Ciliated Cell Differentiation Research Articles

Cystic fibrosis airway epithelium remodelling: involvement of inflammation.

Chronic inflammation is a hallmark of cystic fibrosis (CF) lung disease and airway epithelium damage and remodelling are important components of lung pathology progression in CF. Whether this remodelling is secondary to deleterious infectious and inflammatory mediators, or to alterations of CF human airway epithelial (HAE) cells, such as their hyper inflammatory phenotype or their basic cystic fibrosis transmembrane conductance regulator (CFTR) default, remains debated. In this study, we evaluated the involvement of alterations of CF HAE cells in airway epithelium remodelling. HAE cells from non-CF and CF patients were cultured in an air-liquid interface, with and without inflammatory stimulation, along the regeneration process, and the remodelling of the reconstituted epithelium was analysed. We confirmed that CF HAE cells showed a hyperinflammatory phenotype which was lost with time. In comparison to non-CF epithelium, CF epithelium regeneration in the absence of exogenous inflammation was higher and exhibited basal cell hyperplasia. This remodelling was mimicked by inflammatory stimulation of non-CF cells and was absent when CF HAE cells were no longer hyperinflamed. Moreover, the number of goblet cells was similar in non-CF and CF cultures and increased equally under inflammatory stimulation. Finally, whatever the inflammatory environment, CF cultures showed a delay in ciliated cell differentiation. In conclusion, alterations of CF HAE cells partly regulate airway epithelium remodelling following injury and regeneration. This remodelling, together with goblet cell hyperplasia induced by exogenous inflammation and alteration of ciliated cell differentiation, may worsen mucociliary clearance impairment, leading to injury.

Early events in the pathogenesis of chronic obstructive pulmonary disease. Smoking-induced reprogramming of airway epithelial basal progenitor cells.

The airway epithelium is the primary site of the earliest pathologic changes induced by smoking, contributing to the development of chronic obstructive pulmonary disease (COPD). The normal human airway epithelium is composed of several major cell types, including differentiated ciliated and secretory cells, intermediate undifferentiated cells, and basal cells (BC). BC contain the stem/progenitor cell population responsible for maintenance of the normally differentiated airway epithelium. Although inflammatory and immune processes play a significant role in the pathogenesis of COPD, the earliest lesions include hyperplasia of the BC population, suggesting that the disease may start with this cell type. Apart from BC hyperplasia, smoking induces a number of COPD-relevant airway epithelial remodeling phenotypes that are likely initiated in the BC population, including mucous cell hyperplasia, squamous cell metaplasia, epithelial-mesenchymal transition, altered ciliated and nonmucous secretory cell differentiation, and suppression of junctional barrier integrity. Significant progress has been recently made in understanding the biology of human airway BC, including gene expression features, stem/progenitor, and other functions, including interaction with other airway cell types. Accumulating evidence suggests that human airway BC function as both sensors and cellular sources of various cytokines and growth factors relevant to smoking-associated airway injury, as well as the origin of various molecular and histological phenotypes relevant to the pathogenesis of COPD. In the context of these considerations, we suggest that early BC-specific smoking-induced molecular changes are critical to the pathogenesis of COPD, and these represent a candidate target for novel therapeutic approaches to prevent COPD progression in susceptible individuals.

Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation.

Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.

Submersion and hypoxia inhibit ciliated cell differentiation in a notch-dependent manner.

The epithelium that lines the conducting airways is composed of several distinct cell types that differentiate from common progenitor cells. The signals that control fate selection and differentiation of ciliated cells, a major component of the epithelium, are not completely understood. Ciliated cell differentiation can be accomplished in vitro when primary normal human bronchial epithelial (NHBE) cells are cultured at an air-liquid interface, but is inhibited when NHBE cells are cultured under submerged conditions. The mechanism by which submersion prevents ciliogenesis is not understood, but may provide clues to in vivo regulation of ciliated cell differentiation. We hypothesized that submersion creates a hypoxic environment that prevents ciliated cell differentiation by blocking the gene expression program required for ciliogenesis. This was confirmed by showing that expression of multicilin and Forkhead box J1, key factors needed for ciliated cell differentiation, was inhibited when NHBE cells were cultured in submerged and hypoxic conditions. Multicilin and Forkhead box J1 expression and ciliated cell differentiation were restored in submerged and hypoxic cells upon treatment with the γ-secretase inhibitor, N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT), which suggested that Notch signaling was involved. Overexpression of Notch intracellular domain inhibited differentiation in the presence of DAPT, confirming the role of Notch signaling. These results indicate that submersion and hypoxia prevent ciliated cell differentiation by maintaining Notch signaling, which represses genes necessary for ciliogenesis. These data provide new insights into the molecular mechanisms that control human bronchial differentiation.

Nasal Epithelial Cells Can Act as a Physiological Surrogate for Paediatric Asthma Studies

IntroductionDifferentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures.MethodsPaired nasal and bronchial epithelial cells from asthmatic children (n = 9) were differentiated for 28 days under unstimulated and IL-13-stimulated conditions. Morphological and physiological markers were analysed using immunocytochemistry, transepithelial-electrical-resistance, Quantitative Real-time-PCR, ELISA and multiplex cytokine/chemokine analysis.ResultsPhysiologically, nasal epithelial cells from asthmatic children exhibit similar cytokine responses to stimulation with IL-13 compared with paired bronchial epithelial cells. Morphologically however, nasal epithelial cells differed significantly from bronchial epithelial cells from asthmatic patients under unstimulated and IL-13-stimulated conditions. Nasal epithelial cells exhibited lower proliferation/differentiation rates and lower percentages of goblet and ciliated cells when unstimulated, while exhibiting a diminished and varied response to IL-13.ConclusionsWe conclude that morphologically, nasal epithelial cells would not be a suitable surrogate due to a significantly lower rate of proliferation and differentiation of goblet and ciliated cells. Physiologically, nasal epithelial cells respond similarly to exogenous stimulation with IL-13 in cytokine production and could be used as a physiological surrogate in the event that bronchial epithelial cells are not available.

Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro

The purpose of this study was to compare cell growth characteristics, ciliated cell differentiation, and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures. Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods. Epithelial cell growth characteristics were observed by inverted phase contrast microscopy. Ciliated cell differentiation was detected by β-tubulin IVand ZO-1 immunocytochemistry. Basal and ATP-stimulated ciliary beat frequency (CBF) was measured using a highspeed digital microscopic imaging system. Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition, with both types of cultures comprising ciliated and non-ciliated epithelial cells. Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures. In both culture systems, the highest ciliated cell density appeared at 7th-10th culture day and declined with time, with the lifespan of ciliated cells ranging from 14 to 21 days. Overall, 10% of the cells in explant cultures and 20% of the cells in the dissociated tissue cultures were ciliated. These two cultures demonstrated similar ciliary beat frequency values at baseline (7.78 ± 1.99 Hz and 7.91 ± 2.52 Hz, respectively) and reacted equivalently following stimulation with 100 μM ATP. The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells, which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

Localized Fgf10 expression is not required for lung branching morphogenesis but prevents differentiation of epithelial progenitors

Localized Fgf10 expression in the distal mesenchyme adjacent to sites of lung bud formation has long been thought to drive stereotypic branching morphogenesis even though isolated lung epithelium branches in the presence of non-directional exogenous Fgf10 in Matrigel. Here, we show that lung agenesis in Fgf10 knockout mice can be rescued by ubiquitous overexpression of Fgf10, indicating that precisely localized Fgf10 expression is not required for lung branching morphogenesis in vivo. Fgf10 expression in the mesenchyme itself is regulated by Wnt signaling. Nevertheless, we found that during lung initiation simultaneous overexpression of Fgf10 is not sufficient to rescue the absence of primary lung field specification in embryos overexpressing Dkk1, a secreted inhibitor of Wnt signaling. However, after lung initiation, simultaneous overexpression of Fgf10 in lungs overexpressing Dkk1 is able to rescue defects in branching and proximal-distal differentiation. We also show that Fgf10 prevents the differentiation of distal epithelial progenitors into Sox2-expressing airway epithelial cells in part by activating epithelial β-catenin signaling, which negatively regulates Sox2 expression. As such, these findings support a model in which the main function of Fgf10 during lung development is to regulate proximal-distal differentiation. As the lung buds grow out, proximal epithelial cells become further and further displaced from the distal source of Fgf10 and differentiate into bronchial epithelial cells. Interestingly, our data presented here show that once epithelial cells are committed to the Sox2-positive airway epithelial cell fate, Fgf10 prevents ciliated cell differentiation and promotes basal cell differentiation.

RFX3 Modulation of FOXJ1 regulation of cilia genes in the human airway epithelium

BackgroundCiliated cells play a central role in cleansing the airways of inhaled contaminants. They are derived from basal cells that include the airway stem/progenitor cells. In animal models, the transcription factor FOXJ1 has been shown to induce differentiation to the ciliated cell lineage, and the RFX transcription factor-family has been shown to be necessary for, but not sufficient to induce, correct cilia development.MethodsTo test the hypothesis that FOXJ1 and RFX3 cooperatively induce expression of ciliated genes in the differentiation process of basal progenitor cells toward a ciliated cell linage in the human airway epithelium, primary human airway basal cells were assessed under conditions of in vitro differentiation induced by plasmid-mediated gene transfer of FOXJ1 and/or RFX3. TaqMan PCR was used to quantify mRNA levels of basal, secretory, and cilia-associated genes.ResultsBasal cells, when cultured in air-liquid interface, differentiated into a ciliated epithelium, expressing FOXJ1 and RFX3. Transfection of FOXJ1 into resting basal cells activated promoters and induced expression of ciliated cell genes as well as both FOXJ1 and RFX3, but not basal cell genes. Transfection of RFX3 induced expression of RFX3 but not FOXJ1, nor the expression of cilia-related genes. The combination of FOXJ1 + RFX3 enhanced ciliated gene promoter activity and mRNA expression beyond that due to FOXJ1 alone. Corroborating immunoprecipitation studies demonstrated an interaction between FOXJ1 and RFX3.ConclusionFOXJ1 is an important regulator of cilia gene expression during ciliated cell differentiation, with RFX3 as a transcriptional co-activator to FOXJ1, helping to induce the expression of cilia genes in the process of ciliated cell differentiation of basal/progenitor cells.

Expression of cilium-associated genes defines novel molecular subtypes of idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is an untreatable lung disease with a median survival of only 3–5 years that is diagnosed using a combination of clinical, radiographic and pathologic criteria. Histologically,...

Apc deficiency alters pulmonary epithelial cell fate and inhibits Nkx2.1 via triggering TGF-beta signaling

Wnt signaling is critical for cell fate specification and cell differentiation in many organs, but its function in pulmonary neuroendocrine cell (PNEC) differentiation has not been fully addressed. In this study, we examined the role of canonical Wnt signaling by targeting the gene for Adenomatous Polyposis Coli (Apc), which controls Wnt signaling activity via mediating phosphorylation of beta-catenin (Ctnnb). Targeting the Apc gene in lung epithelial progenitors by Nkx2.1-cre stabilized Ctnnb and activated canonical Wnt signaling. Apc deficiency altered lung epithelial cell fate by inhibiting Clara and ciliated cell differentiation and activating Uchl1, a marker of neuroendocrine cells. Similar to PNEC in normal lung, Uchl1positive cells were innervated. In mice with targeted inactivation of Ctnnb by Nkx2.1-cre, PNEC differentiation was not interrupted. These indicate that, after lung primordium formation, Wnt signaling is not essential for PNEC differentiation; however, its over-activation promotes PNEC features. Interestingly, Nkx2.1 was extinguished in Apc deficient epithelial progenitors before activation of Uchl1. Examination of Nkx2.1 null lungs suggested that early deletion of Nkx2.1 inhibits PNEC differentiation, while late repression does not. Nkx2.1 was specifically inhibited in Apc deficient lungs but not in Ctnnb gain-of-function lungs indicating a functional difference between Apc deletion and Ctnnb stabilization, both of which activate Wnt signaling. Further analysis revealed that Apc deficiency led to increased TGF-beta signaling, which inhibited Nkx2.1 in cultured lung endodermal explants. In contrast, TGF-beta activity was not increased in Ctnnb gain-of-function lungs. Therefore, our studies revealed an important mechanism involving Apc and TGF-beta signaling in regulating the key transcriptional factor, Nkx2.1, for lung epithelial progenitor cell fate determination.

Matrix Metalloproteinase–7 Coordinates Airway Epithelial Injury Response and Differentiation of Ciliated Cells

Matrix metalloproteinase-7 (MMP7) expression is quickly up-regulated after injury, and functions to regulate wound repair and various mucosal immune processes. We evaluated the global transcriptional response of airway epithelial cells from wild-type and Mmp7-null mice cultured at an air-liquid interface. The analysis of differentially expressed genes between genotypes after injury revealed an enrichment of functional categories associated with inflammation, cilia, and differentiation. Because these analyses suggested that MMP7 regulated ciliated cell formation, we evaluated the recovery of the airway epithelium in wild-type and Mmp7-null mice in vivo after naphthalene injury, which revealed augmented ciliated cell formation in the absence of MMP7. Moreover, in vitro studies evaluating cell differentiation in air-liquid interface cultures also showed faster ciliated cell production under Mmp7-null conditions compared with wild-type conditions. These studies identified a new role for MMP7 in attenuating ciliated cell differentiation during wound repair.

CCAAT/Enhancer Binding Protein–α Regulates the Protease/Antiprotease Balance Required for Bronchiolar Epithelium Regeneration

Many transcription factors that regulate lung morphogenesis during development are reactivated to mediate repairs of the injured adult lung. We hypothesized that CCAAT/enhancer binding protein-α (C/EBPα), a transcription factor critical for perinatal lung maturation, regulates genes required for the normal repair of the bronchiolar epithelium after injury. Transgenic Cebpα(Δ/Δ) mice, in which Cebpa was conditionally deleted from Clara cells and Type II cells after birth, were used in this study. Airway injury was induced in mice by the intraperitoneal administration of naphthalene to ablate bronchiolar epithelial cells. Although the deletion of C/EBPα did not influence lung structure and function under unstressed conditions, C/EBPα was required for the normal repair of terminal bronchiolar epithelium after naphthalene injury. To identify cellular processes that are influenced by C/EBPα during repair, mRNA microarray was performed on terminal bronchiolar epithelial cells isolated by laser-capture microdissection. Normal repair of the terminal bronchiolar epithelium was highly associated with the mRNAs regulating antiprotease activities, and their induction required C/EBPα. The defective deposition of fibronectin in Cebpα(Δ/Δ) mice was associated with increased protease activity and delayed differentiation of FoxJ1-expressing ciliated cells. The fibronectin and ciliated cells were restored by the intratracheal treatment of Cebpα(Δ/Δ) mice with the serine protease inhibitor. In conclusion, C/EBPα regulates the expression of serine protease inhibitors that are required for the normal increase of fibronectin and the restoration of ciliated cells after injury. Treatment with serine protease inhibitor may aid in the recovery of injured bronchiolar epithelial cells, and prevent common chronic lung diseases.

Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.

Sec14l3 Is Specifically Expressed in Mouse Airway Ciliated Cells

Airway epithelium is a key component for airway integrity. Previously, we found that expression of the Sec14l3 gene that encodes a 45-kDa secretory protein is inversely associated with the progression of experimentally induced airway inflammation and degeneration/necrosis of alveolar epithelium. In this report, using in situ hybridization we demonstrated that the ciliated cells in mouse lung selectively express Sec14l3 mRNA. In a three-dimensional culture of mouse tracheal epithelial cells, levels of the Sec14l3 mRNA correlated with the differentiation of ciliated cells. Intranasal infection of adult mice with influenza virus resulted in a 20-fold, progressive decrease in Sec14l3 mRNA expression over 10days post infection. These results enhance the potential value of Sec14l3 as a ciliated epithelial cell-specific biomarker for the progression of airway inflammations such as airway viral infection and asthma.

Sox2 Activates Cell Proliferation and Differentiation in the Respiratory Epithelium

Sox2, a transcription factor critical for the maintenance of embryonic stem cells and induction of pluripotent stem cells, is expressed exclusively in the conducting airway epithelium of the lung, where it is required for differentiation of nonciliated, goblet, and ciliated cells. To determine the role of Sox2 in respiratory epithelial cells, Sox2 was selectively and conditionally expressed in nonciliated airway epithelial cells and in alveolar type II cells in the adult mouse. Sox2 induced epithelial cell proliferation within 3 days of expression. Epithelial cell proliferation was associated with increased Ki-67 and cyclin D1 staining. Expression of cell cycle genes, including FoxM1, Ccna2 (Cyclin A2), Ccnb2 (Cyclin B2), and Ccnd1 (Cyclin D1), was increased. Consistent with a role in cell proliferation, Sox2 activated the transcription of FoxM1 in vitro. In alveoli, Sox2 caused hyperplasia and ectopic differentiation of epithelial cells to those with morphologic and molecular characteristics of conducting airway epithelium. Sox2 induced the expression of conducting airway epithelial specific genes, including Scgb1a1, Foxj1, Tubb3, and Cyp2f2. Although prolonged expression of Sox2 caused cell proliferation and epithelial hyperplasia, Sox2 did not induce pulmonary tumors. Sox2 induces proliferation of respiratory epithelial cells and, subsequently, partially reprograms alveolar epithelial cells into cells with characteristics of the conducting airways.

Inhibitory Smad proteins promote the differentiation of mouse embryonic stem cells into ependymal-like ciliated cells

Motile cilia play crucial roles in the maintenance of homeostasis in vivo. Defects in the biosynthesis of cilia cause immotile cilia syndrome, also known as primary ciliary dyskinesia (PCD), which is associated with a variety of complex diseases. In this study, we found that inhibitory Smad proteins, Smad7 and Smad6, significantly promoted the differentiation of mouse embryonic stem (ES) cells into ciliated cells. Moreover, these Smad proteins specifically induced morphologically distinct Musashi1-positive ciliated cells. These results suggest that inhibitory Smad proteins could be important regulators not only for the regulation of ciliated cell differentiation, but also for the subtype specification of ciliated cells during differentiation from mouse ES cells.

Primaryin vitroculture of porcine tracheal epithelial cells in an air-liquid interface as a model to study airway epithelium andAspergillus fumigatusinteractions

Since the airway epithelium is the first tissue encountered by airborne fungal spores, specific models are needed to study this interaction. We developed such a model using primary porcine tracheal epithelial cells (PTEC) as a possible alternative to the use of primary human cells. PTEC were obtained from pigs and were cultivated in an air-liquid interface. Fluorescent brightener was employed to quantify the internalization of Aspergillus fumigatus conidia. Potential differences (Vt) and transepithelial resistances (Rt) after challenge with the mycotoxin, verruculogen, were studied. Primers for porcine inflammatory mediator genes IL-8, TNF-alpha, and GM-CSF were designed for a quantitative real-time PCR procedure to study cellular responses to challenges with A. fumigatus conidia. TEM showed the differentiation of ciliated cells and the PTEC ability to internalize conidia. The internalization rate was 21.9 ± 1.4% after 8 h of incubation. Verruculogen (10(-6) M) significantly increased Vt without having an effect on the Rt. Exposure of PTEC to live A. fumigatus conidia for 24 h induced a 10- to 40-fold increase in the mRNA levels of inflammatory mediator genes. PTEC behave similarly to human cells and are therefore a suitable alternative to human cells for studying interaction between airway epithelium and A. fumigatus.

Sox2 Is Required for Maintenance and Differentiation of Bronchiolar Clara, Ciliated, and Goblet Cells

The bronchioles of the murine lung are lined by a simple columnar epithelium composed of ciliated, Clara, and goblet cells that together mediate barrier function, mucociliary clearance and innate host defense, vital for pulmonary homeostasis. In the present work, we demonstrate that expression of Sox2 in Clara cells is required for the differentiation of ciliated, Clara, and goblet cells that line the bronchioles of the postnatal lung. The gene was selectively deleted in Clara cells utilizing Scgb1a1-Cre, causing the progressive loss of Sox2 in the bronchioles during perinatal and postnatal development. The rate of bronchiolar cell proliferation was decreased and associated with the formation of an undifferentiated, cuboidal-squamous epithelium lacking the expression of markers of Clara cells (Scgb1a1), ciliated cells (FoxJ1 and α-tubulin), and goblet cells (Spdef and Muc5AC). By adulthood, bronchiolar cell numbers were decreased and Sox2 was absent in extensive regions of the bronchiolar epithelium, at which time residual Sox2 expression was primarily restricted to selective niches of CGRP staining neuroepithelial cells. Allergen-induced goblet cell differentiation and mucus production was absent in the respiratory epithelium lacking Sox2. In vitro, Sox2 activated promoter-luciferase reporter constructs for differentiation markers characteristic of Clara, ciliated, and goblet cells, Scgb1a1, FoxJ1, and Agr2, respectively. Sox2 physically interacted with Smad3 and inhibited TGF-β1/Smad3-mediated transcriptional activity in vitro, a pathway that negatively regulates proliferation. Sox2 is required for proliferation and differentiation of Clara cells that serve as the progenitor cells from which Clara, ciliated, and goblet cells are derived.

The PCP Pathway Instructs the Planar Orientation of Ciliated Cells in the Xenopus Larval Skin

Planar cell polarity (PCP) is a property of epithelial tissues where cellular structures coordinately orient along a two-dimensional plane lying orthogonal to the axis of apical-basal polarity. PCP is particularly striking in tissues where multiciliate cells generate a directed fluid flow, as seen, for example, in the ciliated epithelia lining the respiratory airways or the ventricles of the brain. To produce directed flow, ciliated cells orient along a common planar axis in a direction set by tissue patterning, but how this is achieved in any ciliated epithelium is unknown. Here, we show that the planar orientation of Xenopus multiciliate cells is disrupted when components in the PCP-signaling pathway are altered non-cell-autonomously. We also show that wild-type ciliated cells located at a mutant clone border reorient toward cells with low Vangl2 or high Frizzled activity and away from those with high Vangl2 activity. These results indicate that the PCP pathway provides directional non-cell-autonomous cues to orient ciliated cells as they differentiate, thus playing a critical role in establishing directed ciliary flow.

PAR1 specifies ciliated cells in vertebrate ectoderm downstream of aPKC.

Partitioning-defective 1 (PAR1) and atypical protein kinase C (aPKC) are conserved serine/threonine protein kinases implicated in the establishment of cell polarity in many species from yeast to humans. Here we investigate the roles of these protein kinases in cell fate determination in Xenopus epidermis. Early asymmetric cell divisions at blastula and gastrula stages give rise to the superficial (apical) and the deep (basal) cell layers of epidermal ectoderm. These two layers consist of cells with different intrinsic developmental potential, including superficial epidermal cells and deep ciliated cells. Our gain- and loss-of-function studies demonstrate that aPKC inhibits ciliated cell differentiation in Xenopus ectoderm and promotes superficial cell fates. We find that the crucial molecular substrate for aPKC is PAR1, which is localized in a complementary domain in superficial ectoderm cells. We show that PAR1 acts downstream of aPKC and is sufficient to stimulate ciliated cell differentiation and inhibit superficial epidermal cell fates. Our results suggest that aPKC and PAR1 function sequentially in a conserved molecular pathway that links apical-basal cell polarity to Notch signaling and cell fate determination. The observed patterning mechanism may operate in a wide range of epithelial tissues in many species.