Introduction

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The lung contains diverse types of epithelial cells in the proximal (trachea, bronchi, and bronchioles) and distal (alveoli) airways. This diversity allows the airway epithelial cells to carry out a wide variety of functions required for host defense and gas exchange (1, 2). In the developing lung, airway epithelial cell differentiation occurs after the lung bud begins to branch (1, 3). Molecular mechanisms that direct differentiation of airway epithelial cells during development are not well understood. Secreted growth factors such as fibroblast growth factors, transforming growth factor-, and epithelial growth factor appear to be important in branching and epithelial cell differentiation, particularly in the alveolar epithelium (4, 5). Transcription factors, including the homeodomain thyroid transcription factor (TTF)-1 and the forkhead/winged-helix protein hepatocyte nuclear factor (HNF)-3, also have important functions in airway epithelial cell differentiation (6). Both TTF-1 and HNF-3 are expressed in early developing lung epithelial cells of proximal and distal airways, and altered expression of these factors results in a failure of normal airway epithelial cell differentiation (9, 10). These observations suggest that transcription factors play central roles in establishing the general pattern of epithelial cell differentiation in the lung.

Members of the forkhead/winged-helix family of transcription factors are expressed in a tissue-specific manner and have critical functions in cell differentiation and regulation of specialized cellular function (11). For example, forkhead factors BF-1 and BF-2 specify differentiation of distinct regions of the brain, MFH-1 is critical in skeletogenesis, and whn (mutated in the nude mouse) is responsible for hair growth and thymus development (12). In the lung, forkhead factors HNF-3 and  have been shown to regulate the expression of airway epithelial cell-specific genes for surfactant protein B and the Clara cell secretory protein (CCSP) (7, 8). However, HNF-3 and  are widely expressed throughout endoderm-derived cells in most organs, indicating that these proteins alone do not direct lung-specific epithelial cell differentiation (15). Instead, forkhead factors that are more restricted in expression will likely be important for differentiation of specific populations of airway epithelial cells.

We and others have previously described the cloning and expression of HNF-3/forkhead homologue 4 (HFH-4), a third forkhead/winged-helix transcription factor that is expressed in airway epithelial cells. HFH-4 has been cloned from mouse, rat, and human; and, in contrast to HNF-3 and , it has a restricted pattern of expression (16). Northern analysis and in situ hybridization demonstrate that HFH-4 messenger RNA (mRNA) is restricted to the epithelium of lung, oviduct, choroid plexus and ependyma of the brain, and the seminiferous tubules of the testis (17, 19, 21). Previously, we used in situ hybridization to demonstrate that expression of HFH-4 mRNA is developmentally regulated in proximal airway epithelial cells with onset in the mouse lung at embryonic day (E) 14.5 (17). A similar pattern of HFH-4 gene expression occurs in developing human lung (21). Although HFH-4 recognizes a DNA consensus sequence in these tissues, the in vivo gene targets of HFH-4 are not established (19). The restricted pattern of HFH-4 expression suggests that HFH-4 has a specific role in regulation of differentiation of epithelial cells in the airway and other tissues. A role in epithelial cell differentiation is also suggested by recent reports by others and us that targeted deletion of HFH-4 results in an absence of cilia in the airway (22, 23).

The cellular heterogeneity of tissues expressing HFH-4 mRNA makes it difficult to determine the specific type of cell expressing HFH-4. In turn, this makes identification of a function for HFH-4 difficult. For example, the proximal airway of the lung contains ciliated, basal, intermediate, mucus, and Clara cells (1, 24), all or some of which might express HFH-4. Therefore, we developed a polyclonal HFH-4 antibody and used this antibody in combination with other epithelial cell markers to immunolocalize HFH-4 expression. These studies revealed that HFH-4 protein expression is restricted to ciliated cells and temporally related to ciliogenesis. Characterization of expression in developing organs suggests that HFH-4 plays a role in regulation of genes important for epithelial cell differentiation and ciliogenesis.

	Materials and Methods

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Antibody Generation

Rat HFH-4 complementary DNA (cDNA) coding for amino acids 1-117 was subcloned downstream and in-frame with glutathione S-transferase (GST) at the XhoI site of pGEX4T1 (Pharmacia, Uppsala, Sweden) to generate the GST-HFH-4 (1-117) plasmid. The plasmid was used to express GST-HFH-4 (1-117) in Escherichia coli (strain B12), and the fusion protein was purified by affinity chromatography using glutathione sepharose beads (Pharmacia) as described previously (25). The eluted protein was injected into rabbits with 1 ml of Freund's complete adjuvant. Rabbit serum was harvested and the immunoglobulin (Ig) G fraction was isolated by precipitation with caprilic acid (26). Polyclonal anti-HFH-4 antibody was affinity-purified by binding to purified GST-HFH-4 (1-117) protein linked to glutathione sepharose. Antibody concentration was determined using the Bio-Rad protein assay solution (Bio-Rad, Hercules, CA).

Antibody Specificity Testing

A rat HFH-4 cDNA containing the complete open reading frame (1,266 base pairs) was constructed by polymerase chain reaction (PCR) amplification using two overlapping rat HFH-4 cDNA clones (17) as templates, Vent DNA polymerase (New England Biolabs, Beverly, MA), and a 5' oligomer containing an XbaI site (5'-TCTAGAGCAGACATGGCGGAGAGC) and 3' oligomer containing a BamHIsite (5'-GGATCCCTGACTTGAGCACTGTCC). The PCR product was subcloned into pCR3 (Invitrogen, Carlsbad, CA). The cDNA sequences containing the open reading frame of the rat HNF-3 and HNF-3 (kindly provided by J. Darnell, Jr., Rockefeller University, New York, NY) were removed from parent vectors and subcloned into pBSKS II (Strategene, La Jolla, CA) at XhoI and HindIII restriction sites (27). The cDNA sequences for HFH-4, HNF-3, and HNF-3 were in vitro translated in the presence of ³⁵[S]-labeled methionine using rabbit reticulocyte lysates (TNT; Promega, Madison, WI) and the appropriate RNA polymerase (T3 or T7). The appropriate sizes of in vitro-translated proteins were confirmed by polyacrylamide gel electrophoresis (PAGE) and autoradiography. Translated proteins were separated on 7.5% sodium dodecyl sulfate polyacrylamide gels, transferred to nitrocellulose membranes (Hybond-ECL; Amersham, Arlington Heights, IL), blocked (1 h, room temperature [RT]) in Tris-buffered saline with Tween-20 (Tris, 20 mM; NaCl, 137 mM; Tween-20, 0.1%; pH 7.6) containing nonfat milk (5%) and incubated (4°C, overnight) with anti-HFH-4 antibody (1:500) in blocking solution. Primary antibody binding was detected using a chemiluminescence detection system (ECL; Amersham).

Immunohistochemistry

Developmental stage-specific mouse tissues were obtained from timed pregnancies determined using the presence of a vaginal plug at E0.5. Human samples were obtained from the Pathology Department at Barnes-Jewish Hospital with permission of the Human Studies Committee at Washington University (both St. Louis, MO). Mouse tissues for immunohistochemical evaluation were fixed in 10% buffered formalin, embedded in paraffin, and sectioned (6 µM thick). Paraffin-embedded sections were deparaffinized in a D-limonene-based clearing solution (Stephens Scientific, Riverdale, NJ) for 15 min and then rehydrated in graded ethanol. Antigen retrieval was then performed by placing slides containing the sections in a plastic container with antigen unmasking solution (Vector Laboratories, Burlingame, CA). After boiling for 3 min in a microwave oven, samples were subjected to continued low heat to maintain boiling for two consecutive 5-min periods, followed by cooling (1 h) to RT. For peroxidase-based antibody detection, endogenous peroxidase was inactivated by incubation in hydrogen peroxide (3%) in phosphate-buffered saline (PBS) (5 min, RT). Samples were washed twice in PBS and incubated (30 min, RT) in a blocking solution containing 2% fish gel (Sigma, St. Louis, MO) in PBS. The samples were then incubated (4°C, overnight) with control (IgG) antibody or primary antibody in blocking solution. Primary antibodies (and dilutions used) included: rabbit antirat HFH-4 (1:500 dilution); rabbit antirat HNF-3 (28) (1:500; kindly provided by R. Costa, University of Illinois, Chicago, IL); rabbit antimouse CCSP (29) (1:500; kindly provided by F. DeMayo, Baylor University, Houston, TX); goat antimouse olfactory marker protein (OMP) (30) (1:4,000; kindly provided by F. Margolis, University of Maryland, Baltimore, MD); and -tubulin IV (1:250; BioGenex, San Ramon, CA). For light microscopy, sections were washed and incubated (30 min, RT) with a biotinylated secondary antibody that was detected with the avidin-biotin complex using peroxidase or alkaline phosphatase reagents (ABC Elite; Vector Laboratories). Sections were counterstained with hematoxylin.

For immunofluorescent localization, slides containing paraffin-embedded samples were prepared as described above but goat antimouse fluorescein isothiocyanate (FITC)- labeled (1:200 dilution) or donkey antirabbit indocarbocyanine (Cy3)-labeled (1:800 dilution) secondary antibodies (both from Jackson ImmunoResearch Laboratories, West Grove, PA) were used. After washing in PBS, slides were mounted with anti-fade media (Vectashield; Vector Laboratories) and stored at 20°C. In each experiment, parallel samples were incubated with a control antibody (IgG of appropriate species) and enzymatic or fluorescence detection consistently revealed no immunostaining. Photographs of images were electronically digitized by scanning and composed in Photoshop (Adobe Systems, San Jose, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Anti-HFH-4 Antibody Generation

Polyclonal anti-HFH-4 antibody was generated in rabbit by immunization with a GST-HFH-4 fusion protein containing the N-terminal region of rat HFH-4. This HFH-4 amino acid sequence has high homology (93%) to the human HFH-4 sequence and does not include the DNA binding domain (17, 21). To evaluate the specificity of the polyclonal anti-HFH-4 antibody, immunoblot analysis was performed using lysates from in vitro-translated HFH-4, HNF-3, and HNF-3 cDNAs (Figure 1A). In vitro translation of HFH-4 labeled with ³⁵[S]methionine resulted in a 58-kD product. Immunoblots of in vitro-translated HFH-4 (in the absence of methionine labeling) and control forkhead proteins incubated with the anti-HFH-4 antibody demonstrated a specific band at approximately 58 kD in the HFH-4 sample and no cross-reactivity with control forkhead proteins (Figure 1B). No bands were detected when control IgG antibody was substituted for anti-HFH-4 antibody (not shown). These data indicate specific binding of the anti-HFH-4 antibody to HFH-4 protein and not to HNF-3 homologue proteins expressed in lung epithelial cells.

View larger version (43K): [in this window] [in a new window]   Figure 1.   Immunoblot detection of in vitro HFH-4 expression. (A) Lysates containing in vitro-translated cDNA sequences of forkhead proteins HNF-3, HNF-3, or HFH-4 labeled with 35[S]methionine and analyzed by PAGE and autoradiography. (B) Lysates from A of in vitro-translated proteins (in the absence of 35[S]methionine) evaluated by immunoblot analysis using polyclonal anti-HFH-4 antibody demonstrate identification of HFH-4 without cross-reactivity. Protein standards are indicated.

HFH-4 Expression in Adult Lung

To determine whether HFH-4 is expressed in a specific population of epithelial cells in the lung, sections of adult mouse, rat, and human lungs were subjected to immunohistochemical analysis with anti-HFH-4 antibody. HFH-4 was expressed in a subpopulation of proximal airway epithelial cells in the trachea, bronchi, and bronchioles, but not in alveolar epithelial cells (Figures 2A and 2D). A similar pattern of staining was seen in adult rat and human lungs (not shown). HFH-4 expression was more abundant in the nuclei than in the cytoplasm of epithelial cells. The pattern of HFH-4 expression was distinct from expression of HNF-3 that was present in all proximal airway epithelial cells and in distal airway type II pneumocytes (Figure 2B) as previously shown (6). No staining was seen in samples incubated with control IgG antibodies (Figure 2C).

View larger version (89K): [in this window] [in a new window]   Figure 2.   HFH-4 expression in adult mouse lung. Immunolocalization of HFH-4 and localization of HFH-4 with airway epithelial cell-specific markers. Immunoperoxidase staining (brown) demonstrates (A) HFH-4 expression in a subpopulation of proximal airway epithelial cells. (B) HNF-3 expression in all proximal airway cells and in type II pneumocytes (arrows). (C ) Control IgG. (D) Low-magnification view of HFH-4 expression in bronchi (b), and not in alveolar epithelium (a) or endothelium (v). (E ) Immunofluorescent localization of HFH-4 (Cy3) and -tubulin IV (FITC; cilia-specific marker) expression in ciliated cells. (F ) HFH-4 expression (immunoperoxidase) and CCSP expression (alkaline phosphatase; blue) detected in unique populations (F ). Sections counterstained with hematoxylin (A-D). Scale bar in F is 19 µM in A, B, C, and F; 48 µM in D; and 16 µM in E.

Examination of immunoperoxidase localization of HFH-4 by light microscopy revealed that HFH-4 was not detected in endothelium, interstium, or alveolar epithelium (Figure 2D). Cell types expressing HFH-4 appeared to be ciliated cells and not Clara cells (Figures 2A and 2D), as identified by cell morphology (24). To identify cilia on respiratory cells, the expression of -tubulin IV in cilia was used as previously described (31, 32). Comparison of -tubulin IV and HFH-4 expression by immunofluorescence indicated that HFH-4 expression is restricted to ciliated cells (Figure 2E). We also performed immunostaining of mouse lung sections with anti-CCSP and HFH-4 antibodies. Dual localization revealed that expression of HFH-4 and CCSP in the mouse airway are in two mutually exclusive cell populations (Figure 2F). Thus, HFH-4 is expressed in a specific population of proximal airway epithelial cells that are ciliated.

To determine whether HFH-4 may be related to differentiation status associated with malignant transformation of cells derived from the airway epithelium, HFH-4 expression was also evaluated in primary human lung carcinomas. Specimens from surgical resections of primary lung carcinomas, including squamous cell carcinoma (n = 1), adenocarcinoma (n = 5), and bronchoalveolar carcinoma (n = 1), were stained with anti-HFH-4 antibodies. Although HFH-4 expression was detected in bronchial epithelial cells in normal areas of lung, HFH-4 expression was not detected in any of the malignant cell types (not shown). These observations suggest that HFH-4 is expressed specifically in well-differentiated, normal airway epithelial cells.

HFH-4 Expression in Upper Airway of Nose and Paranasal Sinuses

Epithelial cells that line the upper respiratory tract (nose and paranasal sinuses) include ciliated, squamous, and secretory cells that are histologically similar to epithelial cells of the bronchi (33) but have not been previously evaluated for HFH-4 expression by in situ hybridization. We found that HFH-4 protein is expressed in the ciliated cells of the nose and paranasal sinuses in a predominant nuclear pattern, similar to HFH-4 expression in ciliated epithelial cells of the lung (Figures 3A-3C).

View larger version (83K): [in this window] [in a new window]   Figure 3.   HFH-4 expression in nose and paranasal sinuses. Immunoperoxidase or immunofluorescent detection of HFH-4 and other epithelial cell markers. (A) The distribution of HFH-4 expression (immunoperoxidase, brown) and OMP (alkaline phosphatase, blue) in mouse nose and paranasal sinuses. (Boxed areas indicate locations of higher magnification in B and serial section in C ). (B) Immunoperoxidase detection of HFH-4 expression in ciliated epithelial cells of the nose. (C ) Immunofluorescent detection of HFH-4 (Cy3) and -tubulin IV (FITC) expression in ciliated epithelial cells of nose but not in olfactory epithelial cells that contain sensory cilia (not detected by -tubulin IV) and express OMP (arrowheads). (D) HFH-4 expression in ciliated epithelial cells of a human nasal polyp. (E ) HFH-4 expression in nonciliated nasal epithelial cells of an E15.5 mouse. Sections were counterstained with hematoxylin (A, B, D, and E ). Scale bar in E is 140 µM in A, 6 µM in B, 14 µM in C, 19 µM in D, and 70 µM in E.

The nose also contains neuroepithelial cells with bipolar neurons that terminate in an olfactory knob linked to immotile cilia (33). The neuroepithelial olfactory cells were identified by the expression of OMP in the superior regions of the nasal cavity, the vomernasal organ, and the neuronal cells within the vomer bone of the nasal septum (Figure 3A) (30, 34). The sensory, nonmotile cilia of the olfactory epithelium, which have a structure different from the motile cilia of the respiratory epithelium, were not detected by -tubulin IV but were identified by light microscopy (35, 36). Simultaneous anti-HFH-4 and anti-OMP immunostaining demonstrated that olfactory epithelial cells do not express HFH-4 (Figures 3A and 3C). Hence, HFH-4 expression is associated with respiratory cells with motile-type cilia that contain nine outer doublet microtubules with dynein arms and an inner microtubule pair (9 + 2), but not with sensory cilia that contain only the nine outer doublets (without dynein arms or the inner pair; 9 + 0) (2, 36). This indicates a further restriction of HFH-4 expression.

The epithelium of human nasal polyps obtained from patients with advanced chronic sinusitis (Figure 3D) is histologically similar to normal upper airway respiratory epithelium (39). Immunostaining of nasal polyps with anti- HFH-4 antibody demonstrated that the ciliated epithelial cells of the nasal polyp also express HFH-4 (Figure 3D). Thus, although nasal polyps represent abnormally proliferating tissue, the pattern of HFH-4 expression is similar to that in normally differentiated upper airway.

HFH-4 expression in nasal epithelial cells of the mouse was detected at E15.5 (Figure 3E) but cilia were not detected on these cells when examined by light microscopy. However, at birth, the epithelial cells of the nose and paranasal sinuses were ciliated as determined by expression of -tubulin IV (not shown). These observations indicate that HFH-4 can be present in nonciliated epithelial cells, where expression may precede the appearance of cilia on these cells.

HFH-4 Expression in Extrarespiratory Tissues

Other tissues shown to express HFH-4 mRNA include oviduct, choroid plexus, and brain ependymal cells (17, 19). To identify cells in these tissues expressing HFH-4 protein, immunostaining with the anti-HFH-4 antibody was also performed. The oviduct, the most proximal region of the fallopian tube, is lined with epithelial cells containing motile cilia (37). HFH-4 is highly expressed in proximal oviduct within almost every epithelial cell lining the lumen (Figure 4A). In distal oviduct there are fewer ciliated cells, and in these regions HFH-4 expression is found in fewer cells (not shown) (37). As in the respiratory tract, HFH-4 is expressed specifically in ciliated cells and in a predominantly nuclear pattern (Figure 4B). Secretory, nonciliated cells of the oviduct do not express HFH-4 (Figure 4B). Dual localization of HFH-4 and -tubulin IV confirmed the relationship between HFH-4 expression and the ciliated cell phenotype (Figure 4C). A similar pattern of HFH-4 expression is present in human oviduct (not shown).

View larger version (83K): [in this window] [in a new window]   Figure 4.   HFH-4 expression in mouse oviduct and brain. (A) HFH-4 expression (immunoperoxidase, brown) in epithelial cells of oviduct. (B) Detail of oviduct from A demonstrating HFH-4 expression in ciliated but not nonciliated cells. (C ) Immunofluorescent localization of HFH-4 (Cy3) and -tubulin IV (FITC) expression in oviduct epithelial cells (asterisks indicate lumen). (D) Immunoperoxidase detection of HFH-4 expression in brain choroid plexus at E15.5. (E ) HFH-4 expression in adult brain ependymal cells of the lateral ventricle. (F ) Immunofluorescent localization of HFH-4 and -tubulin IV expression in ependymal cells. Sections were counterstained with hematoxylin (A, B, D, and E). Scale bar in F is 19 µM in A; 6 µM in B; 18 µM in C, D, and F; and 13 µM in E.

The motile cilia on the cells of the choroid plexus and ependymal cells that line the ventricles of the brain are thought to assist in local mixing of cerebral spinal fluid (40). In the developing choroid plexus, HFH-4 expression is present at E15.5 (Figure 4D). The ependymal cells lining the ventricles of the brain also express HFH-4 in a predominantly nuclear pattern (Figure 4E). Dual staining of brain tissues to detect HFH-4 and -tubulin IV confirmed that HFH-4 expressing cells are ciliated (Figure 4F). Thus, HFH-4 expression is present in cells that have motile cilia in both respiratory and nonrespiratory tissues.

HFH-4 expression was also evaluated by immunohistochemistry in adult mouse tissues that do not contain motile cilia. HFH-4 expression is not detected in sections from heart, liver, spleen, kidney, skeletal muscle, skin, stomach, small intestine, large intestine, and bladder (data not shown). This further confirms the relationship of HFH-4 to organs containing cells with motile cilia.

HFH-4 Expression in Developing Lung

On the basis of the observation that HFH-4 expression was restricted to ciliated epithelial cells, we next examined the temporal relationship between HFH-4 expression and the appearance of cilia during airway epithelial cell differentiation in developing lung. Previously, in situ hybridization marked the onset of HFH-4 mRNA in the airway epithelium at the late pseudoglandular stage (E14.5) in the mouse lung (17). With anti-HFH-4 antibody, HFH-4 expression was not detected at E14.5 (Figure 5A). In contrast, in a serial section of E14.5 lung, abundant HNF-3 was expressed in the airway epithelial cells (Figure 5B) as previously demonstrated (6). At E14.5, cilia were not present, as indicated by absent expression of -tubulin IV in airway epithelial cells (not shown). One day later, at E15.5, HFH-4 was expressed in approximately half of the proximal airway epithelial cells (Figure 5C). At E15.5 -tubulin IV expression was first detected, restricted to a small number of airway epithelial cells that express HFH-4 (Figure 5D). Tracking HFH-4 and -tubulin IV expression in the developing airway epithelial cells from E17.5 to E18.5 revealed that the number of HFH-4-expressing cells that also express -tubulin IV gradually increased. At birth, HFH-4 was present throughout the proximal airway in a pattern similar to adult lung (Figure 5E) and now almost every cell that expresses HFH-4 also expresses -tubulin IV (Figure 5F). Thus, stage-specific evaluation of HFH-4 and -tubulin IV expression in the developing lung suggests that HFH-4 expression is temporally associated with ciliogenesis.

View larger version (96K): [in this window] [in a new window]   Figure 5.   HFH-4 expression in developing mouse lung. Immunoperoxidase or immunofluorescent detection of HFH-4 and other epithelial cell markers. (A) E14.5 lung evaluated by immunoperoxidase (brown) for HFH-4 shows absent expression. (B) E14.5 serial section at E14.5 immunostained for HNF-3 expression. (C ) HFH-4 expression at E15.5. (D) E15.5 lung (serial section, higher magnification) demonstrating low levels of -tubulin IV (FITC) expression in some HFH-4 (Cy3)-expressing cells. (E ) HFH-4 expression in newborn (NB) lung. (F ) NB lung (serial section, higher magnification) demonstrating more abundant -tubulin IV expression on HFH-4 expressing cells. Sections were counterstained with hematoxylin (A-C and E ). Scale bar in F is 48 µM in A-C and E, and 24 µM in D and F.

HFH-4 Expression during Spermiogenesis

Spermatids develop flagella containing microtubules arranged in a 9 + 2 pattern, identical to that of motile cilia of the airway (37). Sperm maturation occurs in a wavelike fashion within the seminiferous tubules of the testis where spermatocytes mature to become spermatids that develop flagella (spermiogenesis) and are released as spermatozoa. The cycles of sperm maturation were identified in cross sections of tubules using the 14-stage system of Leblond and Clermont based on the morphologic appearance of germ cells (41). HFH-4 was previously shown by in situ hybridization to be expressed in spermatids during stages I to VI within the seminiferous tubules of the testis (17).

Using anti-HFH-4 antibodies, a stage-dependent pattern of nuclear and cytoplasmic expression of HFH-4 was detected during cycles of sperm maturation. Before the maturation of spermatids and formation of flagella, HFH-4 was weakly expressed in the cytoplasm of spermatocytes (Figure 6A). After meiosis, spermatids expressed abundant HFH-4 in the nucleus during stages III through VI (Figures 6B and 6C). Early spermatids with nuclear HFH-4 also expressed cytoplasmic -tubulin IV as the flagella was initially formed (Figure 6F). (At this stage, -tubulin IV was also expressed in the manchette in the innermost layer of more mature spermatids, present simultaneously with the new spermatids [Figure 6F] and at stages XI though XIII [Figure 6H] as previously described [42].) After stage VII, HFH-4 expression in spermatids was extinguished while development of the flagella continued (Figure D). -tubulin IV expression in flagella of mature spermatozoa was present in the central lumen at stage VII, immediately before spermatozoa release (Figure 6G). At these stages, HFH-4 expression in the nucleus of spermatids was no longer present, but was expressed in the cytoplasm of spermatocytes (Figures 6D, 6E, 6G, and 6H). These observations demonstrate that HFH-4 expression in the nuclei of early spermatids precedes the complete formation of flagella in mature spermatids. This suggests that nuclear expression of HFH-4 is associated with early production of flagella but that there is no persistent expression of HFH-4 in maturing spermatids during later flagella development.

View larger version (92K): [in this window] [in a new window]   Figure 6.   Expression of HFH-4 in adult mouse testis. Localization of HFH-4 expression by immunoperoxidase (brown; A-E) or immunofluorescence (Cy3; F-H ) and -tubulin IV (FITC; F- H ) to detect development of flagella in spermatids within seminiferous tubules. (A) HFH-4 is weakly expressed in the cytoplasm of secondary spermatocytes, but not expressed in spermatids (innermost cell layer, stage I) during early maturation. (B) HFH-4 expression in the nucleus of spermatids at stages III through IV. (C ) Less-abundant HFH-4 expression in nucleus of spermatids at stages V to VI. (D) Absent HFH-4 expression in spermatids (inner layer of cells), now present in the cytoplasm of spermatocytes at stages VII to VIII. (E) Absent HFH-4 expression in spermatids and weaker cytoplasmic HFH-4 expression in spermatocytes at stages XI through XIII. (F ) Immunofluorescent localization of HFH-4 and -tubulin IV (FITC) expression demonstrates HFH-4 expression in the nucleus of early spermatids associated with -tubulin IV (FITC) expression in the cytoplasm (arrrowheads). (G) -tubulin IV expression in flagella (tails massed at the center of the tubule) of mature spermatozoa just before release and absent HFH-4 expression in spermatozoa and spermatids at stages VII to VIII. (H ) Absent HFH-4 expression in maturing spermatids with prominent -tubulin IV expression in the manchette (man). HFH-4 is expressed in the cytoplasm of spermatocytes at stages XI through XIII. Sections counterstained with hematoxylin (A-E ). The stage of spermiogenesis in the individual tubules is indicated. Scale bar in E is 46 µm in A, B, C, D, and E; and 29 µm in F, G, and H.

	Discussion

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This study demonstrates that forkhead/winged-helix transcription factor HFH-4 expression is localized to ciliated epithelial cells and is temporally related to ciliogenesis. A role for HFH-4 in ciliogenesis is demonstrated by the temporal relationship of HFH-4 to the appearance of cilia in developing lung and flagella during spermiogenesis. Ciliated epithelial cells are thought to arise in the large airway from undifferentiated columnar cells and basal cells and in bronchioles from undifferentiated cells and Clara cells, but molecular markers to identify progenitor cells have not been identified (43, 44). Ciliogenesis during lung development has previously been identified by -tubulin IV expression at E19 in mice, and by electron microscopy at E20 in rats and at gestational wk 12 to 18 in humans (32, 38, 45). Thus, cilia appearance consistently follows HFH-4 expression in mice and humans (21), consistent with a role for HFH-4 in ciliogenesis. This role is substantiated by the recent observations by others and us that targeted deletion of HFH-4 in the mouse results in an absence of cilia in the airway (22, 23). Thus, HFH-4 is the first reported transcription factor expressed specifically in ciliated cells and related to ciliogenesis. A role for HFH-4 in a fundamental process such as ciliogenesis is consistent with the central biologic functions demonstrated for other winged-helix/ forkhead proteins (11, 13, 14, 46).

The characterization of HFH-4 protein expression extends the previous observations of HFH-4 mRNA in in situ hybridization (17, 19). First, antibody localization of HFH-4 demonstrates that expression is specifically within the epithelial cells of the proximal airway, upper respiratory tract, choroid plexus, ependymal cells, oviduct, and testis. Second, among epithelial cells expressing HFH-4 mRNA, HFH-4 protein expression is localized specifically to ciliated cells. HFH-4 also has a nuclear pattern of expression in these cells that could not be determined by in situ hybridization. Third, localization of HFH-4 expression with other epithelial cell-specific markers demonstrates the temporal relationship of HFH-4 expression to cell differentiation toward the ciliated cell phenotype in the airway. And fourth, HFH-4 protein localization uncovers a complex pattern of cytoplasmic and nuclear HFH-4 expression in the cells of the seminiferous tubules of the testis, strongly suggesting a regulated role in spermiogenesis coincident with the development of flagellaa structural equivalent of the cilia in sperm.

The pattern of HFH-4 protein expression in sperm maturation was more complex than that demonstrated by in situ hybridization. HFH-4 mRNA expression in the testis was detected in the earliest stages of spermatid maturation (stages I through VI), when HFH-4 protein was also detected in the nucleus. However, HFH-4 protein was also present in the cytoplasm of spermatocytes (stages VII through XIV) before differentiation into spermatids. The presence of HFH-4 protein in the cytoplasm of spermatocytes is not consistent with genetic analysis showing absent HFH-4 mRNA in the testis of mutant mice that have defects in meiosis (17, 18). This apparent inconsistency may be accounted for by protein sharing made possible by intercellular bridges between germ cells and demonstrated for other DNA-binding proteins (47). The regulation of nuclear localization of HFH-4 may be through a putative nuclear localization signal (KKRRLPPVH) that is conserved within the DNA-binding domain of other forkhead proteins (48). The transient nuclear expression of HFH-4 in spermiogenesis is also in contrast to the persistent nuclear expression of HFH-4 in ciliated epithelial cells. Persistent nuclear expression may be important for ongoing cell responses or for maintenance of ciliogenesis in cells undergoing continuous low-level loss of ciliaa process not necessary in sperm. Shared and unique functions of HFH-4 in spermiogenesis and ciliogenesis remain to be elucidated.

The in vivo molecular targets for HFH-4 activation are unknown. Although DNA binding sequences for HFH-4 have been identified, these are seemingly not related to proteins involved in ciliogenesis (19). HFH-4 expression is most directly related to cells with motile cilia rather than sensory cilia (36). Sensory or nonmotile cilia lack dynein arms as well as the central microtubule pair (central apparatus) that interacts with the dynein arms, suggesting that the genes coding for central apparatus proteins may be targets for HFH-4 regulation (49). However, more than 23 proteins comprising the central apparatus have been isolated (in the green algae Chlamydamonas), making identification of specific genes difficult (49). Other proteins with roles in ciliogenesis that could be regulated directly by HFH-4 include kinesins, axonemal dyneins, or tubulins (49). On the basis of homology to Chlamydamonas, several axonemal dyneins have recently been cloned from rat and mouse but regulatory regions of these genes have not yet been identified (51). Further characterization of the function of HFH-4 may lead to a greater understanding of the program of ciliogenesis and the treatment of airway diseases characterized by cilia defects (2, 53).