ARTICLE |
Correspondence to: Jeffrey A. Whitsett, Children’s Hospital Medical Center, Div. of Neonatology and Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039.
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Summary |
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HNF-3/forkhead homologue-4 (HFH-4), a transcription factor of the winged-helix/forkhead family, was detected by immunohistochemistry in tissue of the developing mouse. HFH-4 protein was present in epithelial cells of the lung, trachea, oviduct, and embryonic esophagus, and in ependymal cells lining the spinal column and ventricles of the brain. In lung, trachea, and nose, HFH-4 was expressed in a distinct subset of epithelial cells that also expressed ß-tubulin IV, a ciliated cell marker. Cellular sites of HFH-4 and ß-tubulin IV expression were distinct from that of Clara cell secretory protein (CCSP), which was detected in nonciliated epithelial cells in the conducting airway of the lung. HFH-4 and ß-tubulin IV, but not CCSP, were detected in the respiratory epithelium of thyroid transcription factor-1 (TTF-1) gene-targeted mice. The presence of HFH-4 and ß-tubulin IV in TTF-1 gene-targeted mice demonstrates that differentiation of ciliated epithelium does not require TTF-1. Co-localization of HFH-4 and ß-tubulin IV staining in various tissues during mouse development supports a role for HFH-4 in the differentiation of ciliated cell lineages. (J Histochem Cytochem 47:823831, 1999)
Key Words: HFH-4, lung, cilia, ß-tubulin IV
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Introduction |
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HNF-3/FORKHEAD HOMOLOGUE-4 (HNF-4) is a nuclear transcription factor of the winged-helix/forkhead family. Winged-helix/forkhead family members are widely expressed in eukaryotes, where they regulate developmental processes in many tissues (
In vitro, HFH-4 activated the transcription of CCSP and hepatocyte nuclear factor-3 (HNF-3
) genes (
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Materials and Methods |
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Animals Used
FVB/N and heterozygous TTF-1 (+/-) mice were housed in pathogen-free conditions according to institutional and NIH guidelines. Gestational age of fetal mice was determined by weight and crown-to-rump length. Tissues were fixed overnight at 4C in 4% neutral buffered paraformaldehyde, washed in a graded series of ethanols, and embedded in paraffin. Adult mouse lung was inflation-fixed with 4% neutral buffered paraformaldehyde at 20 cm of water pressure for 1 min, followed by fixation overnight at 4C. Five-µm sections were mounted on poly-lysine-coated slides.
Antibodies and Immunohistochemistry
A rabbit polyclonal antibody against a synthetic peptide spanning amino acid residues 1101 of mouse HFH-4 fused to GST was generated as described previously (
Immunohistochemical staining for HFH-4 was carried out as follows. Five-µm paraffin sections were deparaffinized and slides were heated to 90C in 0.1 M citric acid, 0.1 M sodium citrate (pH 6.0) for 20 min. Sections were then treated with 3% hydrogen peroxide in methanol for 15 min, blocked with 2% normal goat serum for 2 hr, and incubated overnight at 4C with HFH-4 primary antibody, and developed with a biotinylated goat anti-rabbit secondary antibody and a Vector Elite ABC kit (Vector Laboratories; Burlingame, CA). Antigen localization was enhanced with Ni-DAB, followed by incubation with Tris-cobalt and counterstaining with Nuclear Fast Red.
For the co-localization studies, double antibody staining was carried out sequentially. HFH-4, CCSP, or TTF-1 immunohistochemistry was performed first as described above, except that slides were not counterstained with Nuclear Fast Red. After enhancement with Tris-cobalt, slides were washed with water, PBS, then PBS0.1% Triton X-100 for 5 min each. The tissues were blocked in 5% normal horse serum for 4 hr, followed by incubation overnight at 4C with the ß-tubulin IV primary antibody. The reaction was developed using a biotinylated horse anti-mouse secondary antibody and the Vector ABCalkaline phosphatase kit with VectorRed as the substrate (Vector Laboratories) and the tissues counterstained with methyl green (Dako; Carpinteria, CA). All immunohistochemical reactions were run with a control reaction lacking the primary antibody to ensure specificity of the staining.
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Results |
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Distribution of HFH-4 Protein in Developing Mouse Tissues
At Day 13.5 p.c., HFH-4 was detected in the nuclei of cells in choroid plexus epithelium (Figure 1A) and ependymal cells lining the lateral ventricle of the brain (Figure 1B). At Day 18.5 p.c., HFH-4 protein was detected in tracheal epithelium (Figure 1C), nasal cavity epithelium (Figure 1E), esophageal epithelium (Figure 1F), and ependymal cells lining the spinal column (Figure 1G). Staining was limited to the nucleus in all tissues. In adult mice, HFH-4 protein was detected in tracheal (Figure 1D) and oviduct epithelium (Figure 1H). HFH-4 was not detected in esophageal epithelium in adult mouse (not shown). HFH-4 was not assessed in brain, nasal epithelium, or testis in adult mice or in developing kidney epithelium.
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Developmental Pattern of HFH-4 Expression in Lung
The distribution of HFH-4 protein in lung was examined from Day 13.5 p.c. to adulthood. HFH-4 was not detected in the lung at Day 13.5 p.c. (Figure 2A), corresponding to the early pseudoglandular stage of lung development. By the canalicular stage of lung development at Day 16.5 p.c., HFH-4 was readily detected in nuclei of columnar cells throughout the conducting airway (Figure 2B), including bronchi and bronchioles. This pattern of expression was also observed in the saccular stage of lung development on Day 18.5 p.c. (Figure 2C) and in lungs of neonates (not shown) and adults (Figure 2D). At all time points, HFH-4 was detected in subsets of columnar or basal cells in the conducting airway epithelium and staining was restricted to nuclei. HFH-4 staining was never observed in the alveolar epithelium.
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Co-localization of HFH-4 and ß-tubulin IV
To identify the cell type expressing HFH-4 in the conducting airway epithelium, immunohistochemistry with HFH-4 and ß-tubulin IV was performed. Double labeling immunohistochemistry demonstrated that HFH-4 and ß-tubulin IV were co-localized in airway epithelial cells. At Day 18.5 p.c., HFH-4 and ß-tubulin IV were co-localized in bronchial (Figure 3A), bronchiolar (not shown), and nasal epithelium (Figure 3B). Although HFH-4 was present in the trachea at Day 18.5 p.c., ß-tubulin IV was not detected (not shown). In the adult mouse, HFH-4 and ß-tubulin IV co-localized in bronchial (Figure 3C), bronchiolar (not shown), and tracheal epithelium (Figure 3D). Staining for HFH-4 and ß-tubulin IV was also present in tracheal submucosal glands (Figure 3D, arrow). Although most cells stained for both HFH-4 and ß-tubulin IV, cells staining for HFH-4 or ß-tubulin IV alone were also detected.
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ß-Tubulin IV and CCSP Are Expressed in Distinct Subsets of Respiratory Epithelial Cells
CCSP is a secreted protein synthesized by nonciliated respiratory epithelial cells (
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Relationships Between TTF-1 and HFH-4
Because TTF-1, a transcription factor of the Nkx family, is required for lung morphogenesis and expression of both CCSP and surfactant proteins (
Mice with a targeted ablation of the TTF-1 gene have cystic lung sacs lined with a poorly differentiated epithelium (
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Discussion |
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This study demonstrates nuclear staining of HFH-4 protein in epithelial cells in various organs of the developing mouse. HFH-4 staining was detected in epithelia at sites consistent with the presence of ciliated cells in various organs, including choroid plexus, nasal cavity, oviduct, tracheal, and lung airway epithelium. This pattern of expression is consistent with a role for HFH-4 in determining ciliated cell differentiation and in the regulation of target genes involved in differentiation of ciliated epithelia. The present findings are consistent with previous studies of HFH-4 mRNA localization (
The developmental expression pattern of HFH-4 was examined more closely in the lung. Nuclear staining of HFH-4 was first detected in airway epithelium of the mouse lung on Day 16.5 p.c. but was not detectable on Day 13.5 p.c., consistent with previous studies demonstrating the presence of HFH-4 mRNA on Day 14.5 p.c. in the mouse (
On close inspection, HFH-4 staining appeared to be found in ciliated cells in the conducting airway. Less frequently, staining of what appeared to be basal cells was also observed. Co-localization with antibodies against HFH-4 and ß-tubulin IV was performed to determine if staining for these two markers could be detected in the same cell. ß-Tubulin IV has been shown to be a marker for ciliated cells in bovine trachea (
The respiratory epithelium is derived from an outpouching of the foregut endoderm, giving rise to a primitive pulmonary epithelium consisting of columnar cells that are believed to be pluripotent. The primitive pulmonary epithelium differentiates into the cells of the conducting airways and alveolar compartment (
Mouse models in which HFH-4 protein is ectopically expressed or genetically ablated support a role for HFH-4 in regulating ciliated cell, but not Clara cell, differentiation. Expression of HFH-4 in the distal respiratory epithelial cells of transgenic mice induces ciliated cell characteristics and blocks differentiation of the Clara cell and alveolar Type II cell lineages (unpublished data). In addition, gene targeting of HFH-4 in mice results in a complete lack of ciliated epithelial cells (
The poorly differentiated lung structure found in TTF-1 (-/-) mice provides a unique system for studying lung development in the absence of a gene known to play an important role in respiratory epithelial cell differentiation (
In summary, HFH-4 is expressed primarily in ciliated cells of the respiratory epithelium and in cells that may serve as precursors of ciliated cells in the fetal and postnatal lung. HFH-4 is detected in ciliated epithelia of other tissues, suggesting a role for HFH-4 in differentiation and gene expression in ciliated cells and their precursors. The presence of HFH-4 and ß-tubulin IV in the conducting airway epithelium of TTF-1 (-/-) mice indicates that HFH-4 marks subsets of cells distinct from Clara cells or alveolar Type II cells in fetal lung development.
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Acknowledgments |
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Supported by NIH grants HL 56387, by the Program of Excellence in Molecular Biology of the Heart and Lung and HL 41496 (JAW), and by a grant from the Council for Tobacco Research (RHC).
We thank Ms Sherri Proffit for excellent technical help with immunohistochemistry.
Received for publication September 4, 1998; accepted January 12, 1999.
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