Role of f-box factor foxj1 in differentiation of ciliated airway epithelial cells

Yingjian You,1 Tao Huang,1 Edward J. Richer,1 Jens-Erik Harboe Schmidt,2 Joseph Zabner,3 Zea Borok,4 and Steven L. Brody1,5

1Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110; 2Department of Biochemistry and Molecular Biology and Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90033; 3Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242; and 4Department of Medicine and Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California 90033

Submitted 29 May 2003 ; accepted in final form 11 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Factors required for commitment of an undifferentiated airway epithelial cell to a ciliated cell are unknown. Cell ultrastructure analysis indicates ciliated cell commitment activates a multistage program involving synthesis of cilia precursor proteins and assembly of macromolecular complexes. Foxj1 is an f-box transcription factor expressed in ciliated cells and shown to be required for cilia formation by gene deletion in a mouse model. To identify a specific role for foxj1 in directing the ciliated cell phenotype, we evaluated the capacity of foxj1 to induce ciliogenesis and direct cilia assembly. In a primary culture model of wild-type mouse airway epithelial cells, foxj1 expression preceded the appearance of cilia and in cultured foxj1 null cells cilia did not develop. Delivery of foxj1 to polarized epithelial cell lines and primary cultured alveolar epithelial cells failed to promote ciliogenesis. Similarly, delivery of foxj1 to wild-type airway epithelial cells did not enhance the total number of ciliated cells. In contrast, delivery of foxj1 to null cells resulted in the appearance of cilia. Analysis revealed that, in the absence of foxj1, null cells contained cilia precursor basal bodies, indicating prior commitment to ciliogenesis. However, the basal bodies were disorganized within the apical compartment and failed to dock with the apical membrane. Reconstitution of foxj1 in null cells restored normal basal body organization, resulting in axoneme growth. Thus foxj1 functions in late-stage ciliogenesis to regulate programs promoting basal body docking and axoneme formation in cells previously committed to the ciliated cell phenotype.

airway; basal body; cilia; differentiation; mouse


COMMITMENT OF AN UNDIFFERENTIATED airway epithelial cell to a ciliated cell and subsequent maturation to the ciliated phenotype occur through undefined molecular pathways. In developing human and mouse lung, ciliated epithelial cells arise from undifferentiated cells in the trachea during the canalicular stage, and, as lung development proceeds, cilia progressively appear in large and then small airways (12). Programs of ciliogenesis have been inferred from analysis of electron micrographs of ultrastructural components in developing and repairing airway cells and gene identification in Chlamydomonas and other invertebrates (10, 27, 30). These studies support a multistep paradigm by which precursor structures called "amorphous bodies" give rise to centrioles that subsequently transform to basal bodies. The basal bodies then migrate and dock at the apical membrane where they give rise to axonemes on the ciliated cell (10, 30). On each cell, ~200 axonemes composed of dynein motor-driven microtubules arranged as nine outer pairs and one linked inner pair (9+2) protrude in the lumen to generate a mechanical wave for airway clearance. Although some regulatory factors have been identified for the assembly of axonemes, these have been associated with putative sensory (primary) cilia mutated in polycystic kidney disease (26), whereas few molecular regulators of ciliogenesis in motile ciliated cells have been identified.

Compared with primary cilia that are expressed in almost all organs, motile cilia are highly localized in specific cell types (36). Motile ciliated cells are not only expressed in the airway of the lung but also within the paranasal sinuses and eustachian tubes of the upper airway, the ependyma and choroid plexus of the brain, and the oviduct and testis in reproductive organs. Furthermore, motile cilia have been identified early in development in the ciliated embryonic node that directs left-right asymmetry patterning and where mutations in dynein arms are proposed to be responsible for situs inversus and defective cilia function in primary ciliary dyskinesia syndrome (15). Thus analysis of genes with a restricted, cell-specific pattern of expression in tissues with motile cilia may reveal regulatory pathways for ciliogenesis.

Foxj1 (previously HFH-4) is a forkhead box (f-box) transcription factor specifically expressed in ciliated cells located in the upper and lower airway, choroid plexus, ependyma, oviducts, testis, and embryonic node (1, 5, 13, 20). During lung development in the mouse, foxj1 expression commences in airway epithelial cells during the late pseudoglandular stage at embryonic day 15.5. This expression immediately precedes the appearance of cilia in foxj1-positive cells (1). Other data also support a direct relationship between foxj1 expression and ciliogenesis. For example, in respiratory virus-induced injury, loss of cilia is associated with loss of foxj1 expression, and during airway repair foxj1 expression is associated with the appearance of ultrastructural components of ciliogenesis (21). Interestingly, a transgenic mouse that expresses foxj1 under control of the surfactant protein C promoter results in ciliated cells in alveolar spaces, suggesting a master gene function in ciliogenesis (32). Compellingly, interruption of the foxj1 gene in a genetically engineered mouse results in absent cilia (5, 7). Furthermore, f-box factors have been shown to play central roles in development and differentiation of specialized cell types (6). Taken together, these observations support the hypothesis that foxj1 is capable of determining the ciliated cell fate in airway epithelial cells. Here, we have tested this hypothesis in vitro by manipulating foxj1 expression in wild-type and foxj1 null cells. We found that foxj1 expression promotes differentiation during late-stage ciliogenesis in airway cells only when the cell is already committed to the ciliated cell phenotype, but not in other cell types. Thus, rather than directing commitment to the ciliated cell phenotye, foxj1 functions in the postcentriologenesis stage by establishing mechanisms for docking of basal bodies at the apical membrane and induction of program(s) of axoneme assembly.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines and primary cell culture. Madin-Darby canine kidney (MDCK) cells (CCL-34; ATCC, Manassas, VA) and transformed human bronchial epithelial BEAS2B cells (kindly provided by D. Look, University of Iowa) were initially cultured in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% FCS and antibiotics. Mouse tracheal epithelial cells (MTEC) were harvested and grown in primary culture on supported membranes at air-liquid interface (ALI) conditions, as described previously (37). Before seeding, purified MTEC were >99% cytokeratin-expressing epithelial cells when immunostained. Rat alveolar epithelial cells (AEC) were isolated, purified, and grown in primary culture, as previously described (2, 3). Freshly isolated cells were assessed for type II cell purity (>90%) by tannic acid staining to identify lamellar bodies (22). Cell viability (>90%) was measured by trypan blue dye exclusion.

Cell differentiation protocols. To induce cell polarization and differentiation, epithelial cells were cultured in growth factor-enriched media on semipermeable membranes (Transwell; Corning-Costar, Corning, NY) at ALI, conditions previously shown to induce ciliogenesis in mouse, rat, and human airway epithelial cells (19, 21, 37). Media were maintained in upper and lower chambers until the transmembrane resistance increased (>1,000 ohms·cm2 in MTEC and 500 ohms·cm2 in AEC), indicating tight junction formation (37). Media were then removed from the upper chamber to establish ALI. Ciliogenesis induction media used in MDCK, BEAS2B, and MTEC was Ham's F-12-DMEM (1:1), supplemented with 2% NuSerum (Becton-Dickinson, Bedford, MA) and antibiotics, previously used in vitro to induce human airway cell and MTEC ciliogenesis (19, 37). Rat AEC were similarly treated with media previously shown to induce ciliogenesis at ALI in rat tracheal epithelial cells (21).

Immunohistochemisty. Cells on supported membranes were fixed with 4% paraformaldehyde in PBS, pH 7.4, for 10 min at room temperature (RT) and processed for immunodetection, as described previously (37). Fixed samples were incubated for 2 h at RT or 18 h at 4°C with isotype-matched control antibody or primary antibody. For detection of {gamma}-tubulin and foxj1, cells were treated with 45 mM PIPES, 45 mM HEPES, 10 mM EGTA, 5 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride (pH 6.8) for 1 min before fixing (23). Primary antibodies and dilutions or concentrations used were as follows: mouse anti-{beta}-tubulin IV (1:250; BioGenex, San Ramon, CA); rabbit anti-foxj1 (1:500; see Ref. 1); and mouse anti-{gamma}-tubulin or rabbit anti-{gamma}-tubulin (both from Sigma Aldrich, St. Louis, MO). Antibody binding was detected using FITC- or indocarbocyanine-labeled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). No detectable staining was observed for isotype-matched control antibodies. Membranes were mounted on slides with media (Vectashield; Vector, Burlingame, CA) containing 4',6-diamidino-2-phenylindole to stain intracellular DNA. Microscopy was performed using a Zeiss laser scanning system with LSM-510 software (Zeiss, Thornwood, NY) for confocal microscopy or an Olympus BX51 (Melville, NY) with a CCD camera interfaced with MagnaFire software (Olympus) for reflected fluorescent microscopy. Images were composed using Photoshop and Illustrator software (Adobe Systems, San Jose, CA). The percentage of cell surface-expressing immunoflourescent staining for {beta}-tubulin IV was measured from photomicrographs using NIH image software (ImageJ 1.29; National Institutes of Health; http://rsb.info.nih.gov/ij). The mean values and SD from independent samples were compared by Student's t-test.

RNA blot analysis. RNA was isolated from cells using guanidinium isothiocyanate lysis and phenol-chloroform extraction (STAT-60; Tel-Test, Friendswood, TX), separated by agarose gel electrophoresis (10 µg/sample), transferred to nylon membranes (Hybond-N; Amersham Pharmacia, Little Chalfont, UK), hybridized with radiolabeled foxj1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes, and detected as previously described (21).

Protein blot analysis. Total cell lysates were resuspended in buffer (50 mM Tris·Cl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholate) containing protease (1 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and phosphatase (10 mM NaF, 1 mM orthovanadate, and 2 mM sodium pyrophosphate) inhibitors. Protein concentrations were estimated using Bio-Rad protein assay reagent (Hercules, CA) and equal amounts resuspended in sample buffer before separation by 7.5% SDS-PAGE. Protein was transferred to polyvinylidene difluoride paper (Millipore, Bedford, MA) and blocked with 5% milk and 0.2% Tween 20 for 1 h at RT or overnight at 4°C. Primary antibody was incubated in blocking solution for 2 h at RT. Horseradish peroxidase-labeled secondary antibody binding was detected by enhanced chemiluminescence (Amersham Pharmacia).

Gene transfer to epithelial cells. Mouse foxj1 cDNA (4) was subcloned in plasmids for production of viruses using standard methods. The foxj1 cDNA was subcloned in retrovirus vector MSCV2.2 (kindly provided by K. Murphy, Washington University, St. Louis, MO), which coexpressed green fluorescent protein (GFP) via an internal ribosome entry site (28). The resulting construct, MSCVfoxj1GFP, was transfected into the Phoenix AMPHO 293T packaging cell line (kindly provided by G. Nolan, Stanford University, Palo Alto, CA) to produce replication-defective virus (18). MDCK and BEAS2B cell lines were transfected with retrovirus-containing supernatant and individual clones selected by expression of GFP after limited dilution. The foxj1 cDNA was subcloned in adenovirus shuttle vector Ad5RSVknpa and a replication-deficient (E1-E4-) adenovirus vector (Adfoxj1) generated by the University of Iowa Gene Transfer Vector Core (35). Adfoxj1 was additionally modified to include enhanced GFP (eGFP) by homologous recombination in the previously deleted E4 site (University of Iowa Gene Transfer Vector Core) to generate Adfoxj1GFP. AdFoxj1 was used at a multiplicity of 20 infectious particles per epithelial cell. In early transfection experiments, adenovirus vectors were delivered at days 4 and 5 after initiating cultures (just before ALI day 0) for 2 h on the apical and basolateral surfaces. In other experiments, MTEC at ALI were pretreated with 6 mM EGTA for 1 h to allow access to basolateral adenovirus receptors before apical delivery (34). For lentivirus vector production, the eGFP cassette was removed from pRRLhCMVGFPsin (referred to as pRRLGFP; generously provided by L. Naldini, University of Turin, Turin, Italy) and replaced with foxj1 cDNA to generate pRRLhCMVfoxj1sin (referred to as pRRLfoxj1). Infectious virus was generated in packaging cell lines, titered, and delivered to primary cultured rat AEC as previously described (3).

Electron microscopy. Cells on membranes were prepared for electron microscopy (EM) as previously described (21). Briefly, samples were fixed with 2.5% glutaraldehyde and stained with 1.25% osmium tetroxide. For transmission EM, cells were counterstained with 2.0% tannic acid, blocked for sectioning, and visualized on a Zeiss 902 model microscope. For scanning EM, cells were processed and visualized on a Hitachi S-450 microscope (Tokyo, Japan).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Foxj1 expression is temporally related to ciliogenesis in primary cultured tracheal epithelial cells. It has been previously shown that foxj1 is expressed in a subpopulation of airway epithelial cells in developing lung before the appearance of cilia and persists in ciliated cells of the adult lung (1, 32). To determine the role of foxj1 in ciliogenesis in vitro, we utilized a primary culture model of airway epithelial cell differentiation that promotes ciliogenesis in ALI conditions (37). MTEC cultures were initiated in growth factor-supplemented media on semipermeable supported membranes. Under these conditions, cells form a confluent layer with tight junctions, permitting establishment of ALI (37). At this time (ALI day 0), {beta}-tubulin IV expression, a protein enriched in cilia, was absent, indicating that ciliary axonemes are not yet formed (Fig. 1A, left). At ALI day 2, {beta}-tubulin IV expression was observed within the cytosol of some cells but was absent in the apical membranes. By ALI day 5, many cells had a diffuse, punctate pattern of {beta}-tubulin IV expression, characteristic of small cilia seen at this time (37). By ALI day 7, mature ciliary axonemes expressing {beta}-tubulin IV were observed. In contrast, apical {beta}-tubulin IV expression was not observed in the foxj1 null cells maintained under identical culture conditions, indicating a requirement for foxj1 for ciliogenesis (Fig. 1A, right).



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Fig. 1. Foxj1 expression is temporally related to ciliogenesis in primary cultured tracheal epithelial cells. A: wild-type (+/+) or foxj1 null (-/-) primary cultured mouse tracheal epithelial cells (MTEC) were differentiated on semipermeable, supported membranes using air-liquid interface (ALI) conditions and then immunostained for {beta}-tubulin IV ({beta}-tub) at indicated days. Nuclei were detected in the same field by 4',6-diamidino-2-phenylindole (DAPI) staining. d, Day. Bar, 10 µm. B: total RNA (10 µg/lane) was purified at indicated days from primary cultured MTEC and analyzed for expression of foxj1 and GAPDH (GAP). C: protein was isolated from MTEC at indicated days and subjected (25 µg/lane) to immunoblot analysis for detection of foxj1 and {beta}-catenin ({beta}-cat). Representative data from 4 independent preparations are shown.

 

The temporal relationship between the appearance of cilia and expression of foxj1 in vitro was determined by RNA and protein blot analyses. In each case, foxj1 expression was absent at ALI day 0 and initially present at ALI day 2, before the earliest appearance of cilia on the cell surface (Fig. 1, B and C). At ALI day 3, foxj1 expression increased and subsequently persisted at high levels throughout analysis to day 14. Thus the temporal relationship of foxj1 expression and ciliogenesis is conserved in this in vitro model of ciliogenesis, and the requirement of foxj1 for cilia formation was similar to in vivo observations (1, 5, 7, 32).

Foxj1 is necessary and sufficient for ciliogenesis in foxj1 null cells but not other epithelial cells. To determine if foxj1 was capable of inducing ciliogenesis, we expressed foxj1 in different cell types using viral vectors to optimize transfection efficiency. Polarized MDCK renal epithelial cells that expressed retrovirus-mediated foxj1 were grown under conditions permissive for induction of ciliogenesis. Clonal populations expressing either high or low amounts of foxj1 (Fig. 2A, left) when evaluated by expression of apical {beta}-tubulin IV did not show cilia development (data not shown). Similarly, expression of foxj1 in polarized, transformed human airway epithelial BEAS2B cells cultured under conditions shown to induce ciliogenesis in primary human tracheal epithelial cells failed to generate cilia (data not shown). To establish that reconstitution of foxj1 in primary foxj1 null cells could rescue the ciliary defect, we delivered foxj1 1 and 2 days before ALI day 0 (before the onset of foxj1 expression in wild-type cells). After 10 days under ALI conditions, apical {beta}-tubulin IV expression was observed (Fig. 2B, top right). Scanning EM demonstrated the appearance of normal ciliated cells (Fig. 2B, bottom right). To determine if reconstitution was dependent on a program of ciliogenesis occurring only within the first 2 days of ALI-associated differentiation, foxj1 null cells cultured at ALI for 20 days were also reconstituted with foxj1 (by EGTA-facilitated Adfoxj1 transfection). When evaluated 10 days later (ALI day 30), apical cilia were present (data not shown), suggesting that foxj1 was not dependent only on factors transiently expressed during a molecular sequence present early in differentiation but also expressed in mature cultures. Together, these observations suggest that foxj1 was both necessary and sufficient for ciliogenesis in foxj1 null cells.



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Fig. 2. Foxj1 is necessary and sufficient for ciliogenesis in foxj1 null cells but not other epithelial cells. A: cells were not transfected (mock) or transfected with indicated gene transfer vectors expressing green fluorescent protein (GFP) and/or foxj1 cDNA. Retrovirus vector (MSCVfoxj1GFP, labeled MSCV-foxj1), adenovirus vectors (AdGFP or Adfoxj1), or lentivirus vectors (pRRLGFP or pRRLfoxj1) were used for gene transfer to indicated cells. After transfection (10 days), cell lysates were subjected to protein blot analysis for foxj1 expression. Arrows indicate 58-kDa foxj1 and nonspecific (ns) signal. B: primary culture wild-type (+/+) or foxj1 null (-/-) MTEC cultured as in Fig. 1 were immunostained for {beta}-tubulin IV or imaged for GFP expression and analyzed for cell morphology by scanning electron microscopy (EM) after mock transfection or gene transfer with the indicated adenovirus vector. Bar, 10 µm (top) and 5 µm (bottom). C: wild-type cells transfected with Adfoxj1GFP were immunostained for {beta}-tubulin IV (red) after 10 days and imaged by confocal microscopy for eGFP (green) to identify cells expressing eGFP and foxj1. Reconstruction of 2 different z-stack series is shown. D: wild-type cells were mock transfected or transfected with the indicated adenovirus vector, differentiated using ALI conditions for 10 days, and then immunostained for {beta}-tubulin IV. The mean and SD of the surface area of cells expressing {beta}-tubulin IV is shown (n = 6). E: primary cultured rat AEC were transfected with pRRLGFP or pRRLfoxj1 and maintained at ALI in media favoring ciliogenesis. After 10 days, cells were immunostained for {beta}-tubulin IV or imaged for eGFP expression. Bar, 10 µm.

 

To assess the capacity of foxj1 to induce ciliogenesis in different pulmonary epithelial cell types, we delivered foxj1 to wild-type MTEC. We have previously shown that, at ALI day 0, MTEC express neither secretory cell marker Clara cell secretory protein (CCSP) nor {beta}-tubulin IV but rapidly differentiate over 7–10 days to express these proteins (37). However, even after ALI day 14, almost half the cells remain undifferentiated (CCSP and {beta}-tubulin IV negative) or express a basal cell marker (Griffonia simplicifolia isolectin B4; see Ref. 37). Thus, if these nonciliated cells were capable of differentiation into ciliated cells through programs initiated by foxj1, the number of ciliated cells should increase after foxj1 delivery. Cells were transfected 1 and 2 days before ALI day 0 with AdGFP, Adfoxj1 (Fig. 2A, middle), or an adenovirus vector expressing both foxj1 and eGFP (Adfoxj1GFP). Transfection efficiency determined at ALI day 10 by quantifying eGFP expression indicated that 30–42% [36.52 ± 5.98% (mean ± SD), n = 3] of the cells expressed the transgene. To identify cells targeted by foxj1 delivery, we used the Adfoxj1GFP vector and evaluated eGFP expression as a surrogate for foxj1. Confocal microscopy showed Adfoxj1GFP vector-mediated expression in ciliated and nondifferentiated cells (Fig. 2C). Dual localization of eGFP and {beta}-tubulin IV showed lack of {beta}-tubulin IV expression in many eGFP expressing cells, suggesting that foxj1 delivery did not induce ciliogenesis in all cell types (Fig. 2C). Comparison of {beta}-tubulin IV expression in MTEC at ALI day 10 that were mock transfected or transfected with AdGFP or Adfoxj1 showed no difference in the number of ciliated cells. In each condition, ~50% of the cell surface was ciliated (Fig. 2D). These observations indicated that foxj1 was not capable of inducing ciliogenesis in Clara or undifferentiated cells. This suggested that, for foxj1 to induce ciliogenesis, other factors important for cilia formation must be present.

The observation that transgenic mice expressing foxj1 under control of the surfactant protein C promoter exhibit ciliated cells lining the alveoli suggested that, in vitro, AEC may be capable of ciliogenesis (32). Therefore, rat AEC in primary culture were transfected with a lentivirus vector expressing eGFP or foxj1 cDNA 1 day after tight junctions establishment. AEC were then cultured for 10 days in ALI conditions using media previously shown to support rat tracheal epithelial cell ciliogenesis (Fig. 2A, right, and 2E). Transfection efficiency determined by expression of eGFP 1 day and 2 days posttransfection was >90%, as previously shown (3). At ALI day 10, ~70% of cells transfected with pRRLGFP expressed eGFP (Fig. 2E). At ALI day 10, AEC transfected with pRRLfoxj1 expressed foxj1 (Fig. 2A). However, at this time, cilia were not found in AEC (Fig. 2E). Thus, taken together with findings in cell lines and wild-type MTEC, these data suggest that foxj1 alone was not sufficient to induce programs of ciliogenesis.

Abnormal localization of basal bodies in foxj1 null cells. Paired {gamma}-tubulin-rich centrioles are an essential part of the microtubule organizing center (MTOC) within the nucleus, but centrioles are additionally present in ciliated cells as precursors to the basal body (11, 23, 24). In ciliated cells, {gamma}-tubulin-expressing basal bodies are localized at the apical membrane, contiguous with axonemes expressing {beta}-tubulin IV (Fig. 3A). Although nuclear doublets of {gamma}-tubulin were present in MDCK, BEAS2B, and AEC within the MTOC, {gamma}-tubulin was not detected in the cytoplasm of those cells (Fig. 3A and data not shown). Thus the presence of apical compartment basal bodies is one fundamental difference between nonciliated and ciliated cells.



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Fig. 3. Abnormal localization of basal bodies in foxj1 null cells. A: wild-type MTEC and Madin-Darby canine kidney (MDCK) cells cultured on membranes under ALI conditions in media favoring ciliogenesis were immunostained for expression of {gamma}-tubulin ({gamma}-tub, red) and {beta}-tubulin IV (green) and counterstained with DAPI (blue) to identify nuclei. Identical fields were merged. B: wild-type (+/+) or foxj1 null (-/-) MTEC were cultured at ALI for 24 days and then imaged by transmission EM. Bar, 50 nm. C: wild-type and foxj1 null MTEC cells from B were immunostained for expression of {gamma}-tubulin (red) and imaged by confocal microscopy. Shown are z-stack reconstructions demonstrating patterns of apical {gamma}-tubulin expression. {gamma}-Tubulin expression in the microtubular organizing center (MTOC) is also seen in two of the foxj1 null cells. Bar, 5 µm.

 

In analysis of the role of foxj1 in ciliogenesis, we have previously shown that airway epithelial cells from foxj1 null mice contained basal bodies within the apical compartment of airway epithelial cells. However, compared with wild-type cells, the basal bodies were disorganized, did not dock at the apical membrane, and lacked the ciliary axoneme (5). The foxj1-deficient mice were typically runted and died within 3 wk of age (or earlier), making it possible that other factors might impair in vivo ciliogenesis. Culture of foxj1 null cells under prolonged, stable conditions favoring ciliogenesis (>3 wk) did not alter the pattern of basal body disorganization observed in vivo (Fig. 3B). When {gamma}-tubulin expression was used to identify basal bodies by immunoflourescence, we detected a characteristic altered expression pattern in foxj1 null cells that was in distinct contrast to wild-type cells cultured for a similar period of time (Fig. 3C). These findings indicated that, compared with other nonciliated epithelial cell types, foxj1 null cells contain cilia precursors and, as such, are committed to the ciliated cell phenotype. The altered pattern of basal bodies seen in foxj1 null cells suggested an interruption in the program of ciliogenesis because of the absence of foxj1.

Foxj1 directs apical basal body localization in late ciliogenesis. To determine a role for foxj1 in relation to basal body localization, we evaluated patterns of foxj1 relative to {gamma}-tubulin expression during in vitro ciliogenesis in MTEC (Fig. 4A). At ALI day 0, foxj1 was absent, and only nuclear localization of {gamma}-tubulin expression was found within the MTOC. At ALI day 2, all cells that expressed foxj1 had a uniform punctate pattern of {gamma}-tubulin in the apical aspect of the cell. By ALI day 5, foxj1 expression was more abundant and accompanied by the characteristic {gamma}-tubulin signal. In contrast, foxj1 null cells contained a dense, globular {gamma}-tubulin signal at the apical region of the cell but lacked the punctate "spray" pattern seen in apical membranes of the wild-type cells (Fig. 4A, right). The presence of the apical punctate pattern associated with foxj1 expression indicated that there was a rapid and uniform apical membrane localization of basal bodies in the wild-type cell during ciliogenesis. The presence of basal bodies in the foxj1 null cells confirmed a prior commitment to ciliogenesis (i.e., cilia precursor structures were present) but impaired basal body localization during normal ciliogenesis.



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Fig. 4. Foxj1 directs apical basal body localization in late ciliogenesis. A: wild-type (+/+) or foxj1 null (-/-) MTEC were cultured in ALI conditions as in Fig. 1. Cells were immunostained for {gamma}-tubulin (red) and foxj1 (green) at indicated time and imaged by reflected immunoflourescence. B: foxj1 null cells were transfected with Adfoxj1 before ALI day 0 and then immunostained for {gamma}-tubulin and foxj1 at ALI day 5 as in A. Arrows indicate foxj1-expressing cells associated with a fine, punctate pattern of apical {gamma}-tubulin. C: wild-type, foxj1 null cells, and null cells transfected with Adfoxj1 (-/- +Adfoxj1) as in B were immunostained for {gamma}-tubulin (red) and {beta}-tubulin IV (green) and imaged by confocal microscopy. Images were reconstructed as a z-stack. *Null cell demonstrating the wild-type pattern of {gamma}-tubulin and {beta}-tubulin IV expression. D: wild-type and foxj1 null cells were cultured using ALI conditions. Cells were analyzed by protein immunoblot for expression of {beta}-tubulin IV and actin at the indicated time. E: model of the role of foxj1 in late ciliogenesis. A pluripotent airway epithelial cell undergoes commitment to ciliogenesis followed by centriologenesis, culminating in axoneme formation. Foxj1 functions in the postcentriologenesis stage.

 

To determine the effect of foxj1 reconstitution on basal body localization and axoneme assembly in foxj1 null cells, we determined the localization of {gamma}-tubulin after Adfoxj1 transfection in foxj1 null cells. The foxj1 expression was associated with the apical punctate {gamma}-tubulin pattern in transfected cells (Fig. 4C). In reconstituted foxj1 null cells, apical {gamma}-tubulin localization was also related to axoneme growth, shown by {beta}-tubulin IV expression (Fig. 4C). To confirm that foxj1 expression was associated with induction of proteins required for axoneme production, we compared expression of {beta}-tubulin IV in wild-type and null cell culture preparations by immunoblot analysis (Fig. 4D). Consistent with the enhanced appearance of {beta}-tubulin IV expression observed by immunoflourescence during ciliogenesis (Fig. 1A), {beta}-tubulin IV expression was upregulated in wild-type compared with foxj1 null cells. Thus, as shown in our model, foxj1 functions in the postcentriologenesis stage of ciliogenesis to direct apical localization of basal bodies and subsequent induction of proteins required for axoneme assembly (Fig. 4E).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Differentiation of the airway epithelium is highly regulated to generate ciliated and secretory cells with unique functions. Identification of molecular factors that determine commitment to a specialized cell type are important for understanding abnormalities in differentiation that characterize airway diseases such as asthma, chronic bronchitis, and bronchogenic carcinoma (16). Developmental models have been a potent source for genetic analysis of regulatory genes, as demonstrated by the identification of "master genes" that direct eye or wing development in Drosophila (14). In mammals, members of the f-box family of transcription factors are expressed in a tissue-specific manner and also have critical functions in cell differentiation and regulation. Specific f-box gene deletion in mice has revealed essential roles for organ formation, including gut, thyroid, and thymus, that are the hallmark of master genes (6). The lack of cilia in the foxj1-deficient mouse and other reports suggesting foxj1 would be capable of inducing the ciliated cell phenotype made it logical to hypothesize that foxj1 was sufficient to initiate ciliogenesis (5, 7, 32). However, data presented here suggest that foxj1 alone could not specify the ciliated cell phenotype and, instead, directs functions in late ciliogenesis.

Several observations indicate that foxj1 does not initiate the commitment of airway epithelial cells to the ciliated cell phenotype. First, expression of foxj1 was not capable of inducing ciliogenesis in polarized airway or nonairway epithelial cell lines when cells were cultured under conditions shown to induce ciliogenesis in primary airway epithelial cells. Second, foxj1 could not enhance ciliogenesis in wild-type airway epithelial cells, even if overexpressed in primary airway epithelial cells of the nonciliated (e.g., secretory or basal) type. Third, foxj1 was only capable of inducing ciliogenesis in foxj1 null cells that contained cilia precursor basal bodies. This was demonstrated by showing reconstitution of foxj1 in null cells shifted the basal bodies from the apical compartment to the apical membrane. The movement of basal bodies to an aligned position triggered axoneme growth demonstrated by {beta}-tubulin IV expression. Thus foxj1 functions in the postcentriologenesis stage, downstream of commitment to the ciliated cell phenotype.

The precise molecular targets of foxj1 are undefined. Although foxj1 recognizes a DNA consensus sequence in vitro, none of the putative target proteins are related to ciliogenesis or intracellular trafficking, and the in vivo gene targets of foxj1 are not established (20). Ultrastructural analysis provided here suggests foxj1 may regulate proteins required for short distant transport of basal bodies within the apical compartment and/or preparation of the apical membrane for docking. Basal bodies are grossly localized to the apical compartment of the foxj1 null cell, indicating that major trafficking and polarity mechanisms are not disturbed. However, proteins pulling cargo for short distances on actin tracks such as unconventional myosin V, previously shown to colocalize {gamma}-tubulin in centrioles, may play a role (33). Alternatively, proteins required for the elaborate organization of the apical membrane required for basal body docking may be regulated by foxj1 (29). Future studies are in progress to elucidate these possibilities.

Interestingly, we found that, after foxj1-induced basal body docking, axoneme growth commenced normally (shown by increased {beta}-tubulin expression). Our foxj1 reconstitution studies cannot separate the process of basal body docking and axoneme growth. The coincidence of synthesis of axoneme elements (e.g., {beta}-tubulin IV) with cilium growth is consistent with regulation of ciliogenesis observed in invertebrates (31). Axoneme assembly has been attributed to several proteins with roles in intraflagellar transport (IFT), including the polycystins (25, 26). These IFT proteins have been identified in sensory (9+0 microtubular arrangement) cilia and are mutated in some forms of polycystic kidney disease (25). Sensory or nonmotile cilia lack dynein arms and the central microtubule pair (central apparatus) that interacts with the dynein arms, and sensory cilia cells do not express foxj1 (1). These findings suggest that genes coding for motor proteins and central apparatus proteins may be targets for foxj1. Alternatively, many of these structural protein complexes, like the basal bodies, may be present but not yet organized at the apical membrane.

Identification of proteins that direct commitment of airway epithelial cells to the ciliated cell phenotype remains elusive, and we cannot fully rule out a role for foxj1 with the current studies. The initiation of ciliogenesis has been linked to mitosis in ciliated invertebrates (31, 11). In this regard, our MTEC in vitro cultures are dependent on a proliferation phase (with establishment of tight junctions) to reach ALI day 0, immediately before ciliogenesis (37). This suggests that a postmitotic signal (in the presence of foxj1) may be important for ciliated cell commitment. It is also possible that in our studies of foxj1 rescue of mature null cells (ALI day 20), cell proliferation was induced by mild injury associated with adenovirus (Adfoxj1) and EGTA delivery (used to access the basolateral adenovirus receptor), leading to the appearance of new ciliated cells in the presence of foxj1. However, our prior studies of in vivo mouse airway epithelial cell repair after Sendai virus injury showed that proliferating cells identified by bromodeoxyuridine incorporation did not colocalize with foxj1 expression, further supporting the notion that foxj1 functions late in a cell already committed to the ciliated phenotype (21). Other previously identified factors required for ciliogenesis may also determine ciliated cell commitment. Studies of primary cultured airway epithelial cells have shown that ciliogenesis requires epidermal growth factor, bovine pituitary hormones, cholera toxin, and retinoic acid in the setting of ALI (8, 17). The effect of these factors on specific stages of ciliogenesis has not been studied using ultrastructural analysis, and it is unclear if one or more of these factors can initiate ciliogenesis. Returning to development of less complex organisms may be instructive. A Notch signaling pathway has recently been identified for determination of ciliated cell fate during Xenopus development, but the role of these proteins in mammalian ciliogenesis has not been reported (9).

In summary, the data presented in these studies indicate that f-box protein foxj1 functions after commitment of the cell to ciliogenesis. Ultrastructural analysis shows that the point of function is postcentriologenesis, i.e., after basal body formation. At this late stage, foxj1 is necessary for ciliogenesis to induce basal body trafficking and docking at the apical membrane with consequent axoneme growth.


    ACKNOWLEDGMENTS
 
We thank D. Look, K. Murphy, and L. Naldini for providing reagents, M. Veith and M. Levy for EM imaging, the Iowa University Gene Transfer Vector Core for assistance, and Mike Walter for helpful discussions.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-56244 and HL-63988 to S. L. Brody, by the American Lung Association, and NHLBI Grants HL-62569 and HL-72231 to Z. Borok.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Steven L. Brody, Washington Univ. School of Medicine, Campus Box 8052, 660 South Euclid Ave., St. Louis, MO 63110 (E-mail: brodys{at}msnotes.wustl.edu.)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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