Departments of 1 Pediatrics and 2 Cell and Developmental Biology, and 3 The Center for Environmental Medicine and Lung Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Ciliogenesis in human fetal airway epithelium occurs from 11 to 24 gestational weeks. Using genetic and antigenic markers specific for human axonemal dynein heavy chain 9, we characterized temporal aspects of axonemal dynein expression associated with large airway epithelial ciliogenesis during human fetal development. Late in the first trimester, an undifferentiated columnar epithelium is characteristic of the large airways, and immunocytochemical studies exhibited focal localization of axonemal dynein antigen on luminal epithelial cell borders at sites consistent with emergent ciliary beds. From 12 to 22 wk, immunocytochemical labeling of new ciliary beds was prominent, and localization within the cytoplasm of epithelial cells suggested avid synthesis of axonemal dynein in advance of ciliogenic events. Quantitative RT-PCR of tracheal RNA and in situ hybridization studies compared favorably with immunocytochemical findings with the earliest expression of axonemal dynein at 9-10 wk gestation. These studies have documented that axonemal dynein is expressed early in human fetal life during airway epithelial maturation and well before histological or ultrastructural evidence of ciliogenesis is apparent.
cilia; ciliogenesis; human fetal airway epithelium
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INTRODUCTION |
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THE STRUCTURAL AND FUNCTIONAL BASIS of ciliary and flagellar motility is similar in all eukaryotic cells, and phylogenetic studies suggest that axonemal structure and composition are highly conserved across the eukaryotic phylogenetic spectrum. The ciliary axoneme is a complex molecular motor that effects motility through the interaction of axonemal dyneins in concert with axonemal microtubules and other structural accessory elements (14, 24, 32, 34). Dyneins imaged by electron microscopy appear as longitudinal arrays of appendages (arms) positioned at discrete intervals along the A subfiber of each of the nine peripheral microtubular pairs of the axoneme (5, 16). In ultrathin section views, inner and outer dynein arms appear as projections from the A subfiber of each microtubular doublet, with the outer arm often exhibiting a prominent "hook."
Each dynein arm is a very large multimeric microtubule-activated ATPase that generates interdoublet forces responsible for ciliary motility. The inner and outer arms are distinct dynein isoforms based upon differences in their structures (reviewed in Ref. 32), biochemical compositions (30, 31, 40), and functions (reviewed in Refs. 3 and 17). In addition to the axonemal dyneins, there are cytoplasmic dyneins that are involved in essential functions of the cell cytoskeleton, including retrograde intracellular transport, mitosis, and maintenance of the Golgi apparatus (reviewed in Ref. 19).
Each functional dynein motor complex is composed of a unique combination of heavy, intermediate, and light chains (reviewed in Ref. 20). The dynein heavy chains (DHCs) are a family of very large (>500 kDa) polypeptides that bear ATPase and microtubule-binding domains. Organisms that employ cilia, flagella, or both express a multitude of DHC isoforms (1, 12, 18, 28, 34, 35, 37, 39). For example, the biflagellate green alga Chlamydomonas is known to express at least 16 DHCs (33). Phylogenetic analyses of partial predicted amino acid sequences of putative DHCs suggest that a given DHC is more closely related to the homologous chain in other species than it is to the other DHCs of the same species (13, 34). This observation reinforces the notion that each functional dynein is defined by its DHCs, whose sequences and functions were highly conserved as species evolved.
The high degree of DHC sequence conservation has led to the identification of a number of putative human DHC genes (6, 7, 22, 23, 28, 29, 36, 38, 39). One of these, DNEL1, has been shown to have a protein product that is a component of cilia and that has biochemical properties characteristic of a DHC (36). Moreover, the expression of DNEL1 mRNA has been temporally associated with airway epithelial ciliogenesis in vitro (36). Determination of the complete predicted amino acid sequence of the DNEL1 gene product as it is expressed in airway epithelium (W. Reed, unpublished observations) has revealed that it is derived from a putative human DHC gene identified previously on the basis of partial cDNA sequences (GenBank Accession numbers U61740 and AJ132088). This gene is the human homolog of rat dynein-like protein 9 (37). It has been given the gene symbol DNAH9. Taken together, the data strongly support the notion that DNAH9/DNEL1 is an axonemal DHC gene whose expression in airway epithelium is restricted to ciliated cells.
In this study, we have examined DNAH9 expression in the large conducting airways during human fetal development by light and electron microscopic immunocytochemistry and quantitative RT-PCR analyses. This study has provided some new insights into temporal aspects of early gene expression as it relates to ciliogenesis in the developing and mature human airways.
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MATERIALS AND METHODS |
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Tissue acquisition procedures. Human fetal trachea and lung were obtained at the time of therapeutic abortion through a tissue procurement agency (Anatomic Gift Foundation, Woodbine, GA). The gestational ages of the specimens ranged from ~8 to 22 wk. The entire extrathoracic trachea from each of two to three specimens from each gestational interval was separated from the remainder of the lung. Because of the small organ size and acquisition procedures, it was not possible to further dissect the intrathoracic trachea or other regions of the parenchymal lung more completely. Normal tracheobronchial epithelium was obtained from healthy, nonsmoking adult male volunteers participating in inhalation toxicology studies in the Environmental Protection Agency Human Studies Facility at the University of North Carolina at Chapel Hill. Epithelium was obtained by forceps biopsy at the time of bronchoscopy. All procedures involving human subjects were approved by an institutional review board, and all volunteers gave informed consent before their participation.
Real-time RT-PCR. Extraction of total RNA and first-strand cDNA synthesis was performed as described previously (9). Relative amounts of mRNAs encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and human ciliary DNAH9 in total RNAs were estimated by quantitative fluorogenic amplification of first-strand cDNAs using a Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA), TaqMan Universal PCR Master Mix (Applied Biosystems), and the primers and fluorophore-labeled probes described at the end of this section. Relative amounts of 18S ribosomal RNA were estimated using TaqMan ribosomal RNA control reagents (Applied Biosystems). Each estimate was performed in a separate amplification using a 2.5-µl aliquot of first-strand cDNAs as template. Real-time fluorescence measurements were used to determine the threshold cycle for each amplification. Serial dilutions of recombinant plasmids bearing the complete coding region of human GAPDH, a fragment of human 18S rRNA, or a fragment of the human DNAH9 cDNA were amplified in parallel reactions and used to construct GAPDH, 18S rRNA, and DNAH9 standard curves relating the threshold cycle to molecules of plasmid standard. The standard curves were used to estimate the relative amounts of GAPDH, 18S rRNA, and DNAH9 cDNAs in each sample relative to their respective standards. The relative amount of DNAH9 cDNA in each sample was normalized to the relative amount of 18S rRNA or GAPDH cDNA, yielding an index proportional to the relative abundance of DNAH9 mRNA in each sample. Primers and fluorophore-labeled probes for GAPDH and DNAH9 were designed using either Primer Express software (Applied Biosystems) or Primer Designer software (Scientific and Educational Software, Durham, NC). Probes were labeled at the 5' end with either 6-carboxyfluorescein (FAM) or 6-carboxy-4',5'-dichloro-2',7-dimethoxyfluorescein (JOE) and at the 3' end with 6-carboxytetramethylrhodamine (TAMRA). Primer and probe sequences were as follows: GAPDH, probe: 5'-JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA-3', sense: 5'-GAAGGTGAAGGTCGGAGTC-3', and antisense: 5'-GAAGATGGTGATGGGATTTC-3'; DNAH9, probe: 5'-FAM-TCTTCAGAAGACCCTCCACCACGTG-TAMRA-3'; sense: 5'-GAGAACCAGGAAGTCAAGGAATG-3', and antisense: 5'-GCTGGACCTGCTGACTCAGA-3'.
Immunoblotting.
A fragment of a putative human axonemal outer arm DHC cDNA
subsequently identified as DNAH9 was cloned by long-range
PCR from a human tracheal cDNA library. A monoclonal antibody
identified as PQD-189 was raised against a 124-amino acid region of
DNAH9. The cloning procedures and subsequent generation and
characterization of the monoclonal antibody have been detailed
elsewhere (36). The specificity of the antibody was
investigated by immunoblotting of ciliary axonemal proteins as follows.
Axonemes were isolated as described (36) from in
vitro differentiated human airway epithelial cultures. The
axonemes were suspended in V1 photolysis buffer containing 30 mM HEPES,
pH 7.4, 5 mM Mg2SO4, 1 mM EGTA, 0.1 mM EDTA, 50 mM NaCl, 140 mM -mercaptoethanol, 1 mM dithiothreitol, 500 µM ATP,
100 µM NaVO3, and 1× protease inhibitor cocktail set III
(Calbiochem, San Diego, CA). One-half of the axoneme suspension was
held on ice, whereas the other one-half was irradiated on ice for 10 min with 254-nm ultraviolet light in a Stratalinker (Stratagene, San
Diego, CA). Samples were dissolved in NuPAGE sample buffer and
separated in 3-8% NuPAGE Tris-acetate gels according to the
supplier's instructions (Invitrogen, San Diego, CA). For gels that
were immunoblotted, each lane was loaded with a volume of axonemes in
NuPAGE sample buffer estimated to be derived from approximately three
well-ciliated 12-mm-diameter cultures (ca. 15 µg protein). Each lane
of gels that were silver stained was loaded with approximately
one-tenth of the volume loaded on immunoblotted gels (ca. 1.5 µg
protein). Electrophoresis was carried out for 6 h to enhance the
separation of DHCs. Gels were stained with a SilverXpress silver
staining kit according to the supplier's instructions (Invitrogen) or
blotted on nitrocellulose as described previously (36).
Blots were probed with a 1:50 dilution of a 50× concentrate of PQD-189
hybridoma supernatant and developed with a 1:5,000 dilution of
horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson
Immunoresearch Laboratories, West Grove, PA) and Super Signal West Pico
chemiluminescence reagents (Pierce, Rockford, IL). Chemiluminescence
was recorded using a Genegnome chemiluminescence imager (Syngene,
Frederick, MD).
In situ hybridization histochemistry. One microgram of linear plasmid DNA (pBluescript SK+, Stratagene, La Jolla, CA) containing 346 bases of the human axonemal dynein coding region was used as a template for generating riboprobes. T7 RNA polymerase (1 U/µl; Boehringer Mannheim) and 1× Digoxigenin-RNA labeling mix (Boehringer Mannheim) were incubated in polymerase buffer (40 mM Tris, pH 7.9 at 37°C, 6 mM magnesium chloride, and 2 mM spermidine) for 2 h at 37°C, and DNA was then degraded with RQ1 (RNAse-free DNase; Promega, Madison, WI). The probe was precipitated, air-dried, and resuspended in sterile water. Labeling efficiency was determined by comparison with a digoxigenin-labeled standard (Boehringer Mannheim).
In situ hybridization histochemistry to localize transcripts was performed as previously described (25) with some modifications. Tissue sections were dewaxed in a 3:1 mixture of Hemo-De (Fisher Scientific, Pittsburgh, PA) and xylenes, hydrated in decreasing ethanol concentrations to distilled water and then dehydrated through increasing ethanol concentrations, and air-dried before hybridization. Hybridization solution [50% deionized formamide, 0.25 mg/ml yeast transfer RNA in 4× SSC (20× SSC in 3 M NaCl, 0.3 M sodium citrate, pH 7.0), and 5 ng digoxigenin-labeled probe/section] was applied and covered with a glass coverslip. Slides were incubated in a humidified chamber at 60°C for 16-18 h. After hybridization, coverslips were removed to 1× SSC, and the sections were washed in 1× SSC for 1 h at 60°C, four times for 15 min at 60°C in 1× SSC, and two times in buffer 1 [150 mM NaCl and 100 mM (pH 7.5) Tris · HCl] at 25°C. Sections were then incubated in buffer 1 containing 1% BSA (Boehringer Mannheim) for 30 min at 25°C. Anti-digoxigenin-horseradish peroxidase (1.5 U/ml; Boehringer Mannheim) was added in the same buffer and incubated for 2 h. This solution was removed by aspiration, and slides were washed four times with buffer 1 at 25°C. Avidin-biotinylated-horseradish peroxidase complex (Vector Laboratories, Burlingame, CA) was added for 60 min at 25°C. Sections were washed as above and detected with 0.05% diaminobenzidine and 0.003% hydrogen peroxide in 0.1 M sodium acetate, pH 5.9, for 6 min. After being washed with distilled water and dehydrated through graded ethanols to xylenes, coverslips were mounted with Cytoseal 60 (Stephens Scientific, Kalamazoo, MI). Omission of the probe was used as a negative control.Immunohistochemistry and electron microscopic immunocytochemistry. Human fetal tracheas from specific gestational ages were fixed in 4% buffered paraformaldehyde for 1 h, rinsed in PBS, dehydrated, and embedded in paraffin. Sections (4 µm) were mounted on Superfrost plus microscope slides (Fisher). Control experiments further tested the specificity of the PQD-189 antibody to axonemal dynein and the immunolocalization procedure. This was accomplished with an immunolocalization protocol using a typically nonciliated tissue (human fetal heart at 22 wk gestation) and probing with antibodies to axonemal dynein (PQD-189) and desmin, a constitutive component of cardiac tissue. For light microscopic localization of axonemal dynein, sections of trachea at the different gestational intervals were probed with serially optimized titers of PQD-189 primary monoclonal antibody from an initial concentration of ~10 mg/ml and stained with Vectastain ABC reagents (Vector Laboratories) and ImmunoPure metal-enhanced 3,3'-diaminobenzidine substrate (Pierce) according to the manufacturer's instructions. Assay controls were performed with buffer alone in the absence of primary antibody.
For electron microscopic immunocytochemistry, fetal tracheas were fixed by immersion in 4% paraformaldehyde plus 0.25% glutaraldehyde in phosphate buffer immediately upon acquisition. The specimens were subsequently dehydrated through a graded ethanol series and embedded in Araldite. Ultrathin sections were incubated with the same primary antisera or ascites as those used for light microscopic studies, followed by washing and labeling with 10-nm colloidal gold-labeled secondary antibody. Control incubations were performed without primary antibody. The sections were poststained with lead citrate, viewed, and photographed on a Zeiss EM900 transmission electron microscope at an accelerating voltage of 50 kV. ![]() |
RESULTS |
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Real-time RT-PCR analyses of total RNA isolated from 9-wk fetal
trachea and lung suggested weak expression of DNAH9 mRNA, indicating that ciliogenesis was underway. However, ciliated cells are
not observed at 9 wk in either trachea or lung, so we investigated the
specificity and sensitivity of the DNAH9 mRNA TaqMan assay. We compared DNAH9 mRNA expression in undifferentiated
(nonciliated) human airway epithelial cell cultures with cultures,
derived from the same donor, that were undergoing mucociliary
differentiation. Amplification of DNAH9 cDNA with templates
from the nonciliated undifferentiated culture yielded a signal ("0"
in Fig. 1A) that was
indistinguishable from the no-template background control (NT in Fig.
1A), whereas signals well above background were observed in
both the nonciliated 4-day-old differentiated culture ("4" in Fig.
1A) and in the 12-day-old culture, which had ~20%
ciliated cells ("12" in Fig. 1A). The absence of signal
in undifferentiated cultures was not the result of scarcity of cDNA,
because the level of GAPDH cDNA in the undifferentiated culture was
comparable to levels in the differentiated cultures (compare 0 with 4 and 12 in Fig. 1B). These observations were reproducible. In
five out of five differentiations of airway epithelial cells from
different donors, DNAH9 mRNA was not detected in
undifferentiated cultures (day 0 in Fig. 1C),
although it was detected in companion cultures undergoing mucociliary
differentiation (days 4, 8, and 12,
Fig. 1C). These observations demonstrated that analysis of a
complex cDNA template from undifferentiated airway epithelial cells
fails to generate a specific signal in the DNAH9 mRNA TaqMan
assay. In contrast to undifferentiated airway epithelial cells,
analysis of the cDNA templates from 9-wk trachea and lung generated
signals that were not only well above background (compare 9 vs. NT,
Fig. 1, D and E) but greater than the signal
generated by amplification of the lowest cDNA standard, which contained
~450 molecules (450, Fig. 1, D and E). Taken
together, the data strongly suggested that DNAH9 mRNA was
present in the total RNAs isolated from 9-wk trachea and lung.
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The kinetics of DNAH9 mRNA expression in developing trachea
and lung are shown in Fig. 2. In trachea,
levels increased dramatically between 9 and 13 wk and then increased
only slightly after that (compare 9, 13, 15, and 18 in Fig.
2A). In lung, levels increased significantly between both 9 and 13 and 13 and 15 wk but were unchanged at 18 wk (compare 9, 13, 15, and 18 in Fig. 2C). Estimates of 18S rRNA levels in
templates from the trachea (Fig. 2B) and lung (Fig.
2D) showed little variation within each group, suggesting that the changes in DNAH9 mRNA are not the result of
differences in the amount of total RNA input into the reverse
transcription or the reverse transcription efficiency.
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The relative abundance of DNAH9 mRNA at each gestational age was estimated using 18S rRNA (Fig. 2E) or GAPDH (Fig. 2F) levels as normalization factors. Both methods of normalization yielded similar kinetics (compare Fig. 2E with Fig. 2F). Both analyses showed that DNAH9 mRNA expression levels in trachea were approximately four (18S rRNA normalization) to ten (GAPDH normalization) times greater than levels in lung at every stage of development assayed. The increase in DNAH9 mRNA abundance was not uniform. Both trachea and lung experienced a phase of rapid expansion in DNAH9 mRNA abundance, but this phase occurred earlier in the trachea. At least 50% of the overall increase in DNAH9 mRNA abundance in the trachea occurred between 9 and 13 wk, whereas only ~12% of the overall increase in lung occurred. More than 80% of the overall increase in DNAH9 mRNA abundance in lung occurred between 13 and 15 wk. These observations suggested that, although ciliated cell differentiation was underway at 9 wk in trachea and lung, differentiation in the trachea was accelerated with respect to lung.
DNAH9 protein has been characterized previously by immunoblotting of
bronchial cell ciliary axonemes using PQD-189, a monoclonal antibody
raised to a fragment of the deduced amino acid sequence of
DNAH9 (36). In those studies, PQD-189 staining
was restricted to proteins of very high molecular weight. However,
there are a number of DHCs with similar molecular weights that migrate
in the region stained by PDQ-189. Consequently, the specificity of PQD-189 was investigated by additional immunoblotting of axonemal proteins separated by extended SDS-PAGE. Although at least five distinct DHCs were resolved in axonemes, only a single immunoreactive species was observed (Fig.
3A). The possibility that two
or more DHCs recognized by PQD-189 were comigrating was investigated in further detail by immunoblotting of axonemes subjected to V1
photolysis. Irradiation of axonemes with ultraviolet light in the
presence of ATP and metavanadate split the DHCs, yielding smaller DHC
fragments that are more easily resolved from one another (Fig.
3A). Only a single V1 photolysis product was immunoreactive
(Fig. 3A). These data supported the notion that PDQ-189 is
specific for a single DHC.
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The results of in situ hybridization histochemistry (Fig.
4) were complementary
to the quantitative PCR data. At 9-11 wk gestation (late 1st
trimester), axonemal dynein expression was not evident in the
tracheal epithelium (Fig. 4A). However, time points in the
second trimester (Fig. 5, B
and C) showed prominent and progressive expression. At 22 wk
gestation, when epithelial growth and differentiation approach
maturity, in situ hybridization again showed a decline in the intensity
of localization.
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Tests of the specificity of the PQD-189 antibody to axonemal dynein for immunolocalization have been reported previously (36) and were documented again in this study by the absence of localization in fetal heart, a typically nonciliated tissue. A procedural control showed positive localization of desmin, a constitutive component of cardiac tissue in the same specimen (Fig. 5, A and B).
Light microscopic examination of human fetal tracheal epithelium at
different gestational ages (Fig. 6,
A, C, and E) revealed organization and
developmental patterns consistent with other reports in the literature
(8, 10, 11, 26, 27). At the earliest stage examined, nine
gestational weeks (Fig. 6A), there was no evidence of
cilia at the light microscopic level. Progressive ciliation was
observed in the second trimester (Fig. 6, C and E), and by 22 gestational weeks the epithelial layer was
fully ciliated and developed and indistinguishable from normal
adult airway epithelium (Fig. 6G).
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In an effort to identify nascent ciliated cells in the developing
trachea and lung, we localized DNAH9 protein by light and electron
microscopic immunocytochemistry. PQD-189 immunoreactivity in human
fetal tissues was observed as early as 10 wk gestation as a dark band
of the chromogen along the luminal border of some epithelial cells
(Fig. 6B). As epithelial maturation progressed, both the
cytoplasm and emergent ciliary beds exhibited marked localization in
the epithelial cell cytoplasm (Fig. 6, D and F). In contrast, normal human adult bronchial epithelium exhibited marked
immunostaining of ciliary beds by PQD-189 (Fig. 6H), but, unlike fetal tissues, no localization was observed in the cytoplasm of
adult tracheal epithelial cells. Electron microscopic
immunocytochemistry was used in an attempt to more specifically
localize the axonemal dynein antigen to the nascent ciliating cells
that exhibit limited morphological evidence of ciliary structure and
ciliogenesis at the light microscopic level. Electron microscopic
immunocytochemistry of human fetal tracheal epithelium revealed
cytoplasmic localization of the antigen by the PQD-189 monoclonal
antibody to human axonemal dynein in the apical cytoplasm of human
fetal tracheal epithelium at 10 gestational weeks (Fig.
7A). Localization of colloidal
gold particles was more extensive in epithelial cell cytoplasm and ciliary axonemes of tissues obtained at later intervals, and, as
epithelial maturation and ciliated cell differentiation proceeded, amorphous aggregates of gold localization were more evident in the
apical cytoplasm (Fig. 8A).
More organized linear arrays of gold particles were evident in close
proximity to luminal membranes (Fig. 8C) and in cytoplasmic
sites containing evident axonemal structures (Fig. 8, B and
D) at later gestational ages.
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DISCUSSION |
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We have applied molecular and morphological approaches to clarify the temporal aspects of expression of a functionally critical and distinctive component of ciliated cells, axonemal dynein, early in human fetal airway development. Our observations indicate that an RNA encoding an axonemal DHC, DNAH9, is detectable as early as 9 wk gestation in human fetal lung and trachea. Furthermore, DNAH9 protein is detectable by 10 gestational weeks in the airway epithelium by immunocytochemical localization at the light and electron microscopic levels. These observations suggest that axonemal dynein gene expression may be underway for 2-3 wk before there is morphological evidence of ciliary axonemes in the fetal airway epithelium.
The kinetics of DNAH9 mRNA relative abundance in developing human trachea and lung that were obtained in this study appear to be valid, on the basis of the following correlations. First, the relative abundance of DNAH9 mRNA was ~4-10 times higher in the trachea (Fig. 2, E and F), in keeping with the greater abundance of ciliated cells in the trachea relative to the lung, in which only the airways are ciliated. Second, the rate of increase in DNAH9 mRNA relative abundance was highest between 9 and 13 wk in the trachea, whereas it was greatest between 13 and 15 wk in lung (Fig. 2, E and F). Although other explanations may be possible, the data are consistent with the proximal-to-distal progression of airway maturation.
The specificity of the anti-DNAH9 antibody, PQD-189, was supported by immunoblotting of human axonemes. It was already known that PQD-189 reacts selectively with one or more very high molecular weight proteins present in human axonemes (36). However, the high sequence similarity and similar molecular weights of DHCs make cross-reactivity with PQD-189 a possibility that had not been ruled out. Cross-reactivity of PQD-189 with human DHCs was investigated by extended one-dimensional SDS-PAGE of axonemes before and after V1 photolysis. Five distinct DHCs were resolved in axonemes (Fig. 3A). Although the number of distinct DHCs present in the axoneme is still uncertain, there are undoubtedly more than five. Enumeration of DHCs in Chlamydomonas suggests that there are at least 14 DHCs that are structural components of its axoneme (33). V1 photolysis of DHCs was used to yield smaller fragments of DHCs that are more likely to be separated (2). The presence of a single immunoreactive species under all conditions suggested that PQD-189 recognizes a single axonemal DHC. Moreover, the data supported the notion that PQD-189 recognizes a human DHC because sensitivity to V1 photolysis is a characteristic of DHCs (15) and confirmed the predicted presence of the epitope recognized by the antibody on the larger carboxy-terminal V1 photolysis fragment of the DHC (shown in Fig. 3B).
Previous studies (8, 10, 11, 26, 27) have documented that ciliation of the human fetal tracheal epithelium occurs from approximately 11 to 20 wk gestation. Before this time, the tracheal epithelium is an undifferentiated columnar epithelium. In the present study, immunohistochemical studies localized the PQD-189 antibody to the luminal borders of nascent ciliating cells before 12 wk gestation, when ciliary beds can be identified by morphological criteria. Once cilia emerged along the luminal borders of the epithelium, PQD-189 clearly localized on the axonemal shafts. Furthermore, the antibody localized to the cytoplasm and cilia in the second trimester, whereas in the bronchial epithelium of a healthy adult human subject, PQD-189 localized only to cilia, suggesting that cytoplasmic localization of axonemal dynein, evidenced by immunostaining with PQD-189 antibody, may be more readily detectable in nascent ciliating cells actively undergoing ciliogenesis. This observation also is consistent with the in situ hybridization studies suggesting that axonemal dynein expression declines as the epithelium approaches maturity.
Previous histological and ultrastructural studies (4, 21) have suggested that turnover of the epithelium in the lower airways is attenuated relative to the nasal epithelium, where environmental irritants and a resident microbial flora may contribute to more rapid turnover. This notion is supported by the observation that ciliogenic profiles in the lower airway epithelium of healthy individuals are rarely observed, whereas such profiles in the nasal epithelium are common. Our comparative immunohistochemical observations of human fetal and adult airway epithelium also suggest that detection of axonemal dynein expression and immunohistochemical localization of antigen appear to be associated with phases of rapid growth and differentiation that subsequently decline with epithelial maturation.
In summary, our observations suggest that axonemal dynein expression occurs as an early event in the differentiation of the ciliated cell component of the developing human airway epithelium and precedes more overt morphological manifestations of ciliogenesis. These studies have further reinforced the specificity of the PQD-189 antibody to human axonemal dynein and suggest its utility as a means of identifying cells in early stages of ciliated cell differentiation. This capability may be useful for future investigations of epithelial remodeling and phenotypic transition that may be associated with acute and chronic respiratory disease processes. Inasmuch as emerging gene-therapeutic technologies may require targeting of specific cell types, this antibody may also find utility as a marker of early ciliated cell differentiation.
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ACKNOWLEDGEMENTS |
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We thank Brian Brighton for expert technical assistance with the in situ hybridization histochemistry studies.
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FOOTNOTES |
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This work has not been subjected to Environmental Protection Agency review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred.
This research was supported in part by National Heart, Lung, and Blood Institute Grants HL-56395, HL-34322, and HL-60280. The United States Environmental Protection Agency, through its Office of Research and Development, partially funded and collaborated in the research described here under Cooperative Agreement no. CR-824915 to Philip A. Bromberg.
Address for reprint requests and other correspondence: J. L. Carson, The Center for Environmental Medicine and Lung Biology, The Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7310 (E-mail: jcarson{at}med.unc.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.
10.1152/ajplung.00147.2001
Received 26 April 2001; accepted in final form 8 November 2001.
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