Ciliary proteins Bbs8 and Ift20 promote planar cell polarity in the cochlea.

Primary cilia have been implicated in the generation of planar cell polarity (PCP). However, variations in the severity of polarity defects in different cilia mutants, coupled with recent demonstrations of non-cilia-related actions of some cilia genes, make it difficult to determine the basis of these polarity defects. To address this issue, we evaluated PCP defects in cochlea from a selection of mice with mutations in cilia-related genes. Results indicated notable PCP defects, including mis-oriented hair cell stereociliary bundles, in Bbs8 and Ift20 single mutants that are more severe than in other cilia gene knockouts. In addition, deletion of either Bbs8 or Ift20 results in disruptions in asymmetric accumulation of the core PCP molecule Vangl2 in cochlear cells, suggesting a role for Bbs8 and/or Ift20, possibly upstream of core PCP asymmetry. Consistent with this, co-immunoprecipitation experiments indicate direct interactions of Bbs8 and Ift20 with Vangl2. We observed localization of Bbs and Ift proteins to filamentous actin as well as microtubules. This could implicate these molecules in selective trafficking of membrane proteins upstream of cytoskeletal reorganization, and identifies new roles for cilia-related proteins in cochlear PCP.

CEP290 alleles in mice disrupt tissue-specific cilia biogenesis and recapitulate features of syndromic ciliopathies.

Distinct mutations in the centrosomal-cilia protein CEP290 lead to diverse clinical findings in syndromic ciliopathies. We show that CEP290 localizes to the transition zone in ciliated cells, precisely to the region of Y-linkers between central microtubules and plasma membrane. To create models of CEP290-associated ciliopathy syndromes, we generated Cep290(ko/ko) and Cep290(gt/gt) mice that produce no or a truncated CEP290 protein, respectively. Cep290(ko/ko) mice exhibit early vision loss and die from hydrocephalus. Retinal photoreceptors in Cep290(ko/ko) mice lack connecting cilia, and ciliated ventricular ependyma fails to mature. The minority of Cep290(ko/ko) mice that escape hydrocephalus demonstrate progressive kidney pathology. Cep290(gt/gt) mice die at mid-gestation, and the occasional Cep290(gt/gt) mouse that survives shows hydrocephalus and severely cystic kidneys. Partial loss of CEP290-interacting ciliopathy protein MKKS mitigates lethality and renal pathology in Cep290(gt/gt) mice. Our studies demonstrate domain-specific functions of CEP290 and provide novel therapeutic paradigms for ciliopathies.

Loss of Bardet-Biedl syndrome protein-8 (BBS8) perturbs olfactory function, protein localization, and axon targeting.

Bardet-Biedl syndrome (BBS) is a pleiotropic, heterogeneous human disease whose etiology lies primarily in dysfunctional basal bodies and/or cilia. Both BBS patients and several BBS mouse models exhibit impaired olfactory function. To explore the nature of olfactory defects in BBS, a genetic ablation of the mouse Bbs8 gene that incorporates a fluorescent reporter protein was created. The endogenous BBS8 protein and reporter are particularly abundant in olfactory sensory neurons (OSNs), and specific BBS8 antibodies reveal staining in the dendritic knob in a shell-like structure that surrounds the basal bodies. Bbs8-null mice have reduced olfactory responses to a number of odorants, and immunohistochemical analyses reveal a near-complete loss of cilia from OSNs and mislocalization of proteins normally enriched in cilia. To visualize altered protein localization in OSNs, we generated a SLP3(eGFP) knock-in mouse and imaged the apical epithelium, including dendritic knobs and proximal cilia, in ex vivo tissue preparations. Additionally, protein reagents that reflect the characteristic neuronal activity of each OSN revealed altered activity in Bbs8-null cells. In addition to previously known defects at the ciliary border, we also observed aberrant targeting of OSN axons to the olfactory bulb; axons expressing the same receptor display reduced fasciculation and project to multiple targets in the olfactory bulb. We suggest that loss of BBS8 leads to a dramatic and variable reduction in cilia, the essential signaling platform for olfaction, which alters the uniformity of responses in populations of OSNs expressing the same receptor, thereby contributing to the observed axon-targeting defects.

Deletion of Cd44 Inhibits Metastasis Formation of Liver Cancer in Nf2-Mutant Mice.

Primary liver cancer is the third leading cause of cancer-related death worldwide. An increasing body of evidence suggests that the Hippo tumor suppressor pathway plays a critical role in restricting cell proliferation and determining cell fate during physiological and pathological processes in the liver. Merlin (Moesin-Ezrin-Radixin-like protein) encoded by the NF2 (neurofibromatosis type 2) gene is an upstream regulator of the Hippo signaling pathway. Targeting of Merlin to the plasma membrane seems to be crucial for its major tumor-suppressive functions; this is facilitated by interactions with membrane-associated proteins, including CD44 (cluster of differentiation 44). Mutations within the CD44-binding domain of Merlin have been reported in many human cancers. This study evaluated the relative contribution of CD44- and Merlin-dependent processes to the development and progression of liver tumors. To this end, mice with a liver-specific deletion of the Nf2 gene were crossed with Cd44-knockout mice and subjected to extensive histological, biochemical and molecular analyses. In addition, cells were isolated from mutant livers and analyzed by in vitro assays. Deletion of Nf2 in the liver led to substantial liver enlargement and generation of hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (iCCAs), as well as mixed hepatocellular cholangiocarcinomas. Whilst deletion of Cd44 had no influence on liver size or primary liver tumor development, it significantly inhibited metastasis formation in Nf2-mutant mice. CD44 upregulates expression of integrin ß2 and promotes transendothelial migration of liver cancer cells, which may facilitate metastatic spreading. Overall, our results suggest that CD44 may be a promising target for intervening with metastatic spreading of liver cancer.

Anti-Inflammatory and Antioxidative Phytogenic Substances against Secret Killers in Poultry: Current Status and Prospects.

Chronic stress is recognized as a secret killer in poultry. It is associated with systemic inflammation due to cytokine release, dysbiosis, and the so-called leaky gut syndrome, which mainly results from oxidative stress reactions that damage the barrier function of the cells lining the gut wall. Poultry, especially the genetically selected broiler breeds, frequently suffer from these chronic stress symptoms when exposed to multiple stressors in their growing environments. Since oxidative stress reactions and inflammatory damages are multi-stage and long-term processes, overshooting immune reactions and their down-stream effects also negatively affect the animal's microbiota, and finally impair its performance and commercial value. Means to counteract oxidative stress in poultry and other animals are, therefore, highly welcome. Many phytogenic substances, including flavonoids and phenolic compounds, are known to exert anti-inflammatory and antioxidant effects. In this review, firstly, the main stressors in poultry, such as heat stress, mycotoxins, dysbiosis and diets that contain oxidized lipids that trigger oxidative stress and inflammation, are discussed, along with the key transcription factors involved in the related signal transduction pathways. Secondly, the most promising phytogenic substances and their current applications to ameliorate oxidative stress and inflammation in poultry are highlighted.

Bbs8, together with the planar cell polarity protein Vangl2, is required to establish left-right asymmetry in zebrafish.

Laterality defects such as situs inversus are not uncommonly encountered in humans, either in isolation or as part of another syndrome, but can have devastating developmental consequences. The events that break symmetry during early embryogenesis are highly conserved amongst vertebrates and involve the establishment of unidirectional flow by cilia within an organising centre such as the node in mammals or Kupffer's vesicle (KV) in teleosts. Disruption of this flow can lead to the failure to successfully establish left-right asymmetry. The correct apical-posterior cellular position of each node/KV cilium is critical for its optimal radial movement which serves to sweep fluid (and morphogens) in the same direction as its neighbours. Planar cell polarity (PCP) is an important conserved process that governs ciliary position and posterior tilt; however the underlying mechanism by which this occurs remains unclear. Here we show that Bbs8, a ciliary/basal body protein important for intraciliary/flagellar transport and the core PCP protein Vangl2 interact and are required for establishment and maintenance of left-right asymmetry during early embryogenesis in zebrafish. We discovered that loss of bbs8 and vangl2 results in laterality defects due to cilia disruption at the KV. We showed that perturbation of cell polarity following abrogation of vangl2 causes nuclear mislocalisation, implying defective centrosome/basal body migration and apical docking. Moreover, upon loss of bbs8 and vangl2, we observed defective actin organisation. These data suggest that bbs8 and vangl2 act synergistically on cell polarization to establish and maintain the appropriate length and number of cilia in the KV and thereby facilitate correct LR asymmetry.

Bardet-Biedl syndrome proteins modulate the release of bioactive extracellular vesicles.

Primary cilia are microtubule based sensory organelles important for receiving and processing cellular signals. Recent studies have shown that cilia also release extracellular vesicles (EVs). Because EVs have been shown to exert various physiological functions, these findings have the potential to alter our understanding of how primary cilia regulate specific signalling pathways. So far the focus has been on lgEVs budding directly from the ciliary membrane. An association between cilia and MVB-derived smEVs has not yet been described. We show that ciliary mutant mammalian cells demonstrate increased secretion of small EVs (smEVs) and a change in EV composition. Characterisation of smEV cargo identified signalling molecules that are differentially loaded upon ciliary dysfunction. Furthermore, we show that these smEVs are biologically active and modulate the WNT response in recipient cells. These results provide us with insights into smEV-dependent ciliary signalling mechanisms which might underly ciliopathy disease pathogenesis.

Non-essential role for cilia in coordinating precise alignment of lens fibres.

The primary cilium, a microtubule-based organelle found in most cells, is a centre for mechano-sensing fluid movement and cellular signalling, notably through the Hedgehog pathway. We recently found that each lens fibre cell has an apically situated primary cilium that is polarised to the side of the cell facing the anterior pole of the lens. The direction of polarity is similar in neighbouring cells so that in the global view, lens fibres exhibit planar cell polarity (PCP) along the equatorial-anterior polar axis. Ciliogenesis has been associated with the establishment of PCP, although the exact relationship between PCP and the role of cilia is still controversial. To test the hypothesis that the primary cilia have a role in coordinating the precise alignment/orientation of the fibre cells, IFT88, a key component of the intraflagellar transport (IFT) complex, was removed specifically from the lens at different developmental stages using several lens-specific Cre-expressing mouse lines (MLR10- and LR-Cre). Irrespective of which Cre-line was adopted, both demonstrated that in IFT88-depleted cells, the ciliary axoneme was absent or substantially shortened, confirming the disruption of primary cilia formation. However no obvious histological defects were detected even when IFT88 was removed from the lens placode as early as E9.5. Specifically, the lens fibres aligned/oriented towards the poles to form the characteristic Y-shaped sutures as normal. Consistent with this, in primary lens epithelial explants prepared from these conditional knockout mouse lenses, the basal bodies still showed polarised localisation at the apical surface of elongating cells upon FGF-induced fibre differentiation. We further investigated the lens phenotype in knockouts of Bardet-Biedl Syndrome (BBS) proteins 4 and 8, the components of the BBSome complex which modulate ciliary function. In these BBS4 and 8 knockout lenses, again we found the pattern of the anterior sutures formed by the apical tips of elongating/migrating fibres were comparable to the control lenses. Taken together, these results indicate that primary cilia do not play an essential role in the precise cellular alignment/orientation of fibre cells. Thus, it appears that in the lens cilia are not required to establish PCP.

Loss of MACF1 Abolishes Ciliogenesis and Disrupts Apicobasal Polarity Establishment in the Retina.

Microtubule actin crosslinking factor 1 (MACF1) plays a role in the coordination of microtubules and actin in multiple cellular processes. Here, we show that MACF1 is also critical for ciliogenesis in multiple cell types. Ablation of Macf1 in the developing retina abolishes ciliogenesis, and basal bodies fail to dock to ciliary vesicles or migrate apically. Photoreceptor polarity is randomized, while inner retinal cells laminate correctly, suggesting that photoreceptor maturation is guided by polarity cues provided by cilia. Deletion of MACF1 in adult photoreceptors causes reversal of basal body docking and loss of outer segments, reflecting a continuous requirement for MACF1 function. MACF1 also interacts with the ciliary proteins MKKS and TALPID3. We propose that a disruption of trafficking across microtubles to actin filaments underlies the ciliogenesis defect in cells lacking MACF1 and that MKKS and TALPID3 are involved in the coordination of microtubule and actin interactions.

A gradient of Bmp7 specifies the tonotopic axis in the developing inner ear.

The auditory systems of animals that perceive sounds in air are organized to separate sound stimuli into their component frequencies. Individual tones then stimulate mechanosensory hair cells located at different positions on an elongated frequency (tonotopic) axis. During development, immature hair cells located along the axis must determine their tonotopic position in order to generate frequency-specific characteristics. Expression profiling along the developing tonotopic axis of the chick basilar papilla (BP) identified a gradient of Bmp7. Disruption of that gradient in vitro or in ovo induces changes in hair cell morphologies consistent with a loss of tonotopic organization and the formation of an organ with uniform frequency characteristics. Further, the effects of Bmp7 in determination of positional identity are shown to be mediated through activation of the Mapk, Tak1. These results indicate that graded, Bmp7-dependent, activation of Tak1 signalling controls the determination of frequency-specific hair cell characteristics along the tonotopic axis.

Cilia, Wnt signaling, and the cytoskeleton.

Primary cilia have recently been highlighted as key regulators in development and disease. This review focuses on current work demonstrating the broad role of cilia-related proteins in developmental signaling systems. Of particular consideration is the importance of the basal body region, located at the base of the cilium, in its role as a focal point for many signaling pathways and as a microtubule organizing center. As the cilium is effectively a microtubular extension of the cytoskeleton, investigating connections between the cilium and the cytoskeleton provides greater insight into signaling and cell function. Of the many signaling pathways associated with primary cilia, the most extensively studied in association with the cytoskeleton and cytoskeletal rearrangements are both canonical and non-canonical Wnt pathways. One of the key concepts currently emerging is a possible additional role for the traditionally 'cilia-related' proteins in other aspects of cellular processes. In many cases, disruption of such processes manifests at the level of the cilium. While the involvement of cilia and cilia-related proteins in signaling pathways is currently being unraveled, there is a growing body of evidence to support the notion that ciliary proteins are required not only for regulation of Wnt signaling, but also as downstream effectors of Wnt signaling. This review summarizes recent advances in our understanding of the involvement of cilia and basal body proteins in Wnt signaling pathways.

Combining Cep290 and Mkks ciliopathy alleles in mice rescues sensory defects and restores ciliogenesis.

Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy Bardet-Biedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290rd16 and Mkksko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.

Patterns of expression of Bardet-Biedl syndrome proteins in the mammalian cochlea suggest noncentrosomal functions.

Bardet-Biedl syndrome is a heterogeneous disorder causing a spectrum of symptoms, including visual impairment, kidney disease, and hearing impairment. Evidence suggests that BBS gene mutations cause defective ciliogenesis and/or cilium dysfunction. Cochlear development is affected by BBS gene deletion, and adult Bbs6(-/-) and Bbs4(-/-) mice are hearing impaired. This study addresses BBS protein expression in the rodent cochlea, to gain a better understanding of its function in vivo. As predicted by in vitro studies, Bbs6 immunofluorescence was localized to the basal bodies of supporting cells and sensory hair cells prior to the onset of hearing. In adult tissue, Bbs6 expression persisted in afferent neurons, including within the dendrites that innervate hair cells, implicating Bbs6 in a sensory neuronal function. Bbs2, which interacts with Bbs6, was also localized to hair cell basal bodies and stereociliary bundles. Additionally, Bbs2 was expressed in supporting cells at their intercellular boundaries, in a spatiotemporal pattern mirroring the development of the microtubule network. Bbs4 localized to cilia and developing cytoplasmic microtubule arrays. Pcm-1, a microtubular protein that interacts with Bbs4 in vitro, showed a comparable expression. Depolymerization of microtubules in slice preparations of the living cochlea resulted in Bbs4 and Pcm-1 mislocalization. Pcm-1 was also mislocalized in Bbs4(-/-) mice. This suggests that Bbs4/Pcm-1 interactions may be important in microtubule-dependent cytoplasmic trafficking in vivo. In summary, our findings indicate that BBS proteins adopt a range of cellular distributions in vivo, not restricted to the centrosome or cilium, and so broaden the possible underlying pathomechanisms of the disease.