Introduction {#s1}
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Joubert syndrome (JBTS) is a rare ciliopathy characterized by a specific midhindbrain malformation presenting as 'molar tooth sign' on axial MRI.  Patients typically have a perturbed respiratory pattern in the neonatal period and pronounced psychomotor delay.  Depending on the genetic subtype, there may be additional retinal degeneration, nephronophthisis, liver fibrosis, and skeletal abnormalities (such as polydactyly).  JBTS is genetically heterogeneous, with recessive mutations reported in more than 20 genes encoding proteins related to the function of cilia and associated structures ([@bib47]; [@bib6]). 

Cilia are axoneme-based organelles which protrude into the extracellular milieu, anchored to the cell by a modified centriole (basal body).  They are present in virtually every cell type ([@bib16]).  Non-motile 'primary' cilia play essential roles in mechanotransduction, chemosensation, and intracellular signal transduction, including Hedgehog (Hh), PDGFα, and WNT pathways, in embryonic development and adult tissue homeostasis ([@bib26]).  In addition, highly modified and specialized cilia constitute the light-sensitive outer segments of retinal photoreceptor cells.  Dysfunction of cilia, centrioles of basal bodies, and centrosomes can lead to a spectrum of developmental single- or multi-organ disorders termed 'ciliopathies' ([@bib12]). 

*KIAA0586* (*TALPID3*; MIM \#610178, MIM \#000979-9031) is essential for vertebrate development and ciliogenesis.  The KIAA0586 (TALPID3) protein is localized at the centrosome in human, chicken, mouse, and zebrafish cells ([@bib61]; [@bib11]; [@bib60]), and in particular, at the distal end of the mother centriole---the basal body of cilia ([@bib35]).  In model organisms, *KIAA0586* null mutations cause failure of basal body docking and loss of cilia, leading to early embryonic lethal phenotypes ([@bib19]; [@bib8]; [@bib11]; [@bib54]).  KIAA0586 (TALPID3) binding partners include PCM1, Cep120, and CP110, which interact with a known JBTS protein, CEP290 ([@bib58]). 

Here, we report three JBTS families with loss-of-function mutations in *KIAA0586*.  Using animal models, we demonstrate that TALPID3 (KIAA0586) is not only essential for transduction of Hedgehog signaling but plays an important role in centrosomal localization, orientation, and length.  Finally, and beyond its established requirement for ciliogenesis, TALPID3 (KIAA0586) plays a key role in cell and tissue polarity. 

Results {#s2}
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Clinical description of patients with *KIAA0586*-associated JBTS {#s2-1}
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The diagnosis of JBTS was based on the presence of a molar tooth sign in all three families. 

Family 1 ([Figure 1A](#fig1){ref-type="fig"}) is a consanguineous Kurdish family from northeast Syria.  The two affected siblings were examined at the age of 6 years and 10 months (MR026-01) and 2 years and 2 months (MR026-04), respectively.  Pregnancy, delivery, and birth parameters of both children were unremarkable.  In the neonatal period, both were hypotonic and weepy.  Motor and speech development in MR026-01 were delayed, and his IQ was estimated to be between 50 and 70.  Further symptoms were severe myopia, scoliosis, brachydactyly, distinct facial characteristics, and recurrent febrile seizures.  Height was reduced (108 cm, −2.6 SD), weight was normal (22 kg, −0.27 SD), and head circumference was increased (57 cm, +2.3 SD).  MR026-04 had not reached any milestones, and at the age of 7 years, she was wheelchair-bound.  Cognitive abilities were weaker than in her brother, with an IQ estimated to be below 35.  MR026-04 had similar physical characteristics as her brother, severe muscular hypotonia, prolonged and therapy-resistant seizures since the age of 14 months, and hypothyroidism.  At the time of examination, her height was 91 cm (1 SD), weight was 11.5 kg (−0.7 SD), and there was macrocephaly (head circumference of 59 cm, +8 SD).10.7554/eLife.08077.003Figure 1.Patients with Joubert syndrome (JBTS) and *KIAA0586* mutations (**A**--**C**).(WT, wildtype; M, mutation).  The 'molar tooth sign' in cranial axial MRI is indicated by arrows. (** A**) Family 1: Homozygosity mapping yielded eight homozygous chromosomal candidate regions (not shown), including the *JBTS23* locus comprising *KIAA0586*.  Patients MR026-01 and MR026-04 carry a homozygous splice site mutation, c.2414-1G\>C. (**B**) Patient MD1 of Family 2 is compound heterozygous for two truncating mutations, including the prevalent c.428delG (p.Arg143Lysfs\*4) allele. (** C**) Family 3: Patient G2 is double heterozygous for c.428delG in *KIAA0586*, and a frameshift mutation in *KIF7* (*JBTS12*; c.811delG, p.Glu271Argfs\*51).  He also carries three potentially pathogenic variants in the ciliopathy genes *CEP41*, *KIF14*, and *WDPCP* (blue). (** D**) Genomic structure of *KIAA0586* with mutations in exons 5 and in/adjacent to exon 18 indicated.  The gel electrophoresis shows the aberrant transcripts due to c.2414-1G\>C. (**E**) Scheme of human KIAA0586 protein and predicted consequences of JBTS-associated mutations.  Orange color: unrelated residues included due to frameshift mutations.  The third coiled-coil domain is the counterpart of the functionally essential fourth coiled-coil domain in chicken (framed in red). (** F**) Chicken TALPID3 (KIAA0586) is highly similar to the human protein.  The *talpid*^*3*^ mutation results in an early frameshift and loss of three coiled-coil domains, including the fourth one.  The in-frame deletion of exons 11 and 12 of mouse *KIAA0586* (*2700049A03Rik*) is depicted above the scheme of the chicken ortholog.**DOI:** [http://dx.doi.org/10.7554/eLife.08077.003](10.7554/eLife.08077.003)10.7554/eLife.08077.004Figure 1---figure supplement 1.Analysis of potential interactions between *Talpid3/TALPID3*, *Kif*7/*KIF*7 and *IFT*57 in the mouse and in chicken. Biallelic *KIF7* mutations cause JBTS type 12 in human ([@bib17]).  Although both the *KIAA0586* mutation c.428delG and the *KIF7* mutation c.811delG were paternally inherited in patient G2, we sought to test for subtle abnormalities resulting from this double heterozygosity.  In addition, we had previously found through a microarray analysis of *talpid*^*3*^ limb buds that IFT57, a protein associated with ciliopathy phenotypes in mice and zebrafish, is downregulated in *talpid*^*3*^ embryos ([@bib8]).  Using in ovo complementation of the *talpid*^*3*^ neural tube, we could not detect induction of ISLET1 expression or ventralized PAX7 in the *wildtype* or *talpid*^*3*^ neural tube by overexpression of *KIF7* or *IFT57* ([Figure 1---figure supplement 1A,B](#fig1s1){ref-type="fig"}).  We then used siRNA constructs against *KIF7* to model a heterozygous loss of *KIF7* in the *TALPID3*^*+/−*^ neural tube.  Knock-down with two siRNA constructs had a weak effect on neural tube patterning compared to the mouse *KIF7*^−/−^ knockout ([@bib15], [@bib39]).  Although the NKX2.2 expression domain could be marginally expanded in *wildtype* embryos (not shown), there was no expansion of ISLET1-positive motorneuron progenitors in *wildtype* or *TALPID3*^*+/−*^ embryos.  PAX7, however, was weakly dorsalized in both *wildtype* and *talpid*^*3+/−*^ embryos ([Figure 1FC](#fig1){ref-type="fig"}).  These results suggested that some *KIF7* function may be cilia-independent as has been suggested ([@bib39]).  To more precisely investigate for a possible epistatic relationship between Kif7 and Talpid3, particularly in the organs primarily affected in JBTS, such as the cerebellum, we undertook a *Talpid3*^*+/−*^ × *Kif7*^*+/−*^ mouse cross in order to determine if double *Talpid3*^*+/−*^*/Kif7*^*+/−*^ heterozygous animals had brain patterning malformations.  We first dissected embryos at E15.5, 16.5, and 17.5 and found that *Talpid3*^*+/−*^*/Kif7*^*+/−*^ embryos were morphologically normal, including size, situs and limb patterning.  MEFs derived from E12.5 embryos were normally ciliated, with the percentage of ciliated cells and cilia length comparable to those seen in *wildtype*, *Talpid3*^*+/+*^*/Kif7*^*+/−*^ and *Talpid3*^*+/−*^*/Kif7*^*+/+*^cells ([Figure 1---figure supplement 1F,G](#fig1s1){ref-type="fig"}).  MRI and sectioning of the brain also showed no brain patterning abnormalities ([Figure 1---figure supplement 1D,E](#fig1s1){ref-type="fig"}).  Subsequently, *Talpid3*^*+/−*^*/Kif7*^*+/−*^ animals were born and grew normally compared to their litter mates and showed no abnormal brain morphology ([Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}).  We conclude that *KIAA0586* (*TALPID3*) and *KIF7* do not act epistatically and hypothesize that additional genetic alterations in ciliopathy genes of patient G2, eventually including those identified in *CEP41* (*JBTS15*), *KIF14*, and *WDPCP*, may contribute to a mutational load that is sufficient to elicit a JBTS phenotype on a *KIAA0586*^*+/−*^*; KIF7*^*+/−*^ background. (** A**) Overexpression of *IFT57* does not have an effect on patterning of the neural tube in the *talpid*^*3*^ chicken. (** B**) Overexpression of *KIF7* does not rescue or alter neural tube patterning in the *talpid*^*3*^ chicken. (** C**) siRNA knockdown of *KIF7* resulted in a weak dorsalization of *PAX7* but no expansion of *ISLET1*. (** D**) *Talpid3*^*+/−*^ *Kif7*^*+/−*^ mice showed no gross anatomical abnormalities, neither were developmental brain defects identified through MRI (**D**) or histology (**E**). (** F**, **G**) No abnormalities were identified in either the percentage of ciliated cells (**F**), nor the length of cilia (**G**) in MEFs derived from wildtype, *Talpid3*^*+/+*^*Kif7*^*+/−*^, *Talpid3*^*+/−*^ *Kif7*^*+/+*^ or *Talpid3*^*+/−*^ *Kif7*^*+/−*^ mice.**DOI:** [http://dx.doi.org/10.7554/eLife.08077.004](10.7554/eLife.08077.004)

Family 2 ([Figure 1B](#fig1){ref-type="fig"}) is of North American origin.  Patient MD1 was born at 34 3/7 weeks gestation following preterm premature rupture of membranes at 26 weeks.  At birth, patient MD1 was found to have cardiac defects including a patent ductus arteriosus (PDA), patent foramen ovale (PFO) and a 3/6 ventricular septal defect (VSD) causing persistent pulmonary hypertension 24 hr after birth.  The PDA and PFO resolved, and VSD was at 2/6 within 22 days.  At 7 months, MD1 was found to have a superior vena cava duplication.  At 2 years of age, MD1 had hypotonia which inhibited motor actions, although she crawled proficiently, used sign language and single words, and self-fed by hand and with utensils.  In addition, she had type I bilateral Duane syndrome with no abduction in either eye, narrowing of the palprebal fissure of the inturned eye, was farsighted, had thin tooth enamel, held her jaw sideways in a cross-bite pattern, and had long fingers with a slight clinodactyly of the fifth finger.  She had a broad forehead, arched eyebrows, ptosis of the right eye, and a triangle-shaped mouth.  Her receptive language was good.  There was intermittent hyperpnea/apnea during awake periods.  Patient MD1 had no liver, kidney, or eye abnormalities at 2 years of age. 

Family 3 ([Figure 1C](#fig1){ref-type="fig"}) is of German origin: patient G2 displayed a relatively mild JBTS phenotype with developmental delay and behavioral abnormalities, but no dysmorphic signs and no renal, retinal, skeletal, or liver abnormalities.  His symptoms were described previously ([Figure 1C](#fig1){ref-type="fig"}, [@bib17]). 

Mutations of *KIAA0586* cause JBTS {#s2-2}
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We have identified *KIAA0586* mutations in three JBTS families ([Figure 1A--D](#fig1){ref-type="fig"}).  Genome-wide SNP genotyping in Family 1 identified eight homozygous chromosomal candidate regions with a total range of 67.1 Mb.  By WES, the homozygous mutation c.2414-1G\>C in intron 17, affecting the invariant consensus of the exon 18 acceptor splice site, was found in the index patient, MR026-01, and his affected sister, MR026-04.  Segregation analysis in the family was compatible with causality ([Figure 1A](#fig1){ref-type="fig"}).  The mutation was absent from 372 healthy Syrian controls, including 92 of Kurdish origin, and not listed in the ExAC database. 

In patient MD1 from Family 2, WES identified compound heterozygosity for the *KIAA0586* mutations c.428delG (p.Arg143Lysfs\*4; rs534542684; MAF of 0.39% in ExAC db) and c.2512C\>T (p.Arg838\*), each inherited from a healthy parent ([Figure 1B](#fig1){ref-type="fig"}), and both resulting in premature stop codons.  Because the coiled-coil domain, which is essential for KIAA0586 function in mouse, chicken, and zebrafish (residues 531--571 and residues 497--530 in human and chicken KIAA0586 (TALPID3), respectively; [Figure 1F](#fig1){ref-type="fig"}), would be lost in a truncated protein derived from the c.428delG mutation, we consider it a loss-of-function mutation (as is the case for the *talpid*^*3*^ chicken mutation which introduces a frameshift 3′ to c.428 in the chicken ortholog, [Figure 1F](#fig1){ref-type="fig"}).  Like the *Talpid3*/*TALPID3* null mutations in mouse and chicken, c.428delG is clearly recessive because the father of the patient is a healthy carrier.  The c.2512C\>T (p.Arg838\*) mutation is predicted to lead to nonsense-mediated decay (NMD) or a truncated protein, but with preservation of the essential coiled-coil domain. 

The simplex patient of Family 3, G2, was a known carrier of a heterozygous N-terminal frameshift mutation in exon 3 of the *JBTS12* gene *KIF7*, c.811delG (p.Glu271Argfs\*51) ([@bib17]).  WES of the family trio (patient G2 and his parents) additionally identified the c.428delG (p.Arg143Lysfs\*4) mutation in *KIAA0586*, in the patient ([Figure 1C](#fig1){ref-type="fig"}).  We hypothesized that disease in patient G2 could be due to biallelic mutations either in *KIF7* (*JBTS12*) or in *KIAA0586* (*JBTS23*), assuming that the 'missing mutation' has escaped detection by sequencing due to an extra-exonic localization.  Genome-wide CGH (Affymetrix 6.0 SNP array) did not reveal structural alterations adjacent to or within *KIF7* ([@bib17]) or *KIAA0586*, thereby largely excluding a large deletion or duplication.  PCR amplification and subsequent sequencing of *KIAA0586* exons from cDNA did not reveal aberrant splicing as a potential hint for a deep intronic splice site mutation.  Because *KIF7* and *KIAA0586* both encode modulators of GLI processing and c.428delG~*KIAA0586*~ and c.811delG~*KIF7*~ likely represent recessive loss-of-function mutations, we investigated the possibility of a potential epistatic effect predisposing to JBTS.  No such interactions were identified in mouse and chicken experiments (details are fully described in [Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}).  Therefore, unidentified mutations are likely to be involved, either mutations in *KIF7*, *KIAA0586* (e.g., deep intronic mutations or alterations in non-coding regulatory regions which would both not be covered by WES) or biallelic mutations in another (yet unknown) JBTS gene.  WES revealed further heterozygous missense variants in three known recessive ciliopathy genes in patient G2 ([Figure 1C](#fig1){ref-type="fig"}), all affecting evolutionarily conserved residues of the respective proteins: (1) c.536 G\>A (p.Arg179His, rs140259402; MAF of 0.001647% in ExAC db) in *CEP41*, the gene associated with *JBTS15* ([@bib36]). ( 2) c.3181A\>G (p.Ile1061Val; MAF of 0.01155% in ExAC db) in *KIF14*, a gene associated with a lethal fetal ciliopathy phenotype ([@bib24]). ( 3) c.1333G\>C (p.Ala445Pro, rs61734466; MAF of 0.6609% in ExAC db) in *WDPCP*, the gene associated with Bardet--Biedl syndrome type 15 (*BBS15*), and a putative contributor to Meckel Gruber syndrome ([@bib33]).  All variants were of paternal origin and rare in the general population except the *WDPCP* allele, which had been maternally inherited and which has been annotated homozygously in five healthy individuals (ExAC db), indicating that this is a benign variant.  Genome-wide CGH (Affymetrix 6.0 SNP array) did not show structural alterations adjacent to or within *CEP41*, *KIF14*, or *WDPCP*.  In addition, we searched the WES data of patient G2 for heterozygous putative loss-of-function (that is, truncating) variants in genes with documented ciliary function.  This revealed a paternally inherited frameshift variant, c.206_207insA (p.Ser70Valfs\*3), in *PLA2G3*, the gene encoding phospholipase A2.  In a functional genomic screen, PLA2G3 was found to be a negative regulator of ciliogenesis and ciliary membrane protein targeting ([@bib34]).  The p.Ser70Valfs\*3~*PLA2G3*~ variant is relatively common, but has not been documented in homozygous state in healthy individuals (MAF of 0.4060 in ExAC db). 

We also filtered for known JBTS genes carrying at least two rare variants in patient G2, but we did not identify such a constellation.  When applying this to all genes captured in the WES approach, there was also no potentially causative double heterozygosity in a gene of known or probable ciliary function.  Filtering for homozygous rare and likely pathogenic variants was negative, compatible with lack of consanguinity in the parents of patient G2. 

Consequences of the *KIAA0586* mutation c.2414-1G\>C on mRNA level {#s2-3}
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The c.2414-1G\>C mutation affects the invariant consensus of the acceptor splice site of exon 18.  RT-PCR and Sanger sequencing of the fragments amplified from cDNA revealed three aberrant splicing products due to usage of alternative exonic acceptor splice sites at AG motifs within exon 18 and due to skipping of exon 18 ([Figure 1D,E](#fig1){ref-type="fig"}): a 13-bp deletion that results in a premature termination codon (alternative acceptor splice site at c.2425/2426AG), a 108-bp in-frame deletion (alternative acceptor splice site at c.2520/2521AG), and a 188-bp deletion due to skipping of exon 18 that results in a premature stop codon.  These aberrant transcripts were present in the cDNA from both patients, but not in the cDNA of a healthy control individual ([Figure 1D](#fig1){ref-type="fig"}).  The mutant mRNA molecules are likely to be degraded by NMD.  If the mutant transcripts were stable, the essential coiled-coil domain ([Figure 1E](#fig1){ref-type="fig"}), which mediates centrosomal localization and function of KIAA0586 protein ([@bib61]; [@bib60]), would be preserved. 

KIAA0586 localizes to the basal body of cultured cells and photoreceptor cells of human and mouse retina {#s2-4}
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KIAA0586 (Talpid3) is a centrosomal protein and localizes to the basal body and the adjacent centriole of primary cilia in human RPE1, IMCD3 cells ([Figure 2A](#fig2){ref-type="fig"}), and other cell types ([@bib35]; [@bib60]).  Immunofluorescence analysis of the retina of *wildtype* C57BL/6 mice allowed us to allocate Talpid3 expression to different retinal layers, namely the photoreceptor layer, the outer and inner plexiform layer, and the ganglion cell layer ([Figure 2B](#fig2){ref-type="fig"}).  Co-staining with the ciliary marker centrin-3 ([@bib57]) demonstrated Talpid3/KIAA0586 localization in the ciliary region at the joint between the inner and outer segment of photoreceptor cells in cryosections through the mouse retina and the retina of a human donor eye ([Figure 2B,C,E](#fig2){ref-type="fig"}).  Higher magnification revealed that Talpid3/KIAA0586 specifically localized at the basal body (mother centriole) and the adjacent centriole as well as between the two centrioles, but not in the connecting cilium of mouse and human photoreceptor cells ([Figure 2D,F](#fig2){ref-type="fig"}).  These findings were confirmed by immunoelectron microscopy of Talpid3 labeling on sections through mouse photoreceptor cilia ([Figure 2G,H](#fig2){ref-type="fig"}).  Immunostaining was found at centrioles and in the pericentriolar region in the apical inner segment of photoreceptor cells.  The spatial distribution of Talpid3/KIAA0586 labeling at the ciliary base of photoreceptor cells is summarized in the scheme of [Figure 2J](#fig2){ref-type="fig"}.10.7554/eLife.08077.005Figure 2.Localization of KIAA0586/Talpid3 in primary cilia and in photoreceptor cilia of mammalian retinas.(**A**) Triple labeling of a ciliated IMCD3 cell demonstrates localization of Talpid3 (green) in the basal body (BB) and the adjacent centriole (Ce) at the base of the primary cilium co-stained by antibodies against Pericentrin-2 (PCNT2, red) and anti-acetylated tubulin (acTub, cyan), a biomarker of the axoneme (Ax). (** B**) Longitudinal cryosections through a mouse retina stained for Talpid3 (green) and counterstained for the ciliary marker Centrin-3 (Cen3, red) and for the nuclear DNA marker DAPI reveal Talpid3 localization in the ciliary region (CR) at the joint between the inner (IS) and the outer segment (OS) of the photoreceptor layer, the outer (OPL) and inner plexiform layer (IPL).  Overlay of DIC (differential interference contrast) image with DAPI (blue) nuclear stain in the outer (ONL) and the inner nuclear layer (INL) and in the ganglion cell layer (GC). (** C**--**F**) Immunostaining of cryosections through the photoreceptor layer of a mouse (**C**) and a human retina (**E**) demonstrates co-localization of KIAA0586/Talpid3 and Cen3 in the CR of photoreceptor cells.  Higher magnification of double-labeled mouse (**D**) and human (**F**) photoreceptor cilium reveals substantial localization of Talpid3/KIAA0586 at the centriole (Ce), the BB and between the Ce and BB of the photoreceptor cilium, but not in the connecting cilium (CC). (** G**, **H**) Immunoelectron microscopy analysis of longitudinal section through the cilium of a mouse rod photoreceptor cell and (**G**) higher magnification of the ciliary base (**H**) labeled for Talpid3 reveals Talpid3 in the periciliary region namely in the Ce and BB. (** J**) Schematic representation of Talpid3/KIAA0586 localization in the photoreceptor cilium.  Scale bars: **A**, 1 μm; **B**, 10 μm; **C**, **E**, 5 μm; **D**, **F**, 0.5 µm; **G**, **H**, 200 nm.**DOI:** [http://dx.doi.org/10.7554/eLife.08077.005](10.7554/eLife.08077.005)

Loss of *TALPID3* (*KIAA0586*) causes abnormal tissue and cell polarity {#s2-5}
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The *talpid*^*3*^/Talpid3^−/−^ phenotype in model animals has thus far been attributed to the role of TALPID3 in ciliogenesis and the subsequent loss of Hh-dependent patterning.  However, the patients in this study did not display any overt defects typical for impaired Hh signaling such as polydactyly or hypotelorism, which have been described in other patients with JBTS ([@bib6]). * Talpid*^*3*^ chicken embryos also have polycystic kidneys ([@bib61]), a phenotype that is frequently ascribed to a loss of oriented cell division ([@bib29]; [@bib14]), as well as cell migration defects ([@bib8]), which may also occur due to loss of cell polarity ([@bib29]; [@bib14]).  To investigate if tissue and cell polarity is impaired by a loss of TALPID3 function, we first examined the patterning of the skin and the inner ear, two highly polarized tissues independent of Hh signaling.  At E10, embryonic chicken feather buds express *β-catenin* in an oriented manner, with a larger domain in the anterior part of the bud ([Figure 3A,C](#fig3){ref-type="fig"}).  While 88% of *wildtype* feather buds at E10 are oriented in this manner (n = 117/133), only 21% of stage-matched *talpid*^*3*^ feather buds were (n = 38/179).  22% of *talpid*^*3*^ buds were oriented in the wrong direction (n = 39/179) and 57% had failed to show any orientation of β-catenin expression (n = 102/179; [Figure 3C′](#fig3){ref-type="fig"}). * Talpid*^*3*^ feather buds also frequently merged (29% of buds; asterisk, [Figure 3B′′](#fig3){ref-type="fig"}).  Thus, the skin of *talpid*^*3*^ embryos did not show the characteristic rostral-caudal polarization of *wildtype* skin.  The hair cells (HCs) of the inner ear (known as the basilar papilla (BP) in chicken) have a highly polarized structure determined by the non-canonical Wnt-PCP signaling pathway.  In the *wildtype* chicken, as in mouse, individual HCs exhibit an orientated actin-based stereocilia bundle, the apex of which lies at the abneural side of the cell, where within an actin-free 'bare zone', a single kinocilium (a microtubule-based true cilium) forms (arrow, [Figure 3D](#fig3){ref-type="fig"}).  HCs are frequently used to assess how cell polarity and ciliogenesis are perturbed in mouse mutants ([@bib26]).  The HCs of *talpid*^*3*^ embryos formed actin filament bundles (curved line, [Figure 3E](#fig3){ref-type="fig"}), but no kinocilium, demonstrating that, as with other tissues studied, loss of TALPID3 impairs ciliogenesis.  Furthermore, although stereocilia were present in *talpid*^*3*^ HCs, stereocilia bundles frequently lacked polarity compared to *wildtype* HCs as indicated by either centrally located stereocilia bundles in SEM or actin filaments throughout the cell (*talpid*^*3*^ n = 1086/1195; *wt* n = 258/502; [Figure 3D--G,L](#fig3){ref-type="fig"}).  Orientation of the polarized stereocilia bundles that did form in *talpid*^*3*^ HCs was also abnormal ([Figure 3E,G,N](#fig3){ref-type="fig"}).  The orientation of stereocilia was determined in relation to their position to the abneural side of the BP ([Figure 3F,G,M](#fig3){ref-type="fig"}).  73% of stereocilia of *wildtype* cells (n = 244) were oriented within 40° of the expected angle (90°, compared to 38% of *talpid*^*3*^ cells (n = 237; [Figure 3M′](#fig3){ref-type="fig"})).  Thus, *talpid*^*3*^ HCs showed disrupted polarity.10.7554/eLife.08077.006Figure 3.Loss of TALPID3 (KIAA0586) causes abnormal tissue and cell polarity and abnormal intracellular organization.(**A**, **A′**, **A′′**) *β-catenin* expression is localized anteriorly within feather buds of the *wildtype* chicken at day 9.5 and (**B**, **B′**, **B′′**) in the *talpid^3^* chicken at day 9.5.  Black circles indicate feather buds with correct polarity; dashed black circles represent no polarity; blue circles represent abnormal polarity (Schematic **C**).  The *talpid*^*3*^ chicken (**B′′**) demonstrates feather buds which lack polarity (blue circles) which is not seen in the *wildtype* chicken (**A′′**).  Asterisks represent merged feather buds. (** C′**) Quantification of the percentage of feather buds with correct, abnormal or no polarity in *wildtype* and *talpid^3^* (**D**, **E**) SEM of the basilar papilla in *wildtype* (**D**) and *talpid*^*3*^ (**E**) chickens.  Arrows indicate cilia.  Curved lines represent the base of stereocilia hair bundles. (** F**--**K**) Actin bundles identified by phalloidin (green) and centriolar localization identified by γ tubulin (red). (** F′**, **G′**) overview of *wildtype* and *talpid^3^* basilar papilla, higher magnification in (**F**, **G**), red circles with line represent orientation of polarized actin bundles in basilar papilla; dashed red circles represent unpolarized actin bundles (Schematic **L**, **M**). (** L′**) Quantification of polarized haircells. (** M′**) Quantification of the angle of polarised hair cells. (** H**--**K**) Dashed white circles represent magnified images (**Ji**--**Kii′**). (** Ji**--**Kii**) White arrows indicate aligned centrosomes; blue arrows indicate unaligned centrosomes (Schematic **N**). (** N′**) Quantification of cells with aligned centrosomes. (** O**, **P**) Orientation based on placement of Golgi (TGN46, red) in comparison to actin indicating the leading edge (phalloidin, green) and nucleus (Dapi, blue, schematic in **Q**) in MEFs.  Asterisks represent areas of higher magnification (not all represented at lower magnification). (** Q′**) Quantification of the angle of orientation of MEF cells in scratch assay.  Scale Bars: **A**, **B** 5 mm; **A′**, **A′′**, **B′**, **B′′** 1 mm; **D**, **E** 1 μm; **F**, **G**, **H**, **I**, **J**, **K** 20 μm; **F′**, **G′** 100 nm; **Ji**, **Ji′**, **Jii**, **Jii′**, **Ki**, **Ki′**, **Kii**, **Kii′** 10 μm; **O**, **P** 100 μm; **Oi**, **Oii**, **Oiii**, **Oiv**, **Ov**, **Ovi**, **Pi**, **Pii**, **Piii**, **Piv**, **Pv**, **Pvi** 25 μm.**DOI:** [http://dx.doi.org/10.7554/eLife.08077.006](10.7554/eLife.08077.006)

Loss of TALPID3 function causes abnormal intracellular organization {#s2-6}
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Loss of TALPID3 function prevents basal body docking ([@bib61]), which we have previously suggested to be due to failure of centrosome migration ([@bib54]).  The migration and subsequent localization and docking of the centriole is crucial to establish polarity and placement of the actin bundle in the HC ([@bib56]), and we therefore hypothesized that disturbed cell polarity may result from defective centrosome migration in *talpid*^*3*^ HCs.  Using antibodies against γ tubulin to determine the localization of the centriole within the actin-negative abneural bare zone in the HCs, 95% of *wildtype* HCs exhibited a basal body (centrosome) within the abneural bare zone (n = 632 from 7 samples, [Figure 3H,J,N](#fig3){ref-type="fig"}).  In contrast, only 49% of *talpid*^*3*^ cells exhibited a centriole within the bare zone (either abneural or abnormally polarized; n = 219 from 6 samples; [Figure 3I,K,N](#fig3){ref-type="fig"}), thus demonstrating that the intracellular organization of *talpid*^*3*^ cells was frequently abnormal.  Furthermore, and in agreement with the failure of correct polarization of the stereocilia, centrioles were frequently observed on the neural side of *talpid*^*3*^ HCs ([Figure 3Kii](#fig3){ref-type="fig"}).  We conclude that failure of centriolar migration in *talpid*^*3*^ cells results in abnormal cell polarization and stereocilia formation in HCs.  Because 49% of *talpid*^*3*^ cells did exhibit a centriole correctly localized yet ciliogenesis was completely disrupted, the failure of ciliogenesis may not only be due to impaired centriolar migration. 

Directional cell migration is also intimately linked to the localization of the centrosome between the leading edge of the migrating cell and the Golgi apparatus. * Talpid3*^*−/−*^ MEFs show abnormal cell migration ([@bib8]), and we therefore examined if the orientation of the Golgi apparatus to the leading edge of migrating cells was also disrupted by a loss of Talpid3 function in mouse, in an in vitro scratch assay ([Figure 3O,P](#fig3){ref-type="fig"}).  The angle between the leading edge and Golgi was taken as the angle of orientation, with an angle of 0° suggesting perfect alignment of the Golgi to the leading edge of the migrating cell ([Figure 3Q](#fig3){ref-type="fig"}).  The angle of orientation was within 40° in 69% of *wildtype* cells and 55% of *Talpid3*^*−/−*^ cells, whilst 20% of *Talpid3*^*−/−*^ cells exhibited orientation angles greater than 60° compared to 11% of *wildtype* cells ([Figure 3Q′](#fig3){ref-type="fig"}; *wildtype* cells = 132, *Talpid3*^*−/−*^ cells = 117 from two experiments; [Figure 3O--Q](#fig3){ref-type="fig"}), suggesting a reduction in intracellular polarization of the Golgi apparatus to the leading edge in the *Talpid3*^*−/−*^ MEFs ([Figure 3O,P](#fig3){ref-type="fig"}).  Thus, KIAA0586 (TALPID3;Talpid3) plays an essential role in the internal organization and polarization of cells, likely through its action on the centrosome. 

Abnormalities of intracellular organization, centriole maturation, and centriolar satellite dispersal in the neuroepithelium {#s2-7}
----------------------------------------------------------------------------------------------------------------------------

JBTS primarily affects the brain of the patients.  The choroid plexus is a highly polarized multiciliated neuroepithelium in which we have previously shown, as now in HCs, a failure of centrosome migration in *talpid*^*3*^ mutant chickens ([@bib54]).  To determine if *talpid*^*3*^ mutant neuroepithelia exhibit cell polarity defects, we examined the intracellular organization of choroid plexus cells in E8 *talpid*^*3*^ mutant chickens. * Wildtype* choroid plexus cells exhibited a distinctive polarization with an apical, centriolar zone (CZ, [Figure 4A](#fig4){ref-type="fig"}) above a separate zone of mitochondria (MZ, [Figure 4A](#fig4){ref-type="fig"}); the most apical mitochondria were found an average of 7 µm from the apical surface ([Figure 4C](#fig4){ref-type="fig"}).  In contrast, the mitochondria in *talpid*^*3*^ choroid plexus are found in the most apical zone, an average of 3 µm from the apical surface (m, [Figure 4B](#fig4){ref-type="fig"}), and centrioles are present throughout the cell (asterisk in [Figure 4B](#fig4){ref-type="fig"}).  We conclude that the neuroepithelium has an abnormal intracellular organization of centrosomes and mitochondria and therefore, like the HCs and migratory fibroblasts, is not correctly polarized.  Although we have previously suggested that a failure of centrosome migration to the apical surface is the primary reason that cilia fail to form ([@bib54]), our analysis of the HCs suggest an additional requirement for TALPID3 during ciliogenesis, independent of the centriole migration.  We therefore investigated the maturation of the mother centriole, crucial for the basal body to dock to the membrane and initiate ciliogenesis.  Subdistal appendages were identified in approximately 40% of *wildtype* and *talpid*^*3*^ centrioles (*wt* n = 35, *talpid*^*3*^ n = 48, [Figure 4D,E,G](#fig4){ref-type="fig"}), whereas distal appendages were noted in 28% of *wildtype* centrioles and only 6% of *talpid*^*3*^ centrioles ([Figure 4D--G](#fig4){ref-type="fig"}).  To determine if there was a loss of distal appendages, we examined localization of CEP164, a protein known to localize to the distal appendages of the mature mother centriole, the basal body.  CEP164 localized correctly at the mother centriole and not at the sister centriole, in both *wildtype* and *talpid*^*3*^ cells of the neuroepithelium and fibroblasts ([Figure 4H--M](#fig4){ref-type="fig"}).  However, CEP164 puncta were smaller, disorganized and frequently orientated away from the apical cell surface in *talpid*^*3*^ cells ([Figure 4K,L](#fig4){ref-type="fig"}).  This confirmed our previous EM analysis ([@bib61]) and data in this study, which demonstrated that centrioles frequently failed to migrate or orientate correctly in *talpid*^*3*^ cells.  Smaller sized CEP164 puncta also suggested that distal appendages were not formed normally in *talpid*^*3*^ cells.  As abnormal or absent distal appendages can result in elongation of the centriole due to improper capping, centriolar length was studied in *wildtype* and *talpid*^*3*^ choroid plexus cells ([Figure 4N--Q](#fig4){ref-type="fig"}).  Centrioles in *wildtype* tissue were on average 0.7 µm in length compared to 0.9 µm in the *talpid*^*3*^ chicken, suggesting that *talpid*^*3*^ centrioles may indeed fail to undergo complete maturation and are subsequently elongated ([Figure 4R](#fig4){ref-type="fig"}).10.7554/eLife.08077.007Figure 4.Loss of TALPID3 causes abnormal intracellular organization and centriolar orientation(**A**, **B**) The chicken choroid plexus at E8 is a highly polarized structure with docked centrioles (asterisk, **A**) identified within a clear centriolar zone apically (CZ, **A**) and a mitochondrial zone (MZ; m indicates mitochondria).  The *talpid*^*3*^ choroid plexus (**B**) lacks these defined zones, with mitochondria identified in the most apical zone (m, **B**) centrioles identified throughout the cell, failing to dock (asterisk, **B**).  Quantification of distance of mitochondria to cell surface (**C**). (** D**--**G**) *talpid*^*3*^ tissue is capable of producing mature centrioles. * Wildtype* centrioles (**D**) and *talpid*^*3*^ centrioles (**E**, **F**) exhibited subdistal appendages (SD), and distal appendages (DA), although DA were less frequently observed on *talpid*^*3*^ centrioles, quantified in (**G**).  CEP164 localizes to the distal mother centriole in *wildtype* and *talpid*^*3*^ choroid plexus neuroepithelium (purple arrow indicated distal mother centriole, green arrow proximal centriole; **H**, **I**, **I′**, **I′′**, **K**, **L**, **L′**, **L′′**) and fibroblasts (**J**, **J′**, **J′′**, **M**, **M′**, **M′′**), but CEP164 puncta are smaller and disorganized in *talpid*^*3*^ choroid plexus and fail to orientate to the apical surface of the cell (arrows **L**).  Centrioles in *wildtype* tissue were on average 0.7 µm (red line indicating centriole/basal body; **N**, **R**) compared to 0.9 µm in the *talpid*^*3*^ choroid plexus (**O**, **P**, **Q**, **R**).  Scale bars: **A**, **B** = 1 μm, **D**, **E**, **F** = 100 nm; **H**, **K** = 10 μm **I**, **J**, **L**, **M** = 5 μm, **N**, **O**, **P**, **Q** = 200 nm.**DOI:** [http://dx.doi.org/10.7554/eLife.08077.007](10.7554/eLife.08077.007)

In human cells, KIAA0586 is also required for centriolar satellite dispersal ([@bib35]).  Compatible with this, we observed electron-dense condensations around the centrioles in the neuroepithelium of *talpid*^*3*^ chicken, which were absent from *wildtype* centrioles (basal body; 80% of wildtype cell exhibited electron-dense clear area around the centriole, whereas only 21% of *talpid*^*3*^ cells did; *wt* n = 35, *talpid*^*3*^ n = 48; [Figure 5A,D,G](#fig5){ref-type="fig"}).  To determine if these were centriolar satellites, we examined the localization of PCM1, a marker for centriolar satellites.  Compared to *wildtype* centrioles ([Figure 5B,C](#fig5){ref-type="fig"}), PCM1 puncta were larger around *talpid*^*3*^ centrioles ([Figure 5E,F](#fig5){ref-type="fig"}), possibly reflecting an increase in centriolar satellites.  Because we observed KIAA0586 immunostaining around the pericentriolar region ([Figure 2G,H](#fig2){ref-type="fig"}), we used the centriolar satellite marker AZI1 in human RPE1 cells to determine if KIAA0586 localized to centriolar satellites ([Figure 5H--J](#fig5){ref-type="fig"}) but found that KIAA0586 and AZI1 did not colocalize.  Thus, as observed in human cell lines, TALPID3 is essential for centriolar satellite dispersal.  As TALPID3 protein does not localize to the centriolar satellites, we assume that this is an indirect consequence of TALPID3 deficiency.10.7554/eLife.08077.008Figure 5.Analysis of centriolar satellites in the *talpid^3^* choroid plexus. An area clear of electron-dense condensations was observed around the basal body in *wildtype* cells (area outlined by dots; **A**), electron-dense condensations were observed adjacent to *talpid*^*3*^ centrioles (indicated by arrows, **D**).  Quantified in (**G**).  Immunostaining for a centriolar satellite marker in the choroid plexus, PCM1 (magnified area outlined by dashed line; PCM1 = red, γ tubulin, green **B**, **B′**, **C**,**C′**, **C′′**, **E**, **E′**, **F**, **F′**, **F′′**).  KIAA0586 protein does not colocalize with AZI1, a satellite protein in human RPE1 cells (KIAA0586 = red, AZI1 = green **H**, **I**, **J**).  Scale bars: **A**, **D** = 500 nm; **B**, **E** 10 = μm; **C**, **F** = 2 μm **H**, **I**, **J** 5 μm.**DOI:** [http://dx.doi.org/10.7554/eLife.08077.008](10.7554/eLife.08077.008)

We conclude that KIAA0586 (TALPID3) is essential for several distinct roles in centriole function, including centriole migration and orientation which can subsequently affect cell and tissue polarity and ciliogenesis, centriole maturation which affects docking of the basal body and ciliogenesis and through an indirect mechanism, centriolar satellite dispersal, which may also affect ciliogenesis. 

Discussion {#s3}
==========

JBTS is a genetically heterogeneous condition, caused by mutations in several genes related to the structure and function of cilia ([@bib47]).  Through homozygosity mapping and WES, we identified a novel disease locus (*JBTS23*), defined by mutations in the *KIAA0586* gene, encoding a centrosomal protein ([@bib5]) ([Figure 1](#fig1){ref-type="fig"}), which is supported by simultaneous concurrent studies ([@bib7]; [@bib48]).  We used *Talpid3/TALPID3*^*−/−*^ mouse and chicken models to understand the corresponding pathomechanisms causing the phenotypes of these patients and discovered centrosome abnormalities and loss of cell polarity. 

We confirm localization of KIAA0586 at centrosomal structures at the basal bodies and the adjacent daughter centrioles of primary cilia of mouse and human photoreceptor cells as well as in pericentriolar regions ([Figure 2](#fig2){ref-type="fig"}).  KIAA0586 has previously been associated with recessive ciliopathy phenotypes in mouse ([@bib8]; [@bib21]), chicken ([@bib19]; [@bib20]) and zebrafish ([@bib11]).  These animal models have either naturally occurring or induced 5′ mutations which disrupt an essential coiled-coil domain, resulting in loss of protein function, consecutive loss of Hh signaling and early embryonic lethality.  The *talpid*^*3*^ chicken is a thoroughly examined animal model with polydactyly, holoprosencephaly, abnormal neural tube patterning, polycystic kidneys, liver fibrosis, short ribs, and endochondral bones with defective ossification ([@bib37]; [@bib13]; [@bib19]; [@bib21]). 

The c.428delG (p.Arg143Lysfs\*4) mutation was identified in heterozygous state in patient MD1, *in trans* to a nonsense mutation ([Figure 1B](#fig1){ref-type="fig"}), and in a patient G2 who is also heterozygous for a *KIF7* (*JBTS12*) frameshift mutation and variants in three other known ciliopathy genes ([Figure 1C](#fig1){ref-type="fig"}) ([@bib17]).  Our experiments did not indicate epistatic interaction between *KIAA0586* and *KIF7*, and a secondary occult mutation in either gene cannot be excluded.  The c.428delG mutation results in a premature termination codon in five human *KIAA0586* isoforms, causing either a major protein truncation 5′ to the essential coiled-coil domain or NMD.  It is comparable to the *talpid*^*3*^ chicken loss-of-function mutation which introduces a frameshift in the orthologous region ([Figure 1F](#fig1){ref-type="fig"}).  The c.428delG mutation is annotated in dbSNP (rs534542684), and its MAF in the general population is surprisingly high (0.39%, 378 out of 96,534 alleles in the ExAC db), reminiscent of the most common deafness (c.35delG in *GJB2*; 0.60% in the ExAC database) or cystic fibrosis (p.Phe508del in *CFTR*; 0.67% in the ExAC database) mutation.  In two concurrent studies reporting *KIAA0586* mutations in patients with JBTS, c.428delG represented the most prevalent mutation ([@bib7]; [@bib48]).  While c.428delG was clearly enriched in patients with biallelic *KIAA0586* mutations in both studies (present in 20 of 24 such patients), only two were homozygous.  Despite its commonness, c.428delG was neither observed in homozygous state in healthy individuals in the TGP, ESP, or ExAC databases.  Such rarity of homozygosity could indicate that it causes embryonic lethality, early death or severe illness leading to underrepresentation of the respective samples.  Embryonic lethality in *talpid*^*3*^ chicken and *Talpid3*^*−/−*^ knockout mice would support such an interpretation.  On the other hand, c.428delG was not found in a simultaneous study that reports biallelic *KIAA0586* mutations in early lethal ciliopathies ([@bib4]).  Of note, a very recent study on rare human knockouts identified in the genomes of 2636 healthy Icelanders lists one individual of 57 years with homozygosity for c.428delG ([@bib55]).  This could either be due to protective modifiers or a low mutational load in the ciliome of the respective person.  Assuming the latter, c.428delG~*KIAA0586*~ could represent a hypomorphic allele that increases susceptibility to develop JBTS, with more severe mutations required either *in trans* (in heterozygous carriers, as in most patients reported by [@bib7]; [@bib48]), or in other genes (in homozygous carriers) for disease manifestation.  The presence of a heterozygous potentially deleterious *C5orf42* (*JBTS17*) variant in the only c.428delG~*KIAA0586*~-homozygous patient reported by [@bib7], and the co-occurence of such variants in four ciliopathy genes in patient G2 (including a truncation in the JBTS gene *KIF7*) support the categorization of c.428delG~*KIAA0586*~ as a hypomorphic mutation of incomplete penetrance.  Of note, no secondary *KIAA0586* mutation was identified in c.428delG-heterozygous JBTS patients in the two other studies ([@bib7]; [@bib48]), which could be due to the contribution of other genes. 

The homozygous mutations c.2414-1G\>C (Family 1) and c.2512C\>T (p.Arg838\*, Family 2) would not disrupt the 5′ functionally essential coiled-coil domain in the consecutive KIAA0586 protein, and partial function may be maintained (possibly due to preserved, albeit truncated, KIAA0586 protein).  We have shown that KIAA0586 has several functions in the centriole, and this may be mediated by different protein residues. 

The occurrence of retinal degeneration in JBTS depends on the genetic subtype, but is variable even within a family.  The localization of KIAA0586 at the ciliary base of retinal photoreceptor cells corresponds to other JBTS proteins.  Proteins of the periciliary compartment at the base of the photoreceptor cilium are thought to be critical for the handover of cargo from the dynein-mediated transport through the inner segment to the kinesin-powered anterograde intraflagellar transport in the ciliary compartment ([@bib46]; [@bib50]).  KIAA0586 may be part of the protein networks implicated in these processes.  The lack of retinal disease in the patients described herein may be due to the less strongly developed structure of the distal appendages and/or the possible functional redundancy in the cilia of retinal photoreceptor cells.  Nevertheless, patients with *KIAA0586*-related JBTS should be investigated for signs of retinal degeneration, and given that mutations in the JBTS gene *CEP290* may cause non-syndromic Leber congenital amaurosis ([@bib22]), *KIAA0586* represents a candidate gene for isolated retinopathies. 

Loss of KIAA0586 (TALPID3) function in animal models results in a failure to produce both primary and motile cilia.  Previously, it has been suggested that this is due to a failure of the centrosome to migrate apically or dock at the plasma membrane ([@bib61]; [@bib54]).  The subsequent failure of cilia formation results in abnormal Hh signaling and disrupted GLI processing ([@bib19]).  Most patients with *KIAA0586*-related JBTS exhibit few classical Hh phenotypes such as polydactyly (this study, [@bib7]; [@bib48]), unlike the corresponding mouse, chicken, and zebrafish models ([@bib19]; [@bib8]; [@bib11]).  We show that, independent of Hh signaling, cell and tissue polarity are disrupted upon loss of *TALPID3*.  JBTS is characterized by cerebellar hypoplasia and loss of decussation of neuronal projections from the cerebellum ([@bib47]).  While Hh signaling is required for controlling the growth of the embryonic cerebellar primordia ([@bib38]), the failure of decussation has been proposed to result from defective axonal guidance ([@bib47]), a process depending on centrosome-guided cell polarity ([@bib52]).  Furthermore, we have shown that in inner ear HCs, cell polarity and ciliogenesis, albeit closely linked, are differentially affected in *talpid*^*3*^ cells.  Thus, loss of decussation may reflect loss of polarity. 

We propose that KIAA0586 exerts a role in intracellular trafficking and cell polarity distinct from its role in docking of the centriole. * Talpid*^*3*^ cells have abnormal microtubule dynamics ([@bib61]).  Microtubules are required for the recruitment of satellites and proteins in the distal centriole ([@bib32]; [@bib49]), a process known to be impaired by loss of KIAA0586.  Abnormal cell polarity in *talpid*^*3*^ cells may be due to the effect of TALPID3 on microtubule dynamics and a direct role in centrosome organization: microtubules are essential for intracellular trafficking, cellular structure, and polarity.  We have shown that localization of the centrosome, mitochondria, and Golgi is disrupted in *talpid*^*3*^ cells.  Moreover, Rab8, a GTPase which binds to the Golgi and is required for vesicular trafficking and ciliogenesis ([@bib41]; [@bib30]; [@bib23]), is mislocalized in *KIAA0586*-depleted cells ([@bib35]).  In JBTS patients with mutations in *AHI1* (*JBTS3*), encoding an interactor of RAB8 ([@bib31]), non-ciliary trafficking from the Golgi and ciliogenesis are impaired.  Of note, Golgi mislocalization in the *talpid*^*3*^ choroid plexus is similar to what has been observed in *Ahi1*^*−/−*^ mice ([@bib31]).  This suggests a similar pathogenesis of *JBTS23* and *JBTS3*, with defective cell polarity, intracellular trafficking, and Hh signaling. 

KIAA0586 interacts with CP110 and Cep120 ([@bib35]), distal centriolar proteins implicated in centriole duplication and maturation, and ciliogenesis.  The predominant expression of Cep120 on the daughter centriole throughout most of the cell cycle depends on Kiaa0586, as indicated by high expression of Cep120 on both centrioles and absence of CP110 from the mother centriole prior to ciliogenesis in *Talpid3*^−/−^ cells ([@bib53]).  Although equal expression of KIAA0586 on the mother and daughter centrioles has been reported (this study, [@bib35]); there is evidence that KIAA0586 predominantly localizes at the mother centriole ([@bib60]).  In addition, loss of chicken *KIAA0586* (*TALPID3*) causes centriole elongation whereas overexpression of Cep120 causes elongation of the mother centriole, suggesting that KIAA0586 (TALPID3) may control centriole length through depletion or suppression of Cep120 on the mother centriole.  Similarly, depletion of CP110 also increases centriole length ([@bib49]), suggesting that KIAA0586 regulates centriolar length through controlling CP110 localization and centriolar capping of the distal mother centriole.  Loss of other centriolar proteins, such as OFD1, likewise results in elongated centrioles and loss of distal appendages ([@bib51]).  Based on the colocalization of KIAA0586, CP110, and Cep164, it has been proposed that KIAA0586 regulates ciliary vesicle docking adjacent to Cep164 localization ([@bib35]), but not distal appendage formation itself, and this is supported by evidence from human patient *KIAA0586*^*−/−*^ cells which show Cep164 within the distal centriole ([@bib4]).  We also find evidence for a vesicle docking defect, demonstrated by an increase in centriolar satellites.  However, we propose that *KIAA0586* loss primarily causes abnormal distal appendages and impaired Cep164 localization, similar to what can be observed in OFD1 mutants ([@bib51]).  In addition, determination of Cep164 expression in cells of highly polarized tissue demonstrates a further centriolar defect not easily distinguished in in vitro assays---the loss of centriole orientation to the apical membrane of the cell.  Whether this defect is due to the depletion of KIAA0586 from the centriole or impairment of another function of KIAA0586 in pericentriolar regions or cytoskeleton remains to be elucidated. 

We have identified *KIAA0586* as a novel gene for JBTS, and we propose that it is not only required for ciliogenesis, but also to establish cell and thus tissue polarity. * JBTS23*, and possibly other JBTS subtypes, may result from impairment of both functions. 

Materials and methods {#s4}
=====================

Patients {#s4-1}
--------

Blood samples for DNA extraction were obtained with written informed consent.  All investigations were conducted according to the Declaration of Helsinki, and the study was approved by the institutional review board of the Ethics Committees of the University of Erlangen-Nürnberg, the University of Bonn, and the University Hospital of Cologne. 

Genetic analysis of human JBTS families {#s4-2}
---------------------------------------

In accordance with the Human Gene Nomenclature Committee (HGNC), we have used *KIAA0586*/KIAA0586 for designation of the human gene and protein, respectively.  In accordance with the Chicken Gene Nomenclature Committee (CGNC), we use *TALPID3*/TALPID3 for designation of the chicken gene and protein, respectively.  Although the current gene symbol for the mouse gene is *2700049A03Rik* (protein: 2700049A03RIK), we use *Talpid3*/Talpid3 as the gene and protein names, respectively.  Where we refer to a generic conclusion on the function of the orthologs of *KIAA0586*, we use *KIAA0586*.  As in previous publications, the chicken model is referred to as *talpid*^*3*^, and the mouse model is referred to as *Talpid3*^*−/−*^. The nomenclature of human *KIAA0586* mutations refers to reference sequence NM_001244189.1 (corresponding protein: NP_001231118.1).  The Exome Aggregation Consortium (ExAC) database (Cambridge, MA, United States; <http://exac.broadinstitute.org>), which aggregates numerous databases including the current versions of the Exome sequencing project (ESP, [@bib25]) and the Thousand Genomes Project (TGP, [@bib59]) was last accessed on 11 July 2015 for the presence and frequency of identified variants in healthy individuals. 

Family 1: genotyping and homozygosity mapping were performed in Family 1 (MR026) as previously reported ([@bib1]).  DNA from patient MR026-01 underwent exome capture and whole-exome sequencing (WES) using the SureSelect Human All Exon 50 Mb Kit (Agilent Technologies, Santa Clara, United States) and a SOLiD4 instrument (Life Technologies, Carlsbad, United States) as described previously ([@bib2]).  Of the targeted regions, 73.2% were covered at least 20×, and 83.4% were covered at least 5×. To validate the results, we also conducted WES in the likewise affected sibling, MR026-04, analogous to previously described disease gene identification approaches ([@bib3]; [@bib45]).  96% of the target sequence were covered at least 20×.

Family 2: samples from the index patient, MD1, and her parents underwent WES at GeneDX (Gaithersburg, MD, United States). 

Family 3: WES and mapping of reads for the index patient (G2) and both parents were carried out as previously described ([@bib9]; [@bib10]).  In brief, filtering and variant prioritization was performed using the varbank database and analysis tool ([https://varbank.ccg.uni-koeln.de](https://varbank.ccg.uni-koeln.de/)) of the Cologne Center for Genomics.  In particular, we filtered for high-quality (coverage \>15-fold; phred-scaled quality \>25), rare (MAF \[minor allele frequency\] ≤0.01) variants (dbSNP build 135, the 1000 Genomes database build 20110521, and the public Exome Variant Server, NHLBI Exome Sequencing Project, Seattle, build ESP6500).  To exclude pipeline-related artifacts (MAF ≤ 0.01), we filtered against variants from in-house WES data sets from 511 patients with epilepsy.  The Affymetrix genome-wide Human SNP Array 6.0 utilizing more than 906,600 SNPs and more than 946,000 copy number probes was used for genome-wide detection of copy number variations in patient G2.  Quantitative data analyses were performed with GTC 3.0.1 (Affymetrix Genotyping Console) using HapMap270 (Affymetrix) as reference file.  In the index patient (G2), all coding *KIAA0586* and *KIF7* exons were Sanger-sequenced in search of a second mutation.  In addition, we amplified and sequenced all *KIAA0586* exons from cDNA (derived from whole blood mRNA, PAXgene Blood RNA Tube, PreAnalytiX, Hombrechtikon, Switzerland) in search of potential hints of aberrant splicing due to extra-exonic variants.  Continuous PCR amplification of *KIF7* exons from whole blood mRNA was not successful.  The sample of patient G2 was analyzed by genome-wide CGH (Affymetrix 6.0 SNP array) to exclude structural alterations adjacent to or within *KIAA0586*, *KIF7*, *CEP41*, *KIF14*, or *WDPCP*.  Confirmation of the identified mutations and segregation analyses were carried out by Sanger sequencing. 

RT-PCR {#s4-3}
------

In Family 1, we isolated mRNA using the RNeasy kit (Qiagen, Hilden, Germany) from lymphoblastoid cell lines that have been established based on standard protocols from patients MR026-01 and MR026-04.  We transcribed mRNA to cDNA using SuperScriptII reverse transcriptase and random primers (Invitrogen; Van Allen Way Carlsbad, California, United States).  To test if the *KIAA0586* mutation c.2414-1G\>C impairs splicing, we used two pairs of primers (*KIAA0586*\_exprF1, 5′-TCCATCTCCTAAGTCCAGACCAC-3′ and *KIAA0586*\_expR1, 5′-TCCAAGTTTGCACAGGAGG-3′, located in exons 16 and 19, and *KIAA0586*\_exprF2, 5′-TCAGGTACATTGGAAGGTCATC-3′ and *KIAA0586*\_expR2, 5′-AACTGGCGGAAATGGAGG-3′, located in exons 17 and 21; NM_001244189.1) and standard PCR methods.  Electrophoresis on standard agarose gel followed by cutting out the DNA bands, purifying the DNA using QIAquick gel extraction kit (QIAgene; Hilden, Germany), and Sanger sequencing were performed. 

Animal models {#s4-4}
-------------

Eggs were obtained from *talpid*^*3*^ flock (MG Davey; *talpid*^*3*^ chicken lines are maintained at the Roslin Institute under UK Home Office license 60/4506 \[Dr Paul Hocking\], after ethical review).  Mice were maintained at the Human Genetics Unit, Western General, Edinburgh, under UK Home Office license PPL 60/4424 (Ian Jackson).  The *Talpid3*^*+/−*^*/Kif7*^*+/−*^ line was produced by crossing of the previously described *Talpid3*^*+/−*^ knockout mouse line ([@bib8]) and the reported *Kif7*^*+/−*^ mouse line ([@bib15]).  Animal experiments carried out at the JGU Mainz corresponded to the statement of the Association for Research in Vision and Ophthalmology (ARVO) as to care and use of animals in research.  Adult mice were maintained under a 12-hr light--dark cycle, with food and water ad libitum. 

Incubation and dissection of animal models {#s4-5}
------------------------------------------

Chicken eggs from *talpid*^*3*^ flock were incubated at 38°C until 12 days at the latest, staged as per [@bib28], dissected into cold PBS, and fixed in 4% PFA/PBS.  Mouse timed matings were established between *Talpid3*^*+/−*^ mice ([@bib8]) and *Kif7*^*+/−*^ mice ([@bib15]) and confirmed by vaginal plug.  Pregnant females were sacrificed at day 10 of pregnancy for production of mouse embryonic fibroblasts (MEFs).  Otherwise between day 12 and 16 of pregnancy and embryos were dissected in cold PBS, decapitated, and fixed immediately in 4% PFA for histological analysis.  Pups were sacrificed between 7 and 21 days after birth by lethal injection and brains were dissected into 4% PFA/PBS. 

Chicken and mouse genotyping {#s4-6}
----------------------------

Embryos used in comparisons were dissected as family groups and genotyped after analysis.  Tissues were collected on dissection, lysed in 10 mM Tris (pH8), 10 mM EDTA (pH 8), 1% SDS, 100 mM NaCl, and 20 mg/ml proteinase K at 55°C overnight before DNA extraction using Manual Phase Lock Gel Tubes (5 Prime) for phenol/chloroform extraction.  For chicken *TALPID3*, sequencing primers used were 5′-TCATTTCATTAGCTCTGCCG-3′ (forward) and 5′-CCATCAAACCAACAGCTCAG-3′ (reverse).  For mouse *Talpid3*, PCR primers were 5′-TGCCATGCAGGGATCATAGC (forward), 5′-GAGCACACTGGAGGAAAGC-3′ (reverse) and 5′-GAGACTCTGGCTACTCATCC-3′, 5′-CCTTCAGCAAGAGCTGGGGAC-3′, respectively.  For mouse *Kif7*, PCR primers were 5′-CACCACCATGCCTGATAAAAC-3′ (P1 forward), 5′-CTATCCCCAATTCAAAGTAGAC-3′ (P1 reverse), 5′-CCAAATGTGTCAGTTTCATAGC-3′ (P2 forward), 5′-TTCTCACCCAAGCTCTTATCC-3′ (P2 reverse). 

Histology {#s4-7}
---------

Fixed samples from mouse brain and chicken legs were embedded in paraffin, sectioned, and stained in haematoxylin and eosin as described previously ([@bib21]). 

Wholemount RNA in situ hybridization {#s4-8}
------------------------------------

Mouse and chicken embryos were rehydrated through a methanol gradient and in situ hybridization carried out for chicken β-catenin (codons 1--127) as previously described ([@bib42]). 

In ovo knockdown of *Kif7* in chicken {#s4-9}
-------------------------------------

The following *Kif7* sequences were targeted for knockdown: Target 1: TTATCGACGAGAACGACCTCAt, Target 2: cATCCAGAACAAAGCGGTGGTG, Target 3: gTCCTCTAACACTAAGAACATT, Target 4: gACAGATGACATAGTCCGTGTG to which 22mer sequences were designed in Genscript and cloned into pRFPRNAiC ([@bib18]) (Dundee Cell Products, Dundee, United Kingdom).  Embryos were electroporated at stage 12HH (as described [@bib61]), observed for RFP expression at stage 24HH, fixed, and prepared for sectioning and immunohistochemistry at stage 22HH as below.  Tissue from embryos was collected and genotyped. 

Cell culture and immunocytochemistry {#s4-10}
------------------------------------

MEFs were prepared from E10.5 eviscerated and decapitated embryos.  Cells were dissociated in trypsin/versin and maintained to passage 2 as per [@bib27] and serum removed from media for 48 hr to induce ciliogenesis.  RPE1 cells (ATCC) were grown in DMEM-F12, 10% FCS Gold, 50 μl hygromycin, 5 ml L-glut.  IMCD3 (mouse inner medullary collecting duct cells) cells were grown in DMEM-F12 10% FCS.  To induce ciliogenesis, RPE1 and IMCD3 cells were starved in DMEM:F12 or Opti-MEM I (Life Technologies, Carlsbad, California, United States) for 72 hr.  Cells were fixed with methanol at −20°C for 2--5 min.  After washing in PBS, cells were immunolabeled with polyclonal antibodies against acetylated tubulin (T7451, Sigma-Aldrich, St. Louis, Missouri, United States), pericentrin-2 (sc-28145, Santa Cruz Biotechnology, Dallas, United States), and KIAA0586 (HPA000846, Atlas Antibodies, Stockholm, Sweden) before incubation with appropriate secondary antibodies conjugated to Alexa 488 (A21206, Molecular Probes, Invitrogen), CF 568 (20106-1, Biotrend, Köln, Germany), and CF 640 (20177, Biotrend), and with DAPI (6335.1, Roth, Karlsruhe, Germany). 

Immunohistochemistry {#s4-11}
--------------------

Eyes from a healthy human donor (\#199-10; 56 years of age, dissection 29 hr post mortem) were obtained from the Department of Ophthalmology, University Hospital of Mainz, Germany, according to the guidelines of the declaration of Helsinki.  After sacrifice, eyeballs from adult C57BL/6J mice were dissected, cryofixed in melting isopentane, cryosectioned and immunostained as previously described ([@bib43]).  Cryosections were incubated with monoclonal antibodies to centrin-3 as a molecular marker for the ciliary apparatus of photoreceptor cells as previously characterized ([@bib57]), and polyclonal antibodies against KIAA0586 (Atlas HPA000846).  Washed cryosections were incubated with appropriate antibodies conjugated to Alexa 488 (Molecular Probes A21206) and Alexa 568 (Molecular Probes A11031) in PBS with DAPI (Roth 6335.1) to stain the nuclear DNA and mounted in Mowiol 4.88 (Hoechst, Germany).  Specimens were analyzed in a Leica DM6000B deconvolution microscope (Leica, Germany).  Image contrast was adjusted with Adobe Photoshop CS using different tools including color correction.  For section immunocytochemistry on chicken tissue, chicken embryos were dissected into PBS, fixed, sectioned, and stained as described ([@bib19]), except for CEP164, in which an antigen retrieval step was undertaken (incubation in 0.1% BME/PBS for 5 min, incubation in 55°C PBS for 4 hr).  For bone sections, legs were dissected at E12.  For immunocytochemistry, cells were fixed as above.  Antibodies were used against: acetylated α-tubulin (Sigma--Aldrich T7451), γ-tubulin (Sigma--Aldrich T5192; T5326), TGN46 (Abcam, Cambridge, United Kingdom, ab16059), PCM1 (Abcam ab72443), AZI1 (kind gift of Jeremy Reiter, UCSF), centrin-3 ([@bib57]), KIAA0586 (Atlas Antibodies, HPA000846, ProteinTech 24421-1-AP), CEP164 (ProteinTech, Manchester, United Kingdom, 22227-1) RFP (Life Technologies R10367), GFP (Life Technologies A-21311), Pax7 (Developmental Studies Hybridoma Bank, Iowa City, United States (DSHB)), ISLET1 (DSHB), NKX2.2 (DSHB), Phalloidin (Life Technologies A12379), Anti-mouse (Life Technologies A11017), anti-rabbit (Life Technologies A21207).  Imagining was undertaken on a Zeiss LSM 710 or a Nikon Air confocal microscope or Leica DMLB. 

Conventional transmission electron microscopy {#s4-12}
---------------------------------------------

Chicken embryos were dissected into PBS at 8 days of incubation, avoiding contamination with yolk, heads were removed and placed into 4% PFA, 2.5% glutaldehdye in 0.1 M cacodylate buffer.  The choroid plexus was immediately removed and placed into fresh fixative (as previous) for 24 hr.  Tissue was prepared and visualized for transmission electron microscopy as described previously ([@bib20]), and axoneme/basal body structure was compared to what was observed and reported previously ([@bib44]). 

Immunoelectron microscopy analysis {#s4-13}
----------------------------------

Anti-KIAA0586 antibody (Atlas Antibodies, HPA000846) was used for pre-embedding labeling in mouse retinas as previously described ([@bib40]; [@bib50]).  Ultrathin sections were cut on a Leica Ultracut S microtome and analyzed with a Tecnai 12 BioTwin transmission electron microscope (FEI, The Netherlands).  Images were obtained with a charge-coupled device SIS Megaview3 SCCD camera (Surface Imaging Systems, Herzogenrath, Germany) and processed with Adobe Photoshop CS. 

Cell polarization and cilia length measurements {#s4-14}
-----------------------------------------------

Angles of proliferation, migration, orientation, and localization were calculated using Axiovision Angle3 software, and cilium length was measured using Zen software (Zeiss, Oberkochen, Germany).  Scratch assays were carried out in *wildtype* and *Talpid3*^*−/−*^ MEFs grown to confluence and serum starved (DMEM + 0.5% FCS) for 48 hr with a p10 pipette tip.  Medium was then renewed and MEFs incubated for four hours before fixation in ice-cold methanol prior to immunofluorescence.  Angles of orientation were then taken as a measurement of the angle from the center of the nucleus, through the center of the leading edge (towards the wound, identified by phalloidin staining for F-actin) and again through the center of the Golgi apparatus (identified by TGN46 antibody staining).  Tiled Z stacks of the scratch/wound were analyzed for greater accuracy. 

The expected orientation of the stereocilia of the basilar papilla hair cells were taken as being at 90° to the abneural edge of the basilar papilla.  The angle of orientation was taken by drawing a line through the cell perpendicular to the abneural edge and a second from the center of the cell, intersecting with both the perpendicular line and center of the actin bundle.  The internal angle was taken to be the angle by which cell orientation deviated from the expected.  Cilium length was measured using Zen software (Zeiss, Oberkochen, Germany). 

Funding Information
===================

This paper was supported by the following grants:

-   BBSRC Career Track Fellowship Funding BB/F024347/1 to Louise A Stephen, Gemma M Davis, Lynn McTeir, Megan G Davey. 

-   European Community FP7/2009/241955 (SYSCILIA) FP7/2009/241955 to Lars Tebbe, Uwe Wolfrum. 

-   FAUN-Stiftung to Uwe Wolfrum. 

-   Foundation Fighting Blindness (FFB) to Lars Tebbe, Uwe Wolfrum. 

-   Imhoff-Stiftung to Hanno J Bolz. 

-   Stiftung Auge (Deutsche Ophthalmologische Gesellschaft) to Hanno J Bolz. 

-   Deutsche Heredo-Ataxie-Gesellschaft to Hanno J Bolz. 

-   Deutsche Forschungsgemeinschaft (DFG) AB393/2-2 to Hasan Tawamie, André Reis, Arif B Ekici, Rami Abou Jamra. 

-   BBSRC ISPG to Louise A Stephen, Gemma M Davis, Lynn McTeir, Amy M Fraser, Megan G Davey. 

-   BBSRC EastBio Studentship to Amy M Fraser. 

-   University Hospital of Cologne to Hanno J Bolz. 

We are indebted to the families who participated in our study.  We thank Prof. Chi-chung Hui, Department of Molecular Genetics, University of Toronto, for the kind gift of the *Kif7* mouse line, Prof. Andrew Forge for help with basal papilla dissection, John James, CHIPs, University of Dundee, United Kingdom, Maurits Jansen of Edinburgh Preclinical Imaging, University/BHF Centre for Cardiovascular Science, University of Edinburgh for technical help, ESRIC for support with advanced imaging, Dr Denis Headon for the kind gift of the β-catenin probe and Dr Jeremy Reiter for the kind gift of the Azi1 antibody.  We thank Elisabeth Sehn, and Gabi Stern-Schneider (both JGU Mainz) for their skillful technical assistance. 

Additional information {#s5}
======================

The authors declare that no competing interests exist. 

LAS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

GMD, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

LMT, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

EAH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

HT, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

PN, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

GN, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

HT, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

MT, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

EB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

SU, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

AMF, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

PM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. 

LT, Acquisition of data. 

OR, Acquisition of data. 

AR, Conception and design, Drafting or revising the article. 

ABE, Acquisition of data, Drafting or revising the article. 

ND, Conception and design, Acquisition of data, Drafting or revising the article, Contributed unpublished essential data or reagents. 

CC, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents. 

UW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents. 

MGD, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents. 

HJB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents. 

RAJ, Clinical examination of patients, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents. 

Human subjects: Blood samples for DNA extraction were obtained with written informed consent.  All investigations were conducted according to the Declaration of Helsinki, and the study was approved by the institutional review board of the Ethics Committees of the University of Erlangen-Nürnberg, the University of Bonn, and the University Hospital of Cologne. 

Animal experimentation: Talpid3 chicken lines are maintained at the Roslin Institute under UK Home Office license 60/4506 \[Dr Paul Hocking\], after ethical review.  Animal experiments carried out at the JGU Mainz corresponded to the statement by the Association for Research in Vision and Ophthalmology (ARVO) as to care and use of animals in research. 

Additional files {#s6}
================

Major datasets {#s6-1}
--------------

The following previously published datasets were used:

Kobayashi T, Kim S, Lin YC, Inoue T, Dynlacht BD[@bib35a], 2014,Homo sapiens KIAA0586 (KIAA0586), transcript variant 1, mRNA,<http://www.ncbi.nlm.nih.gov/nuccore/NM_001244189.1>,Publicly available at the NCBI Gene Expression Omnibus (Accession no: NM_001244189.1). 

Kobayashi T, Kim S, Lin YC, Inoue T, Dynlacht BD[@bib35b], 2014,Homo sapiens KIAA0586 (KIAA0586), transcript variant 3, mRNA,<http://www.ncbi.nlm.nih.gov/nuccore/NM_001244191.1>,Publicly available at the NCBI Gene Expression Omnibus (Accession no: NM_001244191.1). 

10.7554/eLife.08077.009

Decision letter

Dietz

Harry C

Reviewing editor

Howard Hughes Medical Institute and Institute of Genetic Medicine, Johns Hopkins University School of Medicine

,

United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors).  An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown.  Reviewers have the opportunity to discuss the decision before the letter is sent (see [review process](http://elifesciences.org/review-process)).  Similarly, the author response typically shows only responses to the major concerns raised by the reviewers. 

Thank you for submitting your work entitled "*TALPID3* is a regulator of cell polarity and mutated in Joubert syndrome (*JBTS24*)" for peer review at *eLife*.  Your submission has been favorably evaluated by Stylianos Antonarakis (Senior editor) and two reviewers, one of whom, Harry Dietz, is a member of our Board of Reviewing Editors. 

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.  As you will see, both reviewers expressed considerable enthusiasm regarding this study and manuscript, but expressed some suggestions and concerns that need to be addressed prior to formal acceptance. 

While your manuscript nicely documents that deficiency of TALPID3 causes Joubert syndrome and associates will alterations of cellular polarity, it is essential to provide further comment regarding documented or potential links between mechanism and phenotype and to avoid conclusion that your findings document a direct role for TALPID3 in PCP signaling, as opposed to indirect involvement through regulation of centrosome architecture and migration. 

A number of minor points also require your consideration:

1\) The details regarding the various aberrant transcripts induced by the splice site mutation are somewhat unclear.  Why, for example, is the 108bp deletion expected to result in NMD?  Why is the protein shown as truncated for this in-frame deletion, but not for the out-of-frame 188bp deletion (if I am reading [Figure 1](#fig1){ref-type="fig"} correctly)? 

2\) There seems to be excessive discussion about family 3 despite substantial ambiguity regarding the etiology of disease.  I think that this could be downplayed in the text. 

3\) What is the rational for putting Figure 6 in sixth position when it is discussed before [Figure 2](#fig2){ref-type="fig"}?  Although no genetic interaction is observed these are important results for discerning the underlying causal mutations in patient G2 and therefore warrant a more detailed explanation in the results. 

4\) In the second paragraph of the subsection "Loss of TALPID3 disrupts planar cell polarity patterning", the authors note that *talpid*^*3*^ mutant mice lack columnar chondrocytes in the long bones and state that this is similar to loss of PCP signaling.  The long bone phenotype observed in the *talpid*^*3*^ mouse is more akin to Ihh mutants and does not resemble PCP mutants, which are able to form columnar chondrocytes however they are misaligned. 

5\) Unlike in long bones of the *talpid*^*3*^ mouse, the authors report that columnar chondrocytes are present in the *talpid*^*3*^ chicken leg.  Higher magnification images are needed of data given in [Figure 3E and F](#fig3){ref-type="fig"} so it is possible to see this.  The authors also report development of a poorly defined bone collar.  H and E staining is not sufficient to conclude this, Alizarin red would give a definitive answer. 

6\) Please give evidence of *talpid*^*3*^ columnar chondrocytes where cell division angle is abnormal.  Suggest giving higher magnification image of data in [Figure 3G and H](#fig3){ref-type="fig"}.  Please provide numbers of cells counted.  Graph [Figure 3I](#fig3){ref-type="fig"} shows 11% of Wt cells align within 25o of expected angle, while in the Results the authors state that all Wt cells align within 20°. 

7\) In [Figure 3J, K](#fig3){ref-type="fig"}, what are the VANGL2 foci and what is the precedence that this is a marker of polarity in columnar chondrocytes?  Please indicate in the figure examples of cells counted as having one or two foci or dispersed VANGL2 expression, higher magnification images may help.  Please provide numbers of cells counted and statistical analysis. 

8\) In Figure 3M and N, Golgi staining hard to see, orientation of leading edge hard to interpret from images shown.  Please indicate how orientation of Golgi in relation to the leading edge was measured.  Please provide numbers of cells counted and statistical analysis. 

9\) It is unclear from the data shown in [Figure 4C--H](#fig4){ref-type="fig"} how the conclusions were drawn.  We suggest providing higher magnification images clearly marking the axis of the 'expect orientation' and how actin bundles are orientated in relation to this in Wt cells but misorientated in *talpid*^*3*^ cells as well as indicating *talpid*^*3*^ cells with actin across the apical cell surface.  Give higher magnification of centrosomes localized in bare zones in Wt and not in *talpid*^*3*^. Although actin bundles appear to be orientated differently in *talpid*^*3*^ compared to Wt, $\text{γ}$-tubulin staining is present within bare zones in *talpid*^*3*^ in the images shown.  Please provide numbers of cells counted and perform statistical analyses for these experiments. 

10\) Maturation of the mother centriole to a basal body requires assembly of subdistal and distal appendages.  The authors clearly show presence of subdistal appendages in *talpid*^*3*^ mutant centrosomes but evidence of distal appendages given in [Figure 5E](#fig5){ref-type="fig"} is ambiguous.  Immunofluorescence staining with antibodies against distal appendage proteins such as CEP164 would give a definitive answer as to presence of distal appendages on *talpid*^*3*^ basal bodies. 

11\) More extensive description of data given in Figure 7 required in the text.  Consider quantifying PCM1 staining between Wt and *talpid*^*3*^.

10.7554/eLife.08077.010

Author response

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.  As you will see, both reviewers expressed considerable enthusiasm regarding this study and manuscript, but expressed some suggestions and concerns that need to be addressed prior to formal acceptance. 

While your manuscript nicely documents that deficiency of TALPID3 causes Joubert syndrome and associates will alterations of cellular polarity, it is essential to provide further comment regarding documented or potential links between mechanism and phenotype

Our lab has a substantial background in investigating the links between the loss of TALPID3 and the 'disregulation of Hedgehog signaling' seen in animal models.  Previously, we have been able to associate this phenotype with a loss of ciliogenesis (i.e. mechanism).  Our study extends our understanding of the phenotype and mechanism.  In animal models, we demonstrate loss of cell polarity (which we believe prevents ciliogenesis and, consecutively, Hedgehog signaling).  We have increased the discussion of this area. 

The phenotype of the JBTS patients is different from what would be expected from a direct extrapolation from the animal models.  We discuss the potential reasons for this phenomenon in our revised manuscript.  Based on the work in the current study, we are now conducting a follow-up study to investigate if *TALPID3*-related loss of cell polarity alters axonal projections in the central nervous system, which may underlie the 'molar tooth sign' of the patients. 

We have reconfigured [Figure 3 and 4](#fig3 fig4){ref-type="fig"} into a single [Figure 3](#fig3){ref-type="fig"} so as to progressively highlight the link between the disruption of tissue polarity (i.e. skin) to cell polarity (basilar papilla) to centrosome localization (basilar papilla and in cell migration) in *TALPID3* mutant tissue.  We have then examined the centrosome architecture to show abnormal centrosomal length and polarized orientation (using Cep164- newly added) and finally examine pericentriolar abnormalities.  We feel that this strengthens the discussion of the potential pathomechanisms in *KIAA0586*-deficient patients (loss of tissue polarity and abnormal Hedgehog signaling) in relation to centriolar polarity. 

\[...\] and to avoid conclusion that your findings document a direct role for TALPID3 in PCP signaling, as opposed to indirect involvement through regulation of centrosome architecture and migration. 

We have replaced speculations that TALPID3 (KIAA0586) mutant tissue exhibits PCP defects by with a more generalized statement that TALPID3 is required for normal tissue polarity, cell polarity and correct positioning of centrosomes in polarized cells (as appropriate).  In addition, we have removed the work on bone development in which we used the PCP component VANGL2 as a marker of chondrocyte polarity, which we hope will also help avoiding this confusion. 

A number of minor points also require your consideration:

*1) The details regarding the various aberrant transcripts induced by the splice site mutation are somewhat unclear.  Why, for example, is the 108bp deletion expected to result in NMD?  Why is the protein shown as truncated for this in-frame deletion, but not for the out-of-frame 188bp deletion (if I am reading* [*Figure 1*](#fig1){ref-type="fig"} *correctly)?* 

We agree, and we apologize for the confusion.  We have redesigned [Figure 1](#fig1){ref-type="fig"}, now with corrected protein schemes for the three aberrant transcripts that result from the splice site mutation.  The designation of the splice site mutation has also been corrected into c.2414-1G\>C (in the previous version of the manuscript, the designation had erroneously been delineated from another (shorter) *KIAA0586* isoform). 

2\) There seems to be excessive discussion about family 3 despite substantial ambiguity regarding the etiology of disease.  I think that this could be downplayed in the text. 

We agree.  The patient of Family 3 (patient G2), carries the very common allele c.428delG. We have shortened the discussion on this specific patient, but we have modified and extended the general discussion of the c.428delG allele because of very recent literature which indicates that this mutation represents a highly interesting (and possibly underestimated) class of recessive mutations:

The concurrent study of [@bib48] (*eLife*)([@bib48]) and of [@bib6] (Human Mutation) identified this mutation in most of their JBTS patients with biallelic *KIAA0586* mutations -- but very rarely in homozygous state. 

The c.428delG mutation is listed with high frequency in databases of large-scale sequencing projects (ESP, TGP, ExAC), but not in homozygous state.  It was neither found in a recent study that identified biallelic *KIAA0586* mutations in early lethal ciliopathies. 

A very recent study about homozygous knockout alleles identified in healthy Icelanders listed one healthy adult with homozygosity for c.428delG. We speculate that c.428delG may represent a hypomorphic allele that requires a more severe *KIAA0586* mutation in *trans*, as is the case in most patients reported by us, Roosing et al.  and Bachmann-Gargescu et al.,  or additional mutations in other (ciliopathy) genes if c.428delG is present in homozygous state (as in case of the homozygous patient reported by Bachmann-Gargescu et al. 

We speculate that a similar mechanism could apply to patient G2 from Family 3, with double heterozygosity for truncations in two JBTS genes (*KIF7* and *KIAA0586*) and potentially deleterious variants in other ciliopathy genes.  However, we have shortened this part of the Discussion. 

Taken together, we think that the unusually common c.428delG allele may -- at least in some patients -- contribute to oligogenic rather than monogenic JBTS. 

*3) What is the rational for putting Figure 6 in sixth position when it is discussed before* [*Figure 2*](#fig2){ref-type="fig"}*?  Although no genetic interaction is observed these are important results for discerning the underlying causal mutations in patient G2 and therefore warrant a more detailed explanation in the results.* 

We apologize for this mistake.  We should have referred to supplementary figure 1, which is now [Figure 1--figure supplement 1](#fig1s1){ref-type="fig"} in the revised manuscript. 

*4) In the second paragraph of the subsection "Loss of* TALPID*3 disrupts planar cell polarity patterning", the authors note that* talpid^3^ *mutant mice lack columnar chondrocytes in the long bones and state that this is similar to loss of PCP signaling.  The long bone phenotype observed in the* talpid^3^ *mouse is more akin to Ihh mutants and does not resemble PCP mutants, which are able to form columnar chondrocytes however they are misaligned.* 

We agree with the reviewer that the *talpid*^*3*^ chicken mutant bone defect may primarily be an IHH defect, as we have described previously in the *talpid3*^-/-^ mouse conditional limb knockout ([@bib8]).  In the respective paper, we conclude that IHH signalling is severely affected in this ciliopathy mutant, based on comparisons with both IHH knockout mice and IFT knockout mice (Haycraft et al.,  2007).  We would also predict that this is true for the *talpid*^*3*^ chicken mutant -- particularly based on PTCH1/PTCH2 RNA in situ hybridization that shows loss of expression of these genes in *talpid*^*3*^ chicken, suggesting that IHH signaling is misregulated in the *talpid*^*3*^ endochondral bones (Davey, unpublished). 

We believe that the analysis of the *talpid*^*3*^ chicken endochondral bones has validity in describing the cell polarity defects that we observe in this mutant.  However, we agree with the reviewer (Point 4) that we cannot sufficiently distinguish the phenotype caused by the IHH signalling defect from Wnt-driven defects (particularly Wnt5a/b) to conclude that the cell polarity defects are not only due to a loss of IHH signalling. 

However, our results indicate phenotypic differences between the IHH^-/-^ mouse and *talpid3*^-/-^ conditional limb knockout; the bones are not only short as in the Ihh^-/-^ mutant mouse, but wider and fused and comparable to chicken in which the PCP pathway is manipulated (Li and Dudley, 2009)or in Wnt5a^-/-^ mice.  There is also soft tissue syndactyly, which is common in mutants such as Prickle1^-/-^ mice.  Thus, signaling through the Wnt PCP pathway may also been altered.  IHH signaling is upstream of Wnt5a expression, an important regulator of cell polarity in the bone (Gao et al.,  2011) and it is therefore possible that IHH regulates cell polarity through Wnt5a.  This has not been tested, although the IHH^-/-^, Wnt5a^-/-^ mice and other manipulations of the Wnt PCP pathway differ in phenotype an issue which has been raised in other ciliopathy papers (i.e. Haycraft et al.,  2007).  We agree with the reviewer, that we can at present only conclude that loss of cell polarity in *talpid*^*3*^ chicken endochondral bones is likely dependent on IHH through regulation of Wnt5a.  There are also other possibilities beyond of the current IHH and Wnt5a theories; loss of cilia in the developing bone could result in a loss of mechanosensation and consecutive loss of bone polarity.  Because we are currently not able to determine if the loss of IHH, Wnt signalling or mechanotransduction through defective ciliogenesis is primarily responsible for the cell polarity defects in the bone, we have removed these results. 

*5) Unlike in long bones of the* talpid^3^ *mouse, the authors report that columnar chondrocytes are present in the* talpid^3^ *chicken leg.  Higher magnification images are needed of data given in* [*Figure 3E and F*](#fig3){ref-type="fig"} *so it is possible to see this.  The authors also report development of a poorly defined bone collar.  H and E staining is not sufficient to conclude this, Alizarin red would give a definitive answer.* 

We have previously published an analysis of the bone mineralization (Macrae et al.,  2010) in which we show that Alizarin red, TNAP and PHOSPHO1 are all lost in *talpid*^*3*^ chicken endochondral bones.  Data were removed as per above. 

*6) Please give evidence of* talpid^3^ *columnar chondrocytes where cell division angle is abnormal.  Suggest giving higher magnification image of data in* [*Figure 3G and H*](#fig3){ref-type="fig"}*.  Please provide numbers of cells counted.  Graph* [*Figure 3I*](#fig3){ref-type="fig"} *shows 11% of Wt cells align within 25o of expected angle, while in the Results the authors state that all Wt cells align within 20°.* 

We apologize for the ambiguity.  Our results, as written were based on 50 bins of degrees, which was more precise ([Author response image 1](#fig6){ref-type="fig"}).  However we increased the bin size in the graph in the submission for size.  The data has now been removed.10.7554/eLife.08077.011Author response image 1.**DOI:** [http://dx.doi.org/10.7554/eLife.08077.011](10.7554/eLife.08077.011)

*7) In* [*Figure 3J, K*](#fig3){ref-type="fig"}*, what are the VANGL2 foci and what is the precedence that this is a marker of polarity in columnar chondrocytes?  Please indicate in the figure examples of cells counted as having one or two foci or dispersed VANGL2 expression, higher magnification images may help.  Please provide numbers of cells counted and statistical analysis.* 

There is little precedence that VANGL2 is a marker for polarity in columnar chondrocytes and very few studies have examined localization of VANGL2 protein in different tissues.  Until recently, VANGL2 protein was thought to label the proximal side of hair cells of the mammalian inner ear, and this was used extensively in many studies, whereas it has now been shown that localization is in the distal side of the supporting cells (Ezan et al.,  2013).  We have demonstrated subcellular localization of VANGL2 protein in any tissue (Stephen et al.,  2014).  To our knowledge, one other group has demonstrated localization of VANGL2 in the long bones (Supplemental figure 4 of Gao et al.,  2011), but they did not thoroughly describe or examine this expression.  Both this supplemental figure and our own work agree on the localisation of VANGL2 in the proximal long bones of the limb.  This however is very different to the distal localisation shown in the chondrocytes of the digits.  Data removed as per above. 

Because we employed the same techniques in the bone immunohistochemistry that successfully determined VANGL2 localization in the chicken gastrula, we are confident that the bone localization we observe is real.  In addition, we have thoroughly quantitfied this (1213 wildtype cells in 6 samples, 1780 *talpid*^*3*^ cells in 6 samples).  Data removed as per above. 

Subsequently, we have combined [Figure 3](#fig3){ref-type="fig"} and [Figure 4](#fig4){ref-type="fig"} into a combined [Figure 3](#fig3){ref-type="fig"} "Cellular polarity and centrosome localization is disrupted in KIAA0586 null (TALPID3) tissues" (as per above). 

*8) In* [*Figure 3M and N*](#fig3){ref-type="fig"}*, Golgi staining hard to see, orientation of leading edge hard to interpret from images shown.  Please indicate how orientation of Golgi in relation to the leading edge was measured.  Please provide numbers of cells counted and statistical analysis.* 

We have modified these figures accordingly, improving the resolution and adding additional panels to highlight the findings.  We have added a scheme to show how we undertook measurements. 

*9) It is unclear from the data shown in* [*Figure 4C-H*](#fig4){ref-type="fig"} *how the conclusions were drawn.  We suggest providing higher magnification images clearly marking the axis of the 'expect orientation' and how actin bundles are orientated in relation to this in Wt cells but misorientated in* talpid^3^ *cells as well as indicating* talpid^3^ *cells with actin across the apical cell surface.  Give higher magnification of centrosomes localized in bare zones in Wt and not in* talpid^3^*.  Although actin bundles appear to be orientated differently in* talpid^3^ *compared to Wt,* $\gamma$*-tubulin staining is present within bare zones in* talpid^3^ *in the images shown.  Please provide numbers of cells counted and perform statistical analyses for these experiments.* 

We have modified these figures, adding additional magnifications and annotations to highlight what we have described in the text.  We have added a scheme to make these data easier to understand. 

*10) Maturation of the mother centriole to a basal body requires assembly of subdistal and distal appendages.  The authors clearly show presence of subdistal appendages in* talpid^3^ *mutant centrosomes but evidence of distal appendages given in* [*Figure 5E*](#fig5){ref-type="fig"} *is ambiguous.  Immunofluorescence staining with antibodies against distal appendage proteins such as CEP164 would give a definitive answer as to presence of distal appendages on* talpid^3^ *basal bodies.* 

We carried out immunohistochemistry with anti-Ninein, anti-OFD1 and anti-CEP164 antibodies, and have found that the anti-CEP164 antibody reacts very well with chicken tissue.  These additional data were very helpful in understanding the orientation of the centriole in *talpid*^*3*^ cells, and we have included them in the revised manuscript. 

*11) More extensive description of data given in Figure 7 required in the text.  Consider quantifying PCM1 staining between Wt and* talpid^3^*.* 

We have revised the description of these data.  Unfortunately, we were not able to quantify the PCM1 staining. 

[^1]: These authors contributed equally to this work.