DEVELOPMENT 307 RESEARCH ARTICLE INTRODUCTION Cilia are microtubule-based organelles that are expressed on the surface of most cells in the mammalian body. Intraflagellar transport (IFT), the process by which cilia are formed and maintained, was first described in Chlamydomonas, and proteins required for IFT concentrate at the base of cilia, where they assemble into large protein complexes called IFT particles (Kozminski et al., 1995; Piperno and Mead, 1997). The IFT particles are trafficked along the axoneme by a heterotrimeric kinesin-II and a cytoplasmic dynein in the anterograde and retrograde directions, respectively. Cilia and flagella have diverse functions ranging from fluid and cell movement to mechanosensation and sensory perception (Davenport and Yoder, 2005; Scholey, 2003). In mammals, Kif3a is a component of the kinesin-II motor protein complex required for cilia assembly while Ift88 (also known as Tg737 or polaris) is a core component of the IFT particle (Cole et al., 1998; Pazour et al., 2000; Taulman et al., 2001). Mice homozygous for mutations in Kif3a or any of the IFT proteins identified to date, including Ift88, die during mid-gestation and have randomization of the left-right body axis, neural tube closure and patterning defects, as well as polydactyly (Marszalek et al., 1999; Murcia et al., 2000; Nonaka et al., 1998; Takeda et al., 1999). The severe phenotype of homozygous mutants and the expression of cilia on most cells throughout the body have complicated research directed at understanding the function of cilia on specific cell types or during distinct stages of development, as well as their role in normal tissue function in postnatal life. Recent work from several groups has shown that disruption of IFT results in abnormal patterning of the developing murine limb and neural tube and that this is due to impaired sonic hedgehog (Shh) signal transduction (Haycraft et al., 2005; Huangfu and Anderson, 2005; Huangfu et al., 2003; Liu et al., 2005). Hedgehog signal transduction is both positively and negatively regulated, and disruption of this pathway leads to severe developmental defects (Huangfu and Anderson, 2006; Ingham and McMahon, 2001). In the absence of ligand, the pathway is repressed through the inhibition of the signal transducer smoothened (Smo) by the hedgehog receptor patched (Ptch1). The Glioma family of transcription factors, Gli1, Gli2 and Gli3, are the main transducers of signaling. In the absence of ligand, Gli3 is proteolytically processed to generate a potent transcriptional repressor (Gli3R) of the pathway (Ding et al., 1999; Dunaeva et al., 2003; Stone et al., 1999; Wang et al., 2000). Whereas the major role of Gli3 appears to be repression of target gene transcription in the absence of ligand, Gli2 is predicted to act as the main transcriptional activator upon pathway induction (Bai and Joyner, 2001; Litingtung and Chiang, 2000; Persson et al., 2002; Ruiz i Altaba, 1999). Unlike Gli2 and Gli3, which are regulated post-translationally, Gli1 is predicted to act only as a transcriptional activator after pathway activation (Park et al., 2000). Normal IFT function is required in the Shh signaling pathway, as Smo, the Gli transcription factors and Sufu have all been localized to the cilium axoneme (Corbit et al., 2005; Haycraft et al., 2005; May et al., 2005). While Sufu and the Gli proteins are found at the distal tip of cilia, Smo translocation to the cilium axoneme is induced in response to pathway activation (Corbit et al., 2005; Haycraft et al., 2005). In mice with congenital loss of Kif3a or Ift proteins required for anterograde trafficking, such as Ift88, the Shh signaling pathway remains inactive, despite the fact that the processing of Gli3 to the repressor form is severely impaired Intraflagellar transport is essential for endochondral bone formation Courtney J. Haycraft 1, *, Qihong Zhang 1, * ,† , Buer Song 2 , Walker S. Jackson 3,‡ , Peter J. Detloff 3 , Rosa Serra 1 and Bradley K. Yoder 1,§ While cilia are present on most cells in the mammalian body, their functional importance has only recently been discovered. Cilia formation requires intraflagellar transport (IFT), and mutations disrupting the IFT process result in loss of cilia and mid-gestation lethality with developmental defects that include polydactyly and abnormal neural tube patterning. The early lethality in IFT mutants has hindered research efforts to study the role of this organelle at later developmental stages. Thus, to investigate the role of cilia during limb development, we generated a conditional allele of the IFT protein Ift88 (polaris). Using the Cre-lox system, we disrupted cilia on different cell populations within the developing limb. While deleting cilia in regions of the limb ectoderm had no overt effect on patterning, disruption in the mesenchyme resulted in extensive polydactyly with loss of anteroposterior digit patterning and shortening of the proximodistal axis. The digit patterning abnormalities were associated with aberrant Shh pathway activity, whereas defects in limb outgrowth were due in part to disruption of Ihh signaling during endochondral bone formation. In addition, the limbs of mesenchymal cilia mutants have ectopic domains of cells that resemble chondrocytes derived from the perichondrium, which is not typical of Indian hedgehog mutants. Overall these data provide evidence that IFT is essential for normal formation of the appendicular skeleton through disruption of multiple signaling pathways. KEY WORDS: Cilia, Limb patterning, Hedgehog, Bone development, IFT, Mouse Development 134, 307-316 (2007) doi:10.1242/dev.02732 1 Department of Cell Biology, 2 Department of Pathology and 3 Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA. *These authors contributed equally to this work † Present address: Department of Pediatrics, University of Iowa, Iowa City, IA, USA ‡ Present address: Whitehead Institute for Biomedical Research, Cambridge, MA, USA § Author for correspondence (e-mail: Byoder@uab.edu) Accepted 7 November 2006