ARTICLE |
Correspondence to: Jeffrey A. Whitsett, Children's Hospital Medical Center, Div. of Neonatology and Pulmonary Biology, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail: jeff.whitsett@chmcc.org
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Summary |
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Expression of sonic hedgehog (Shh) is required for normal development of the lung during embryogenesis. Loss of Shh expression in mice results in tracheoesophageal fistula, lung hypoplasia, and abnormal lung lobulation. To determine whether Shh may play a role later in lung morphogenesis, immunostaining for Shh was performed in mouse lung from embryonic day (E) 10.5 to postnatal day (PD) 24. Shh was detected in the distal epithelium of the developing mouse lung from E10.5 to E16.5. From E16.5 until PD15, Shh was present in epithelial cells in both the peripheral and conducting airways. Although all cells of the developing epithelium uniformly expressed Shh at E10.5, Shh expression was restricted to subsets of epithelial cells by E16.5. Between E16.5 and PD15, non-uniform Shh staining of epithelial cells was observed in the conducting airways in a pattern consistent with the distribution of non-ciliated bronchiolar cells (i.e., Clara cells) and the Clara cell marker CCSP. Shh did not co-localize with hepatocyte nuclear factor/forkhead homologue-4 (HFH-4), ß-tubulin, or with the presence of cilia. These results support the concept that Shh plays a distinct regulatory role in the lung later in morphogenesis, when it may influence formation or cytodifferentiation of the conducting airways.
J Histochem Cytochem 49:15931603, 2001)
Key Words: morphogenesis, immunohistochemistry, Nkx2.1, ß-tubulin, HFH-4, CCSP
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Introduction |
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Morphogenesis of the mammalian lung begins as a ventral outpouching of the foregut endoderm into the surrounding splanchnic mesoderm. Dichotomous branching of the lung primordium forms the left and right lung buds, which undergo subsequent generations of dichotomous branching to generate the lobes of the lung, the bronchopulmonary segments, and the respiratory tubules that will form the mature lung. Branching morphogenesis of the lung buds is dictated by the underlying mesenchyme, which provides signals that induce unique genetic programs at different locations in the lung (see
One of the signaling molecules implicated in this complex process is sonic hedgehog (Shh), the vertebrate homologue of the Drosophila hedgehog gene (Hh). In the lung, Shh mRNA is expressed in the distal epithelium during early embryonic stages. The proposed Shh receptor, patched (Ptc), is expressed in the juxtaposed mesenchyme. Shh is autoproteolytically cleaved to produce N-terminal and C-terminal peptides. The N-terminal peptide is secreted and attaches to the cell membrane of the expressing cells, whereas the C-terminal remains unattached after secretion. The N-terminal fragment is believed to mediate signal transduction. At present, no biological function has been attributed to the C-terminal peptide. In the absence of Shh, the Ptc receptor prevents transcription of Shh-responsive genes by actively inhibiting smoothened (Smo), a transmembrane protein involved in relaying the Shh signal to the Gli family of transcriptional regulators. Secreted Shh binds to Ptc, relieving its inhibition of Smo, thereby allowing activating-Gli to translocate into the nucleus and bind to its consensus sequence in Shh-responsive genes (for review see
Several mouse models have been developed to examine the role of Shh during development. Lung-specific overexpression of Shh using the human 3.7-kD surfactant protein C (SP-C) enhancer/promoter leads to overexpression of Shh in the distal epithelium (
Homozygous null mice for Shh exhibited tracheoesophageal fistulas, tracheal or esophageal atresia, lung hypoplasia, fistula-like fusion of the alimentary and respiratory tract, and abnormal digit formation, demonstrating its importance early in branching morphogenesis (
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Materials and Methods |
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Animal Maintenance
FVBN mice were housed under pathogen-free conditions in accordance with institutional guidelines. Embryonic day (E) 1 was defined as the day when formation of a vaginal plug was first detected.
Antibodies
A rabbit polyclonal antibody to rat thyroid transcription factor-1 (Nkx2.1) was generated as described previously (
Immunohistochemistry
Immunohistochemical staining for Nkx2.1 was carried out using 5-µm paraffin sections that were deparaffinized, rehydrated, and heated at 90C in 0.1 M citric acid in 0.1 M sodium citrate (pH 6.0) for an initial 15 min and three successive 5-min periods (
The Shh immunolabeling procedure was similar to that described above, consisting of deparaffinization and rehydration of 5-µm sections, incubation in 3% hydrogen peroxide and methanol, heating in 0.1 M citric acid in 0.1 M sodium citrate, and blocking in 2% horse serum with subsequent overnight incubation with the primary antibody. Control tissues were incubated overnight at 4C in blocking serum only.
For the competition experiments, three applications were used. One set of slides from different timepoints was treated with Shh primary antibody (1:100 dilution) as described above. The second set was treated as a control with no primary antibody application. The third application consisted of incubating the primary antibody (1:100 dilution) for 15 min with Shh recombinant peptide such that the concentration of the peptide was 100-fold that of the antibody. After incubation, the antibody/peptide solution was applied to the slides similarly to the primary antibody application. Thereafter, the slides were treated as described above.
Immunolabeling using monoclonal antibodies against HFH-4 and ß-tubulin was carried out using the Vector M.O.M. Immunodetection Kit (Vector Laboratories) according to the manufacturer's directions, as previously described (
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Results |
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Developmental Pattern of Shh Staining in Mouse Lung
The distribution of Shh in the mouse lung was determined from E10.5 to PD15 by immunohistochemistry. At E10.5, distribution of the Shh peptide was detected in all epithelial cells of the mouse lung buds and was co-localized with Nkx2.1 expression (Fig 1). Shh staining was detected in the epithelial cells of the distal primordial tubules on E13.5, at which time cells from the proximal regions of the tubules were clearly devoid of expression (Fig 2). This proximaldistal difference in Shh expression was maintained through E14.5 and E15.5 (Fig 3). Low-level staining of Shh was first detected in the bronchial epithelium on E16.5 at the end of the pseudoglandular stage (Fig 3). Unlike the uniform staining detected earlier in lung development, staining of Shh during these latter stages of development was detected only in subsets of cells (Fig 3). Localization of Shh in the bronchiolar and bronchial epithelium on E16.5, E17.0/17.5, and E18.0 was consistent with sites and abundance of non-ciliated epithelial cells (Fig 3). The intensity of Shh immunostaining peaked in the bronchiolar and bronchial epithelium on E17.0/17.5 and was maintained at this level on E18.0 (Fig 4). Shh staining remained intense in the distal respiratory epithelium (i.e., terminal saccules) during E17.0/17.5 and E18.0 (Fig 4). However, at PD1, when high levels of Shh were detected in the epithelium of the conducting airways, there was a marked decrease in Shh immunostaining in the peripheral respiratory epithelium (Fig 4). Low-level Shh staining was detected in the alveolar epithelium during the postnatal period (PD5PD15; not shown) and was maintained at detectable levels in the bronchial epithelium until PD15. By PD24, the alveolar and bronchial epithelia were devoid of Shh staining (not shown).
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Specificity of Shh Staining in the Mouse Lung
To determine the specificity of the immunostaining, competition binding studies using the N-terminal Shh peptide were carried out with lung sections from E15.5, E17.0/17.5, E18.0, and PD1 (Fig 5). At all time points, the presence of the N-terminal peptide prevented staining in the lung epithelium, indicating that the immunostaining was specific for Shh.
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Correlation of Shh Expression with Cell-specific Molecular Markers
To determine the epithelial cell type in the conducting airway that expressed Shh, serial sections of mouse lung at E18.0 and PD1 were taken and immunostained for Shh, ß-tubulin, HFH-4, and CCSP (Fig 6). Expression of Shh was consistently detected in subsets of cells in the conducting epithelium that corresponded to non-ciliated cells, which protrude out into the lumen of the conducting tubules (Fig 6D). These dome-shaped cells stained for CCSP, a Clara cell marker (Fig 6B). The nuclei of ciliated cells stained with the HFH-4 antibody, which co-localized with ß-tubulin immunostaining (Fig 6E and Fig 6F) and the presence of cilia. The expression pattern of Shh was distinct from that of HFH-4 (Fig 6F) or ß-tubulin (Fig 6E) and did not co-localize with the ciliated cells (Fig 6C). Sites of Shh staining were similar to that of CCSP (Fig 6B), indicating that Shh was expressed in non-ciliated epithelial cells in the conducting airways (i.e., Clara cells).
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Discussion |
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Shh has been proposed to play a mitogenic role in several systems during embryonic development of several organs. In the vertebrate limb, Shh is required for patterning along the anterioposterior (A/P) axis and distal outgrowth of the limb bud (
Shh mRNA has been detected as early as E9.5 in the tracheal diverticulum (
During early lung development (around E10), Shh is expressed in the epithelium of the primordial tubules along with Nkx2.1 and BMP-4 (for review see
The Shh receptor Ptc is expressed at E11.5 in the mesenchyme surrounding the distal region of the primordial tubules (
At E16.5, Shh was detected at low levels in a subset of epithelial cells in the bronchiolar tubules, the non-ciliated bronchiolar epithelial cells. The appearance of Shh in non-ciliated cells of the conducting airways occurs in association with cytodifferentiation of ciliated and non-ciliated cells in the conducting airways. Expression of HFH-4 is required for formation of ciliated cells in the conducting airways (
Postnatally, there was a significant decrease in Shh staining in the alveolar epithelium, whereas strong staining was maintained in the non-ciliated cells of the bronchiolar epithelium. This pattern of Shh staining persisted until PD15, when staining of the respiratory epithelium was not detectable and that detected in the bronchiolar epithelium was significantly reduced. Shh staining was not detected in either the respiratory or bronchiolar epithelium at PD24. The saccular stage of lung morphogenesis begins at E17.4 and continues until PD5. During this stage, there is a decrease in cell proliferation and further development of the respiratory and bronchiolar systems with the differentiation of Clara cells, ciliated cells, and Type I alveolar cells (
The retinoic acid (RA) signaling pathway has recently been shown to be both spatially and temporally regulated (
Ongoing expression of Shh in the respiratory epithelium, with induction of Shh expression in the bronchiolar and bronchial epithelium during the later stages of development, suggests that Shh plays a role in lung morphogenesis or cytodifferentiation throughout lung development.
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Acknowledgments |
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Supported by funding from NIH grant SCOR HL56387.
We thank Ms Paula Blair for preparation of embryonic lungs for paraffin embedding and sectioning and Ms Sherri Profitt for technical assistance with immunohistochemistry.
Received for publication February 26, 2001; accepted June 27, 2001.
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