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

Developmental Biology 345 (2010) 215–225

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Developmental Biology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / d e v e l o p m e n t a l b i o l o g y

Bbs8, together with the planar cell polarity protein Vangl2, is required to establish left–right asymmetry in zebrafish Helen L. May-Simera a,1,2, Masatake Kai b,1,3, Victor Hernandez a, Daniel P.S. Osborn a, Masazumi Tada b, Philip L. Beales a,⁎ a b

Molecular Medicine Unit, Institute of Child Health, University College London, WC1N 1EH, UK Department of Cell and Developmental Biology, University College London, WC1E 6BT, UK

a r t i c l e

i n f o

Article history: Received for publication 22 March 2010 Revised 8 July 2010 Accepted 9 July 2010 Available online 17 July 2010 Keywords: BBS Wnt/PCP pathway Left–right asymmetry Kupffer's vesicle Cilium Basal body

a b s t r a c t 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. © 2010 Published by Elsevier Inc.

Introduction Situs inversus totalis is observed in ~1 in 8500 persons; a condition in which there is complete right to left reversal of the thoracic and abdominal organs. Whilst this is usually of no medical consequence, many more are born with incomplete reversal of organs (laterality or heterotaxia) which can cause significant problems associated with cardiac or gut development for example. The problem is not confined to humans and is seen in almost all vertebrates in whom left–right (LR) asymmetry is a necessary component of embryogenesis. The underlying sequence of events that culminates in breakage of early embryonic symmetry has now been worked out and in general terms, involves the establishment of unidirectional flow by cilia within an

⁎ Corresponding author. Fax: +44 20 7404 6191. E-mail address: [email protected] (P.L. Beales). 1 These authors contributed equally to the work. 2 Present address: Section on Developmental Neuroscience, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA. 3 Present address: National Institute for Basic Biology, Okazaki, 444-8585, Japan. 0012-1606/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.ydbio.2010.07.013

organising centre (e.g. node in mammals or Kupffer's vesicle in fish). Disruption of this flow can be one of a number of causes of a failure to successfully establish LR asymmetry (Essner et al., 2005). Bardet–Biedl syndrome (BBS) is a rare genetic multisystem disorder with a prevalence of 1 in 100,000–160,000 live births in the USA and Northern Europe. It is clinically heterogeneous with primary features that include: age-related retinal dystrophy, obesity, polydactyly, renal dysplasia, reproductive tract anomalies and cognitive impairment. To date 14 loci have been associated with the disease (BBS1-14) (Tobin and Beales, 2009). Identification of mutations in BBS8 in patients with situs inversus implicated the cilium in the etiology of the disease (Ansley et al., 2003). BBS is an archetypal ciliopathy, an emerging class of syndromes resulting from disruption of the structure or function of cilia and/or basal bodies. BBS proteins have been shown to play a role in the Wnt/planar cell polarity (PCP) pathway, one of the non-canonical Wnt signalling pathways important for the establishment of polarized epithelia in a variety of tissues as well as directed migration of cell groups during development (Gerdes et al., 2007; Ross et al., 2005). Additionally other ciliary components have been shown to be involved in Wnt/PCP signalling. For example Wnt/PCP effectors inturned and fuzzy have

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been shown to control the orientation of ciliary microtubules in Xenopus laevis (Park et al., 2006). Localisation of PCP proteins such as Dishevelled, Inturned, Inversin and Vangl2 (Park et al., 2006; 2008; Ross et al., 2005) to the cilium further strengthens the involvement of this organelle in the Wnt/PCP signalling pathway. Furthermore, it has been proposed that the cilium might act as a regulatory switch between canonical and non-canonical Wnt signalling, by controlling the level of Dsh through Inversin (Simons et al., 2005). Several signalling pathways are implicated in LR asymmetry, notably nodal, hedgehog, Wnt/PCP and most recently FGF (Hong and Dawid, 2009; Regan et al., 2009). Evidence for the role of the Wnt/PCP pathway is indirect and limited to a few studies; an epistatic interaction of Bardet–Biedl syndrome proteins with Vangl2 (Ross et al., 2005), the discovery of the mediator of Wnt signalling, duboraya (Oishi et al., 2006), and the requirement of two PCP effector proteins, inturned and fuzzy for ciliogenesis (Park et al., 2006). Intriguingly the zebrafish gene seahorse has been shown to be required for LR asymmetry, highly enriched in ciliated tissues, including Kupffer's vesicle, the otic vesicle, the pronephric duct, and the floor plate of the neural tube but not required for ciliogenesis. During gastrulation seahorse has been shown to promote the Wnt/PCP pathway, possibly through its association with Inversin and Dishevelled (Antic et al., 2010; Hashimoto et al., 2010; Kishimoto et al., 2008). In the zebrafish, the Kupffer's vesicle (KV) is a ciliated transient structure originating from dorsal forerunner cells during gastrulation (Melby et al., 1996). It is proposed to have a similar function to the node of chick and mouse in establishment of LR asymmetry during development (Essner et al., 2002) (Bisgrove et al., 2005). Electron microscopy has shown that a single cilium protrudes into the lumen from each cell lining the KV (Brummett and Dumont, 1978). These cilia rotate in concert to generate a consistent anti-clockwise fluid flow (Okabe et al., 2008). It has been proposed that this fluid flow functions to activate non-motile mechanosensory cilia which initiate an asymmetric calcium influx, triggering signalling pathways resulting in the asymmetric expression of genes necessary for proper LR development, in a similar fashion to the mouse node (McGarth et al., 2003; Nonaka et al., 2002). In the zebrafish, ciliated KV cells are required during early somitogenesis for subsequent correct LR patterning in the brain, heart and gut (Essner et al., 2005) (KramerZucker et al., 2005). As previous work had suggested a disruption to the KV upon bbs gene knock-down in zebrafish (Yen et al., 2006), we sought to investigate this further. Whilst recent studies have alluded to the involvement of cilia in Wnt/PCP signalling, here we show a direct interaction of cilia with the Wnt/PCP pathway. We observed a synergistic effect of bbs8 and trilobite (tri), the zebrafish orthologue of vangl2. Down-regulation of these two genes in zebrafish caused laterality defects, due to aberrant cilia structure and function at the Kupffer's vesicle. Finally we observed a physical interaction between these two proteins in vitro. These findings reveal a mechanism by which BBS proteins contribute to establishment of LR asymmetry and show that Vangl2, a core PCP protein, is directly required for Kupffer's vesicle function and establishment of asymmetry.

was adjusted to 25 °C or 31 °C to decrease/increase the speed of development accordingly.

Materials and methods

Nuclear to apical membrane distance for Kupffer's vesicle

Embryo culture and zebrafish stocks

Using ImageJ software, background exposure of Alexa488 signal was increased and contrast was adjusted to highlight the transition between the tissue and vesicle. Measurements were taken from the nearest nuclei to the apical membrane (n = minimum of 28 measurements per condition). Using Graphpad Prism software, data was pooled from several individuals per experimental group and analysed for significance using Tukey's one-way analysis of variance at a 99.9% confidence level. Mean and SEM (μm) values were plotted on a bar chart.

Zebrafish, Danio rerio, were maintained by the UCL Fish Facility at 28.5 °C on a 14 h/10 h light/dark cycle. Embryos were collected from matings, cultured and staged by developmental time and morphological criteria. TL wild-type and trilobitem209 (trim209) allele animals were used for analyses. Mutant embryos were obtained by incrossing heterozygous carriers. To allow embryos to develop normally they were incubated at 28.5 °C. Incubation temperature

Morpholino antisense oligonucleotide injection Translational morpholino against the start ATG of bbs8 (ttc8: BC062872) was designed by and obtained from Gene Tools (Oregon). Morpholino antisense oligo sequence for bbs8 was 5′-GATCACTGTCTGCGTATATTGTCGA-3′. Diluted morpholino was injected into the yolk just under the blastoderm of 1–2 cell stage embryos or into the centre of the yolk at midblastula stages. 1 nl volume of 4–6 ng of morpholino was delivered using a micro-injector. To test specificity we also injected a scrambled morpholino whereby five bases were substituted with the following sequence — 5′-GAACACTCTCTGAGTATAGTGTAGA-3′. RNA in situ hybridisation and immunohistochemistry Antisense RNA probes were synthesised with a digoxigenin RNAlabelling kit (Roche), using plasmids containing cDNA for myoD (Weinberg et al., 1996), krox20 (Oxtoby and Jowett, 1993) and southpaw (Long et al., 2003). Whole-mount in situ hybridisation was carried out as described previously (Barth and Wilson, 1995). For immunolocalisation of the Kupffer's vesicle, immunohistochemistry was performed on whole-mount 8-somite stage embryos. A monoclonal anti-acetylated tubulin antibody (Sigma) was used to stain the cilia. Primary antibodies were detected using a tagged antimouse secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes). Post dechorionation, embryos were fixed in 80% methanol/20% DMSO overnight at 4 °C. They were then rehydrated through 75%, 50% and 25% methanol/PBS before being washed several times in PBST (0.5% Triton X-100/PBS). Embryos were blocked over night in blocking reagent (10% goat serum [heat inactivated]/1% DMSO/PBST) at 4 °C. Embryos were incubated with primary antibody, diluted in blocking reagent overnight at 4 °C. Following several vigorous washes with PBST at room temperature and under shaking, embryos were further blocked for 2 h at room temperature. Incubation with secondary antibody made up in blocking reagent was done overnight at 4 °C. Following a further round of vigorous washing several times with PBST, embryos were stained with DAPI solution for 2 h at 4 °C. Embryos were stored in PBS at 4 °C for a maximum of one week before being dissected and mounted. Dissections were done in PBS under a dissection microscope to remove the yolksac and orientate the Kupffer's vesicle upright before mounting in 1% agarose. Imaging Live and fixed embryos were mounted in 3% methylcellulose or 70% glycerol respectively for orientation, and imaged using a digital camera (Zeiss Axio Cam MRm). Images were processed using Adobe Photoshop. A Lecia Confocal microscope was used for confocal microscopy imaging of the Kupffer's vesicle. Images were processed using Volocity software (Improvision).

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Visualisation of fluid flow in the Kupffer's vesicle Embryos were cultured till 4–5-somite stages before they were dechorinated and mounted in 1.2% agarose with the Kupffer's vesicle positioned upright. Approximately 0.5 μl of 1:50 diluted fluosphere beads (ø = 0.02 μm, Sigma) was injected via a micro-injector. The Kupffer's vesicle and rotating beads were imaged using an Axioplan 2 microscope (Zeiss) with 63× water immersion lens and captured at approximately 16 frames per second, using Orca ER digital camera (Hamamatsu) and Volocity software (Improvision). Co-immunoprecipitation and GST pull-down assays For co-immunoprecipitation assay, the full-length open reading frames of human BBS8 and mouse Vangl2 were co-transfected into HEK293 cells using Lipofectamine (Invitrogen) and harvested after 48 h by scraping into 1 ml ice-cold RIPA buffer (50 mM Tris–HCl [pH 7.6], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Roche). Cell lysates were spun down and a 30 μl aliquot was removed to confirm fusion protein expression by Western analysis with mouse monoclonal anti-myc (9E10, Sigma) or rabbit polyclonal anti-EGFP (Clontech) antibody. The remainder of the lysate was immunoprecipitated overnight with rocking at 4 °C with 3 μl rabbit polyclonal anti-EGFP (Clontech) and 45 μl slurry of protein G magnetic beads (Invitrogen). Beads were washed three times with 1 ml fresh RIPA buffer, and resuspended in 2 × SDS-PAGE sample buffer. SDS-PAGE was then performed and immunoblotting was carried out using monoclonal anti-myc antibody as probe. Membranes were blocked with 5% skimmed milk, probed with the appropriate antibodies, and detected by ECL (Amersham). For GST pull-down assay, the full-length ORFs of human BBS8 and mouse Vangl2 were cloned into modified pCS2+ plasmid vector to generate pCS2-BBS8-GST and pCS2-Myc-Vangl2. HEK293 cells were transfected to co-express BBS8-GST and Myc-Vangl2. Total cellular proteins were extracted with lysis buffer (50 mM Tris–HCl [pH7.5], 150 mM NaCl, 1% NP-40 supplemented with protease inhibitors [Roche]). The extracts were incubated with Glutathione Sepharose 4B (GE Healthcare) beads for 2 h at 4 °C with gentle mixing. The beads were subsequently washed four times with the lysis buffer. The retained proteins were solubilised in SDS-PAGE sample buffer and separated by SDS-PAGE. Myc-tagged proteins were detected by Western blotting using anti-c-Myc (J1507, Santa Cruz) antibody. Results Bbs8 epistatically interacts with Vangl2 to influence cell movements, actin organisation and otolith formation during zebrafish development We previously observed phenotypes linked with Wnt/PCP signalling defects in Bbs1, Bbs4 and Bbs6 mutant mice, including abnormally rotated stereociliary bundles and neural tube defects (Ross et al., 2005). Trans-heterozygotes for Bbs4 and Vangl2 also displayed abnormal stereociliary bundles and exencephaly indicative of a genetic interaction. This was further supported by knock-down of bbs4 or bbs6 using an antisense morpholino (MO) in vangl2/trilobite (tri) mutant zebrafish, resulting in enhanced cell movement defects in tri zebrafish embryos (Ross et al., 2005). Mutations in BBS8 give rise to a typical BBS phenotype in patients and in some cases situs inversus totalis (Ansley et al., 2003). In view of the phenotypic similarities of BBS8 patients with cases caused by mutations in other BBS genes and the paucity of knowledge of the function of BBS8, we explored the possibility that BBS8 might also influence Wnt/PCP signalling. To test this hypothesis we used a previously validated bbs8 translation-blocking MO in zebrafish (Tobin et al., 2008). All embryos injected with 4–6 ng of bbs8-MO exhibited a pronounced phenotype. At 30 h post fertilisation (hpf) all embryos developed an abnormal

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curve at the end of their tail in conjunction with poor somitic definition (Fig. 1B). A lack of pigmentation was consistently observed amongst all embryos. The brain ventricles were poorly defined. In addition the reduced size of the telencephalon produced a partial cyclopic appearance. The yolk sac extension was variable. Knockdown of bbs8 in zebrafish embryos also reduced the size of the eyes, as previously described (Tobin et al., 2008). The bbs8-MO was then injected into tri mutant embryos to determine if convergence and extension (C&E) cell movements of tri homozygotes (tri−/−) was defective. Embryos from tri+/− × tri+/− matings were injected with bbs8-MO at the 1–2-cell stages and the phenotype was scored at 30 hpf. bbs8-MO enhanced the C&E defects (whereas a control MO had no effect — data not shown) in tri−/− mutants, in addition to the bbs8 phenotype. All injected tri−/− (22/22) had more severe body axis compression than tri alone (Figs. 1A–F). Moreover, narrower eyespacing in tri−/− was enhanced by bbs8 knock-down, to give rise to cyclopic phenotype (Figs. 1E and F insets). This further supports synergism between bbs8 and vangl2/tri in zebrafish development. To confirm this enhanced body axis compression arises from defective cell movements during gastrulation, we examined early stage embryos (8-somite stage [8ss]), using markers myoD and krox20, which highlighted the somites and the positions and shapes of rhombomeres 3 and 5, respectively. Expression of either marker was not affected in tri siblings (1/3 expected wild-type and 2/3 expected heterozygotes), however homozygous tri mutants injected with bbs8MO had more severely compressed somites, a wider presumptive neural tube, and a shorter relative distance between rhombomere 5 and the first somite compared with uninjected homozygous tri mutants (Figs. 1G–N). This indicated that the body axis compression results from an enhancement of abnormal C&E movements during gastrulation. These data confirm an epistatic interaction between bbs8 and vangl2/tri in cell movement during gastrulation. To understand the underlying cellular mechanisms, we investigated the organisation of F-actin in the somites in tri−/− embryos and bbs8-morphants at 48 hpf by phalloidin staining (Figs. 2A–D). Compared to chevron shaped somites and organised actin bundles in WT (Fig. 2A), bbs8-morphants had less defined somites with disrupted actin bundles (Fig. 2B). In tri−/− embryos, dorsal somites were broad and rectangular with an altered pattern of actin bundles, while ventral somites were totally disorganised (Fig. 2C). Injection of bbs8-MO into tri−/− resulted in very severe perturbation of actin and disorganization of somites, suggesting combinatorial roles for bbs8 and vangl2/tri in regulation of actin organisation. Furthermore, knock-down of vangl2/tri or bbs8 affected the number of otoliths found in the zebrafish inner ear at 48 hpf (Figs. 2E–M). In addition to two otoliths as seen in the control (100%, n = 5; Fig. 2E), one or three otoliths were observed in tri−/− (60%, n = 5) and bbs8-morphant (30%, n = 10) embryos (Figs. 2F, H, I, K and L). In tri−/− mutants injected with bbs8-MO, in addition to a variable number of otoliths the size of each individual otolith was also reduced (100%, n = 10). As otolith formation in the zebrafish inner ear is known to be dependent on the presence of functional cilia (Colantonio et al., 2009), these results indicated that bbs8 and vangl2/tri might also act on cilia function. Reduced expression of bbs8 and tri results in laterality defects Loss of bbs8 and vangl2/tri expression during embryogenesis also culminated in cardiac abnormalities. Injection of bbs8-MO into wildtype zebrafish did not cause any overt cardiac defects at 30 hpf. tri−/− mutants exhibited a cardiac phenotype, such that 10.5% (n = 57) of the embryos developed an abnormal heart tube, which included the absence of looping or reversed looping (leftward L-looping, as opposed to normal rightward D-looping) (Figs. 3A–C). Strikingly, in tri−/− embryos injected with bbs8-MO (4–6 ng), only 40.7% (n = 27)

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Fig. 1. Bbs8 interacts with Vangl2 during zebrafish development. Injection of bbs8-MO at the 1–2-cell stages into trilobite (tri) zebrafish. A–F) At 30 hpf, defective CE cell movement of tri−/− was enhanced after injection of bbs8-MO, resulting in severe body axis compression. Inset C–F) Ventral view of zebrafish embryos at 48 hpf. Note varied spacing between eyes. G–N) Visualisation of somites (myoD) and rhombomeres 3 and 5 (krox20) by in situ hybridisation at the 8-somite stage. Patterning is unaltered in tri siblings. bbs8-MO injected tri−/− embryos had more severe CE defects as evidenced by compressed somites and a wider presumptive neural tube, and a shorter distance between rhombomere 5 and the first somite. Views: lateral (A–G, K, I, M), dorsal (H, L, J, N).

showed normal D-looping (Fig. 3D). The effect in tri sibling embryos was also evident. 98.5% (n = 139) of uninjected animals exhibited Dlooping, this was reduced to 84.8% (n = 86) after injection of bbs8-MO (Fig. 3D). We next tested the expression of the nodal-related gene, southpaw (spaw) normally expressed in the left lateral mesoderm, in 15–16ss embryos (Figs. 3E–H). In bbs8-MO injected embryos we observed laterality defects; 7% showed inverted (right) spaw expression, whilst 46.5% (n = 43) displayed a bilateral signal, with either expression on both sides (double left) or no expression (double right). These defects were further enhanced in tri embryos, as injection of bbs8-MO randomised the expression of spaw as summarised in Fig. 3H. Injection of bbs8-MO into tri−/− embryos resulted in 47.4% (n = 19) bilateral, and equal percentages of both reversed and normal spaw expression (26.3%; n = 19). Injection into tri siblings resulted in 56.1% (n = 57) embryos with bilateral spaw expression, 15.8% (n = 57) had reversed spaw expression and only

28.1% (n = 57) had normal (left) spaw expression. These data suggest that the epistasis between bbs8 and vangl2/tri is dosage dependent extending to almost all tri heterozygotes. This led us to speculate that bbs8 interacts with vangl2/tri to establish correct laterality. Symmetry of somitogenesis was not affected in these animals, as expression patterns of both myoD (differentiated somites) at 8ss (Figs. 1G–N) and her1 (presomitic mesoderm) at 6ss (Supplementary Fig. 1) were evenly distributed around the midline. This is similar to other cilia mutants that also have symmetrical somitogenesis (Kishimoto et al., 2008). BBS8 and Vangl2 are required for normal ciliogenesis and Kupffer's vesicle structure To evaluate whether our observed laterality defects were caused by disruption to the Kupffer's vesicle (KV), we targeted bbs8-MO in

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Fig. 2. Bbs8 and Vangl2 act on normal actin organisation and otolith formation. A–D) Visualisation of F-actin in the somite region of zebrafish embryos at 48 hpf by phalloidin. A) Control embryo. B) Injection of bbs8-MO caused undulation of actin filaments. C) In tri−/− embryo the dorsal somites became broad and rectangular with an altered pattern of actin bundles, while the ventral somites were disorganised. D) Injection of bbs8-MO into tri−/− background totally disrupted actin organisation. E–M) Observation of otoliths in zebrafish inner ear at 48 hpf. 1×, 2×, and 3× represent 1, 2 or 3 otoliths respectively. E) Control embryo with two otoliths. H, K) tri−/− embryos showed varied number of otoliths (two or three). F, G, I, J, L, M) Injection of bbs8-MO into tri−/− (G, J, M) or siblings (F, I, L) also resulted in varying numbers of otolith (one to three). Otoliths also appeared to be smaller (G, J, M) than normal. A–D) Lateral view, anterior to the left. A–D) Scale bar: 200 μM; E–M) Scale bar: 100 μM.

the KV by injecting 250–1000-cell stage embryos, directly into the yolk. Morpholino injection at this stage specifically targets the KV (Bisgrove et al., 2005). To ensure accurate targeting of the morpholino, we co-injected fluorescein to facilitate exclusion of those embryos where signal leaked into other organs (data not shown). From the same batch of tri zebrafish, bbs8-MO was injected into embryos at either the 1-cell stage (WE) or the 250–1000-cell stage (KV) (Fig. 4A). spaw expression was randomised in the 250–1000-cell injected embryos, similar to the 1-cell injected embryos (Fig. 4A), suggesting that laterality defects were, at least in part, due to disruption at the KV.

To further investigate the effect of bbs8 depletion on the Kupffer's vesicle, we immunolabeled cilia with an antibody against αacetylated tubulin in 8ss embryos (Figs. 4B–E). At this stage the KV appears as a hollow sphere lined with long cilia. We observed that injection of bbs8-MO alone had no significant effect on the number of cilia present; however the length of the individual cilium was significantly reduced (Figs. 4F–G). Surprisingly, animals homozygous for the tri mutation alone showed both significantly fewer and shorter cilia (p b 0.05). In tri−/− embryos injected with bbs8-MO the number of cilia was further reduced (by almost 70%), confirming a synergistic action of vangl2/tri and bbs8.

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Fig. 3. Reduced expression of bbs8 and vangl2/tri results in laterality defects. Cardiac looping and the laterality marker expression. A–D) Cardiac looping at 30 hfp. A) Normal looping (D-loop). B) Reversed looping (L-loop). C) Absent looping. The heart tube is highlighted by a red dotted line (A–C). D) At 30 hpf, injected tri−/− embryos exhibited increased percentages of abnormal cardiac looping. The fractions of embryos of each category are noted in percent along the X-axis. E–G) spaw expression (arrowheads) in 15–16-somite embryos. E) Normal spaw expression on the left lateral mesoderm. F) Bilateral and G) inverted spaw expressions. H) Quantitative representation of spaw expression. In tri embryos injected with bbs8-MO, the expression is randomised. Views: ventral (A–C), dorsal (E–G).

Further image analysis of cilia and their basal bodies in both the pronephric duct and the otic vesicle in these animals shows a similar disturbance of morphology in tri embryos injected with bbs8-MO as opposed to either the effect of the tri mutation or bbs8-MO alone (Supplementary Fig. 2). These data demonstrate that other ciliated structures are affected in morphants.

Intracellular organisation at the Kupffer's vesicle is disrupted in tri−/− zebrafish On closer inspection of the position of the Kupffer's vesicle cilia in relation to the corresponding cell nuclei, we found a significant increase in the distance of the nucleus in tri−/− embryos compared to

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Fig. 4. BBS8 and Vangl2 are required for normal Kupffer's vesicle structure. A) Percentage spaw expression (at 15–16-somite) after bbs8-MO injection targeted to the whole embryo (WE) or the Kupffer's vesicle (KV). Expression was randomised under both conditions. B–E) Visualisation of cilia (Ac-Tub) in the Kupffer's vesicle in 8-somite embryos in bbs8-MO injected tri embryos. The number and/or length of the cilia are altered in the experimental conditions. F–G) Table depicting average cilia numbers and cilia length (μm) in injected and uninjected tri embryos.

either bbs8-MO or sibling controls. Additionally, many of the basal bodies appeared to be more detached from the apical membrane of the KV of these animals, suggesting that intracellular organisation is disrupted in tri embryos. No significant difference was observed with bbs8-MO alone (Fig. 5). ZO-1 staining indicated that apical specification in these embryos appears to be intact (Supplementary Fig. 3).

BBS8 and Vangl2 are required for normal function of the Kupffer's vesicle A combination of cilia motility and fluid dynamics inside Kupffer's vesicle has been proposed as the driving force for breaking left–right symmetry during embryogenesis (Okabe et al., 2008). As motile cilia are responsible for producing fluid flow within the KV, we investigated

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Fig. 5. Positions of the nucleus and basal body in Kupffer's vesicle cells. A, B, E, F) Measurements of the nearest nuclei to the apical membrane. In tri−/− embryos, the distance of the nucleus (DAPI) and the apical membrane is increased compared to either bbs8-morphants or sibling controls. C, D) Basal bodies (γ-Tub) and cilia (Ac-Tub) appear to be more detached from the apical membrane of the KV in tri−/− embryos. G) Table depicting nuclear distance from the apical membrane (μm) in injected and uninjected tri embryos.

if this might be disrupted in tri mutants, bbs8 morphants or in combination. Using adaptations of methods originally described by Nonaka et al. (1998), we injected microbeads into wild-type and mutant/morphant KVs (Figs. 6A–B). Bead movement was tracked as a constant anti-clockwise circular movement throughout the KV in WT embryos (Supplementary Movie 1; Fig. 6C). In tri−/− mutants beads moved slowly and erratically in an often incomplete anti-clockwise direction. Many of these spin in small circles along their path (Supplementary Movie 2; Fig. 6D). In bbs8-morphants there was also an irregular motion in which beads lacked an overall net direction within the vesicle, sometimes travelling in opposite directions (Supplementary Movie 3; Fig. 6E). Finally, in tri−/− mutants injected with bbs8-MO there was no vectorial movement of beads other than that attributable to Brownian motion (Supplementary Movie 4; Fig. 6F). These results indicate that a critical combination of KV cilia length, number and positioning is required for effective fluid flow. Abrogation of vangl2/tri and bbs8 together abolishes this flow completely which in turn is responsible for the observed laterality defects.

Discussion In this study we set out to understand the role of BBS8 in Wnt/PCP signalling. We discovered that the core PCP protein Vangl2, acting together with BBS8, is needed for the establishment and maintenance of vertebrate LR asymmetry. Furthermore, we demonstrate for the first time a direct requirement of Wnt/PCP signalling for cilia formation and function. Abrogation of vangl2/tri in zebrafish embryos resulted in reduction of the length and number of cilia in the KV. In addition, we found that vangl2/tri is necessary for correct positioning of the nucleus in epithelial cells lining the KV. bbs8 also appears to be responsible for maintaining normal cilium length in the KV but not cilia number. Double knock-down of bbs8 and vangl2/tri led to complete inhibition of rotatory flow within the KV and randomised LR asymmetry, indicating that bbs8 and vangl2/tri act synergistically to establish and maintain the appropriate length and number of cilia in the KV.

Basal body and nuclear position BBS8 and Vangl2 proteins interact in vitro In view of the epistatic relationship between bbs8 and vangl2/tri and the prior localization of Bbs8 and Vangl2 to the basal body (Ansley et al., 2003; Ross et al., 2005), we next tested if Vangl2 and BBS8 physically interact in vitro. Plasmid vectors containing the full-length open reading frames of Vangl2-GFP and BBS8-myc were co-transfected into HEK293 cells. BBS8-myc was co-precipitated with Vangl2GFP by anti-GFP antibody, demonstrating a physical interaction of the two proteins (Fig. 7A). To further verify this interaction we performed GST pull-down assay with BBS8 and Vangl2. BBS8-GST and Myc-Vangl2 were coexpressed in HEK293 cells, and the total cell extracts were incubated with glutathione sepharose beads. myc-Vangl2 was pulled-down by BBS8-GST (Fig. 7B), further confirming the physical interaction between BBS8 and Vangl2.

Two further ciliopathy-related proteins, MKS1 and meckelin have also been shown to be important for centrosome migration to the apical cell surface during ciliogenesis (Dawe et al., 2007b). Furthermore, migration of the centrosome during early cell polarization is a crucial step for primary cilia formation (Dawe et al., 2007a; (Adams et al., 2008). Following migration and docking of the centrosome at the apical cell surface, the centrosome matures to form the basal body which in turn serves as the template for the ciliary axoneme. Recent evidence demonstrates that meckelin interacts with nesprin-2, a nuclear envelope protein which binds actin and is important for nuclear positioning (Dawe et al., 2009). Here we show that Vangl2 and cell polarization are also required for nuclear positioning and centrosome migration. The exact relationship of Vangl2 with nesprin is at present unclear but the observations that PCP effector proteins (see below) govern apical actin assembly may provide clues.

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Fig. 6. Bbs8 and Vangl2 are required for normal fluid flow in the Kupffer's vesicle. A) Schematic representation of the site of bead injection into the KV for fluid flow analysis. B) Bright field image of the KV with injected beads (red circles). C–F) Tracked bead movements in live bbs8-MO injected tri embryos at 4–5-somite stages. C) A constant anti-clockwise circular movement as seen from the dorsal side was observed in WT embryos. D, E) Bead movement was disrupted in both bbs8-morphants and tri−/− mutants. F) Abrogation of both vangl2/ tri and bbs8 abolishes this bead movement completely.

Planar cell polarity and cilia function Intact Wnt/PCP signalling in vertebrates is necessary for convergence and extension, a process whereby cells elongate, intercalate between one another and rearrange along their short axis, thereby contributing to extension of a tissue to one direction (Tada and Kai, 2009) (Roszko et al., 2009). PCP signalling has also been demonstrated to direct actin assembly and basal body docking at the apical cell surface; thereby controlling polarized cell behaviour (Park et al., 2008). Here, Park et al. (2006) reported that the PCP effector proteins inturned and fuzzy are required for ciliogenesis through a process that

governs apical actin assembly thus controlling the orientation, but not assembly, of ciliary microtubules. The same group has recently determined that Dishevelled (another “core” PCP protein) and Inturned mediate the activation of Rho GTPase at basal bodies, and that these three proteins together mediate the docking of basal bodies to the apical plasma membrane (Park et al., 2008). Indeed, both Fuz and Intu mouse mutants display features consistent with predicted cellular roles which include neural tube defects, abnormal dorsal/ ventral patterning of the spinal cord, and defective anterior/posterior patterning of the limb buds (a consequence of SHH disruption secondary to defective ciliogenesis) (Gray et al., 2009; Heydeck et al.,

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Fig. 7. In vitro binding analysis of BBS8 and Vangl2. A) Co-immunoprecipitation of BBS8-myc with Vangl2-GFP using an anti-GFP antibody for the immunopercipitation and an antimyc antibody for the Western blot. Empty-GFP, empty-myc and a random GFP construct were used as negative controls. BBS2-GFP, a known binding partner of BBS8 was used as a positive control. Positive co-IP bands are only observed with Vangl2-GFP and BBS8-myc and in the positive control. B) GST pull-down assay of BBS8 and Vangl2. myc-Vangl2 was pulled-down with BBS8-GST and concentrated, as compared to GST-only control.

2009; Zeng et al., 2010). Our own observations suggesting that vangl2 dictates the number and length of cilia in zebrafish are consistent with these prior PCP studies. Moreover, we have previously determined that vangl2 localises to the basal body and proximal ciliary axoneme suggesting that it may play a role in actin organization (Ross et al., 2005). These observations do however, differ from the recent study of Borovina et al. (2010) in which they did not observe an effect on the length of cilia in maternal-zygotic vangl2/tri mutant. This could be explained by the different methodology employed whereby they expressed GFP-tagged Arl13b to visualise the cilia in vivo which may not label the entire population of ciliary axonemes. It should also be noted that the alleles of vangl2/tri mutation differ between the two studies (tk50f in Borovina et al. (2010) and m109 in this study). Planar cell polarity in development Disruption of Vangl2 is known to cause misorientation of the uniform arrangement of actin-containing stereociliary bundles (SCB) in Looptail mutant cochlea hair cells (Montcouquiol et al., 2003). It has been shown that during mammalian embryonic development the single hair cell kinocilium coordinates final position and formation of the “W”-shaped bundle (Denman-Johnson and Forge, 1999). In turn we surmise that the underlying centrosome migration, maturation into a basal body and docking are precise events dependent upon PCP. Such defects have also been observed in ciliopathy mutants such as Ift88, Bbs1, Bbs4 and Bbs6 mice which display rotated stereociliary bundles in the cochlear hair cells, reminiscent of the looptail animals (Jones et al., 2008; Ross et al., 2005). Nodal and Kupffer's vesicle flow The manner in which mouse nodal cilia effect a leftward flow has been elegantly shown to depend on the orientation of the basal body at the base of the cilium (which lies at an angle of 15° to 35° posterior tilt) (Hashimoto et al., 2010; Hirokawa et al., 2006; Nonaka et al., 2005). In this way the cilium does not rotate in a single plane but rather rises vertically generating maximal flow in one direction and then sweeps horizontally on the recovery stroke. During zebrafish embryogenesis, cilia inside the KV are motile and create a directional fluid flow just prior to the onset of asymmetric gene expression in lateral cells (Essner et al., 2005). Moreover, flow disruption studies indicate that ciliated KV cells are required during early somitogenesis for subsequent LR patterning in the brain, heart and gut (Basu and Brueckner, 2008).

BBS8, a basal body component, is proposed to function as part of the BBSsome, a functional protein complex comprised of several BBS proteins. In C. elegans it has been demonstrated that BBS8 plays an important role in coordinating the velocity of the kinesin molecular motors during anterograde transport along the ciliary axoneme (Ou et al., 2005). In the absence of BBS8 the distal portion of the cilium is not constructed, leading to shortening consistent with the findings reported herein. Defects in anchoring the basal body/cilium in the tri embryos evidenced by the increased distance between cilium and nucleus, serve to explain KV cilia disruption. In addition, loss of bbs8 alone enhances the tri convergence and extension phenotype and results in low level laterality defects. However, together with perturbation of vangl2/tri function, the effect is magnified culminating in a compound lack of rotary flow at the KV and consequent developmental defects. Recent evidence, in Xenopus and zebrafish, following abrogation of Vangl2 function, disrupts the posterior cellular localization of motile cilia in the gastrocoel roof plate (GRP) and the posterior tilt of cilia in the KV respectively (Antic et al., 2010; Borovina et al., 2010). The mechanism by which this arises is unknown. Additionally it was recently shown that Vangl2 is also required for left– right asymmetry in the chick embryo (Zhang and Levin, 2009). Here, we suggest that this mechanism arises through combinatorial actions of BBS8 and Vangl2 upon actin organisation and basal body migration. This hypothesis is further supported by recent data from the Hamada laboratory demonstrating that the basal body of node ciliated cells is initially positioned centrally but then gradually shifts toward the posterior side of the node cells (Hashimoto et al., 2010). A key question remains as to the precise processes which govern centrosome migration and maintenance of basal body position. A positive feedback mechanism which governs polarity and direction of motion of cilia, has been proposed (Mitchell et al., 2007). These researchers show that the planar orientation of Xenopus multiciliate cells is disrupted when components in the PCP signalling pathway are altered and that this occurs in a non-cell autonomous fashion (Mitchell et al., 2007). Interaction of Bbs8 with Vangl2 We were surprised to find that Bbs8 can interact directly with Vangl2 but is nevertheless consistent with the epistatic relationship we observed between these two genes. Previously we have determined that Bbs4 interacts genetically with Vangl2 in both mice and zebrafish (Ross et al., 2005) and that Bbs4 functions as an adapter protein to load cargo onto IFT complexes in preparation for retrograde transport (Kim et al., 2004). We now know that Bbs4 and Bbs8

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participate in the BBSome; a protein complex involved in the transport of proteins to the cilium (Nachury et al., 2007; Roselli et al., 2002). Therefore it remains to be confirmed if other components of the BBSome also interact with Vangl2 and/or other core PCP proteins and furthermore, to determine if perturbation of this complex collectively alters PCP or vice-versa. Acknowledgments We thank Dr. M Kelley for providing reagents. We are grateful to Steve Wilson and members of the UCL zebrafish group for fruitful discussions. MK and MT were supported by grants from the MRC and the Royal Society. HMS was supported by a grant from the MRC. PLB is a Wellcome Trust Senior Research Fellow. DO and VH are supported by an EU FP7 EUCILIA grant (HEALTH-F2-2007-201804). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ydbio.2010.07.013. References Adams, M., Smith, U.M., Logan, C.V., Johnson, C.A., 2008. 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