BBS10 encodes a vertebrate-specific chaperonin-like protein and is a major BBS locus

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous ciliopathy. Although nine BBS genes have been cloned, they explain only 40-50% of the total mutational load. Here we report a major new BBS locus, BBS10, that encodes a previously unknown, rapidly evolving vertebrate-specific chaperonin-like protein. We found BBS10 to be mutated in about 20% of an unselected cohort of families of various ethnic origins, including some families with mutations in other BBS genes, consistent with oligogenic inheritance. In zebrafish, mild suppression of bbs10 exacerbated the phenotypes of other bbs morphants.     

Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet-Biedl syndrome

Meckel-Gruber syndrome (MKS) is a genetically heterogeneous, neonatally lethal malformation and the most common form of syndromic neural tube defect (NTD). To date, several MKS-associated genes have been identified whose protein products affect ciliary function. Here we show that mutations in MKS1, MKS3 and CEP290 (also known as NPHP6) either can cause Bardet-Biedl syndrome (BBS) or may have a potential epistatic effect on mutations in known BBS-associated loci. Five of six families with both MKS1 and BBS mutations manifested seizures, a feature that is not a typical component of either syndrome. Functional studies in zebrafish showed that mks1 is necessary for gastrulation movements and that it interacts genetically with known bbs genes. Similarly, we found two families with missense or splice mutations in MKS3, in one of which the affected individual also bears a homozygous nonsense mutation in CEP290 that is likely to truncate the C terminus of the protein. These data extend the genetic stratification of ciliopathies and suggest that BBS and MKS, although distinct clinically, are allelic forms of the same molecular spectrum.     

Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates

The evolutionarily conserved planar cell polarity (PCP) pathway (or noncanonical Wnt pathway) drives several important cellular processes, including epithelial cell polarization, cell migration and mitotic spindle orientation. In vertebrates, PCP genes have a vital role in polarized convergent extension movements during gastrulation and neurulation. Here we show that mice with mutations in genes involved in Bardet-Biedl syndrome (BBS), a disorder associated with ciliary dysfunction, share phenotypes with PCP mutants including open eyelids, neural tube defects and disrupted cochlear stereociliary bundles. Furthermore, we identify genetic interactions between BBS genes and a PCP gene in both mouse (Ltap, also called Vangl2) and zebrafish (vangl2). In zebrafish, the augmented phenotype results from enhanced defective convergent extension movements. We also show that Vangl2 localizes to the basal body and axoneme of ciliated cells, a pattern reminiscent of that of the BBS proteins. These data suggest that cilia are intrinsically involved in PCP processes.     

AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1

The Wnt signaling pathway is essential for development and organogenesis. Wnt signaling stabilizes beta-catenin, which accumulates in the cytoplasm, binds to 1-cell factor (TCF; also known as lymphocyte enhancer-binding factor, LEF) and then upregulates downstream genes. Mutations in CTNNB1 (encoding beta-catenin) or APC (adenomatous polyposis coli) have been reported in human neoplasms including colon cancers and hepatocellular carcinomas (HCCs). Because HCC5 tend to show accumulation of beta-catenin more often than mutations in CTNNB1, we looked for mutations in AXIN1, encoding a key factor for Wnt signaling, in 6 HCC cell lines and 100 primary HCC5. Among the 4 cell lines and 87 HCC5 in which we did not detect CTNNB1 mutations, we identified AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs. In cell lines containing mutations in either gene, we observed increased DNA binding of TCF associated with beta-catenin in nuclei. Adenovirus mediated gene transfer of wild-type AXINI induced apoptosis in hepatocellular and colorectal cancer cells that had accumulated beta-catenin as a consequence of either APC, CTNNB1 or AXIN1 mutation, suggesting that axin may be an effective therapeutic molecule for suppressing growth of hepatocellular and colorectal cancers.     

Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome

Protein-protein interaction analyses have uncovered a ciliary and basal body protein network that, when disrupted, can result in nephronophthisis (NPHP), Leber congenital amaurosis, Senior-L?ken syndrome (SLSN) or Joubert syndrome (JBTS). However, details of the molecular mechanisms underlying these disorders remain poorly understood. RPGRIP1-like protein (RPGRIP1L) is a homolog of RPGRIP1 (RPGR-interacting protein 1), a ciliary protein defective in Leber congenital amaurosis. We show that RPGRIP1L interacts with nephrocystin-4 and that mutations in the gene encoding nephrocystin-4 (NPHP4) that are known to cause SLSN disrupt this interaction. RPGRIP1L is ubiquitously expressed, and its protein product localizes to basal bodies. Therefore, we analyzed RPGRIP1L as a candidate gene for JBTS and identified loss-of-function mutations in three families with typical JBTS, including the characteristic mid-hindbrain malformation. This work identifies RPGRIP1L as a gene responsible for JBTS and establishes a central role for cilia and basal bodies in the pathophysiology of this disorder.     

The Bardet-Biedl protein BBS4 targets cargo to the pericentriolar region and is required for microtubule anchoring and cell cycle progression

BBS4 is one of several proteins that cause Bardet-Biedl syndrome (BBS), a multisystemic disorder of genetic and clinical complexity. Here we show that BBS4 localizes to the centriolar satellites of centrosomes and basal bodies of primary cilia, where it functions as an adaptor of the p150(glued) subunit of the dynein transport machinery to recruit PCM1 (pericentriolar material 1 protein) and its associated cargo to the satellites. Silencing of BBS4 induces PCM1 mislocalization and concomitant deanchoring of centrosomal microtubules, arrest in cell division and apoptotic cell death. Expression of two truncated forms of BBS4 that are similar to those found in some individuals with BBS had a similar effect on PCM1 and microtubules. Our findings indicate that defective targeting or anchoring of pericentriolar proteins and microtubule disorganization contribute to the BBS phenotype and provide new insights into possible causes of familial obesity, diabetes and retinal degeneration.     

Deregulated expression of c-Myc depletes epidermal stem cells

The beta-catenin/TCF signaling pathway is essential for the maintenance of epithelial stem cells in the small intestine. c-Myc a downstream target of beta-catenin/TCF (ref. 2), can induce differentiation of epidermal stem cells in vitro. To determine the role of c-Myc in epidermal stem cells in vivo, we have targeted expression of human MYC2 to the hair follicles and the basal layer of mouse epidermis using a keratin 14 vector (K14.MYC2). Adult K14.MYC2 mice gradually lose their hair and develop spontaneous ulcerated lesions due to a severe impairment in wound healing; their keratinocytes show impaired migration in response to wounding. The expression of beta1 integrin, which is preferentially expressed in epidermal stem cells is unusually low in the epidermis of K14.MYC2 mice. Label-retaining analysis to identify epidermal stem cells reveals a 75% reduction in the number of stem cells in 3-month-old K14.MYC2 mice, compared with wildtype mice. We conclude that deregulated expression of c-Myc in stem cells reduces beta1 integrin expression, which is essential to both keratinocyte migration and stem cell maintenance.     

Effective targeted gene 'knockdown' in zebrafish

The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted 'knockdown' technology in this model vertebrate. We show here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode, all zebrafish cells readily respond to this technique. We also developed also morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene result in embryos with hepatoerythropoietic porphyria. We also used morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos.     

Wnt-beta-catenin signaling initiates taste papilla development

Fungiform taste papillae form a regular array on the dorsal tongue. Taste buds arise from papilla epithelium and, unusually for epithelial derivatives, synapse with neurons, release neurotransmitters and generate receptor and action potentials. Despite the importance of taste as one of our five senses, genetic analyses of taste papilla and bud development are lacking. We demonstrate that Wnt-beta-catenin signaling is activated in developing fungiform placodes and taste bud cells. A dominant stabilizing mutation of epithelial beta-catenin causes massive overproduction of enlarged fungiform papillae and taste buds. Likewise, genetic deletion of epithelial beta-catenin or inhibition of Wnt-beta-catenin signaling by ectopic dickkopf1 (Dkk1) blocks initiation of fungiform papilla morphogenesis. Ectopic papillae are innervated in the stabilizing beta-catenin mutant, whereas ectopic Dkk1 causes absence of lingual epithelial innervation. Thus, Wnt-beta-catenin signaling is critical for fungiform papilla and taste bud development. Altered regulation of this pathway may underlie evolutionary changes in taste papilla patterning.     

CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium

Tubulin glutamylation is a post-translational modification that occurs predominantly in the ciliary axoneme and has been suggested to be important for ciliary function. However, its relationship to disorders of the primary cilium, termed ciliopathies, has not been explored. Here we mapped a new locus for Joubert syndrome (JBTS), which we have designated as JBTS15, and identified causative mutations in CEP41, which encodes a 41-kDa centrosomal protein. We show that CEP41 is localized to the basal body and primary cilia, and regulates ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mice and results in glutamylation defects in the ciliary axoneme. Our data identify CEP41 mutations as a cause of JBTS and implicate tubulin post-translational modification in the pathogenesis of human ciliary dysfunction.     

Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways

Cystic renal diseases are caused by mutations of proteins that share a unique subcellular localization: the primary cilium of tubular epithelial cells. Mutations of the ciliary protein inversin cause nephronophthisis type II, an autosomal recessive cystic kidney disease characterized by extensive renal cysts, situs inversus and renal failure. Here we report that inversin acts as a molecular switch between different Wnt signaling cascades. Inversin inhibits the canonical Wnt pathway by targeting cytoplasmic dishevelled (Dsh or Dvl1) for degradation; concomitantly, it is required for convergent extension movements in gastrulating Xenopus laevis embryos and elongation of animal cap explants, both regulated by noncanonical Wnt signaling. In zebrafish, the structurally related switch molecule diversin ameliorates renal cysts caused by the depletion of inversin, implying that an inhibition of canonical Wnt signaling is required for normal renal development. Fluid flow increases inversin levels in ciliated tubular epithelial cells and seems to regulate this crucial switch between Wnt signaling pathways during renal development.     

Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation leading to abnormal endoplasmic-reticulum-to-Golgi trafficking

Cranio-lenticulo-sutural dysplasia (CLSD) is an autosomal recessive syndrome characterized by late-closing fontanels, sutural cataracts, facial dysmorphisms and skeletal defects mapped to chromosome 14q13-q21 (ref. 1). Here we show, using a positional cloning approach, that an F382L amino acid substitution in SEC23A segregates with this syndrome. SEC23A is an essential component of the COPII-coated vesicles that transport secretory proteins from the endoplasmic reticulum to the Golgi complex. Electron microscopy and immunofluorescence show that there is gross dilatation of the endoplasmic reticulum in fibroblasts from individuals affected with CLSD. These cells also exhibit cytoplasmic mislocalization of SEC31. Cell-free vesicle budding assays show that the F382L substitution results in loss of SEC23A function. A phenotype reminiscent of CLSD is observed in zebrafish embryos injected with sec23a-blocking morpholinos. Our observations suggest that disrupted endoplasmic reticulum export of the secretory proteins required for normal morphogenesis accounts for CLSD.     

Notch1 functions as a tumor suppressor in mouse skin

Notch proteins are important in binary cell-fate decisions and inhibiting differentiation in many developmental systems, and aberrant Notch signaling is associated with tumorigenesis. The role of Notch signaling in mammalian skin is less well characterized and is mainly based on in vitro studies, which suggest that Notch signaling induces differentiation in mammalian skin. Conventional gene targeting is not applicable to establishing the role of Notch receptors or ligands in the skin because Notch1-/- embryos die during gestation. Therefore, we used a tissue-specific inducible gene-targeting approach to study the physiological role of the Notch1 receptor in the mouse epidermis and the corneal epithelium of adult mice. Unexpectedly, ablation of Notch1 results in epidermal and corneal hyperplasia followed by the development of skin tumors and facilitated chemical-induced skin carcinogenesis. Notch1 deficiency in skin and in primary keratinocytes results in increased and sustained expression of Gli2, causing the development of basal-cell carcinoma-like tumors. Furthermore, Notch1 inactivation in the epidermis results in derepressed beta-catenin signaling in cells that should normally undergo differentiation. Enhanced beta-catenin signaling can be reversed by re-introduction of a dominant active form of the Notch1 receptor. This leads to a reduction in the signaling-competent pool of beta-catenin, indicating that Notch1 can inhibit beta-catenin-mediated signaling. Our results indicate that Notch1 functions as a tumor-suppressor gene in mammalian skin.     

Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia

Primary ciliary dyskinesia most often arises from loss of the dynein motors that power ciliary beating. Here we show that DNAAF3 (also known as PF22), a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes before their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in individuals from families with situs inversus and defects in the assembly of inner and outer dynein arms. Knockdown of dnaaf3 in zebrafish likewise disrupts dynein arm assembly and ciliary motility, causing primary ciliary dyskinesia phenotypes that include hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a PF22-null mutant cannot assemble any outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests that DNAAF3 (PF22) acts at a similar stage as other preassembly proteins, for example, DNAAF2 (also known as PF13 or KTU) and DNAAF1 (also known as ODA7 or LRRC50), in the dynein preassembly pathway. These results support the existence of a conserved, multistep pathway for the cytoplasmic formation of assembly competent ciliary dynein complexes.     

Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency

Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.     

The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome

Cerebello-oculo-renal syndrome (CORS), also called Joubert syndrome type B, and Meckel (MKS) syndrome belong to the group of developmental autosomal recessive disorders that are associated with primary cilium dysfunction. Using SNP mapping, we identified missense and truncating mutations in RPGRIP1L (KIAA1005) in both CORS and MKS, and we show that inactivation of the mouse ortholog Rpgrip1l (Ftm) recapitulates the cerebral, renal and hepatic defects of CORS and MKS. In addition, we show that RPGRIP1L colocalizes at the basal body and centrosomes with the protein products of both NPHP6 and NPHP4, known genes associated with MKS, CORS and nephronophthisis (a related renal disorder and ciliopathy). In addition, the RPGRIP1L missense mutations found in CORS individuals diminishes the interaction between RPGRIP1L and nephrocystin-4. Our findings show that mutations in RPGRIP1L can cause the multiorgan phenotypic abnormalities found in CORS or MKS, which therefore represent a continuum of the same underlying disorder.     

A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1

Cell adhesion to extracellular matrix (ECM) proteins is crucial for the structural integrity of tissues and epithelial-mesenchymal interactions mediating organ morphogenesis. Here we describe how the loss of a cytoplasmic multi-PDZ scaffolding protein, glutamate receptor interacting protein 1 (GRIP1), leads to the formation of subepidermal hemorrhagic blisters, renal agenesis, syndactyly or polydactyly and permanent fusion of eyelids (cryptophthalmos). Similar malformations are characteristic of individuals with Fraser syndrome and animal models of this human genetic disorder, such as mice carrying the blebbed mutation (bl) in the gene encoding the Fras1 ECM protein. GRIP1 can physically interact with Fras1 and is required for the localization of Fras1 to the basal side of cells. In one animal model of Fraser syndrome, the eye-blebs (eb) mouse, Grip1 is disrupted by a deletion of two coding exons. Our data indicate that GRIP1 is required for normal cell-matrix interactions during early embryonic development and that inactivation of Grip1 causes Fraser syndrome-like defects in mice.     

EphB-ephrin-B interactions suppress colorectal cancer progression by compartmentalizing tumor cells

The genes encoding tyrosine kinase receptors EphB2 and EphB3 are beta-catenin and Tcf4 target genes in colorectal cancer (CRC) and in normal intestinal cells. In the intestinal epithelium, EphB signaling controls the positioning of cell types along the crypt-villus axis. In CRC, EphB activity suppresses tumor progression beyond the earliest stages. Here we show that EphB receptors compartmentalize the expansion of CRC cells through a mechanism dependent on E-cadherin-mediated adhesion. We demonstrate that EphB-mediated compartmentalization restricts the spreading of EphB-expressing tumor cells into ephrin-B1-positive territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1-expressing intestinal cells at the onset of tumorigenesis.