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.     

Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling

The vertebrate planar cell polarity (PCP) pathway has previously been found to control polarized cell behaviors rather than cell fate. We report here that disruption of Xenopus laevis orthologs of the Drosophila melanogaster PCP effectors inturned (in) or fuzzy (fy) affected not only PCP-dependent convergent extension but also elicited embryonic phenotypes consistent with defective Hedgehog signaling. These defects in Hedgehog signaling resulted from a broad requirement for Inturned and Fuzzy in ciliogenesis. We show that these proteins govern apical actin assembly and thus control the orientation, but not assembly, of ciliary microtubules. Finally, accumulation of Dishevelled and Inturned near the basal apparatus of cilia suggests that these proteins function in a common pathway with core PCP components to regulate ciliogenesis. Together, these data highlight the interrelationships between cell polarity, cellular morphogenesis, signal transduction and cell fate specification.     

Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells

The planar cell polarity (PCP) signaling system governs many aspects of polarized cell behavior. Here, we use an in vivo model of vertebrate mucociliary epithelial development to show that Dishevelled (Dvl) is essential for the apical positioning of basal bodies. We find that Dvl and Inturned mediate the activation of the Rho GTPase specifically at basal bodies, and that these three proteins together mediate the docking of basal bodies to the apical plasma membrane. Moreover, we find that this docking involves a Dvl-dependent association of basal bodies with membrane-bound vesicles and the vesicle-trafficking protein, Sec8. Once docked, basal bodies again require Dvl and Rho for the planar polarization that underlies directional beating of cilia. These results demonstrate previously undescribed functions for PCP signaling components and suggest that a common signaling apparatus governs both apical docking and planar polarization of basal bodies.     

Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway

The mammalian auditory sensory organ, the organ of Corti, consists of sensory hair cells with uniformly oriented stereocilia on the apical surfaces and has a distinct planar cell polarity (PCP) parallel to the sensory epithelium. It is not certain how this polarity is achieved during differentiation. Here we show that the organ of Corti is formed from a thicker and shorter postmitotic primordium through unidirectional extension, characteristic of cellular intercalation known as convergent extension. Mutations in the PCP pathway interfere with this extension, resulting a shorter and wider cochlea as well as misorientation of stereocilia. Furthermore, parallel to the homologous pathway in Drosophila melanogaster, a mammalian PCP component Dishevelled2 shows PCP-dependent polarized subcellular localization across the organ of Corti. Taken together, these data suggest that there is a conserved molecular mechanism for PCP pathways in invertebrates and vertebrates and indicate that the mammalian PCP pathway might directly couple cellular intercalations to PCP establishment in the cochlea.     

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.     

Complex interactions between genes controlling trafficking in primary cilia

Cilia-associated human genetic disorders are striking in the diversity of their abnormalities and their complex inheritance. Inactivation of the retrograde ciliary motor by mutations in DYNC2H1 causes skeletal dysplasias that have strongly variable expressivity. Here we define previously unknown genetic relationships between Dync2h1 and other genes required for ciliary trafficking. Mutations in mouse Dync2h1 disrupt cilia structure, block Sonic hedgehog signaling and cause midgestation lethality. Heterozygosity for Ift172, a gene required for anterograde ciliary trafficking, suppresses cilia phenotypes, Sonic hedgehog signaling defects and early lethality of Dync2h1 homozygotes. Ift122, like Dync2h1, is required for retrograde ciliary trafficking, but reduction of Ift122 gene dosage also suppresses the Dync2h1 phenotype. These genetic interactions illustrate the cell biology underlying ciliopathies and argue that mutations in intraflagellar transport genes cause their phenotypes because of their roles in cilia architecture rather than direct roles in signaling.     

A Gata6-Wnt pathway required for epithelial stem cell development and airway regeneration

Epithelial organs, including the lung, are known to possess regenerative abilities through activation of endogenous stem cell populations, but the molecular pathways regulating stem cell expansion and regeneration are not well understood. Here we show that Gata6 regulates the temporal appearance and number of bronchioalveolar stem cells (BASCs) in the lung, its absence in Gata6-null lung epithelium leading to the precocious appearance of BASCs and concurrent loss in epithelial differentiation. This expansion of BASCs was the result of a pronounced increase in canonical Wnt signaling in lung epithelium upon loss of Gata6. Expression of the noncanonical Wnt receptor Fzd2 was downregulated in Gata6 mutants and increased Fzd2 or decreased beta-catenin expression rescued, in part, the lung epithelial defects in Gata6 mutants. During lung epithelial regeneration, canonical Wnt signaling was activated in the niche containing BASCs and forced activation of Wnt signaling led to a large increase in BASC numbers. Moreover, Gata6 was required for proper lung epithelial regeneration, and postnatal loss of Gata6 led to increased BASC expansion and decreased differentiation. Together, these data demonstrate that Gata6-regulated Wnt signaling controls the balance between progenitor expansion and epithelial differentiation required for both lung development and regeneration.     

A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition

Mutations affecting ciliary components cause ciliopathies. As described here, we investigated Tectonic1 (Tctn1), a regulator of mouse Hedgehog signaling, and found that it is essential for ciliogenesis in some, but not all, tissues. Cell types that do not require Tctn1 for ciliogenesis require it to localize select membrane-associated proteins to the cilium, including Arl13b, AC3, Smoothened and Pkd2. Tctn1 forms a complex with multiple ciliopathy proteins associated with Meckel and Joubert syndromes, including Mks1, Tmem216, Tmem67, Cep290, B9d1, Tctn2 and Cc2d2a. Components of this complex co-localize at the transition zone, a region between the basal body and ciliary axoneme. Like Tctn1, loss of Tctn2, Tmem67 or Cc2d2a causes tissue-specific defects in ciliogenesis and ciliary membrane composition. Consistent with a shared function for complex components, we identified a mutation in TCTN1 that causes Joubert syndrome. Thus, a transition zone complex of Meckel and Joubert syndrome proteins regulates ciliary assembly and trafficking, suggesting that transition zone dysfunction is the cause of these ciliopathies.     

Defective planar cell polarity in polycystic kidney disease

Morphogenesis involves coordinated proliferation, differentiation and spatial distribution of cells. We show that lengthening of renal tubules is associated with mitotic orientation of cells along the tubule axis, demonstrating intrinsic planar cell polarization, and we demonstrate that mitotic orientations are significantly distorted in rodent polycystic kidney models. These results suggest that oriented cell division dictates the maintenance of constant tubule diameter during tubular lengthening and that defects in this process trigger renal tubular enlargement and cyst formation.     

Loss-of-function mutations in the EGF-CFC gene CFC1 are associated with human left-right laterality defects

All vertebrates display a characteristic asymmetry of internal organs with the cardiac apex, stomach and spleen towards the left, and the liver and gall bladder on the right. Left-right (L-R) axis abnormalities or laterality defects are common in humans (1 in 8,500 live births). Several genes (such as Nodal, Ebaf and Pitx2) have been implicated in L-R organ positioning in model organisms. In humans, relatively few genes have been associated with a small percentage of human situs defects. These include ZIC3 (ref. 5), LEFTB (formerly LEFTY2; ref. 6) and ACVR2B (encoding activin receptor IIB; ref. 7). The EGF-CFC genes, mouse Cfc1 (encoding the Cryptic protein; ref. 9) and zebrafish one-eyed pinhead (oep; refs 10, 11) are essential for the establishment of the L-R axis. EGF-CFC proteins act as co-factors for Nodal-related signals, which have also been implicated in L-R axis development. Here we identify loss-of-function mutations in human CFC1 (encoding the CRYPTIC protein) in patients with heterotaxic phenotypes (randomized organ positioning). The mutant proteins have aberrant cellular localization in transfected cells and are functionally defective in a zebrafish oep-mutant rescue assay. Our findings indicate that the essential role of EGF-CFC genes and Nodal signalling in left-right axis formation is conserved from fish to humans. Moreover, our results support a role for environmental and/or genetic modifiers in determining the ultimate phenotype in humans.     

Loss of BBS proteins causes anosmia in humans and defects in olfactory cilia structure and function in the mouse

Defects in cilia are associated with several human disorders, including Kartagener syndrome, polycystic kidney disease, nephronophthisis and hydrocephalus. We proposed that the pleiotropic phenotype of Bardet-Biedl syndrome (BBS), which encompasses retinal degeneration, truncal obesity, renal and limb malformations and developmental delay, is due to dysfunction of basal bodies and cilia. Here we show that individuals with BBS have partial or complete anosmia. To test whether this phenotype is caused by ciliary defects of olfactory sensory neurons, we examined mice with deletions of Bbs1 or Bbs4. Loss of function of either BBS protein affected the olfactory, but not the respiratory, epithelium, causing severe reduction of the ciliated border, disorganization of the dendritic microtubule network and trapping of olfactory ciliary proteins in dendrites and cell bodies. Our data indicate that BBS proteins have a role in the microtubule organization of mammalian ciliated cells and that anosmia might be a useful determinant of other pleiotropic disorders with a suspected ciliary involvement.     

The stress kinase MKK7 couples oncogenic stress to p53 stability and tumor suppression

Most preneoplastic lesions are quiescent and do not progress to form overt tumors. It has been proposed that oncogenic stress activates the DNA damage response and the key tumor suppressor p53, which prohibits tumor growth. However, the molecular pathways by which cells sense a premalignant state in vivo are largely unknown. Here we report that tissue-specific inactivation of the stress signaling kinase MKK7 in KRas(G12D)-driven lung carcinomas and NeuT-driven mammary tumors markedly accelerates tumor onset and reduces overall survival. Mechanistically, MKK7 acts through the kinases JNK1 and JNK2, and this signaling pathway directly couples oncogenic and genotoxic stress to the stability of p53, which is required for cell cycle arrest and suppression of epithelial cancers. These results show that MKK7 functions as a major tumor suppressor in lung and mammary cancer in mouse and identify MKK7 as a vital molecular sensor to set a cellular anti-cancer barrier.     

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.     

Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation

Wnt signaling defines the colonic epithelial progenitor cell phenotype, and mutations in the gene adenomatous polyposis coli (APC) that activate the Wnt pathway cause the familial adenomatous polyposis coli (FAP) syndrome and most sporadic colon cancers. The mechanisms that regulate the transition of epithelial precursor cells into their differentiated derivatives are poorly characterized. We report that Indian hedgehog (Ihh) is expressed by mature colonocytes and regulates their differentiation in vitro and in vivo. Hedgehog (Hh) signaling restricts the expression of Wnt targets to the base of the colonic crypt in vivo, and transfection of Ihh into colon cancer cells leads to a downregulation of both components of the nuclear TCF4-beta-catenin complex and abrogates endogenous Wnt signaling in vitro. In turn, expression of Ihh is downregulated in polyps of individuals with FAP and expression of doxycycline-inducible dominant negative TCF4 (dnTCF4) restores Ihh expression in APC mutant DLD-1 colon cancer cells. These data identify a new Wnt-Hh axis in colonic epithelial renewal.     

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.     

Unraveling adaptive evolution: how a single point mutation affects the protein coregulation network

Understanding the mechanisms of evolution requires identification of the molecular basis of the multiple (pleiotropic) effects of specific adaptive mutations. We have characterized the pleiotropic effects on protein levels of an adaptive single-base pair substitution in the coding sequence of a signaling pathway gene in the bacterium Pseudomonas fluorescens SBW25. We find 52 proteomic changes, corresponding to 46 identified proteins. None of these proteins is required for the adaptive phenotype. Instead, many are found within specific metabolic pathways associated with fitness-reducing (that is, antagonistic) effects of the mutation. The affected proteins fall within a single coregulatory network. The mutation 'rewires' this network by drawing particular proteins into tighter coregulating relationships. Although these changes are specific to the mutation studied, the quantitatively altered proteins are also affected in a coordinated way in other examples of evolution to the same niche.     

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.