Jeremy F. Reiter

Vertebrate Smoothened functions at the primary cilium

The unanticipated involvement of several intraflagellar transport proteins in the mammalian Hedgehog (Hh) pathway has hinted at a functional connection between cilia and Hh signal transduction. Here we show that mammalian Smoothened (Smo), a seven-transmembrane protein essential for Hh signalling, is expressed on the primary cilium. This ciliary expression is regulated by Hh pathway activity; Sonic hedgehog or activating mutations in Smo promote ciliary localization, whereas the Smo antagonist cyclopamine inhibits ciliary localization. The translocation of Smo to primary cilia depends upon a conserved hydrophobic and basic residue sequence homologous to a domain previously shown to be required for the ciliary localization of seven-transmembrane proteins in Caenorhabditis elegans. Mutation of this domain not only prevents ciliary localization but also eliminates Smo activity both in cultured cells and in zebrafish embryos. Thus, Hh-dependent translocation to cilia is essential for Smo activity, suggesting that Smo acts at the primary cilium.

Kif3a constrains β-catenin-dependent Wnt signalling through dual ciliary and non-ciliary mechanisms

Primary cilia are microtubule-based organelles involved in signal transduction and project from the surface of most vertebrate cells. Proteins that can localize to the cilium, for example, Inversin and Bardet-Biedl syndrome (BBS) proteins, are implicated in both β-catenin-dependent and -independent Wnt signalling. Given that Inversin and BBS proteins are found both at the cilium and elsewhere in the cell, the role of the cilium itself in Wnt signalling is not clear. Using three separate mutations that disrupt ciliogenesis (affecting Kif3a, Ift88 and Ofd1), we show in this study that the primary cilium restricts the activity of the canonical Wnt pathway in mouse embryos, primary fibroblasts, and embryonic stem cells. Interestingly, unciliated cells activate transcription only in response to Wnt stimulation, but do so much more robustly than ciliated cells. Loss of Kif3a, but not other ciliogenic genes, causes constitutive phosphorylation of Dishevelled (Dvl). Blocking the activity of casein kinase I (CKI) reverses this constitutive Dvl phosphorylation and abrogates pathway hyper-responsiveness. These results suggest that Kif3a restrains canonical Wnt signalling both by restricting the CKI-dependent phosphorylation of Dvl and through a separate ciliary mechanism. More generally, these findings reveal that, in contrast to its role in promoting Hedgehog (Hh) signalling, the cilium restrains canonical Wnt signalling.

Primary cilia can both mediate and suppress Hedgehog pathway–dependent tumorigenesis

Primary cilia are present on most mammalian cells and are implicated in transducing Hedgehog (Hh) signals during development; however, the prevalence of cilia on human tumors remains unclear, and the role of cilia in cancer has not been examined. Here we show that human basal cell carcinomas (BCCs) are frequently ciliated, and we test the role of cilia in BCC by conditionally deleting Kif3a (encoding kinesin family member 3A) or Ift88 (encoding intraflagellar transport protein 88), genes required for ciliogenesis, in two Hh pathway–dependent mouse tumor models. Ciliary ablation strongly inhibited BCC-like tumors induced by an activated form of Smoothened. In contrast, removal of cilia accelerated tumors induced by activated Gli2, a transcriptional effector of Hh signaling. These seemingly paradoxical effects are consistent with a dual role for cilia in mediating both the activation and the repression of the Hh signaling pathway. Our findings demonstrate that cilia function as unique signaling organelles that can either mediate or suppress tumorigenesis depending on the nature of the oncogenic initiating event.

The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization

Both the basal body and the microtubule‐based axoneme it nucleates have evolutionarily conserved subdomains crucial for cilium biogenesis, function and maintenance. Here, we focus on two conspicuous but underappreciated regions of these structures that make membrane connections. One is the basal body distal end, which includes transition fibres of largely undefined composition that link to the base of the ciliary membrane. Transition fibres seem to serve as docking sites for intraflagellar transport particles, which move proteins within the ciliary compartment and are required for cilium biogenesis and sustained function. The other is the proximal‐most region of the axoneme, termed the transition zone, which is characterized by Y‐shaped linkers that span from the axoneme to the ciliary necklace on the membrane surface. The transition zone comprises a growing number of ciliopathy proteins that function as modular components of a ciliary gate. This gate, which forms early during ciliogenesis, might function in part by regulating intraflagellar transport. Together with a recently described septin ring diffusion barrier at the ciliary base, the transition fibres and transition zone deserve attention for their varied roles in forming functional ciliary compartments.

THE PRIMARY CILIUM: AT THE CROSSROADS OF MAMMALIAN HEDGEHOG SIGNALING

Cilia function as critical sensors of extracellular information, and ciliary dysfunction underlies diverse human disorders including situs inversus, polycystic kidney disease, retinal degeneration, and Bardet-Biedl syndrome. Importantly, mammalian primary cilia have recently been shown to mediate transduction of Hedgehog (Hh) signals, which are involved in a variety of developmental processes. Mutations in several ciliary components disrupt the patterning of the neural tube and limb bud, tissues that rely on precisely coordinated gradients of Hh signal transduction. Numerous components of the Hh pathway, including Patched, Smoothened, and the Gli transcription factors, are present within primary cilia, indicating that key steps of Hh signaling may occur within the cilium. Because dysregulated Hh signaling promotes the development of a variety of human tumors, cilia may also have roles in cancer. Together, these findings have shed light on one mechanism by which primary cilia transduce signals critical for both development and disease.

Scoring a backstage pass: Mechanisms of ciliogenesis and ciliary access

Cilia are conserved, microtubule-based cell surface projections that emanate from basal bodies, membrane-docked centrioles. The beating of motile cilia and flagella enables cells to swim and epithelia to displace fluids. In contrast, most primary cilia do not beat but instead detect environmental or intercellular stimuli. Inborn defects in both kinds of cilia cause human ciliopathies, diseases with diverse manifestations such as heterotaxia and kidney cysts. These diseases are caused by defects in ciliogenesis or ciliary function. The signaling functions of cilia require regulation of ciliary composition, which depends on the control of protein traffic into and out of cilia.

Ofd1, a Human Disease Gene, Regulates the Length and Distal Structure of Centrioles

Centrosomes and their component centrioles represent the principal microtubule organizing centers of animal cells. Here, we show that the gene underlying orofaciodigital syndrome 1, Ofd1, is a component of the distal centriole that controls centriole length. In the absence of Ofd1, distal regions of centrioles, but not procentrioles, elongate abnormally. These long centrioles are structurally similar to normal centrioles but contain destabilized microtubules with abnormal posttranslational modifications. Ofd1 is also important for centriole distal appendage formation and centriolar recruitment of the intraflagellar transport protein Ift88. To model OFD1 syndrome in embryonic stem cells, we replaced the Ofd1 gene with missense alleles from human OFD1 patients. Distinct disease-associated mutations cause different degrees of excessive or decreased centriole elongation, all of which are associated with diminished ciliogenesis. Our results indicate that Ofd1 acts at the distal centriole to build distal appendages, recruit Ift88, and stabilize centriolar microtubules at a defined length.

Polycomb-like 2 Associates with PRC2 and Regulates Transcriptional Networks during Mouse Embryonic Stem Cell Self-Renewal and Differentiation

Polycomb group (PcG) proteins are conserved epigenetic transcriptional repressors that control numerous developmental gene expression programs and have recently been implicated in modulating embryonic stem cell (ESC) fate. We identified the PcG protein PCL2 (polycomb-like 2) in a genome-wide screen for regulators of self-renewal and pluripotency and predicted that it would play an important role in mouse ESC-fate determination. Using multiple biochemical strategies, we provide evidence that PCL2 is a Polycomb Repressive Complex 2 (PRC2)-associated protein in mouse ESCs. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics, defects in differentiation, and altered patterns of histone methylation. Integration of global gene expression and promoter occupancy analyses allowed us to identify PCL2 and PRC2 transcriptional targets and draft regulatory networks. We describe the role of PCL2 in both modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation.

Wounding mobilizes hair follicle stem cells to form tumors.

A wide variety of human cancers are associated with injury. Although stem cells participate in tissue regeneration after wounding, it is unclear whether these cells also contribute to epithelial tumors. Human basal cell carcinomas (BCCs) are associated with misactivation of Hedgehog (Hh) signaling, commonly through acquisition of mutations in Smoothened (Smo). We have found that expression of an activated form of Smo by stem cells of the hair-follicle bulge and secondary hair germ does not induce robust Hh signaling or produce BCCs. However, wounding recruits these cells from the follicle to the wound site, where downstream Hh signal transduction is derepressed, giving rise to superficial BCC-like tumors. These findings demonstrate that BCC-like tumors can originate from follicular stem cells and provide an explanation for the association between wounding and tumorigenesis.

Disruption of a ciliary B9 protein complex causes Meckel syndrome.

Nearly every ciliated organism possesses three B9 domain-containing proteins: MKS1, B9D1, and B9D2. Mutations in human MKS1 cause Meckel syndrome (MKS), a severe ciliopathy characterized by occipital encephalocele, liver ductal plate malformations, polydactyly, and kidney cysts. Mouse mutations in either Mks1 or B9d2 compromise ciliogenesis and result in phenotypes similar to those of MKS. Given the importance of these two B9 proteins to ciliogenesis, we examined the role of the third B9 protein, B9d1. Mice lacking B9d1 displayed polydactyly, kidney cysts, ductal plate malformations, and abnormal patterning of the neural tube, concomitant with compromised ciliogenesis, ciliary protein localization, and Hedgehog (Hh) signal transduction. These data prompted us to screen MKS patients for mutations in B9D1 and B9D2. We identified a homozygous c.301A>C (p.Ser101Arg) B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. Unlike wild-type B9D2 mRNA, the p.Ser101Arg mutation failed to rescue zebrafish phenotypes induced by the suppression of b9d2. With coimmunoprecipitation and mass spectrometric analyses, we found that Mks1, B9d1, and B9d2 interact physically, but that the p.Ser101Arg mutation abrogates the ability of B9d2 to interact with Mks1, further suggesting that the mutation compromises B9d2 function. Our data indicate that B9d1 is required for normal Hh signaling, ciliogenesis, and ciliary protein localization and that B9d1 and B9d2 are essential components of a B9 protein complex, disruption of which causes MKS.