of basal body function and/or ciliogenesis that permits normal Gli activator function but blocks formation of Gli3R. Further studies may pinpoint whether the Ofd1 mutation separates the functions of the cilium and the basal body in the regulation of Gli protein activity in the limb.
Most interesting is that some cilia-associated proteins have different roles in Hh signaling than seen in the kinesin, dynein and IFT complex B proteins described above. For example, in direct contrast to loss of complex B IFT proteins where Gli activator function is disrupted, a null mutation in mouse Ift122
, which encodes a complex A protein, leads to constitutive activation of the Hedgehog pathway and a ventralization of the neural tube (J. Qin and JTE, in preparation). Genetic studies show that mouse IFT122 functions at a step in the pathway downstream of Shh and Smoothened, but upstream of the transcription factor Gli2. In contrast to complex B Ift
mutant cells, Ift122
mutant cells have primary cilia, but these cilia are morphologically abnormal and exhibit pronounced accumulation of IFT proteins within the ciliary axoneme and at its tip, a phenotype similar to that of mutants that lack the retrograde IFT dynein motor (Huangfu and Anderson, 2005
), suggesting that retrograde transport is severely impaired in Ift122
Studies in C. elegans and Chlamydomonas
have also distinguished between the functions of complex A and complex B proteins. While loss of complex B proteins leads to absence of flagella, disruption of complex A proteins causes less severe effects on cilia length (Brazelton et al., 2001
; Piperno et al., 1998
; Perkins et al., 1986
; Blacque et al., 2006
). In mutants defective for complex A, flagella show swellings or bulges, accompanied by accumulation of IFT proteins within or at the tips of axonemes (Perkins et al., 1986
, Blacque et al., 2006
). A temperature sensitive allele of Chlamydamonas fla15
, which exhibits significant depletion of complex A IFT proteins yet normal levels of complex B IFT proteins, shows normal rates of anterograde but reduced rates of retrograde transport at the permissive temperature (Piperno et al., 1998
). These data have lead to the hypothesis that complex B IFT proteins are important for anterograde transport, whereas complex A IFT proteins may be dedicated to retrograde IFT (Piperno et al, 1998
, Blacque et al, 2006
Nevertheless, the neural patterning phenotype of the Ift122
mutants is surprising because mutant embryos deficient for Dynch2, a subunit of the retrograde IFT motor, fail to activate Hh target genes, similar to complex B mutants, which is the opposite phenotype to that of Ift122
mutants. Recent data suggest that complex A and B could each participate in several aspects of the transport process. For example, complex A IFT proteins can associate with Kinesin II in a complex B-independent manner (Pederson et al., 2006; Ou et al., 2005
). Thus it is possible that complex B and complex A both have roles in anterograde transport and have different cargo specificities. Complex B may be generally required for anterograde transport of cargo used to assemble the cilium (e.g. tubulin dimers, radial spoke proteins, and other structural components of the cilium), whereas complex A IFT proteins may be important for the transport of machinery used for turnaround at the distal tip or retrograde transport. As a result, loss of IFT122 could lead to constitutive activation of the pathway because of a defect in anterograde transport of a specific factor that antagonizes Gli function within the cilium.
Arl13b (formerly called Arl2l1, the homologue of the zebrafish scorpion
gene; Sun et al., 2004
) is a small GTPase of the Arl/Arf subgroup of the Ras superfamily and plays a unique role of formation of the cilium and in Hh signaling. The Arl13b protein is localized to the shaft of the cilium, and mouse mutants that lack Arl13b have short cilia of abnormal structure, in which the B-tubule of the outer doublet is open (T. Caspary and KVA, submitted). Coupled to this defect in cilia structure, the mutant embryos show a novel defect in neural patterning, in which both the most dorsal and the most ventral cell types are not specified and the domain of motor neurons, which arise at an intermediate position, is greatly expanded. Analysis of double mutants with the core components of the Hh pathway indicate that Gli3 repressor activity is normal in the Arl13b mutant embryos, but Gli activators are constitutively active at low levels. Thus while normal cilia are required for both activator and repressor activities of Gli proteins, those events are separated in the Arl13b cilia, which can regulate Gli repressor but not activator.
The phenotypes of the Ift122
mutants suggest that the cilium is more than a sensory antenna where Hh pathway components are concentrated. Instead, the structure and dynamics of the cilium appear to have a more integral role in Hh signaling. Additional studies will be needed to address how proteins in the cilium interact with and regulate Hh signaling components, and how the spatially regulated expression of genes encoding IFT proteins and other components of the cilium (e.g. Huangfu and Anderson, 2005
) modulates Hh signaling. A number of other vertebrate-specific genes have been identified that affect Hh signaling at the same step as IFT proteins, downstream of Smo and upstream of Gli proteins. These include both positive and negative regulators of the pathway (mouse Rab23, Fkbp8, and Sil, zebrafish Iguana and chicken Talpid3; Eggenschwiler et al., 2001
; Bulgakov et al., 2004
; Izraeli et al., 2001
; Sekimizu et al., 2004
; Wolff et al., 2004
; Davey et al., 2006), and could also affect cilia. Studies on the structure of cilia in these mutants and of the subcellular localization of these proteins will be necessary to see if these proteins also affect Hh signaling through cilia.