ARTICLE |
Correspondence to: Teresa E. Wagner, 325 Ninth Ave., Box 359640, Seattle, WA 98104. E-mail: twagner@u.washington.edu
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Summary |
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The complex interplay between cells and extracellular matrix (ECM) proteins is critical for lung development. Integrins are key modulators of this interaction. The integrin subunit 8 associates with the ß1-subunit to form an RGD-binding integrin. We previously showed that, in adult lung,
8 is expressed in contractile interstitial cells and smooth muscle cells and is upregulated in lung injury. To gain insight into the function of
8 during lung development, we examined the spatiotemporal expression of
8 throughout murine lung development. We compared the distribution of
8 with
-smooth muscle actin (
SMA), fibronectin (
8 ligand), and cytokeratin.
8 co-localized with
SMA and fibronectin in the peribronchial and perivascular regions. In all stages,
8 immunoreactivity was detected diffusely in the mesenchyme except for cells surrounding distal, newly forming airways.
8,
SMA, and fibronectin co-localized at tips of secondary septae in the alveolar stage. We conclude that
8 is marker for lung mesenchymal cells starting early in development.
8 is also a marker for smooth muscle cells, expressed as early as
SMA. Co-localization of
8 with fibronectin suggests a role in branching morphogenesis. Furthermore,
8 may participate in secondary septation by modulating signals from the extracellular matrix to alveolar myofibroblasts. (J Histochem Cytochem 13071315, 2003)
Key Words:
lung, development, organogenesis, integrin, 8ß1
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Introduction |
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THE COMPLEX INTERPLAY between cells and extracellular matrix (ECM) proteins is critical for cell growth, migration, and differentiation in the developing lung (-subunit noncovalently associated with a ß-subunit. Integrinligand interactions result in activation of signal transduction pathways and alterations in the cytoskeleton. The resulting events include cell adhesion, migration, proliferation, and differentiation.
The integrin subunit 8 associates with the ß1-subunit to form an arginineglycineaspartic acid (RGD)-binding integrin. Its ligands include fibronectin, vitronectin, tenascin-C, osteopontin, and nephronectin (
8 is expressed in cells with contractile properties including vascular and visceral smooth muscle cells, mesangial cells, and lung interstitial cells (
8 is expressed in contractile interstitial cells, including alveolar myofibroblasts, lipid-containing fibroblasts, and pericytes (
8 expression is increased after injury in models of pulmonary fibrosis, hepatic fibrosis, and glomerulonephritis (
SMA. These events are followed by remodeling of injured tissues and organ fibrosis. Because the molecular pathways of wound healing and development may be similar, we hypothesized that
8 plays an important role in development. The expression of
8 in lung development is unknown.
Our aim was to examine the spatiotemporal distribution of 8 to gain insight into its role in lung development. Because we had previously found co-expression of
SMA with
8 in activated alveolar myofibroblasts in a model of lung injury, we examined the relationship between
8 and
SMA expression in lung development. We also compared the distribution of
8 to one of its ligands, fibronectin, believed to play a role in murine lung development.
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Materials and Methods |
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Tissue Specimens
This study was approved by the University of Washington Institutional Animal Care and Use Committee. C57BL/6 female mice were mated with C57BL/6 males and checked every morning for a vaginal plug to determine day 0.5 of gestation. Pregnant females at 12.5, 13.5, 14.5, 15.5, 16.5, and 17.5 days of gestation (E12.5E17.5) were sacrificed and embryos were extracted via laparotomy. Gestational age was further confirmed by examination of external features. C57BL/6 pups at postnatal days 2 and 8 (PN2 and PN8) were also examined. Lungs were dissected and immersed at 4C in serial concentrations of sucrose in PBS: 5%, 10%, 15%, and 20%. After sucrose preservation, tissues were placed in OCT embedding medium (Tissue-Tek; Sakura Finetek, Torrance, CA), frozen in methylbutane that had been chilled in liquid nitrogen, and stored at -80C. Five-µm cryostat sections of tissues were obtained and placed on slides. In preparation for immunohistochemistry (IHC), sections were thawed, fixed for 3 min in acetone at -20C, allowed to air-dry at room temperature (RT) for 30 min, and rehydrated in PBS.
IHC for Light Microscopy
Slides from each time point were blocked sequentially with Peroxo-block (Zymed; San Francisco, CA) for 30 sec, 3% normal goat serum (Vector; Burlingame, CA) in PBS for 1 hr at RT, and Endogenous Avidin and Biotin Blocking Reagents (Zymed) for 10 min each. Generation of rabbit anti-8 peptide polyclonal antiserum was previously described (
8 antibody (polyclonal antiserum 1:16,0001:32,000 dilution) followed by 1-hr RT incubation with biotinylated goat anti-rabbit IgG antibody (1.5 µg/ml) (Vector). Immunoperoxidase complexes were formed using a Vectastain Elite ABC Kit (Vector). Color development was performed with DAB (Sigma Chemical; St Louis, MO) in the dark for 10 min, followed by DAB enhancer (Zymed) for 1 min. Sections were counterstained with hematoxylin (Vector), serially dehydrated, and mounted with Permount (Sigma). Control sections were incubated with pre-immune rabbit serum (Vector) in place of rabbit anti-
8 polyclonal antiserum or incubated with
8 antiserum preadsorbed with peptide used to generate the antiserum as previously described (
IHC for SMA was performed using the Mouse on Mouse (MOM) Peroxidase kit (Vector). The anti-
SMA monoclonal antibody (clone 1A4; Sigma) concentration used was 0.64 µg/ml. Color development was performed with DAB as described above. Control sections for
SMA were performed with the omission of the primary antibody.
IHC for fibronectin was performed as described for 8 using anti-fibronectin antibody (F3648; Sigma), concentration 0.044 µg/ml. Control sections were performed with the omission of the primary antibody.
Photographs were taken through a Nikon Labophot microscope (Tokyo, Japan) using x20 and x40 lenses with a Spot Insight Color digital camera (model 3.2.0; Diagnostic Instruments, Sterling Heights, MI).
Immunofluorescence
Immunofluorescence was performed to examine co-localization between 8 and
SMA. Sections were blocked for endogenous peroxidase for 30 sec with Peroxoblock (Zymed), then incubated with Cy3-conjugated
SMA (clone 1A4; Sigma) (5.5 µg/ml in 3% normal goat serum) for 1 hr at RT. This was followed by avidin and biotin blocking reagents (Zymed) for 10 min each. Sections were incubated overnight at 4C with
8 antiserum (1:300,000 dilution) followed by 1-hr RT incubation with biotinylated goat anti-rabbit IgG antibody (0.3 µg/ml; Vector). The signal was amplified using the TSA Biotin System (PerkinElmer Life Sciences; Boston, MA) by incubating the slides with streptavidin-conjugated horseradish peroxidase in PBS (1:100) for 1 hr, followed by TSAbiotin (1:50) in TSA Amplification Diluent for 4 min. Sections were then incubated for 1 hr at RT with streptavidin-conjugated Alexa 488 (Molecular Probes; Eugene, OR) at 5 µg/ml in PBS. The nuclei were counterstained with To-Pro-3 at 0.67 µg/ml (Molecular Probes) for 10 min at RT. Slides were mounted with Vectashield Hardset Medium (Vector).
To differentiate epithelial cells from mesenchymal cells, double immunofluorescence was performed with pan-cytokeratin and 8 antibodies. Immunofluorescent staining for
8 was performed as described above. The sections were then permeabilized with 0.1% Triton X-100 in PBS (PBT) for 15 min at RT. Monoclonal anti-pan cytokeratin antibody (mixture of clones C-11, PCK-26, CY-90, KS-1A3, M20 and A53-B/A2) (Sigma) was dialyzed using the Slide-A-Lyzer Mini-Dialysis Unit (Pierce; Rockford, IL) to remove phenol red to avoid quenching of the Alexa dyes. The pan-cytokeratin antibody was labeled with the Zenon One Mouse IgG1 Alexa 568 Labeling Kit (Molecular Probes) by incubating 1 µl of dialyzed antibody with 7.5 µl of the Zenon Alexa 568-labeled Fab fragments and 10 µl of PBS for 5 min at RT. Excess Fab fragments were adsorbed with 7.5 µl of IgG block for 5 min. The pan-cytokeratin antibody Zenon Fab Alexa 568 complex was diluted in PBT to a final volume of 100 µl and applied to the sections for 1 hr at RT. After washing, tissues were fixed in 4% paraformaldehyde for 10 min at RT to stabilize the Zenon FabFc interaction. The slides were counterstained with To-Pro-3 and mounted as described above.
Immunofluorescent photographs were obtained using a Leica DM-R upright epifluorescent microscope (Carlsbad, IL) using x20 lenses and x40 oil-immersion lenses with a Leica TCS/SP Confocal Scanner equipped with argon, krypton, and helium/neon lasers. Images were superimposed with the help of ImagePro plus (version 4.0; Media Cybernetics, Carlsbad, CA) and processed with Adobe Photoshop (version 6.0; San Jose, CA).
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Results |
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In the early pseudoglandular stage, lungs are primarily composed of mesenchymal cells (m) with interspersed airways (a) surrounded by peribronchial cells (Fig 1A1F). The proportion of epithelial to mesenchymal cells increases dramatically during branching morphogenesis, ending around E16.5 (Fig 1G1L and Fig 2A2C). In the canalicular stage, the distal airways (da) can be distinguished from the mesenchyme by delineation of the basement membrane by fibronectin (arrows in 2A and 2D). Postnatally, during the saccular stage, the interstitium has thinned significantly and gas exchange occurs in saccules (s in Fig 2G2I). True alveoli (al) are formed within saccules during the alveolar stage by secondary septation (arrowheads) (Fig 2J2L).
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Prenatal Peribronchial and Perivascular Expression
8 immunoreactivity was present in the peribronchial and perivascular regions throughout lung development in a pattern similar to that of
SMA (Fig 1 and Fig 2). To further characterize the localization of
8 and
SMA, we performed co-immunofluorescence and found that
8 co-localized with
SMA in the peribronchial region throughout smooth muscle cell development at all time points examined (Fig 3). Fibronectin, a ligand for
8, was prominently expressed in the basement membrane at the epithelialmesenchymal interface throughout lung development (arrows in Fig 1A, Fig 1D, Fig 1G, Fig 1J, Fig 2A, and Fig 2D). Fibronectin was also present in the peribronchial regions beyond the epithelialmesenchymal interface. In the early pseudoglandular stage, the peribronchial expression of fibronectin was more limited compared to that of
8 and
SMA (Fig 1A1F). However, from the mid-pseudoglandular stage and beyond, fibronectin staining was more similar to that of
8 and
SMA (Fig 1G1L and Fig 2A2F).
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Prenatal Mesenchymal Expression
Prenatally, 8 was expressed diffusely throughout the lung mesenchyme (Fig 1B, Fig 1E, Fig 1H, Fig 1K, Fig 2B, and Fig 2E). There appeared to be some mesenchymal cells that did not express
8 but it was difficult to define the pattern of mesenchymal
8 expression using light microscopy alone. We used a pan-cytokeratin antibody to identify epithelial cells and performed co-immunofluorescence for
8 and cytokeratin. Cytokeratin staining revealed that many of the
8-negative cells visualized by light microscopy were actually clusters of small airways (Fig 4). Furthermore, we found that all mesenchymal cells were immunoreactive for
8 except for mesenchymal cells surrounding newly forming, distal airways (da) (Fig 3I and Fig 4). Fibronectin was expressed as diffusely as
8 in the mesenchyme during the mid-pseudoglandular stage (Fig 1G and Fig 1J) but less prominently during all other prenatal stages (Fig 1A, Fig 1D, Fig 2A, and Fig 2D). In contrast to
8 and fibronectin, there is minimal mesenchymal expression of
SMA until birth (Fig 1C, Fig 1F, Fig 1I, Fig 1L, Fig 2C, and Fig 2F).
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Postnatal Expression
Postnatally, during the later saccular and alveolar stages, the mesenchymal (interstitial) distribution of 8 continued to be diffuse Fig 2H, Fig 2K, Fig 3G, and Fig 3H). Fibronectin was also expressed diffusely in the interstitium during the late saccular stage (Fig 2G), then less diffusely in the alveolar stage (Fig 2J). During the late saccular stage,
SMA was expressed in alveolar myofibroblasts at sites of future secondary septum formation (arrows in Fig 2I and Fig 3G), then at the leading tip of secondary septae in the alveolar stage (arrowheads in Fig 2L and Fig 3H).
8 co-localized with
SMA in alveolar myofibroblasts at these sites (Fig 3G, Fig 3H, and Fig 3J). Fibronectin also localized to the tips of secondary septae in the alveolar stage at PN8 (Fig 2J), suggesting co-localization of fibronectin,
8, and
SMA at these sites.
Although yellow fluorescence indicated co-localization of 8 and
SMA in the peribronchial and vascular regions (Fig 3A3F and Fig 3I), there was no yellow fluorescence to indicate co-localization in alveolar myofibroblasts (Fig 3G and Fig 3H). However, the intracellular location of
8 and
SMA is different (
8 is a transmembrane protein and
SMA is a cytoskeletal protein). Examination under higher magnification confirmed localization of
8 and
SMA in the same cells (Fig 3J).
8 immunoreactivity was not present on endothelial or epithelial cells at any of the time points examined.
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Discussion |
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We investigated the spatiotemporal expression pattern of the integrin subunit 8 in the developing murine lung and examined its co-localization with
SMA, fibronectin, and cytokeratin.
Murine lung development begins at approximately 9.5 days of gestation, when the trachea and mainstem bronchi develop from the foregut followed by lateral branching to the segmental level (embryonic stage) (
SMA has been identified as one of the earliest cytoskeletal markers for smooth muscle cell differentiation (
8 co-localizes with
SMA in developing smooth muscle cells as early as E12.5, demonstrating that it is also an early marker of smooth muscle cell differentiation. Fibronectin is prominently expressed in the basement membrane at the epithelialmesenchymal interface throughout development. Starting in the mid-pseudoglandular stage, fibronectin co-localized with
8 and
SMA in peribronchial and perivascular regions. In vitro, fibronectin has been shown to affect smooth muscle cell proliferation and activation of focal adhesion kinase (FAK) (
8 may play a role in mediating signals from the extracellular matrix to developing smooth muscle cells during lung development.
Fibronectin is thought to play an important role in branching morphogenesis in the lung (8-positive except for those surrounding distal newer airways. Decreased fibronectin associated with lack of
8 expression at the sites of distal airways may provide a permissive environment for airway budding. Conversely,
8fibronectin interactions may influence cleft formation and airway bifurcation.
Similar to lung development, kidney development occurs by branching morphogenesis. In an 8 knockout model, homozygotic mice displayed renal agenesis or dysgenesis and most died within the first few days of birth (
8 plays a critical role in the mesenchymalepithelial interaction in kidney development. The initial report showed grossly normal lungs (
8-null lung explants, branching was reduced by 3355% and the pattern was irregular. Redundancy between the function of
8 and that of other integrins, such as
5, may account for more subtle abnormalities in lung development in surviving mice. Our
8 expression data also support a role for
8 in branching morphogenesis.
By the late saccular stage and the alveolar stage, the distribution of 8 was diffuse throughout the interstitium (Fig 3G and Fig 3H), similar to the pattern seen in adult tissues (
8 in mature alveolar walls localizes to contractile interstitial cells (
8-positive mesenchymal cells during development probably represent precursors to contractile interstitial cells. Furthermore,
8 co-localized with
SMA in alveolar myofibroblasts during secondary septation. Co-localization in these cells has also been described in alveolar myofibroblasts, which are present in the adult lung in response to injury (
8- and
SMA-positive alveolar myofibroblasts are located. Therefore,
8 is in a position to mediate signals from the extracellular matrix (fibronectin) to alveolar myofibroblasts and hence to contribute to secondary septation.
In summary, integrin subunit 8 is expressed in the mesenchyme throughout lung development and co-localizes in the peribronchial and perivascular regions with
SMA. Along with
SMA,
8 is an early marker for smooth muscle cells.
8 may interact with fibronectin in the peribronchial regions and play a role in smooth muscle cell differentiation.
8 is also an early marker for lung mesenchymal cells. Decreased fibronectin expression in distal budding airways is associated with
8 negativity of mesenchymal cells in the same regions. Considering the data from the
8 knockout model (
8 may play a role in branching morphogenesis by interacting with ECM components, such as fibronectin, to coordinate airway growth, cleft formation, and airway bifurcation. Furthermore,
8 may mediate signals from the ECM to alveolar myofibroblasts and participate in secondary septation.
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Acknowledgments |
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Supported by an AHA grant-in-aid, Northwest Affiliate (LMS).
We thank Jeremy Ehly for technical support on the confocal microscope.
Received for publication July 31, 2002; accepted June 26, 2003.
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