Deletion of CEP164 in mouse photoreceptors post-ciliogenesis interrupts ciliary intraflagellar transport (IFT).

Centrosomal protein of 164 kDa (CEP164) is located at distal appendages of primary cilia and is necessary for basal body (BB) docking to the apical membrane. To investigate the function of photoreceptor CEP164 before and after BB docking, we deleted CEP164 during retina embryonic development (Six3Cre), in postnatal rod photoreceptors (iCre75) and in mature retina using tamoxifen induction (Prom1-ETCre). BBs dock to the cell cortex during postnatal day 6 (P6) to extend a connecting cilium (CC) and an axoneme. P6 retina-specific knockouts (retCep164-/-) are unable to dock BBs, thereby preventing formation of CC or outer segments (OSs). In rod-specific knockouts (rodCep164-/-), Cre expression starts after P7 and CC/OS form. P16 rodCep164-/- rods have nearly normal OS lengths, and maintain OS attachment through P21 despite loss of CEP164. Intraflagellar transport components (IFT88, IFT57 and IFT140) were reduced at P16 rodCep164-/- BBs and CC tips and nearly absent at P21, indicating impaired intraflagellar transport. Nascent OS discs, labeled with a fluorescent dye on P14 and P18 and harvested on P19, showed continued rodCep164-/- disc morphogenesis but absence of P14 discs mid-distally, indicating OS instability. Tamoxifen induction with PROM1ETCre;Cep164F/F (tamCep164-/-) adult mice affected maintenance of both rod and cone OSs. The results suggest that CEP164 is key towards recruitment and stabilization of IFT-B particles at the BB/CC. IFT impairment may be the main driver of ciliary malfunction observed with hypomorphic CEP164 mutations.

TAp73 is a central transcriptional regulator of airway multiciliogenesis.

Motile multiciliated cells (MCCs) have critical roles in respiratory health and disease and are essential for cleaning inhaled pollutants and pathogens from airways. Despite their significance for human disease, the transcriptional control that governs multiciliogenesis remains poorly understood. Here we identify TP73, a p53 homolog, as governing the program for airway multiciliogenesis. Mice with TP73 deficiency suffer from chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance. Organotypic airway cultures pinpoint TAp73 as necessary and sufficient for basal body docking, axonemal extension, and motility during the differentiation of MCC progenitors. Mechanistically, cross-species genomic analyses and complete ciliary rescue of knockout MCCs identify TAp73 as the conserved central transcriptional integrator of multiciliogenesis. TAp73 directly activates the key regulators FoxJ1, Rfx2, Rfx3, and miR34bc plus nearly 50 structural and functional ciliary genes, some of which are associated with human ciliopathies. Our results position TAp73 as a novel central regulator of MCC differentiation.

RPGRIP1L is required for stabilizing epidermal keratinocyte adhesion through regulating desmoglein endocytosis.

Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway.

Centrosomal protein Dzip1l binds Cby, promotes ciliary bud formation, and acts redundantly with Bromi to regulate ciliogenesis in the mouse.

The primary cilium is a microtubule-based organelle required for Hedgehog (Hh) signaling and consists of a basal body, a ciliary axoneme and a compartment between the first two structures, called the transition zone (TZ). The TZ serves as a gatekeeper to control protein composition in cilia, but less is known about its role in ciliary bud formation. Here, we show that centrosomal protein Dzip1l is required for Hh signaling between Smoothened and Sufu. Dzip1l colocalizes with basal body appendage proteins and Rpgrip1l, a TZ protein. Loss of Dzip1l results in reduced ciliogenesis and dysmorphic cilia in vivo Dzip1l interacts with, and acts upstream of, Cby, an appendage protein, in ciliogenesis. Dzip1l also has overlapping functions with Bromi (Tbc1d32) in ciliogenesis, cilia morphogenesis and neural tube patterning. Loss of Dzip1l arrests ciliogenesis at the stage of ciliary bud formation from the TZ. Consistent with this, Dzip1l mutant cells fail to remove the capping protein Cp110 (Ccp110) from the distal end of mother centrioles and to recruit Rpgrip1l to the TZ. Therefore, Dzip1l promotes ciliary bud formation and is required for the integrity of the TZ.

CEP164 is essential for efferent duct multiciliogenesis and male fertility.

Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. In the male reproductive tract, multicilia in efferent ducts (EDs) move in a whip-like motion to prevent sperm agglutination. Previously, we demonstrated that the distal appendage protein CEP164 recruits Chibby1 (Cby1) to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in multiciliated cells (MCCs) (FoxJ1-Cre;CEP164fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility; however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. We found that multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164fl/fl mice. In FoxJ1-Cre;CEP164fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that male infertility in FoxJ1-Cre;CEP164fl/fl mice is caused by sperm agglutination and obstruction of EDs due to loss of multicilia. Our study, therefore, unravels an essential role of the distal appendage protein CEP164 in male fertility.

Roles of steroid hormones in oviductal function

The oviduct (known as the fallopian tube in humans) is the site for fertilization and pre-implantation embryo development. Female steroid hormones, estrogen and progesterone, are known to modulate the morphology and function of cells in the oviduct. In this review, we focus on the actions of estrogen and progesterone on secretory, ciliated, and muscle cell functions and morphologies during fertilization, pre-implantation embryo development, and embryo transport in humans, laboratory rodents and farm animals. We review some aspects of oviductal anatomy and histology and discuss current assisted reproductive technologies (ARTs) that bypass the oviduct and their effects on embryo quality. Lastly, we review the causes of alterations in secretory, ciliated, and muscle cell functions that could result in embryo transport defects.

Conditional knockout mice for the distal appendage protein CEP164 reveal its essential roles in airway multiciliated cell differentiation.

Multiciliated cells of the airways, brain ventricles, and female reproductive tract provide the motive force for mucociliary clearance, cerebrospinal fluid circulation, and ovum transport. Despite their clear importance to human biology and health, the molecular mechanisms underlying multiciliated cell differentiation are poorly understood. Prior studies implicate the distal appendage/transition fiber protein CEP164 as a central regulator of primary ciliogenesis; however, its role in multiciliogenesis remains unknown. In this study, we have generated a novel conditional mouse model that lacks CEP164 in multiciliated tissues and the testis. These mice show a profound loss of airway, ependymal, and oviduct multicilia and develop hydrocephalus and male infertility. Using primary cultures of tracheal multiciliated cells as a model system, we found that CEP164 is critical for multiciliogenesis, at least in part, via its regulation of small vesicle recruitment, ciliary vesicle formation, and basal body docking. In addition, CEP164 is necessary for the proper recruitment of another distal appendage/transition fiber protein Chibby1 (Cby1) and its binding partners FAM92A and FAM92B to the ciliary base in multiciliated cells. In contrast to primary ciliogenesis, CEP164 is dispensable for the recruitment of intraflagellar transport (IFT) components to multicilia. Finally, we provide evidence that CEP164 differentially controls the ciliary targeting of membrane-associated proteins, including the small GTPases Rab8, Rab11, and Arl13b, in multiciliated cells. Altogether, our studies unravel unique requirements for CEP164 in primary versus multiciliogenesis and suggest that CEP164 modulates the selective transport of membrane vesicles and their cargoes into the ciliary compartment in multiciliated cells. Furthermore, our mouse model provides a useful tool to gain physiological insight into diseases associated with defective multicilia.

Chibby is a weak regulator of ß-catenin activity in gastric epithelium.

The canonical Wnt-ß-catenin pathway is important in normal development. Mutations in ß-catenin or proteins involved with regulating its phosphorylation or localization result in its nuclear accumulation where it activates its target genes and stimulates cell proliferation. This pathway is dysregulated in many different types of cancer, including gastric cancer (GC). Chibby (Cby) is a 14-kDa protein that inhibits ß-catenin localization to the nucleus and represses ß-catenin-induced transcriptional activity. In the current study, we examined the expression and function of Cby in normal and cancerous human gastric tissue. Reverse-transcription polymerase chain reaction and immunohistochemistry revealed that Cby is expressed in human stomach and localized to glandular elements. Immunohistochemical staining intensity of Cby was decreased in GC tissue when compared with normal gastric epithelium. In AGS cells, a human gastric carcinoma cell line, Cby expression was low. Stable AGS cell transfectants overexpressing Cby were prepared. Cby overexpression did not affect proliferation rates or ß-catenin levels. However, confocal microscopy and subcellular fractionation studies revealed that Cby overexpression resulted in a small decrease in nuclear ß-catenin. Moreover, Cby overexpression caused a molecular weight shift in nuclear ß-catenin and resulted in decreased ß-catenin signaling in AGS cells as measured by the TopFlash assay. However, Cby overexpression did not affect c-Myc protein levels. To conclude, Cby expression was decreased in GC samples and Cby expression altered ß-catenin localization in cultured GC cells. However, Cby did not affect cell proliferation rates or ß-catenin-induced protein expression. Cby may be involved in the early events in the pathogenesis of GC.

Estrogen receptor α is required for oviductal transport of embryos.

Newly fertilized embryos spend the first few days within the oviduct and are transported to the uterus, where they implant onto the uterine wall. An implantation of the embryo before reaching the uterus could result in ectopic pregnancy and lead to maternal death. Estrogen is necessary for embryo transport in mammals; however, the mechanism involved in estrogen-mediated cellular function within the oviduct remains unclear. In this study, we show in mouse models that ciliary length and beat frequency of the oviductal epithelial cells are regulated through estrogen receptor α (ESR1) but not estrogen receptor ß (ESR2). Gene profiling indicated that transcripts in the WNT/ß-catenin (WNT/CTNNB1) signaling pathway were regulated by estrogen in mouse oviduct, and inhibition of this pathway in a whole oviduct culture system resulted in a decreased embryo transport distance. However, selective ablation of CTNNB1 from the oviductal ciliated cells did not affect embryo transport, possibly because of a compensatory mechanism via intact CTNNB1 in the adjacent secretory cells. In summary, we demonstrated that disruption of estrogen signaling in oviductal epithelial cells alters ciliary function and impairs embryo transport. Therefore, our findings may provide a better understanding of etiology of the ectopic pregnancy that is associated with alteration of estrogen signals.-Li, S., O'Neill, S. R. S., Zhang, Y., Holtzman, M. J., Takemaru, K.-I., Korach, K. S., Winuthayanon, W. Estrogen receptor α is required for oviductal transport of embryos.

Abrogation of ß-catenin signaling in oligodendrocyte precursor cells reduces glial scarring and promotes axon regeneration after CNS injury.

When the brain or spinal cord is injured, glial cells in the damaged area undergo complex morphological and physiological changes resulting in the formation of the glial scar. This scar contains reactive astrocytes, activated microglia, macrophages and other myeloid cells, meningeal cells, proliferating oligodendrocyte precursor cells (OPCs), and a dense extracellular matrix. Whether the scar is beneficial or detrimental to recovery remains controversial. In the acute phase of recovery, scar-forming astrocytes limit the invasion of leukocytes and macrophages, but in the subacute and chronic phases of injury the glial scar is a physical and biochemical barrier to axonal regrowth. The signals that initiate the formation of the glial scar are unknown. Both canonical and noncanonical signaling Wnts are increased after spinal cord injury (SCI). Because Wnts are important regulators of OPC and oligodendrocyte development, we examined the role of canonical Wnt signaling in the glial reactions to CNS injury. In adult female mice carrying an OPC-specific conditionally deleted ß-catenin gene, there is reduced proliferation of OPCs after SCI, reduced accumulation of activated microglia/macrophages, and reduced astrocyte hypertrophy. Using an infraorbital optic nerve crush injury, we show that reducing ß-catenin-dependent signaling in OPCs creates an environment that is permissive to axonal regeneration. Viral-induced expression of Wnt3a in the normal adult mouse spinal cord induces an injury-like response in glia. Thus canonical Wnt signaling is both necessary and sufficient to induce injury responses among glial cells. These data suggest that targeting Wnt expression after SCI may have therapeutic potential in promoting axon regeneration.

The Cby3/ciBAR1 complex positions the annulus along the sperm flagellum during spermiogenesis.

Proper compartmentalization of the sperm flagellum is essential for fertility. The annulus is a septin-based ring that demarcates the midpiece (MP) and the principal piece (PP). It is assembled at the flagellar base, migrates caudally, and halts upon arriving at the PP. However, the mechanisms governing annulus positioning remain unknown. We report that a Chibby3 (Cby3)/Cby1-interacting BAR domain-containing 1 (ciBAR1) complex is required for this process. Ablation of either gene in mice results in male fertility defects, caused by kinked sperm flagella with the annulus mispositioned in the PP. Cby3 and ciBAR1 interact and colocalize to the annulus near the curved membrane invagination at the flagellar pocket. In the absence of Cby3, periannular membranes appear to be deformed, allowing the annulus to migrate over the fibrous sheath into the PP. Collectively, our results suggest that the Cby3/ciBAR1 complex regulates local membrane properties to position the annulus at the MP/PP junction.

Essential Roles of Efferent Duct Multicilia in Male Fertility.

Cilia are microtubule-based hair-like organelles on the cell surface. Cilia have been implicated in various biological processes ranging from mechanosensation to fluid movement. Ciliary dysfunction leads to a plethora of human diseases, known as ciliopathies. Although non-motile primary cilia are ubiquitous, motile multicilia are found in restricted locations of the body, such as the respiratory tract, the oviduct, the efferent duct, and the brain ventricles. Multicilia beat in a whip-like motion to generate fluid flow over the apical surface of an epithelium. The concerted ciliary motion provides the driving force critical for clearing airway mucus and debris, transporting ova from the ovary to the uterus, maintaining sperm in suspension, and circulating cerebrospinal fluid in the brain. In the male reproductive tract, multiciliated cells (MCCs) were first described in the mid-1800s, but their importance in male fertility remained elusive until recently. MCCs exist in the efferent ducts, which are small, highly convoluted tubules that connect the testis to the epididymis and play an essential role in male fertility. In this review, we will introduce multiciliogenesis, discuss mouse models of male infertility with defective multicilia, and summarize our current knowledge on the biological function of multicilia in the male reproductive tract.

Sustained glymphatic transport and impaired drainage to the nasal cavity observed in multiciliated cell ciliopathies with hydrocephalus.

BACKGROUND: Hydrocephalus (increased ventricular size due to CSF accumulation) is a common finding in human ciliopathies and in mouse models with genetic depletion of the multiciliated cell (MCC) cilia machinery. However, the contribution of MCC to CSF dynamics and, the mechanism by which impaired MCC function leads to hydrocephalus remains poorly understood. The aim of our study was to examine if defects in MCC ciliogenesis and cilia-generated CSF flow impact central nervous system (CNS) fluid homeostasis including glymphatic transport and solute waste drainage. METHODS: We used two distinct mouse models of MCC ciliopathy: MCC-specific CEP164 conditional knockout mice (FOXJ1-Cre;CEP164fl/fl (N = 10), 3-month-old) and p73 knock-out (p73-/- (N = 8), 5-month-old) mice. Age-matched, wild-type littermates for each of the mutants served as controls. Glymphatic transport and solute drainage was quantified using in vivo T1 mapping by magnetic resonance imaging (MRI) after CSF infusion of gadoteric acid. Brain morphometry and aquaporin 4 expression (AQP4) was also assessed. Intracranial pressure (ICP) was measured in separate cohorts. RESULTS: In both of the two models of MCC ciliopathy we found the ventriculomegaly to be associated with normal ICP. We showed that FOXJ1-Cre;CEP164fl/fl mice with hydrocephalus still demonstrated sustained glymphatic transport and normal AQP4 expression along capillaries. In p73-/- mice glymphatic transport was even increased, and this was paralleled by an increase in AQP4 polarization around capillaries. Further, solute drainage via the cribriform plate to the nasal cavity was severely impaired in both ciliopathy models and associated with chronic rhinitis and olfactory bulb hypoplasia. CONCLUSIONS: The combination of sustained glymphatic transport, impaired solute drainage via the cribriform plate to the nasal cavity and hydrocephalus has not previously been reported in models of MCC ciliopathy. Our data enhance our understanding of how different types of ciliopathies contribute to disruption of CNS fluid homeostasis, manifested in pathologies such as hydrocephalus.

nNOS regulates ciliated cell polarity, ciliary beat frequency, and directional flow in mouse trachea.

Clearance of the airway is dependent on directional mucus flow across the mucociliary epithelium, and deficient flow is implicated in a range of human disorders. Efficient flow relies on proper polarization of the multiciliated cells and sufficient ciliary beat frequency. We show that NO, produced by nNOS in the multiciliated cells of the mouse trachea, controls both the planar polarity and the ciliary beat frequency and is thereby necessary for the generation of the robust flow. The effect of nNOS on the polarity of ciliated cells relies on its interactions with the apical networks of actin and microtubules and involves RhoA activation. The action of nNOS on the beat frequency is mediated by guanylate cyclase; both NO donors and cGMP can augment fluid flow in the trachea and rescue the deficient flow in nNOS mutants. Our results link insufficient availability of NO in ciliated cells to defects in flow and ciliary activity and may thereby explain the low levels of exhaled NO in ciliopathies.

Loss of the ciliary protein Chibby1 in mice leads to exocrine pancreatic degeneration and pancreatitis.

Primary cilia protrude from the apical surface of many cell types and act as a sensory organelle that regulates diverse biological processes ranging from chemo- and mechanosensation to signaling. Ciliary dysfunction is associated with a wide array of genetic disorders, known as ciliopathies. Polycystic lesions are commonly found in the kidney, liver, and pancreas of ciliopathy patients and mouse models. However, the pathogenesis of the pancreatic phenotype remains poorly understood. Chibby1 (Cby1), a small conserved coiled-coil protein, localizes to the ciliary base and plays a crucial role in ciliogenesis. Here, we report that Cby1-knockout (KO) mice develop severe exocrine pancreatic atrophy with dilated ducts during early postnatal development. A significant reduction in the number and length of cilia was observed in Cby1-KO pancreta. In the adult Cby1-KO pancreas, inflammatory cell infiltration and fibrosis were noticeable. Intriguingly, Cby1-KO acinar cells showed an accumulation of zymogen granules (ZGs) with altered polarity. Moreover, isolated acini from Cby1-KO pancreas exhibited defective ZG secretion in vitro. Collectively, our results suggest that, upon loss of Cby1, concomitant with ciliary defects, acinar cells accumulate ZGs due to defective exocytosis, leading to cell death and progressive exocrine pancreatic degeneration after birth.

Chibby promotes adipocyte differentiation through inhibition of beta-catenin signaling.

The canonical Wnt/beta-catenin signaling pathway plays diverse roles in embryonic development and disease. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and in mice. Here, we report that the beta-catenin antagonist Chibby (Cby) is required for adipocyte differentiation. Cby is expressed in adipose tissue in mice, and Cby protein levels increase during adipogenic differentiation of 3T3-L1 cells. Ectopic expression of Cby induces spontaneous differentiation of these cells into mature adipocytes to an extent similar to that of dominant-negative Tcf-4. In contrast, depletion of Cby by RNA interference potently blocks adipogenesis of 3T3-L1 and mouse embryonic stem cells. In support of this, embryonic fibroblasts obtained from Cby-deficient embryos display attenuated differentiation to the adipogenic lineage. Mechanistically, Cby promotes adipocyte differentiation, in part by inhibiting beta-catenin, since gain or loss of function of Cby influences beta-catenin signaling in 3T3-L1 cells. Our results therefore establish Cby as a novel proadipogenic factor required for adipocyte differentiation.

Crystal structure of a full-length beta-catenin.

beta-catenin plays essential roles in cell adhesion and Wnt signaling, while deregulation of beta-catenin is associated with multiple diseases including cancers. Here, we report the crystal structures of full-length zebrafish beta-catenin and a human beta-catenin fragment that contains both the armadillo repeat and the C-terminal domains. Our structures reveal that the N-terminal region of the C-terminal domain, a key component of the C-terminal transactivation domain, forms a long alpha helix that packs on the C-terminal end of the armadillo repeat domain, and thus forms part of the beta-catenin superhelical core. The existence of this helix redefines our view of interactions of beta-catenin with some of its critical partners, including ICAT and Chibby, which may form extensive interactions with this C-terminal domain alpha helix. Our crystallographic and NMR studies also suggest that the unstructured N-terminal and C-terminal tails interact with the ordered armadillo repeat domain in a dynamic and variable manner.

Chibby forms a homodimer through a heptad repeat of leucine residues in its C-terminal coiled-coil motif.

BACKGROUND: The Wnt/beta-catenin signaling pathway plays crucial roles in embryonic development and in maintenance of organs and tissues in adults. Chibby (Cby) is an evolutionarily conserved molecule that physically interacts with the key downstream coactivator beta-catenin and represses its transcriptional activation potential. Although Cby harbors a predicted coiled-coil motif in the C-terminal region, its molecular nature and functional importance remain largely unexplored. RESULTS: Here we report that Cby forms a stable complex with itself. Alanine substitutions of two or more of four critical leucine residues within the C-terminal heptad repeats completely eliminate the Cby-Cby interaction. The Cby oligomer predominantly exists as a homodimer. Furthermore, we found that dimerization-deficient Cby mutants still retain the ability to bind to beta-catenin and to repress beta-catenin-dependent gene activation. More importantly, Cby homodimerization is required for its efficient interaction with the nuclear import receptor importin-alpha and subsequent nuclear translocation. CONCLUSION: Our comprehensive mutational analysis of the Cby coiled-coil domain reveals that the four heptad leucine residues play an essential role in mediating Cby homodimerization. Although monomeric Cby is sufficient to bind to beta-catenin and block beta-catenin-mediated transcriptional activation, homodimer formation of Cby is indispensable for its efficient nuclear import.

Dapper, a Dishevelled-associated antagonist of beta-catenin and JNK signaling, is required for notochord formation.

Dapper was isolated in a screen for proteins interacting with Dishevelled, a key factor in Wnt signaling. Dapper and Dishevelled colocalize intracellularly and form a complex with Axin, GSK-3, CKI, and beta-catenin. Overexpression of Dapper increases Axin and GSK-3 in this complex, resulting in decreased soluble beta-catenin and decreased activation of beta-catenin-responsive genes. Dapper also inhibits activation by Dishevelled of c-Jun N-terminal kinase (JNK), a component of beta-catenin-independent Frizzled signaling. Inhibition of Dapper activates both beta-catenin-responsive genes and an AP1-responsive promoter, demonstrating that Dapper is a general Dishevelled antagonist. Depletion of maternal Dapper RNA from Xenopus embryos results in loss of notochord and head structures, demonstrating that Dapper is required for normal vertebrate development.