Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes.

Joubert syndrome (JBTS), related disorders (JSRDs) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and CORS2 (JBTS2) loci are allelic and caused by mutations in TMEM216, which encodes an uncharacterized tetraspan transmembrane protein. Individuals with CORS2 frequently had nephronophthisis and polydactyly, and two affected individuals conformed to the oro-facio-digital type VI phenotype, whereas skeletal dysplasia was common in fetuses affected by MKS. A single G218T mutation (R73L in the protein) was identified in all cases of Ashkenazi Jewish descent (n=10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in mutant fibroblasts or after knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 formed a complex with Meckelin, which is encoded by a gene also mutated in JSRDs and MKS. Disruption of tmem216 expression in zebrafish caused gastrulation defects similar to those in other ciliary morphants. These data implicate a new family of proteins in the ciliopathies and further support allelism between ciliopathy disorders.

The neurological features of JSRD include hypotonia, ataxia, psychomotor delay, irregular breathing pattern and oculomotor apraxia and are variably associated with multiorgan involvement, mainly retinal dystrophy, nephronophthisis (NPH) and congenital liver fibrosis. JSRD are genetically heterogeneous, and all known genes encode proteins localized at or near the primary cilium1. We previously mapped the JBTS2 (also known as CORS2) locus to chromosome 11p12-q13.3 in a large Sicilian family and in three consanguineous pedigrees from the Middle East2-3. Aligning the two datasets suggested a minimal candidate interval between D11S1344 and D11S1883 (46.123-63.130 Mb)4 (Fig. 1a).

Figure 1

Mutations in the TMEM216 gene in patients linked to the JBTS2 and MKS2 loci. (a) Chromosomal location of the JBTS2 and MKS2 loci on Chr. 11cent. (b) TMEM216 genomic organization, depicting start and stop codon, and location of identified base changes. (c) The longest splice isoform encodes for a 148 aa tetraspan membrane protein. Patient mutations predominate towards the middle, with one prevalent p.R73 change occurring repeatedly. Missense, nonsense and splice mutations were identified. (d) Evolutionary conservation of mutated amino acids. (e) Patient mutations lead to unstable protein products. Western blot of whole lysate of cells transfected with a cDNA encoding wild type (WT) vs. patient missense mutations, compared with control (p.V71L). Each mutation resulted in the production of 40-50% of WT protein levels, compared with α-tubulin loading control.

Overlapping with JSRD is MKS, characterized by occipital encephalocele and other posterior fossa defects, cystic dysplastic kidneys, hepatic bile duct proliferation and polydactyly, and the two conditions are known to be allelic at four loci5-8. The MKS2 locus was initially mapped in families of North African and Middle Eastern ancestry to a chromosome 11q region telomeric to JBTS2, but our subsequent identification of additional families, as well as SNP re-analysis of the initial family, indicated allelism with JBTS2 between rs1113480 and rs953894 (48.014-62.518 Mb) (Supplementary Fig. 1). Because JSRD and MKS are considered ciliopathies, of the 200 total candidate genes, we first sequenced the exons and splice sites of genes listed in the cilia proteome databases10-11 in one affected subject from each JBTS2/MKS2 family, but no mutations were identified.

Tetraspan transmembrane proteins are characterized by four hydrophobic, putative transmembrane domains (TM1-TM4), forming two extracellular and one intracellular loop, which regulate signaling and trafficking properties of their partner proteins in multiple cellular contexts12. While little is known about their function, they can act with Wnt receptors13, and their ability to form complexes with a wide variety of membrane and cytosolic proteins14 suggests that they may participate in the formation of membrane domains that regulate signaling and sorting processes. Transmembrane proteins also represented attractive candidates, due to similarities to MKS3/TMEM67 encoding Meckelin, which is mutated both in JSRD and MKS5,15. Therefore we additionally sequenced the eight genes encoding transmembrane proteins, eventually identifying homozygous deleterious mutations in TMEM216 in six of the 12 JSRD/MKS families compatible with linkage to the locus (Table 1). Interestingly, residue p.R73 was mutated both in a Sicilian family with JSRD (COR000, p.R73L) and in a Turkish family, in which MKS and JSRD coexisted in the same sibship (COR114/F37, p.R73H). The p.G77A mutation in two Palestinian families (F56, F58) resulted from a substitution (c.230G>C) that affects the first base of exon 5, leading to the use of an alternative splice site in intron 4, the inclusion of an additional 46bp and resultant premature protein termination (p.T78KfsX30) (Supplementary Fig. 2). None of these mutations were identified in over 500 controls from ethnically matched cohorts.

Table 1

Clinical and molecular data of TMEM216 mutated families

Family data			Clinical data					Genetic data
Fam	Age (sex)	Origin	CNS	Eye	Kidney	Liver	Other	Nucleotidechanges	Proteinalterations
Joubert syndrome related disorders
COR000	11y, M 15y, F 20y, F 29y, M	Italian	MTS	-	NPH	ELE	-	c.218G>T	p.R73L
	15y, F		MTS	-	NPH	-	-
	20y, F		MTS	-	NPH	-	-
	29y, M		MTS	-	NPH	-	-
COR284	22y, F	Italian	MTS		NPH			c.218G>T	p.R73L
COR114/F37	1m, M	Turkish	MTS	MicroC			PD	c.218G>A	p.R73H
	13w, M*		Ec		CK	BDP	PD, BLB
F401		New Zealand	MTS	+	NPH			c.217C>Tc.398T>G	p.R73Cp.L133X
COR076	1y, Ffetus	Ashk	MTS, PMG	-			ToF	c.218G>T	p.R73L
COR287	3m, M	Ashk	MTS				PD	c.218G>T	p.R73L
MTI005	13y, M	Euro	MTS	OMA	NPH?	-	CD	c.218G>T	p.R73L
	3y, F		MTS	OMA		-	CD
MTI161	4y, M	Ashk	MTS	OMA, Nys	-	-	PD, TT, MP	c.218G>T	p.R73L
MTI214	4y, F	Ashk	MTS	OMA	-	-	CMD	c.218G>;T	p.R73L
MTI467	fetus	Ashk	MTS, DWM		-	-	PD, MOF,CD, HYPT	c.218G>;T	p.R73L
MTI585	1y, F	Ashk	MTS	Co	ACMD	-	CMD	c.218G>;T	p.R73L
MTI658	8y, F	Ashk	MTS	Nys	NPH?	-	PD, CMD	c.218G>;T	p.R73L
	5y, F		MTS	Nys	NPH?	-	PD, CMD
	2 fetuses						PD
MTI1006	9y, M	Ashk	MTS	OMA, Nys	-	-	-	c.218G>;T	p.R73L
	1y, M (cousin)		MTS	OMA, Nys	-	-	PD
MTI1008	4y, F	Ashk	MTS, DWM	OMA			RTP	c.218G>;T	p.R73L
Meckel syndrome
F2	21w, M	Tunisian	Mc		CK	BDP	PD, CP, BLB	c.341T>G	p.L114R
	14w, F		An		CK	BDP	PD, BLB
F5	24w, M	Tunisian	Mc	MicroO	CK	BDP	PD, CP, IUGR,BLB, HypoG	c.341T>G	p.L114R
F56	fetus	Palestinian	An		CK			c.230G>C	p.G77A→splice:p.T78KfsX30
	15w		DW, Ec		CK		IUGR
F58	SB, M	Palestinian	Ec		CK	BDP	PD, CP	c.230G>C	p.G77A→splice:p.T78KfsX30
	1day		Ec		CK	BDP	PD
F154	22w SB	Palestinian	Mc		CK			c.230G>C	p.G77A→splice:p.T78KfsX30
A2423	21w, M	British	Ec		CK	BDP	PD, CP, VSD, IM, BLB	c.253C>T	p.R85X
	12w, M		Ec		CK	-	CH, Omph

Legend: -: not affected; ACMD: abnormal cortico-medullary differentiation; An: anencephaly; Ashk: Ashkenazi Jewish; BDP: bile ducts proliferation; BLB: bowing of long bones; CD: clinodactyly; CH: cystic hygroma; CK: cystic kidneys; CMD: camptodactyly; Co: chorioretinal coloboma; CP: cleft palate; CVA: cerebellar vermis agenesis; DW: Dandy-Walker malformation; Ec: encephalocele; ELE: elevated liver enzymes; Euro: European; F: female; HypoG: hypoplastic external genitalia; HYPT: hypertelorism; IM: intestinal malrotation; IUGR: intrauterine growth retardation; M: male; m: months; IUGR: intrauterine growth retardation; Mc: meningocele; MEc: meningoencephalocele; MOF: multiple oral frenulae; MicroC: microcornea; MicroO: microphthalmia; MP: micropenis; MTS: molar tooth sign; NPH: nephronophthisis; Nys: nystagmus; OMA: oculomotor apraxia; Omph: omphalocele; PD: polydactyly; PMG: polymicrogyria; RTP: rhythmic tongue protrusions; SB: stillbirth; ToF: tetralogy of Fallot; TT: tongue tumors; VSD: ventricular septal defect; w: gestational weeks; y: years. Empty cells denote unavailable clinical information;

MKS fetus.

We next screened an additional 460 JSRD and 132 MKS probands () and identified mutations in 12 and two further cases, respectively (Table 1). Twelve of 14 JSRD families shared the same homozygous p.R73L founder mutation, including two families from Sicily and ten families of Ashkenazi Jewish descent. Saturation of the region surrounding the p.R73L mutation with 17 SNP/microsatellite markers indicated that these families shared the same ancestral haplotype, spanning 472 Kb around the mutation (Supplementary Fig. 3), that could be dated back at least 20 generations. The carrier frequency in the Ashkenazi population was determined to be about 1:100, as we identified two heterozygous healthy unrelated carrier individuals among a screened cohort of 212 Ashkenazi individuals, making carrier detection possible at least in this population. A similar carrier frequency of 1:92 has also been determined for the p.R73L mutation in a distinct study on eight Ashkenazi JBTS2 families and 2766 unaffected controls16. Microsatellite analysis also detected shared haplotypes in the two Palestinian (F56, F58) and in the two Tunisian families (F2, F5), homozygous for the same mutations (Supplementary Fig. 1).

Overall, 20 JSRD patients and 11 MKS fetuses carried TMEM216 mutations (Fig. 1b,​ Table 1). All of the nonsynonymous changes occurred in evolutionarily conserved residues (Fig. 1c-d), and led to unstable protein when transfected into heterologous cells (Fig. 1e,​ Supplementary Fig. 4). Although truncating mutations were identified in both the middle and end of the protein, p.R73 transversions predominated (Fig. 1c), with the p.R73L clearly a founder mutation. Among JSRD, the phenotype was characterized by frequent occurrence of NPH (9/20) and polydactyly (9/20), while retinal dystrophy and congenital hepatic fibrosis were never observed. In keeping with this, sequence analysis of 96 patients with Bardet-Biedl syndrome identified no homozygous mutations, since retinopathy is a key feature of this condition. In two JSRD patients (MTI161 and MTI467), polydactyly was associated with either tongue tumors or multiple oral frenula, corresponding to the Oro-Facio-Digital type VI (or Varadi-Papp) syndrome16 (OMIM%277170), indicating that TMEM216 mutations are the first known identified cause. In the 11 MKS fetuses with TMEM216 mutations, distinctive clinical features were skeletal dysplasia, including intrauterine growth retardation or bowing of the long bones in six fetuses, cleft palate in four, and anencephaly in two (Table 1,​ Supplementary Fig. 5).

TMEM216 is a poorly annotated gene, with RefSeq predicting a protein of just 86 aa, suggesting potential alternative splicing. To characterize this mRNA we performed Northern analysis with a commercial human fetal blot, and found a single major mature isoform at about 1.4 Kb (Supplementary Fig. 6a), agreeing with the predicted 1.3 Kb of the longest representative cDNAs. To interrogate splicing we designed primers complementary to the furthest 5′ and 3′ regions of the known cDNA, and sequenced 48 cloned PCR products from a 20 week gestation human fetal brain library. We identified four major splice isoforms, the longest and most prevalent predicting a protein of 148 aa (Supplementary Fig. 6b), which we consider to be the full-length mRNA. There is also extensive alternative splicing, encoding very short proteins (Supplementary Fig. 6b), the functions of which were not evaluated further. Importantly, we did not find any mutations in any of the putative UTRs.

To elucidate roles for TMEM216 in human development, we first examined its expression in human embryonic tissues. In situ hybridization analysis in human embryos confirmed expression in the central nervous system, limb bud, kidney and cartilage (Supplementary Fig. 6c-h), which is similar to the broad and relatively low-level expression pattern of other JSRD/MKS genes. We next raised an anti-TMEM216 polyclonal affinity-purified antibody against aa 81-90, demonstrated specificity (Supplementary Fig. 7a-b), and immunostained two different ciliated cell lines (inner medullary collecting duct [IMCD3] and retinal pigment epithelium [hRPE]). We observed localization with the base of the primary cilium or adjacent basal body in the majority of cells, as marked by either acetylated or glutamylated tubulin staining (Fig. 2a-d). TMEM216 antibody also reacted strongly to the base of cilia in organs like kidney containing ciliated cells (Fig. 2c,​ Supplementary Fig. 7c), but failed to react with these structures in hTERT-immortalized fetal TMEM216 p.R85X homozygous mutant fibroblasts (Supplementary Fig. 7d). Epitope-tagged TMEM216 showed similar localization to the base of cilia and other microtubule structures (i.e. mitotic spindle in cells undergoing late telophase, Supplementary Fig. 8).

Figure 2

Ciliary localization of TMEM216. (a-d) Overlapping localization of endogenous TMEM216 (green) and acetylated α-tubulin or GT335 (glutamylated tubulin) (red) at the base of the primary cilium (arrows) in IMCD3 (a, b), proximal renal tubules (c) or hRPE cells (d). White dashed line indicates the tubule lumen. Boxes show insets at magnification x10. Scale bar 5 μm.

In TMEM216 p.R85X mutant fibroblasts, we noticed a failure in ciliogenesis following 48 hr serum starvation (Fig. 3a) compared with controls. Western analysis of whole cell lysates from control fibroblasts identified a band at 19 kD (Fig. 3b), matching the predicted 148 aa full length protein, whereas this band was attenuated or lost in TMEM216 p.R85X fibroblasts or in IMCD3 cells in which Tmem216 was knockded down.

Figure 3

TMEM216 mutation or knockdown results in impaired ciliogenesis and centrosome docking. (a) Two different TMEM216-mutated patient fibroblasts lines show defective ciliogenesis and impaired centrosome docking (marked by γ-tubulin). Scale bar: left 20 μm; right 1 μm. (b) TMEM216 antisera reacts with a 19 kDa band in control cells, which is reduced in TMEM216 p.R85X fibroblasts (some residual is apparent likely due to read-through from geneticin treatment), as well as in siRNA1-treated IMCD3 cells. Fibro. = fibroblasts; Non-transf. = non-transfected; scr. = scrambled. (c) Transfected IMCD3 cells showing effect of Tmem216 siRNA treatment, with reduced ciliogenesis and centrosome docking (note lack of cilia and lack of apically located centrosomes following knockdown). Top is x-y, and bottom is x-z projection, scale bar 10 μm. (d) Percent ciliated cells (defined as cilia > 1 μm length) is reduced following Tmem216 siRNA treatment. Percent cells with apical basal bodies (defined as most superior 1.0 μm sections compared to nuclear position) is similarly reduced. *p<0.01, **p<0.001, chi-squared test. (e) Shows method of quantification at 72 hrs. Scale bars: white, most apical 1.0 μm; grey, basal 1.5 μm.

To determine the basis of the ciliogenesis defect, we performed transient transfection of monolayers of IMCD3 cells with two separate Tmem216 siRNA duplexes. Tmem216 knockdown prevented ciliogenesis in polarized cells, and blocked correct docking of centrosomes at the apical cell surface (Fig. 3c), as seen previously for Meckelin and MKS117. This ciliogenesis defect was quantified by comparing the percentage of cells with cilia (defined as > 1 μm length) vs. those without cilia (< 1μm length), and by analyzing the percentage of cells with centrosomes located apical to the nucleus. In cells in which Tmem216 was knocked down, we observed a striking defect in both of these measurements compared with two separate control transfections (Fig. 3d-e, chi-squared test, p<0.001, for 350 cells from each condition), suggesting its requirement in centrosome docking.

The similarities in cellular phenotypes of Mks3 and Tmem216 knockdown, and subcellular localizations of Meckelin and TMEM216, prompted us to ask if the two proteins could interact. Firstly, GFP-tagged TMEM216 was immunoprecipitated with antibodies to either N- or C-terminal portions of Meckelin (Fig. 4a) and, secondly, the reciprocal IP experiment used α-GFP antibody to pull down Meckelin (Fig. 4b). Both assays detected a complex between TMEM216 and Meckelin.

Figure 4

TMEM216 complexes with Meckelin, and their loss results in Rho hyperactivation and actin cytoskeleton remodelling. (a) TMEM216-GFP (~37 kDa; arrow) is immunoprecipitated (IP) with anti-Meckelin antisera against either the N- or C-termini from whole cell extract (input WCE), but not in control IPs with an irrelevant antibody (irr. Ab) or the preimmune antiserum (preimm.). Arrowhead is IgG heavy chain. (b) IP of TMEM216-GFP by α-GFP pulls down a 60 kDa C-terminal containing isoform of endogenous Meckelin (arrow), but not in control IPs with no antibody (no MAb) or an irrelevant antibody (irr. MAb). Arrowhead is IgG heavy chain. (c) MKS2 fibroblast (fibro.) WCE has increased levels of activated RhoA-GTP compared to normal control. (d) siRNA knockdown of Tmem216 and Mks3 in IMCD3 cells increased RhoA activation, compared with scrambled control (scr.). Total RhoA and β-actin are loading controls. Positive control (+) is loading with non-hydrolyzable GTPγS, negative control (−) is loading with GDP. (e) RhoA (red) localizes to the basal bodies (γ-tubulin, green) in IMCD3 cells following 24 hr treatment with scrambled siRNA, but mislocalizes to regions adjacent to the basal bodies (arrows; and inset, magnification x5) and at basolateral surfaces (arrowheads) following Tmem216 knockdown. Mislocalization of γ-tubulin is also apparent (bottom inset). Scale bar 10 μm. (f) Subcellular phenotypes of fibroblasts cultured from undiseased control and two MKS fetuses mutated in TMEM216 [p.R85X homozygous] and MKS3 [p.R217X]+[p.M261T], as indicated. Actin stress fibers in both mutated cells (arrowheads) are detected by phalloidin staining. Scale bar 10 μm.

Many aspects of actin-dependent polarized cell behavior, including morphogenetic cell movements18 and ciliogenesis19, are mediated by the planar cell polarity (PCP) pathway of non-canonical Wnt signaling20. We therefore first examined RhoA, since the Rho family of small GTPases are key mediators of this pathway20-21. Consistent with previous results following MKS3 loss22, we found that RhoA signaling was hyperactived in both TMEM216 p.R85X fibroblasts or following Tmem216 knockdown (Fig. 4c-d), despite normal total amounts of RhoA in these cells. Centrosome docking at the apical cell surface is prevented by the interruption of actin remodeling23, and is dependent on both RhoA activation and regulation by the core PCP protein Dishevelled (Dvl)24. We confirmed that RhoA is localized to the basal body in confluent IMCD3 cells but, following Tmem216 knockdown for 24 hr, RhoA was mislocalized to peripheral regions of the basal body and to basolateral cell-cell contacts (Fig. 4e), consistent with translocation of ectopically-activated RhoA to the cytosol25. Tmem216 knockdown also showed evidence of a mislocalization of γ-tubulin at the centrosome/basal body for this timepoint, which suggests a defect in γ-tubulin nucleation, one of the earliest steps in ciliogenesis26. The established role of RhoA in modulating the actin cytoskeleton in the PCP pathway then led us to evaluate MKS2 patient fibroblast lines for alterations. We found a co-localization of actin stress fibers and the actin cross-linker filamin-A in the cytoplasm of these mutant cells, which was absent in control (Fig. 4f).

We next looked at Dvl signaling in cells, since cilia negatively regulate Dvl activation27, and Dvl mediates Rho activation at the apical surface of ciliated epithelial cells24. We found that loss of TMEM216 increased phosphorylation of Dvl1 (Fig. 5a​ left and right panel), implying that TMEM216 modulates hyper-responsiveness of signaling pathways mediated by Dvl and RhoA. We found that Rho inhibition also increased the Dvl1 phosphorylation in ciliated cells, supporting the existence of feedback mechanism between Rho and Dvl (Fig. 5a, right panel). Unexpectedly, the constitutive Dvl1 phosphorylation associated with TMEM216 loss was blocked by Rho inhibition (Fig. 5a, right panel), suggesting that this loss in ciliated cells can modify the feedback mechanism. Although this possibility warrants further investigation, our data nevertheless suggest a working model in which Dvl1, RhoA and TMEM216 may serve as part of a complex in the pericentrosomal compartment to mediate cellular polarization and centrosomal apical docking. Previous studies have shown that Dvl and Rho contribute to a core framework for regulating the apical docking of centrosomes24, and we also see evidence of a common complex containing TMEM216, Dvl1 and RhoA in TMEM216-transfected cells (Fig. 5b). Since we saw no difference in the localization of Dvl1 following Tmem216 knockdown (Supplementary Fig. 9), these data predict that the hyperactivation of Rho in the absence of TMEM216 might be responsible for the centrosome docking defect at the apical cellular surface. As expected, we found that the impaired centrosome docking caused by Tmem216 knockdown was rescued in a dose-dependent fashion using Rho inhibitor (Fig. 5c).

Figure 5

TMEM216 disruption results in Dvl1 phosphorylation, and planar cell polarity-like phenotypes in zebrafish. (a) siRNA knockdown of Tmem216 (left panel) and TMEM216 p.R85X patient fibroblasts (right panel) cause an increase in the upper (phosphorylated) isoform (P-Dvl1) compared to scrambled control (scr.). Treatment with Rho inhibitor exoenzyme-C3-transferase (2 μg/ml) alone increased Dvl1 phosphorylation, but increases in P-Dvl1 by TMEM216 loss are reversed by Rho inhibition (right panel). (b) Co-immunoprecipitation of both Dvl1 and RhoA with TMEM216 in TMEM216-GFP transfected cells. Arrowhead is IgG heavy chain. (c) Dose-dependent rescue of centrosome/basal body docking phenotype in Tmem216 siRNA-treated cells following += 0.5, ++= 1.0, +++= 2.0 μg/ml Rho inhibitor treatment. *p<0.01; **p<0.001 for chi-squared test. (d) Injection of translation-blocking morpholino (MO) to tmem216 vs. scrambled MO causes a ciliary defect phenotype in injected zebrafish embryos (>50 each condition). Injection of human TMEM216 RNA causes no phenotype in WT embryos, but allows partial, dose-dependent rescue of the MO phenotype. (e) Lateral (top) and dorsal (bottom) views of zebrafish embryos injected with tmem216 or mks3 MO at 8-somite stage had ciliopathy features. (f) Representative 11-somite stage embryos hybridized with krox20, pax2, and myoD riboprobes. Convergence to the midline is measured by the width at the fifth rhombomere (horizontal arrow), and extension along the anterior-posterior (AP) axis by notochord length (vertical arrow) (n=12-15 embryos/injection). Suppression of the tmem216 or mks3 morphant gastrulation defect causes significant differences in width and length compared to controls (*p<0.005). Pheno.=phenotype; embry.=embryonic; rhomb.=rhombomere; Bars=standard error of means.

Meckelin is proposed to regulate centrosomal docking through the RhoA signaling pathway22, and bears similarity to the Frizzled family of transmembrane Wnt receptors15. Direct evidence of a role for Meckelin in PCP signaling stems from zebrafish embryo morphant phenotypes following morpholino knock-down of mks3. These included defects in gastrulation movement (a shortened body axis, broad notochords and misshapen somites), which are typical of defects in non-canonical (PCP) Wnt signaling, and have been observed in numerous ciliary and basal body morphants28-29. We observed identical ciliary phenotypes in tmem216 morphants, which were largely rescued by RNA encoding human TMEM216 (Fig. 5d), and fully rescued by RNA encoding non-targetable zebrafish tmem216 (not shown). We therefore directly compared the tmem216 and mks3 morphant phenotypes in zebrafish, and noted similar defects in both live embryos and in embryros in which pronephric mesoderm, anterior neural structures, adaxial mesodermal cells, and somites were labeled with a krox20, pax2, and myoD riboprobe cocktail (Fig. 5e-f). Quantification demonstrated alteration of convergence to the midline and extension along the AP axis consistent with a PCP defect, although the AP extension defect was more pronounced in the mks3 compared to the tmem216 morphant.

Recent work has implicated the tetraspanin TSPAN12 in the regulation of Norrin signaling by the Wnt receptor Frizzled-4 and coreceptor LRP513. We therefore speculate that TMEM216, a novel tetraspan protein, forms a non-canonical Wnt receptor-coreceptor complex with Meckelin. Our data support a role for both proteins in mediating PCP signaling through the RhoA pathway to cause actin cytoskeleton rearrangements, although whether Rho functions upstream or downstream of Dvl1 remains to be determined. In apical regions of the cell, such actin reorganization would be an essential step before the centrosome/basal body could dock correctly and initiate ciliogenesis. The identification of mutations in TMEM216 as a cause of JSRD and MKS therefore further emphasizes the interrelationship between cell polarity, cellular morphogenesis and signal transduction pathways.