Role of Intraflagellar Transport and Primary Cilia ... - Wiley Online Library

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blets around the outer edge of the axoneme but is miss- ing the central two ... *Correspondence to: Rosa Serra, Department of Cell Biology,. University of ...
THE ANATOMICAL RECORD 291:1049–1061 (2008)

Role of Intraflagellar Transport and Primary Cilia in Skeletal Development ROSA SERRA* Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama

ABSTRACT Primary cilia are nonmotile microtubule-based appendages extending from the surface of almost all vertebrate cells. The process of intraflagellar transport (IFT) is responsible for building and maintaining the structure and function of primary cilia. Disruption of Kif3a, a component of the Kinesin-II motor complex, disables anterograde IFT and leads to failure in the formation and maintenance of cilia. Likewise, the absence of IFT88/Tg737/Polaris, a core component of the IFT particle, results in the loss of cilia. Although primary cilia were described on chondrocytes almost 40 years ago, only recently has the functional significance of IFT and cilia in skeletal development been uncovered through the use of mouse models containing mutations or deletions in genes required to make and maintain cilia. Together, the results indicate that primary cilia/ IFT are involved in coordinating multiple signaling pathways within the skeleton. Anat Rec, 291:1049–1061, 2008. Ó 2008 Wiley-Liss, Inc.

Key words: intraflagellar transport; cilia; IFT88; Kif3a; mouse models

WHAT ARE CILIA? Cilia are organelles that project from the surface of most eukaryotic cells (Davenport and Yoder, 2005; Pan et al., 2005; Bisgrove and Yost, 2006; Satir and Christensen, 2007). The cilium is made of a microtubule-based axoneme that is covered by a specialized plasma membrane. It extends from a basal body, a centriole-derived microtubule organizing center, that is localized at the base of the cilia structure. There are two basic types of cilium: the motile cilium and nonmotile or primary cilium. This review will focus on the primary cilium, which when seen in cross-section, has a 910 microtubule pattern. The primary cilium contains nine microtubule doublets around the outer edge of the axoneme but is missing the central two microtubules, necessary to generate motile force, that are seen in most motile cilia with a 912 pattern (Fig. 1). Cilia are generated through the process of intraflagellar transport (IFT; Fig. 1) (Davenport and Yoder, 2005; Pan et al., 2005; Bisgrove and Yost, 2006; Satir and Christensen, 2007). Cilia elongate from the basal body by the addition of new components from the base to the distal tip. Protein synthesis does not occur in cilia so the proteins that make up the structure must be transported up and down the cilia along the microtubules. IFT particles are organized into two complexes. Complex A Ó 2008 WILEY-LISS, INC.

cartilage;

bone;

mediates retrograde transport of cargoes from the tip to the base of the cilia, and complex B mediates anterograde transport of specific cargoes from the base to the tip. Anterograde transport is driven by heteromeric kinesin 2 motors, which are composed of Kif3a and Kif3b motor subunits. Retrograde transport is mediated by a different motor, dynein 1B. Disruption of Kif3a disables anterograde IFT and leads to failure in the formation and maintenance of cilia (Lin et al., 2003). Likewise, the absence of IFT88/Tg737/Polaris, a core component of the complex B IFT particle, results in the loss of cilia (Yoder et al., 2002b; Haycraft et al., 2007). Intensive studies in the past decade have found that defects in the normal structure or function of the pri-

Grant sponsor: NIH; Grant numbers: R01 AR053860 and R01 AR045605. *Correspondence to: Rosa Serra, Department of Cell Biology, University of Alabama at Birmingham, 1918 University Blvd., 660 MCLM, Birmingham, AL 35294-0005. Fax: 205-975-5648. E-mail: [email protected] Received 4 October 2007; Accepted 29 October 2007 DOI 10.1002/ar.20634 Published online in Wiley InterScience (www.interscience.wiley. com).

Fig. 1. Intraflagellar transport (IFT) and cilia structure. A: Diagram of the cross-section of a typical motile (left) or nonmotile (right) cilium. The motile cilium is composed of a 912 microtubule pattern with nine doublet microtubules around the periphery. Two singlet microtubules are in the center. The nonmotile cilium has a 910 pattern as it is missing the two singlet microtubules in the center. Most nonmotile cilia do not have dynein arms connecting the doublet microtubules. The exception to this rule is the 910 cilia in the node of very early

embryos, which are motile. B: Diagram of a typical cilium demonstrating IFT. The microtubules of the ciliary axoneme elongate from the basal body located in the cytoplasm at the base of the cilium. Kinesin motors and IFT complex B proteins mediate anterograde transport of cargoes from the cytoplasm to the tip of the cilium. Dynein and IFT complex A proteins mediated retrograde transport. Pc1/Pc2 complexes are found in the ciliary membrane.

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mary cilia are associated with various congenital human diseases, including polycystic kidney disease (PKD), situs-inversus, and retinal degeneration (Davenport and Yoder, 2005; Pan et al., 2005; Bisgrove and Yost, 2006). Pleiotrophic syndromes such as Senior-Loken syndrome, Jeune syndrome, Kartagener syndrome, and BardetBiedl syndrome are characterized by various combinations of pathological changes in the kidney, retina, and skeleton that are also associated with mutations in ciliary genes (Davenport and Yoder, 2005; Pan et al., 2005; Bisgrove and Yost, 2006). Juene’s asphyxiating thoracic dystrophy is associated with a mutation in IFT80, part of the IFT complex B (Beales et al., 2007). The mutation results in missing or shortened cilia and severe constriction of the ribs. More recently, a novel protein mutated in chondroectodermal dysplasia Ellis-van Creveld syndrome (EVC) was shown to be localized to the base of the cilia, and disruption of this gene in humans and mice results in a variety of skeletal and craniofacial abnormalities as well as alterations in the teeth and nails (Ruiz-Perez et al., 2007). Cilia were first identified on chondrocytes approximately 40 years ago (Scherft and Daems, 1967). Ultrastructural studies confirmed that each chondrocyte has one cilium and that the cilium has the 910 microtubule pattern associated with nonmotile cilia (Scherft and Daems, 1967; Wilsman and Fletcher, 1978). Chondrocyte cilia vary in length from 1 to 4 microns and are only 0.2 microns in width (Scherft and Daems, 1967; Poole et al., 2001). The microtubules of the cilia are enriched with detyrosinated and acetylated tubulin, forming a more stable structure than that generally found in the cytoplasm (Poole et al., 2001). The use of antibodies to these modified forms of tubulin have been used to immunolocalize cilia in a variety of cells, including chondrocytes (Poole et al., 1997, 2001). Preliminary studies using confocal and widefield microscopy suggest that cilia on articular chondrocytes point away from the articular surface while cilia on columnar chondrocytes protrude from the center of the cell between the Golgi and nucleus (McGlashan et al., 2007; Song et al., 2007). Recently developed mathematical models in combination with multiphoton microscopy will help clarify the orientation of cilia in different types of cartilage (Ascenzi et al., 2007).

SIGNALING PATHWAYS IN THE SKELETON THAT POTENTIALLY INVOLVE CILIA Mechanical Signals Cilia are involved in transmitting both chemical and mechanical signals. The role of cilia in transmitting mechanical signals has been most well characterized in the kidney, where it has been shown that the protein products for genes associated with PKD are localized to cilia (Yoder et al., 2002a; Nauli and Zhou, 2004). Autosomal dominant PKD results from mutations in Pkd1 or Pkd2, which encode polycystin 1 (Pc1) and polycystin 2 (Pc2). Pc1 and Pc2 are integral membrane proteins that form a regulatory protein and a Ca12 channel in the primary cilia of renal epithelial cells. Cilia on renal cells are sensitive to fluid shear stress and respond with changes in the concentration of cytosolic Ca12 (Praetorius and Spring, 2001). Direct bending of the cilia with a pipette

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also results in an increase in Ca levels and cells without cilia or cells in which the function of Pc1 or Pc2 has been blocked do not respond to mechanical stimulation (Praetorius et al., 2003; Praetorius and Spring, 2003a,b). Pc1 and Pc2 are not required for the formation or the maintenance of cilia, but their localization to the cilia is key for their function. Pc1 also regulates G-protein signaling and activation of the JAK-STAT pathway (Nauli and Zhou, 2004). Recently, it was shown that in the absence of fluid flow, Pc1 itself is cleaved and its C-terminal fragment translocates to the nucleus where it activates the expression of specific target genes leading to dedifferentiation and growth of cells (Low et al., 2006). The PKD studies indicate that mechanical signals transmitted through the cilia are required for normal renal cell polarity, growth, and differentiation. It has also been suggested that mechanical forces are transmitted through the cartilage extracellular matrix to chondrocytes by bending of the cilium (Jensen et al., 2004). Ultrastructural studies have shown that the chondrocyte cilium projects into the extracellular matrix and is tightly associated with the Golgi apparatus (Poole et al., 1997). A combination of imaging techniques, including electron, confocal, and tomographic microscopy indicate that chondrocyte cilia display various bending patterns in association with the surrounding pericellular matrix (Poole et al., 2001; Jensen et al., 2004). Some of the bending patterns fit with a model of shear stress, whereas others suggested deflection by the matrix. Furthermore, the close anatomical association of the cilium with the microtubule cytoskeleton, Golgi apparatus, and microtubule organizing centers within the cell support a model of direct signaling between the matrix, the cilia, and the inside of the cell. Recently, it was shown that primary cilia can mediate mechanical signals in bone cells (Malone et al., 2007). It was shown that bone cells in culture contain primary cilia that project into the culture medium and bend in response to dynamic fluid flow. Pharmacological and siRNA approaches indicated that cilia were required to mediate the effects of fluid flow in culture on osteogenesis and bone resorption. Unlike in kidney cells, cilia were not required to stimulate Ca12 flux in response to fluid flow.

Hedgehog Signaling Of the chemical or growth factor signals involving cilia, the Hedgehog (Hh) pathway is the most well-characterized (Scholey and Anderson, 2006). The vertebrate Hh proteins are required during many stages of development in multiple organs (Lum and Beachy, 2004; Kalderon, 2005; Huangfu and Anderson, 2006). There are three members of the vertebrate Hh family, including Sonic, Indian, and Desert Hh. Of these, Indian Hh (Ihh) is the most important regulator of endochondral bone formation. Ihh is expressed in cells that are committed to become hypertrophic and acts in a negative feedback loop with parathyroid hormone–related protein (PTHrP) and other factors to sense and regulate the rate of chondrocyte differentiation (Lanske et al., 1996; Vortkamp et al., 1996). Sonic Hh (Shh) is required early in limb development to regulate the pattern of the limb (Riddle et al., 1993).

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Figure 2.

CILIA IN THE SKELETON

Hh signaling is complex (Lum and Beachy, 2004; Kalderon, 2005; Huangfu and Anderson, 2006). Hh proteins bind to an integral membrane protein called Patched 1 (Ptc1) on the cell surface (Fig. 2). This binding causes de-repression of another integral membrane protein, Smoothened (Smo). Activation of Smo results in the activation of glioma transcription factors (Gli1, Gli2), which stimulate the expression of Hh responsive genes. Another factor, Gli3, is special in that, in the absence of Hh, Gli3 is proteolytically processed to a repressor form that blocks Hh-mediated transcription. In the presence of Hh, the formation of the Gli3 repressor is blocked. Gli1 and Ptc1 are also direct transcriptional targets of Hh signaling, and their expression can be used as a marker for cells that are responding to Hh. The first clue that IFT/cilia were involved in Hh signaling came from genetic screens in mice. Mouse mutants with defects in ciliogenesis resulting from mutations in IFT encoding genes demonstrated phenotypes in early neural tube and limb development that were similar to those in mice harboring mutations in Shh (Huangfu et al., 2003; Huangfu and Anderson, 2005). Further genetic analysis suggested that IFT acted down-stream of Smoothed but upstream of Gli in the Hh signaling cascade. Subsequently, it was shown that many components of the Hh signaling pathway, including Gli1, Gli2, and Gli3, are localized to the cilia (Haycraft et al., 2005). Furthermore, Smo translocates to the cilium in the presence of Hh ligand resulting in the activation of Gli2 (Corbit et al., 2005). Cilia are also required for processing Gli3 to the repressor form resulting in generalized misregulation of Hh-responsive genes in the absence of cilia (Haycraft et al., 2005; Liu et al., 2005). IFT mutants can display a loss-of-Shh phenotype in cells in which Gli activators have a major role or a gain-of-Shh function phenotype in cells where the Gli3 repressor plays a major role. It has been suggested that cilia provide a platform where the signaling molecules are enriched and signaling can be easily coordinated (Corbit et al., 2005; Haycraft et al., 2005; Liu et al., 2005).

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(Dsh), acts to inhibit the activity of GSK-3b, which is in a complex with the adenomatous polyposis coli protein (APC) and Axin resulting in stabilization of the b-catenin protein and its subsequent translocation to the nucleus. Nuclear b-catenin associates with the lymphoid enhancer factor/T-cell-specific transcription factor (LEF/TCF) family of transcription factors and activates transcription of Wnt target genes. Among the noncanonical pathways, IFT and cilia have been implicated in the regulation of the planar cell polarity pathway (PCP; Dabdoub and Kelley, 2005; Ross et al., 2005; Park et al., 2006). PCP regulates cell polarity, migration, and orientation of cells during convergent extension movements in development. The PCP pathway results in cytoskeletal changes and cell movement by means of activation of RhoA, and c-Jun N-terminal kinase (JNK; Katoh, 2005). Defects in the formation of cilia have been correlated with alterations in PCP signaling in Xenopus development (Park et al., 2006). The disruption of convergent extension movements in frogs is correlated with the loss of cilia resulting from the reduction of two ciliary proteins, inturned and fuzzy. Furthermore, recent evidence suggests that IFT/cilia function as a molecular switch between canonical and PCP Wnt signaling. A key ciliary protein in this switch is Inversin (Invs; Simons et al., 2005). Overexpression of Invs inhibits canonical Wnt signaling and knockdown results in loss of PCP signaling suggesting Invs promotes PCP. Invs forms a complex with Dsh that targets Dsh for degradation thus inhibiting canonical signaling. In response to a signal from the cilia, for example, fluid flow in the kidney, Invs and Dsh localize to the membrane where they can interact with other PCP signaling molecules, thus, promoting PCP signaling (Simons et al., 2005).

FUNCTIONS OF IFT/CILIA IN SKELETAL DEVELOPMENT Mouse Models

The Wnt family of proteins consists of at least 19 members whose functions contribute to the regulation of a wide range of cellular processes, including proliferation and differentiation (Veeman et al., 2003; Kikuchi et al., 2006). Wnts activate many signaling cascades that can be broadly divided into two general categories (1) the canonical, b-catenin pathway and (2) the noncanonical pathways that do not involve b-catenin. Canonical Wnts transmit their signals by binding to a subset of members within a family of seven-pass-transmembrane-spanning receptors, termed Frizzled receptors. In the absence of Wnt, glycogen synthase kinase-3b (GSK-3b) is active and phosphorylates b-catenin, targeting it for degradation. In the presence of Wnt, the cytoplasmic protein, dishelleved

Several mouse models have been used to uncover the role of IFT and cilia in various aspects of skeletal development (Table 1). Large ENU and insertional-mutagenesis screens have yielded several mutations in IFT related genes including hypomorphic alleles of IFT88/Tg737/ Polaris (flexo (IFT88fxo) and Oak Ridge Polycystic Kidney (IFT88orpk) as well as a strong hypomorphic allele of Pkd1 (Pkd1m1Bei), all of which demonstrate skeletal abnormalities (Yoder et al., 1995; Herron et al., 2002; Huangfu et al., 2003). Likewise, targeted germline disruption of Pkd1 results in alterations in endochondral bone formation (Pkd1D17-21bgeo; Pkd1null; Boulter et al., 2001; Lu et al., 2001). Recently, the EvC gene, coding a novel protein that is localized to the base of the cilia, was disrupted in the germline resulting in skeletal abnormalities reminiscent of those seen in patients with Ellis-van Creveld syndrome (Ruis-Perez et al., 2007).

Fig. 2. Hedgehog (Hh) signaling. A: In the absence of Hh ligand, Ptc represses the activity of Smo. Gli3 is processed to the repressor form and the expression of Hh target genes is repressed. Gli transcription factors are localized to the cilia and processing of Gli3 is dependent on intraflagellar transport (IFT) and cilia. In the absence of IFT and cilia, Gli3 is not processed to the repressor form, resulting in mis-

regulation of Hh target genes. B: Binding of Hh to Ptc results in localization of Smo to the cilia and derepression of Smo activity. Processing of Gli3 to the repressor form is blocked and Gli2 is converted to a transcriptional activator. In the absence of cilia, Smo cannot accumulate in the cilia and Gli activators are not processed, resulting in a block to Shh signaling.

Wnt Signaling

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TABLE 1. Mouse models used to determine the role of IFT/cilia in the skeleton Gene alteration

Strain name

IFT88/ Tg737/ Polaris Hypomorphic allele

IFT88orpk

Null allele

IFT88flexo IFT88D2-3ßgal;IFT88tmRpw

Cre-LoxP conditional

Prx1Cre;IFT88LoxP/LoxP Col2aCre;IFT88LoxP/LoxP

Kif3a Cre-LoxP conditional

Prx1Cre;Kif3aLoxP/LoxP

Zhang et al., 2003 Haycraft et al., 2005 McGlashan et al., 2007 Liu et al., 2005 Zhang et al., 2003 Haycraft et al., 2005 Liu et al., 2005 Haycraft et al., 2007 Song et al., 2007

Dermo1-Cre;Kif3aLoxP/LoxP Wnt1-Cre;Kif3aLoxP/LoxP

Haycraft et al., 2007 Kolpakova-Hart et al., 2007 Song et al., 2007 Koyama et al., 2007 Kolpakova-Hart et al., 2007 Kolpakova-Hart et al., 2007

Pkd1null Pkd1m1Bei Pkd1D17-21ßgeo

Lu et al., 2001 Xiao et al., 2006 Boulter et al., 2001

EvCnull/null

Ruiz-Perez et al., 2007

Col2aCre;Kif3aLoxP/LoxP

Pkd1 Null allele Strong hypomorphic/Null Truncation EvC Null allele

References

Targeted germline disruption of IFT88/Tg737/Polaris (IFT88D2-3bgal; IFT88tmRpw) results in skeletal abnormalities, but the mice die in midgestation making complete characterization of the skeletal phenotypes in these mice impossible (Murcia et al., 2000). More recently, conditional deletion using Cre-LoxP recombination has been used (Sauer, 1998). Cre is the 38-kDa product of the cre (cyclization recombination) gene of bacteriophage P1 and is a site-specific DNA recombinase of the INT family. Cre recognizes a 34-bp site on the P1 genome called loxP (locus of X-over of P1) and efficiently catalyzes reciprocal conservative DNA recombination between pairs of loxP sites. Mice generated with a loxP-flanked gene may be mated to transgenic mice with Cre under the control of a promoter with a desired temporal or tissue specificity. This mating will yield a double transgenic mouse where the loxP-modified gene has been deleted in those tissues in which the Cre transgene has been expressed. LoxP alleles of Kif3a (Kif3aloxP) and IFT88/Tg737/Polaris (IFT88loxP) have been generated (Lin et al., 2003; Haycraft et al., 2007). Cre expressed under the control of the Col2a promoter or the Prx1 promoter has been used to target deletion of IFT and cilia to chondrocytes and limb mesenchyme, respectively (Haycraft et al., 2007; Song et al., 2007). Prx1-Cre is expressed in limb mesenchyme starting at embryonic day (E) 9.5 days of gestation and thus targeted DNA deletion is present in both chondrocytes and perichondrium (Logan et al., 2002). In contrast, Col2a-Cre is expressed later in the limbs, after cells have committed to the chondrocyte lineage and the perichondrium is not efficiently targeted (Ovchinnikov et al., 2000). More recently, mice were described in which Kif3a was conditionally deleted using Wnt1-Cre and Dermo1-cre expressing mice (Kolpakova-Hart et al., 2007). Wnt-1 Cre is expressed in neural crest cells and thus directs disruption of cilia in many of the bones of the head and face (Chai et al., 2000). During skeletal

development, Dermo1 is expressed in condensed mesenchyme before expression of Type II collagen. It is also expressed in perichondrial and periosteal cells surrounding cartilage (Yu et al., 2003). In addition, Dermo-1 is expressed in progenitors of osteoblasts. Mice with targeted deletion of IFT and cilia have a wide variety of skeletal phenotypes that provide interesting insights into the how the cilia function.

Limb Patterning The limb is patterned in three-dimensions. It has proximal–distal, dorsal–ventral, and anterior–posterior polarity (Niswander, 2003; Tickle, 2003). The outgrowth of the limb is coordinated by the apical ectodermal ridge (AER), which is a thick ectoderm overlying the limb mesoderm. Anterior–posterior patterning of the limb is controlled by a region of mesoderm in the posterior end of the bud called the zone of polarizing activity (ZPA). The activity of the ZPA is primarily due to the action of Shh (Riddle et al., 1993). Synthesis of Shh is localized to this area and ectopic Shh placed at the anterior of the bud results in digit duplication and polydactyly. A role for cilia in limb patterning was first indicated in mice harboring the IFT88orpk hypomorphic allele (Zhang et al., 2003). These mice demonstrated several defects in skeletal development, including craniofacial abnormalities, cleft palate, supernumerary teeth, and preaxial duplication of the digits. Dermo1-Cre;Kif3aloxP/loxP mice also demonstrated preaxial polydactyly with duplication of digit 1 (Kolpakova-Hart et al., 2007). Mice with a null mutation in IFT88 die in midgestation but a pronounced expansion of the limb field with as many as eight digits was evident before the mice died (Zhang et al., 2003; Haycraft et al., 2005). Subsequently, it was shown that cilia are present on both the ectoderm and mesoderm components of the limb; however, only cilia in

CILIA IN THE SKELETON

the mesoderm are required for proper limb patterning (Haycraft et al., 2005, 2007). The limbs of mice in which IFT88loxP was deleted using an ectoderm specific Cre (Msx2-Cre) were completely normal while deletion of IFT88loxP in the mesoderm using Prx1-Cre resulted in a phenotype similar to that seen in mice with the null mutation (Fig. 3A,B). That is, extensive polydactyly with up to eight unpatterned digits on each limb. Proximal– distal, and dorsal–ventral patterning appeared normal in these mice suggesting that IFT/cilia are only required to generate the anterior–posterior axis. Because anterior–posterior polarity in the limb is determined by the ZPA and Shh, it was hypothesized that there may be alterations in Shh localization or signaling that would explain the subsequent polydactyly. Localization of Shh mRNA was normal; however, expression of Ptc1 and Gli1, direct targets of Shh activity, were reduced, suggesting that the Shh signaling pathway was repressed in the limb field (Haycraft et al., 2005; Liu et al., 2005). In addition, primary cells lacking IFT88 were unable to respond to exogenously added Shh (Haycraft et al., 2005). However, loss of Shh signaling could not account for the severe polydactyly observed because mice with a null mutation in Shh have only one digit not polydactyly (Chiang et al., 2001). It was previously shown that mutations in Gli3 result in polydactyly (Wang et al., 2000; te Welscher et al., 2002). As mentioned above, in the absence of Hh signaling, Gli3 is processed to a repressor form, suppressing transcription of Hh target genes. It was shown in the limb that IFT/cilia are required for processing of Gli3 to the repressor form so that loss of cilia affects both Shh-related activator and repressor functions (Haycraft et al., 2005; Liu et al., 2005). The expression pattern of two genes, gremlin and hand2, that are normally repressed by Gli3 were expanded in the limbs of IFT/cilia mutants (Haycraft et al., 2007; Liu et al., 2005). Thus, in the case of limb patterning, the loss of the Gli3 repressor and overall misexpression of Shh target genes resulted in polydactyly.

Endochondral Bone Formation and Postnatal Growth Plates Overview of the embryonic and postnatal growth plates. Most of the bones in the body develop through a process called endochondral bone formation, which starts with embryonic mesenchymal cells condensing at the sites where the skeletal elements will form (Cancedda et al., 1995; van der Eerden et al., 2003; Colnot, 2005). The condensed mesenchymal cells then undergo differentiation to become chondrocytes forming the cartilaginous anlagen of the future bone. The chondrocytes proliferate and differentiate toward their terminally differentiated form, the hypertrophic chondrocyte. Hypertrophic chondrocytes do not proliferate and change the profile of their extracellular matrix deposition so that their matrix can be mineralized. The hypertrophic cells secrete factors that attract vasculature to bone anlagen and promote the formation of the bone collar from the adjacent perichondrium. The bone collar represents a type of intramembranous bone formation because the mesenchymal cells of the perichondrium differentiate directly into osteoblasts. Finally, hypertrophic chondrocytes undergo apoptosis, and the matrix is

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replaced with osteoblasts forming trabecular bone. It has been shown that Ihh regulates proliferation of chondrocytes, the rate of hypertrophic differentiation, as well as the formation of the bone collar (St-Jacques et al., 1999). Regulation of proliferation is thought to be a direct effect of Ihh on chondrocytes, while hypertrophic differentiation is mediated though the action of PTHrP (Long et al., 2001). The formation of the bone collar is thought to involve canonical Wnt signaling in addition of that of Ihh (Hu et al., 2005). It has been shown that early osteoblast differentiation requires canonical Wnt signaling, whereas chondrocyte differentiation occurs in the absence of canonical signaling in the presence of noncanonical Wnt signals (Day et al., 2005; Hill et al., 2005). After birth, the growth plate allows for continued longitudinal growth of the bone through continued proliferation, differentiation, and replacement by bone. The cells in the growth plate are aligned into columns representing a continuum of differentiation. The cells are maintained in columns through the process of rotation. Rotation was described more than 70 years ago (Doods, 1930). During cell division, chondrocytes divide perpendicular to the long axis of the bone then undergo a series of cell shape changes and movements that result in the careful positioning of flat cells one on top of the other. Very little is known about this process. It was recently suggested that integrins and signaling through the surrounding matrix are involved (Aszodi et al., 2003).

Cilia and IFT in embryonic endochondral In addition to polydactyly, mice bone formation. with mutations in IFT88 demonstrate shortening of the bones in the limbs due to alterations in endochondral bone formation (Zhang et al., 2003; Haycraft et al., 2007; McGlashan et al., 2007; Song et al., 2007). Mice with conditional deletion of Ift88 or Kif3a in limb mesenchyme (Prx1-Cre;IFT88loxP/loxP or Prx1-Cre;Kif3aloxP/loxP) demonstrated shortening of the bones in the embryonic stages (Fig. 3A,B) (Haycraft et al., 2007). Histological analysis revealed accelerated hypertrophic differentiation as well as a delay in vascularization of the primary ossification center, resulting in the persistence of hypertrophic cells in the growth plate. The phenotype resembled that seen in mice with germline mutations in Ihh (St-Jacques et al., 1999). In support of a role for IFT/cilia in mediating Ihh signaling during embryonic endochondral bone formation, Ptc1 and Gli1 mRNA were dramatically reduced in the long bones of Prx1-Cre;IFT88loxP/loxP and Prx1Cre;Kif3aloxP/loxP mice relative to controls. Expression of Ptc1 and Gli1 was reduced in both chondrocytes and perichondrial cells, suggesting that Ihh signaling was disrupted in both compartments. Nevertheless, PTHrP expression was maintained perhaps due to the loss of Gli3 repressor function. Mice in which Kif3a was deleted using Dermo1-Cre had a similar, although milder, phenotype to the Prx1-Cre mutants that also appeared to be a result of altered Ihh signaling (Kolpakova-Hart et al., 2007). In contrast to the results obtained by the characterization of Prx1-Cre and Dermo1-Cre mutant mice, mice in which IFT88 or Kif3a were deleted using Col2aCre (Col2a-Cre;IFT88loxP/loxP or Col2a-Cre;Kif3aloxP/loxP) did not demonstrate any alterations in embryonic endochondral bone formation, even though the cilia were clearly deleted and Ptc1 expression was dramatically

Fig. 3. Conditional deletion of intraflagellar transport (IFT) and cilia from the skeleton alters embryonic and postnatal skeletal development. A,B: Alizarin red stained limbs from postnatal day (P) 11 day Prx1Cre;IFT88LoxP/LoxP (B) and control (A) mice are shown. Note polydactyly (arrowhead) in the autopod and extreme shortening of the long bones in the limb. Images were provided by Dr. C.J. Haycraft, Medical University of South Carolina. C,D: Alcian blue/alizarin red stained limbs from newborn control (C) and Col2aCre;Kif3aLoxP/LoxP (D) mice. Differences were not detected in the length or area of mineralization in the control and mutant limbs at this stage. E,F: Alizarin red stained limbs from

P30 day control (E) and Col2aCre;Kif3aLoxP/LoxP (F) hind limb joints. The growth plate was still visible in control mice but was absent in the mutants. Also note that the overall shape of the joint and articular surface is altered in the mutants. G,H: Hematoxylin and eosin (H&E)stained sections of the hind limb joint from P30 day control (G) and Col2aCre;Kif3aLoxP/LoxP (H) mice. The growth plate is present in the control (arrowhead) but missing in the mutant. Although aggrecan expression was maintained in the articular cartilage, some alterations in the histology of the mutant tissue were noted.

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reduced in chondrocytes by E15.5 days (Fig. 3C,D; Song et al., 2007). As expected, Ptc1 expression was maintained in the perichondrium because Col2a-Cre does not efficiently target these cells. The differences in the mouse models can be explained by differences in the timing and localization of Cre expression as described above. Prx1Cre and Dermo1-Cre target the perichondrium while Col2a does not. The combined results suggest an important role for perichondrial IFT/cilia in regulating embryonic endochondral bone formation. In support of this model, there is significant evidence indicating the importance of perichondrial Ihh signaling in the regulation of hypertrophic differentiation (Vortkamp et al., 1996; Long et al., 2001; Alvarez et al., 2002; Colnot, 2005). EVC is a novel protein that is mutated in human Ellisvan Creveld syndrome, a chondroectodermal dysplasia characterized by various skeletal abnormalities (RuizPerez et al., 2007). Recently, it was shown that EVC localizes to the base of the cilia but it is not required for the formation or the maintenance of cilia structure (RuizPerez et al., 2007). Evc mRNA is normally expressed in the mouse skeleton and in the developing face (RuizPerez et al., 2007). In the mouse growth plate, Evc is expressed in the perichondrium as well as in resting and proliferating chondrocytes. Expression was not detected in late prehypertophic or hypertrophic cells. Mice with germline deletion of Evc demonstrate chondrodysplasia that is similar to that seen in Ellis-van Creveld patients (Ruiz-Perez et al., 2007). That is, short ribs, short limbs, and dental abnormalities. Shortened limbs in the Evcnull mice were due to accelerated hypertrophic differentiation, alterations in proliferation or early stages of differentiation were not observed. Defects in the growth plate were associated with diminished Ihh signaling as measured by a reduction in the expression of Ptc1 and Gli1; however, processing of Gli3 to the repressor form was normal. In contrast to what is seen when the cilia are deleted all together and both activator and repressor functions of Hh are affected, the results suggest that Evc is specifically involved in Hh-dependent activation of gene expression. Phenotypic differences in Evc-null mice and mice with conditional mutations in Kif3a or Ift88 can in part be accounted for by intact Gli3 function in the Evc mutant mice (Ruiz-Perez et al., 2007).

Postnatal growth plate. Although Col2a-Cre; IFT88loxP/loxP and Col2a-Cre;Kif3aloxP/loxP mice did not demonstrate defects in embryonic endochondral bone formation, the mice demonstrated postnatal dwarfism due to a progressive loss of the growth plate between postnatal day (P) 7 and P15 (Fig. 3E–H; Song et al., 2007). At P7 days, a decrease in the length of the proliferation zone was apparent. This was associated with reduced cell proliferation. By P10 days, the columnar organization of the growth plate was severely altered along with the localization of activated focal adhesion kinase (FAK) and organization of the actin cytoskeleton suggesting a defect in the process of chondrocyte rotation. There was also an increase in the area of histologically hypertrophic cells within the growth plate, suggesting accelerated hypertrophy. The phenotype of Col2a-Cre;IFT88loxP/loxP and Col2aCre;Kif3aloxP/loxP mice had some similarities to mice with conditional deletion of Ihh induced in postnatal cartilage (Col2a-CreER;IhhloxP/loxP; Maeda et al., 2007). Nevertheless, alterations in Ptc1 expression were not detected,

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suggesting the phenotype in Col2a-Cre; Kif3a mice was independent of Ihh signaling. It was proposed that defects in rotation could be the result of alterations in cell adhesion and shape mediated by mechanical stimulation, integrins, and/or noncanonical Wnt PCP signaling, all of which have been linked in some way to IFT/ cilia and/or chondrocyte rotation (Aszodi et al., 2003; Jensen et al., 2004; Park et al., 2006). As described above, it has been suggested that cilia transmit mechanical forces through their interaction with the surrounding ECM (Jensen et al., 2004). It is interesting to note that, although cilia are dramatically reduced in the cartilage of Col2a-Cre;Kif3afl/fl mice as early as E15.5 days, alterations in the organization of the growth plate were not observed until 7 to 10 days after birth. This is the time when the young mice are starting to become subjected to significant mechanical load. In addition, integrins are present on the chondrocyte cilium as well as the cell body (Praetorius et al., 2004; McGlashan et al., 2006). Deletion of chondrocyte b1 integrin results in defects in rotation that include alterations in cell shape and orientation with some similarities to that seen in mice with disrupted cilia (Aszodi et al., 2003). Furthermore, it was recently shown that deletion of Pkhd1, a ciliary protein mutated in autosomal recessive PKD, in cultured renal epithelial cells results in alterations in cell adhesion and activation of FAK, suggesting a link between the primary cilia and integrin mediated cellECM interactions in the kidney (Mai et al., 2005). Finally, many aspects of chondrocyte rotation are similar to the process of convergent extension that occurs during embryonic gastrulation (Keller, 2002; Jones and Chen, 2007). PCP has been shown to regulate convergent extension and recently it was shown that defects in cilia disrupt PCP and convergent extension-like movements in Xenopus Meckel’s cartilage (Park et al., 2006). Mice harboring the IFT88orpk mutation also demonstrate short limbs at P4 (McGlashan et al., 2007). Minor alterations were seen in the shape and organization of growth plate chondrocytes. In contrast to Col2aCre;IFT88loxP/loxP and Col2a-Cre;Kif3aloxP/loxP mice, IFT88orpk mutants demonstrated a delay in hypertrophic differentiation as measured by a reduction in the expression domain of Type X collagen protein with continued expression of Type II collagen. The differences in the IFT88orpk and Col2aCre models may reflect differences between the hypomorphic and null alleles or systemic effects of germline mutation versus conditional deletion. Synchondroses consist of two mirror image growth plates found in the bones at the base of the skull that are important for development and growth of the cranial base. Synchondroses have some similarities and differences to growth plates in the long bones. The synchondroses in Col2a-Cre;Kif3aloxP/loxP mice were recently characterized at postnatal stages and compared with mice with inducible and conditional deletion of Ihh (Col2a-CreER; IhhloxP/loxP; Koyama et al., 2007). Similar to what was seen in the postnatal long bone, the growth plates were disorganized and there was a significant reduction in chondrocyte proliferation most clearly seen by P7. Although there was an increase in histologically hypertrophic cells, the expression of Col10a mRNA was dramatically reduced, indicating a delay in complete hypertrophic differentiation. There was also a substantial reduction in Ptc1 and Gli1 present in the chondrocytes,

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suggesting that Hh signaling was disrupted in the cranial base growth plates. The cranial base in mice deficient in Ihh only minimally resembled the cranial base of mice in which the cilia were disrupted, suggesting that IFT/cilia also have unique roles in synchondroisis development (Koyama et al., 2007).

Perichondrium and bone collar. Col2a-Cre; Kif3aloxP/loxP mice also demonstrated an increase in intramembranous bone collar formation in the perichondrium along the cranial base growth plates resulting in bony bridges across the growth plate visible by microcomputed tomography (Koyama et al., 2007). Ectopic balls of cartilage were also found in some areas of the perichondrium. Similar ectopic cartilages were found in the perichondrium of Prx1-Cre;IFT88loxP/loxP and Dermo1-Cre; Kif3aloxP/loxP long bones (Haycraft et al., 2007; KolpakovaHart et al., 2007). In contrast to the synchondroses, intramembranous bone collar formation was significantly delayed in Prx1-Cre mutant long bones. It has been shown that development of the bone collar is dependent on both Ihh secreted from the early hypertrophic chondrocytes and canonical Wnt signaling in the perichondrium (Hu et al., 2005). The bone collar is missing in Ihhnull mice, but ectopic cartilage is not found (St-Jacques et al., 1999). Conditional disruption of canonical Wnt signaling in the limbs blocks osteoblast differentiation and promotes chondrocyte differentiation resulting in the formation of ectopic cartilage (Day et al., 2005; Hill et al., 2005; Hu et al., 2005). It was proposed that loss of cilia in the perichondrium also alters Wnt signaling resulting in the formation of cartilage instead of bone (Haycraft et al., 2007). This would suggest that loss of cilia blocks canonical Wnt signaling in the perichondrium, but the mechanism is not known. In contrast, excessive bone formation in the Col2a-Cre mutant synchondroses is likely due to increased Hh signaling in the perichondrium. For the most part, the cilia in the perichondrium of the cranial base growth plates were intact (Koyama et al., 2007). There was an increase in the length of the expression domain for Ptc1 and Gli1 in the perichondrium along the growth plate, suggesting ectopic Hh signaling in these cells. Because Ihh has been shown to promote the development of bone collar in perichondrium, it is likely that the extension of the bone collar is a direct result of this ectopic Ihh signaling. It was proposed that reduced levels of Ptc1 and syndecan-3 in chondrocytes, which normally bind to Ihh and restrict its range of action, resulted in a broader domain of Ihh activity leading to excessive bone formation (Koyama et al., 2007). Pkd1. Mutations in Pkd1 are associated with autosomal dominant PKD and mice with mutations in Pkd1 demonstrate defects in embryonic endochondral bone formation as well as osteopenia in adults (Boulter et al., 2001; Lu et al., 2001; Xiao et al., 2006). Pkd1 mRNA is expressed in early condensing mesenchyme and later in prechondrogenic tissue (Guillaume et al., 1999). As skeletal development progresses, it becomes enriched in the perichondrium and intervertebral disc. Recently, expression was shown in osteoblast and osteocyte cell lines as well as in cultures of primary cells (Xiao et al., 2006). Pkd1 mutant mice demonstrate shortened long bones as well as defects in the formation of the vertebrae (spina bifida occulta; Lu et al., 2001). A delay in hypertrophic

differentiation, vascularization, and skeletal mineralization were also noted (Boulter et al., 2001; Lu et al., 2001; Xiao et al., 2006). The bone collar was narrow in embryonic mice, and there was a reduction in the expression of a master regulator of osteoblast differentiation, Runx2, as well as a reduction in a range of markers of later osteoblast differentiation (Xiao et al., 2006). Adult mice demonstrated osteopenia, and immortalized Pkd1 mutant osteoblasts demonstrated defects in differentiation in culture (Xiao et al., 2006). It is known that mechanical stress plays an important role in bone formation and adaptation. Cilia on osteoblasts/osteocytes were proposed as candidates for the elusive mechanosensor in bone that may sense fluid flow within the canuliculi similar to the way renal epithelial cells sense fluid in the renal ducts (Xiao et al., 2006). In support of the hypothesis, it was recently shown that cilia on bone cells in culture mediate expression of osteopontin and other genes related to osteoblast function in response to dynamic fluid flow (Malone et al., 2007). Nevertheless, it is also likely that cilia regulate the transmission of growth factor signals in addition to mechanical signals in the bone.

Articular Cartilage IFT88orpk mice demonstrated alterations in the shape of cells in the superficial layers of the articular cartilage (McGlashan et al., 2007). In addition, reduced toluidine blue staining and reduced Type II collagen protein were evident. Although the general shape of the joint was altered in Col2a-Cre;Kif3aloxP/loxP mice (Fig. 3E–G), expression of aggrecan was maintained up to P30 (Song et al., 2007). Knee joint surfaces of Dermo-1Cre; Kif3aloxP/loxP mice were severely misshapen due to early patterning defects in the articular cartilage (KolpakovaHart et al., 2007). Severe systemic defects in the IFT88orpk mouse and perinatal lethality in Dermo-1Cre;Kif3aloxP/loxP mice preclude comprehensive studies on osteoarthritis. Formation or progression of osteoarthritis in the Col2aCre;Kif3aloxP/loxP line has not been addressed, although based on the potential role of cilia in sensing mechanical load, this should be an important area for future study.

Craniofacial Development IFT88orpk mice demonstrate craniofacial abnormalities including cleft palate and supernumerary teeth (Zhang et al., 2003). Mice with conditional deletion of Kif3a in Wnt1 expressing neural crest cells demonstrate a more severe craniofacial phenotype, including severe frontonasal dysplasia and shortening of the lower jaw as well as profound cleft secondary palate (Kolpakova-Hart et al., 2007). Patterning defects in the midline of the face, including midfacial clefting, were also observed. In addition, Kif3a-deficient animals were missing the tongue and incisors. The skeletal alterations observed suggested that ablation of Kif3a in cranial neural crest cells had an effect on both endochondral and intramembranous bones of the head and face. As expected, structures derived from cephalic mesoderm were not affected. The phenotype of the Wnt1-Cre;Kif3aloxP/loxP mice had some similarities to those seen in mice with conditional deletion of Smo in Wnt1 expressing cells, suggesting that Hh signaling was altered in the absence of Kif3a (Jeong et al., 2004). In control mice, Gli1 was expressed

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in both the epithelial and mesenchymal components of the midface region at E12.5 days (Kolpakova-Hart et al., 2007). However, in Wnt1-Cre mutant mice, Gli1 mRNA was limited to the epithelial structures and was absent from the mesenchyme, suggesting that Hh signaling was disrupted in the neural crest derived mesenchyme leading to at least a subset of the defects observed. In contrast, midfacial clefting, like that seen in the Wnt1-Cre; Kif3a mutants, was not observed in Smo mutants. It was suggested that defects in midline patterning were due to disruptions in the Hh-independent repressor function of Gli3, although alterations in other signaling pathways could not be excluded (Kolpakova-Hart et al., 2007).

SUMMARY AND CONCLUSIONS In summary, mice with mutations in IFT and cilia related genes have a wide variety of skeletal phenotypes that provide interesting insights into how the cilia function. Many lines of evidence indicate that IFT/cilia are essential for Ihh signaling during limb patterning and endochondral bone development; however, alterations in Ihh signaling cannot account for all of the defects observed in IFT/cilia mutant mice. Alterations in the bone collar in cilia deleted limbs suggest alterations in Wnt signaling. Localization of integrins to cilia and defects in chondrocyte rotation implicate signaling through integrins or perhaps alterations in noncanonical PCP signaling. Furthermore, based on the known actions of primary cilia in the kidney, recent imaging studies on chondrocyte cilia and, the reported alterations in the postnatal growth plate of cilia deleted mice as well as osteopenia in Pkd1 mutant mice, it is reasonable to propose a role for primary cilia in mechanotransduction in cartilage and bone. It is likely that cilia play multiple roles in the skeleton, and future experiments will surely sort out the mechanisms of cilia action in cartilage and bone.

ACKNOWLEDGMENT I thank Dr. C.J. Haycraft for thoughtful discussion and providing some of the images used in this review.

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