Thyroid hormone affects distal airway formation during the late pseudoglandular period of mouse lung development

Molecular Genetics and Metabolism 80 (2003) 242–254 www.elsevier.com/locate/ymgme

Thyroid hormone affects distal airway formation during the late pseudoglandular period of mouse lung development MaryAnn V. Volpe,a,* Heber C. Nielsen,a Kwanchai Archavachotikul,a Terrigi J. Ciccone,a and Mala R. Chinoyb a

Department of Pediatrics, Division of Newborn Medicine, New England Medical Center, Box 44, 750 Washington St., Boston, MA 02111, USA b Lung Development Research Program, Department of Surgery, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA Received 3 June 2003; received in revised form 31 July 2003; accepted 4 August 2003

Abstract We recently showed that T3 treatment of cultured gestational day 11.5 early pseudoglandular period mouse lungs, accelerated terminal airway development at the expense of decreased branching morphogenesis. As the ability of T3 to influence epithelial cell differentiation increases with advancing development, we hypothesized that in the late pseudoglandular period, T3 would cause further premature changes in the morphology of the distal airways leading to abnormal saccular development. Gestational day 13.5 embryonic mouse lungs were cultured for 3 and 7 days without or with added T3 . Increasing T3 dose and time in culture resulted in progressive development of thin walled, abnormal saccules, an increase in cuboidal and flattened epithelia and airway space with a concomitant decrease in mesenchymal cell volume. Consistent with increased cuboidal and flattened epithelial cell volume identified by morphometry, immunostaining suggested increased cell proliferation detected by localization of proliferating cell nuclear antigen (PCNA) in epithelial cells of T3 treated lungs. T3 decreased mesenchymal expression of Hoxb-5 protein and caused progressive localization of Nkx2.1 and SP-C proteins to distal cuboidal epithelia of early abnormal saccules, evidence that T3 prematurely and abnormally advanced mesenchymal and epithelial cell differentiation. Western blot showed a T3 -dependent decrease in Hoxb-5 and a trend towards decreased Nkx2.1 and SP-C, after 3 and 7 days of culture, respectively. We conclude that exogenous T3 treatment during the late pseudoglandular period prematurely and abnormally accelerates terminal saccular development. This may lead to abnormal mesenchymal and epithelial cell fate. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Thyroid hormone; Lung morphogenesis; Hoxb-5; Nkx2.1; Surfactant protein C

Introduction Lung development begins at gestational day 9.5 (Gd9.5) in the mouse embryo. The embryonic and pseudoglandular periods (Gd9.5–Gd16.5) are characterized by the development of the tracheobronchial tree. During the canalicular and terminal sac periods from Gd16.5-Postnatal day 2 (P2) the air exchanging regions of the lung begin to form from the distal ends of the tracheobronchial tree. In most species, the alveolar period of lung development begins only late in fetal development

* Corresponding author. Fax: 1-617-636-4233. E-mail address: [email protected] (M.A.V. Volpe).

1096-7192/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2003.08.018

or not until the postnatal period [1,2]. During all periods of lung development, lung morphology and cell differentiation are influenced by the precise timing of paracrine, autocrine and juxtacrine signals which act through mesenchymal–epithelial cell interactions [3–5]. Triiodothyronine (T3 ), the active form of thyroid hormone, influences lung development through binding to nuclear T3 hormone receptors [6–8]. Both in vivo and in vitro studies have demonstrated that T3 affects various aspects of lung development including alveolar development by accelerating epithelial differentiation and surfactant phospholipid synthesis in late fetal life and by influencing alveolar septation in the postnatal period [8–10]. It has been questioned whether acceleration of these effects and synergism between thyroid hormone

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and other substances, in particular, corticosteroids, could be beneficial to the advancement of lung maturation in infants at risk for respiratory distress syndrome (RDS), bronchopulmonary dysplasia and other pulmonary dysmaturity syndromes [4,11,12]. However, animal and human studies of the effect of thyroid hormone, alone or in combination with corticosteroids have produced variable and in some cases concerning results. The exact molecular pathways involved in these processes remain unknown [8,13–17]. In order to understand the regulatory role of T3 in lung development, we recently studied the effect of exogenous T3 on cultured embryonic mouse lung during the early pseudoglandular period (Gd11.5, term is 19 days), when active airway branching of the tracheobronchial tree is occurring [18]. We showed that T3 treatment accelerated epithelial cell differentiation at the expense of decreased branching morphogenesis. Hoxb–5 protein expression, which normally remains high throughout the pseudoglandular period until the completion of branching morphogenesis, was decreased coincident with decreased branching morphogenesis in the T3 treated lungs. Nkx2.1 (also called TTF1, TITF1), which is normally diffusely expressed in columnar and cuboidal epithelium, became prematurely restricted to distal cuboidal epithelia. SP-C protein, which is regulated by Nkx2.1, followed a similar pattern becoming restricted to distal epithelial cells. These changes in the cellular and spatial expression patterns of Hoxb-5, Nkx2.1 and SP-C are characteristic of changes normally observed in late fetal lung development heralding the onset of alveolar formation [19–22]. This premature alteration in the expression patterns of Hoxb-5 and Nkx2.1 is important because Hoxb-5 and Nkx2.1 are necessary for normal branching morphogenesis to occur [23,24]. Premature changes in the cellular expression patterns of these proteins may prevent proper epithelial branching, and alter the normal progressive maturation of the mesenchymal and epithelial cell components. The other known effects of T3 , including distal airway epithelial maturation and surfactant phospholipid synthesis occur later in gestation and postnatally. In this study we hypothesized that T3 treatment of embryonic mouse lungs during the late pseudoglandular period would cause further premature distal airway morphologic development. This would be marked by abnormal expression of terminal saccular epithelial characteristics. To test this hypothesis, we have evaluated the impact of T3 on lung morphogenesis and epithelial differentiation by studying embryonic mouse lung in vitro development starting in the late pseudoglandular period and progressing into the canalicular and terminal sac periods (during development of the air exchanging regions of the lung). Late pseudoglandular period embryonic mouse lungs (Gd13.5) were cultured in the presence or absence of increasing doses of T3 for 3 and 7 days. Changes in

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mesenchymal and epithelial cell fate were evaluated by assessment of spatial and quantitative changes in Hoxb5, Nkx2.1 and SP-C protein expression. Changes in lung structural development were objectively evaluated using lung morphometry. Changes in lung cell proliferation were studied using proliferating cell nuclear antigen (PCNA) immunostaining.

Materials and methods Reagents BGJb medium was purchased from Gibco (Grand Island, NY) and supplemented with penicillin G 100 U/ ml, streptomycin 0.1 mg/ml, amphotericin B 0.25 mg/ml, and sodium ascorbate 1 mg/L) to a pH of 7.4 [25]. T3 was purchased from Sigma–Aldrich (St. Louis, MO), Nkx2.1 antibody from Neomarkers (Fremont, CA). Hoxb-5 antibody was previously developed in our laboratory [20]. SP-C antibody used for immunostaining was a gift from M. Beers (Univ. of Penn) and SP-C antibody used for Western blot analysis was a gift from J. Whitsett (Univ. of Cincinnati). Both SP-C antibodies recognize pro-SP-C and are rabbit polyclonal antimouse antibodies [22,26]. Antibody against PCNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunostaining reagents were purchased from Vector (Burlingame, CA). Chemiluminescent Western blot analysis reagents were purchased from Amersham (Arlington Heights, IL). All other reagents were from Sigma–Aldrich (St. Louis, MO) unless otherwise specified. Animals The animal study protocol was approved by the Institutional Animal Research Committee. Principles of laboratory animal care (NIH publication 86-23, revised 1985) were followed. Timed pregnant CD-1 mice were purchased from Charles River Labs (Wilmington, MA). CD-1 mice are an outbreed stock of Swiss Webster mice. The mice were sacrificed by an overdose of halothane on gestational day 13.5 (Gd13.5 with Gd0.5 defined as the morning of the vaginal plug and term at Gd19). The fetuses were harvested by sterile laparotomy and placed in ice cold HankÕs solution. Using a dissecting microscope, a median sternotomy was performed and the fetal heart, lung and trachea were excised en bloc, followed by removal of the fetal heart and trachea prior to culturing of the lungs. Whole lung organ culture Using methods that we have previously described, Gd13.5 embryonic mouse lungs were placed onto

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0.45 lM pore size, 5 mm2 membranes (Gelman Sciences, Ann Arbor, MI) and cultured at the air–liquid interface for 3 and 7 days in serum- and hormone-free BGJb medium with or without added T3 at concentrations of 2, 10, and 100 nM [18,19,27]. These culture end points were chosen to assess the short-term effects of T3 treatment ending during the early canalicular period of lung development (3 day treatment) and the effects of T3 treatment extending to the terminal sac period of lung development (7 day treatment). The concentrations of T3 used in this study span the range of circulating endogenous fetal levels and represent the range of exogenous T3 doses that we and others have shown to influence lung branching morphogenesis, surfactant phospholipid synthesis and alveolar development in cultured fetal lung [8,18,28,29]. Lungs were evaluated daily under a dissecting microscope for gross changes in morphologic development. After 3 and 7 days of culture, lungs were harvested and processed for evaluation of overall histology, immunocytochemistry or Western blot analysis of Nkx2.1, Hoxb-5, and SP-C proteins, and PCNA immunostaining. Morphometric analysis was performed on lung sections processed for immunocytochemistry.

incubation with diaminobenzadine (DAB) reagent for 5 min. The same sections were then prepared to detect Hoxb-5 protein localization by washing in TBST pH 8.0 for 30 min at 4 °C, followed by blocking with avidin, biotin and 5% normal goat serum plus 3% normal mouse serum. Experimental sections were incubated overnight with rabbit polyclonal anti-mouse Hoxb-5 IgG antibody (1:200). Control sections were incubated with preimmune rabbit serum in place of primary antibody. The next day, sections were warmed to room temperature and washed in TBST pH 8.0, and sequentially incubated with 0.5% goat anti-rabbit biotinylated IgG antibody plus 2% normal goat serum and 3% normal mouse serum (60 min), then with avidin–biotin complex conjugated to alkaline phosphatase [20]. Sections were then reacted with alkaline phosphatase substrate (Vector Blue) for detection of specific Hoxb-5 protein labeling. Sections were counterstained with nuclear fast red, dehydrated through 95 and 100% ethanol washes and cleared with Histoclear (National Diagnostics, Atlanta, GA). Sections were cover slipped and analyzed using an inverted light microscope.

Double label immunocytochemistry for Nkx2.1 and Hoxb-5 proteins

Using a HRP detection method modified from Beers et al. [26], SP-C protein immunolocalization was evaluated in separate lung sections from the same lungs in which Nkx2.1 and Hoxb-5 were studied. After sectioning at
24 °C, sections were washed with PBS (0.1 M anhydrous sodium phosphate, 0.8% NaCl, pH 7.4). Endogenous peroxidase activity was quenched by washing sections in a hydrogen peroxide/methanol solution. Non-specific sites were then blocked with sequential incubations in 5% normal goat serum, avidin and biotin. Experimental sections were then reacted with rabbit polyclonal-anti-mouse antibody to pro-SP-C (1:800, 1.5 h) and immunostaining control sections with normal rabbit serum followed by incubations with 0.5% goat anti-rabbit secondary antibody with 1.5% normal goat serum (30 min) and avidin–biotin complex-conjugated to HRP (30 min). The SP-C antibody-specific sites were then detected with DAB reagent (3 min). Sections were counterstained with methyl green, dehydrated through graded alcohols, cleared with Histoclear (National Diagnostics, Atlanta, GA), cover slipped and analyzed using an inverted light microscope.

Representative lungs from each culture condition (2 lungs/condition) were processed for Nkx2.1 and Hoxb-5 double immunostaining procedures. Lungs were placed in freshly prepared 4% paraformaldehyde (4 °C) for 2 h followed by immersion in 30% sucrose overnight (4 °C). Lungs were then embedded in OCT frozen specimen medium (Miles, Elkhart, IN) and sectioned (6 lm) at
24 °C. Serial sections were mounted onto superfrost plus slides (Fisher Scientific, Pittsburgh, PA). Mounted sections were processed at 4 °C. Sections were washed for 30 min in 10 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20 (TBST), pH 7.5, at 4 °C. All subsequent washes between incubations were done in TBST, pH 7.5, for 10 min at 4 °C. Non-specific binding sites were blocked by sequential incubations with avidin (15 min), biotin (15 min) and then with 5% normal horse serum (20 min). Experimental sections were incubated with mouse monoclonal IgG antibody to Nkx2.1 (1:200 dilution) and control sections with normal mouse serum overnight at 4 °C. The following day, sections were warmed to room temperature and washed in TBST pH 7.5 and sequentially incubated at room temperature with 0.5% horse anti-mouse biotinylated IgG antibody for 30 min, then with avidin–biotin complex conjugated to horseradish peroxidase (HRP)(30 min). Endogenous peroxidase activity was eliminated by washing in a solution of hydrogen peroxide and methanol (1:4) for 30 min. Specific peroxidase activity was then detected by

SP-C immunostaining

PCNA immunostaining Using an ABC-alkaline phosphatase detection method (Vector Laboratories, Burlingame, CA), PCNA immunostaining was studied in lung sections (6 lM) from Gd13.5 embryonic lungs (2 lungs/condition) cultured for 7 days without or with added T3 . As changes in lung morphology were most profound in the lungs

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treated for 7 days, PCNA immunostaining was performed on lung sections from these experiments. Randomly picked sections were evaluated for each condition. Lungs were prepared as noted above for Hoxb-5/Nkx2.1 double immunostaining. Six micron mounted lung sections were washed with PBST (0.1 M anhydrous sodium phosphate, 0.8% NaCl, pH 7.4 with 0.05% Tween 20). Non-specific sites were blocked with sequential incubations in avidin, biotin and normal rabbit serum (1.5%) followed by two hour room temperature incubation with Goat Polyclonal IgG antibody to PCNA. Sections were then reacted with rabbit–antigoat secondary antibody, and avidin–biotin complex conjugated to alkaline phosphatase. PCNA-labeled specific sites were detected with Vector black alkaline phosphatase chromagen. Sections were counterstained with methyl green and analyzed using an inverted light microscope.

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total protein concentration measured by Bradford protein microassay as described earlier [28,30]. The protein homogenates were mixed with lysis buffer (62 mM Tris–HCl, pH 6.8, 2.3% SDS, 10% glycerol, 5% b-mercaptoethanol) and heated at 80 °C for 5 min. Total lung protein (20 lg) was separated by SDS– polyacrylamide gel electrophoresis followed by transfer to PVDF membranes. PVDF membranes were then blocked with 5% milk and incubated with polyclonal rabbit–anti-mouse Hoxb-5 antibody (1:200), mouse monoclonal Nkx2.1 antibody (1:150) or polyclonal rabbit–anti-mouse SP-C antibody (1:1500), followed by reaction with HRP-linked secondary antibody. Antigen–antibody sites were then detected by chemiluminescence and quantified by densitometry. Hoxb-5, Nkx2.1, and SP-C levels were evaluated from the same lungs for each culture condition and the results from untreated and T3 -treated lungs compared by densitometry.

Morphometry Lung sections from the immunocytochemistry studies were morphometrically evaluated using an eyepiece micrometer at 40 magnification. Lung sections used for morphometric evaluation were separated by 3–10 sections (18–60 lm) and were representative of the overall morphology observed in the untreated and T3 treated lungs. Criteria for columnar, cuboidal or flattened epithelial cells, mesenchymal cells and airway space were predefined and based on published descriptions of lung histology [1,2]. In each lung section, the percentage of grid points intersecting columnar epithelium, cuboidal/flattened epithelium, airway and mesenchyme were statistically compared in untreated and T3 -treated lungs. Analyses were performed by the same observer. Evaluation of grid points for each lung section were performed without knowledge of results from other control or T3 -treated lung sections. When all morphometric counts were completed, the percentage of grid points was calculated and comparisons between untreated and T3 -treated conditions performed. Western blot analysis Western blot analysis was performed to evaluate quantitative differences in Hoxb-5, Nkx2.1, and SP-C proteins in untreated and T3 -treated lungs. Western blot analysis for Nkx2.1, Hoxb-5, and SP-C proteins were performed as previously described [19,20]. Lungs from each culture condition and from in vivo lungs at Gd13.5, Gd15.5, Gd18.5 and neonatal lungs (<12 h post-birth) (3 lungs per condition for each of 3–10 observations) were homogenized on ice in PBS (pH 7.5) in the presence of protease inhibitors (aprotinin 1 lg/ml, antipain 2 lg/ml, and leupeptin 2 lg/ml) and

Statistical analysis For morphometric evaluations, the percentage of grid points from each lung section that intersected columnar epithelium, cuboidal/flattened epithelium, airway and mesenchyme from the untreated and T3 treatment conditions were compared by ANOVA with Bonferroni multiple comparison post-test, with a value of p 6 0:05 considered significant. For Western blot analysis, densitometry results for untreated and T3 -treated lung cultures were compared by StudentÕs t test, with p 6 0:05 considered significant.

Results Whole lung organ culture All lungs were evaluated daily by light microscopy. Both treated and untreated lungs continued to grow over the time in culture. Gd13.5 lungs cultured in the presence of different doses of T3 (2, 10, and 100 nM) showed differential gross morphology. As compared to untreated lungs, lungs treated with the higher doses of T3 (10 and 100 nM) remained smaller in size. However, in lungs treated with the lower dose of T3 (2 nM), more airway branching was observed and the lungs appeared larger in size than untreated lungs or lungs treated with 10 and 100 nM T3 . These morphologic findings are depicted in Fig. 1 (representative sections from 7 day cultures). T3 treated lungs showed progressive thinning of mesenchyme and increased dilation of distal airways and early saccules (Fig. 1, compare control lung section, A, to T3 treated lung sections, B–D).

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Double label immunocytochemistry for Nkx2.1 and Hoxb-5 proteins Gd13.5 embryonic mouse lungs cultured for 3 days To assess the effect of short term T3 treatment during the late pseudoglandular period, Gd13.5 were cultured for 3 days in the absence or presence of the different doses of T3 as discussed in Methods. Untreated Gd13.5 embryonic mouse lungs cultured for 3 days (Figs. 2A and B) showed airway development typical of the late pseudoglandular period. Airway branches progressed from proximal conducting airways lined by columnar epithelia to distal airways lined by cuboidal epithelia. The distal airways were characteristic of terminal branches destined to become the acinar regions of the lung. Nkx2.1 protein expression remained fairly homogeneous, being expressed in nuclei of both columnar and cuboidal epithelia (Fig. 2A). Hoxb-5 protein was expressed in the nuclei of many mesenchymal cells but with a predilection to subepithelial mesenchymal cells around airways lined by columnar epithelia (Fig. 2B). In contrast, with increasing dose of T3 , changes in lung structure, development of distal airways and associated changes in Nkx2.1 and Hoxb-5 protein localization were observed (Figs. 2C and D). Consistent with the visual morphology of lungs noted after 3 days of culture with 2 nM T3 (Figs. 2C), distal airway lumens appeared widened and mesenchymal tissue density appeared decreased. Hoxb-5 staining of mesenchymal cell nuclei appeared reduced but remained localized to subepithelial mesenchyme. There was no apparent difference in columnar or cuboidal epithelial staining for Nkx2.1. After treatment with the higher doses of T3 , as shown by a lung section from a 100 nM T3 treated lung (Fig. 2D), there were progressive changes in the development of the distal airways with formation of thin walled early saccular-like structures. Hoxb-5 protein expression further waned in distal mesenchyme around these saccules and remained localized to a minority of subepithelial mesenchymal cells. At this higher dose of T3 , Nkx2.1 pro-

tein cellular expression was more intermittent in columnar epithelia and decreased in flattened epithelia of early saccular-like structures (Fig. 2D). The specificity of Hoxb-5 and Nkx2.1 immunostaining was confirmed by omission of either one or both primary antibodies. In the absence of either primary antibody, no specific staining was seen for Hoxb-5 (Fig. 2A) or Nkx2.1 (Fig. 2B). In the absence of both primary antibodies, no staining was seen with the HRP or alkaline phosphatase reactions (data not shown). Gd13.5 fetal mouse lungs cultured for 7 days To evaluate the effects of prolonged T3 treatment starting in the late pseudoglandular period and extending into the terminal sac period of lung development, Gd13.5 mouse lungs were cultured and treated for 7 days with varying doses of T3 as described in Materials and Methods. The histology of these lungs were compared to untreated lungs and to lungs treated with a short course of T3 (3 day treatment). Untreated lungs showed development of distal airway branches resembling terminal and respiratory bronchioles, alveolar ducts and early presumptive saccular structures (Fig. 3A). The cellular expression of Hoxb-5 protein was appropriately decreased compared to lungs cultured for 3 days and remained mostly localized to subepithelial mesenchyme underlying low columnar and cuboidal epithelia of distal bronchioles. Nkx2.1 protein expression waned in low columnar and cuboidal epithelia of proximal airways but remained localized to nuclei of distal cuboidal epithelia. With low dose T3 -treatment, there was thinning of mesenchyme, a concomitant decrease in the cells expressing Hoxb-5 protein, and increased development of terminal airway-like structures lined by cuboidal epithelia whose nuclei remained positive for Nkx2.1 protein (Fig. 3B). With increasing dose of T3 there was further mesenchymal thinning, and the distal airways became progressively more dilated. At the highest dose of T3 (Fig. 3C and D) these distal airways were markedly dilated, suggestive of abnormal terminal

c Fig. 2. Hoxb-5 and Nkx2.1 immunostaining in Gd13.5 embryonic mouse lungs cultured for 3 days: Bar ¼ 25 lM. Untreated lung with Nkx2.1 immunostaining (A) or Hoxb-5 immunostaining (B); Hoxb-5 and Nkx2.1 double immunostaining in representative lung sections from (C) 2 nM T3 (low dose); (D) 100 nM T3 (high dose) treated lungs. Untreated lung (A, B) showed airway branching which progressed from proximal airways lined by columnar epithelial cells (arrow) to distal airways (asterisk) lined by cuboidal epithelial cells (arrowhead) with fairly homogeneous epithelial staining for Nkx2.1 (brown nuclei) as shown in (A) and diffuse mesenchymal staining for Hoxb-5 (blue nuclei) as shown in (B). With increasing dose of T3 (C, D) there was a dose dependent thinning of the mesenchyme and increased development and widening of the terminal airways (asterisks). Hoxb-5 protein expression waned and become more localized to subepithelial fibroblasts around airways lined by columnar and cuboidal epithelial cells. At the highest dose of T3 (D), Nkx2.1 waned in flattened epithelial cells (arrowheads) of early saccules (asterisk) and remained present but intermittent in cuboidal and columnar epithelia (arrow). Fig. 3. Hoxb-5 and Nkx2.1 immunostaining in Gd13.5 embryonic mouse lungs cultured for 7 days: (Bar ¼ 25 lM in A, B, D); (Bar ¼ 50 lM in C). Untreated lung (A); (B) 2 nM T3 ; (C, D) 100 nM T3 . Untreated lung (A) showed orderly development of airway branches, alveolar ducts and distal saccules (asterisk). Hoxb-5 (blue nuclei) was localized to subepithelial mesenchyme. Nkx2.1 nuclear staining (brown nuclei) was minimal in low columnar and cuboidal epithelia of proximal airways (arrow) but was positive in distal cuboidal epithelia (arrowheads). With increasing dose of T3 (B–D) there was thinning of mesenchymal tissue and increased terminal airway development (asterisks) which at the highest dose (C, D) showed impressive dilatation. Hoxb-5 expression waned to negligible levels. At the highest dose of T3 (C, D) Nkx2.1 expression was weak in cuboidal epithelia (arrowhead) and columnar epithelia (arrow).

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Fig. 1. Hematoxylin and eosin stained lung sections from Gd13.5 embryonic mouse lungs cultured for 7 days: low power view of lung histology. Bar ¼ 50 lM (A) Un Lu 7d ctx: untreated Gd13.5 lung cultured for 7 days; Gd13.5 lung treated for 7 days with (B) T3L ¼ 2 nM T3 ; (C) T3M ¼ 10 nM T3 ; (D) T3H: ¼ 100 nM T3 . Low power view of lung sections from untreated and T3 treated lungs shows the overall histology of modest numbers of airway lumens separated by mesenchymal cells in untreated lungs (A). Lungs treated with low dose T3 (B) show apparent increased numbers of airway lumens, whereas lungs treated with the higher doses of T3 (C and D) show progressive dilation of airway lumens (asterisks) and thinning of surrounding mesenchyme tissue.

Fig. 2.

Fig. 3.

saccular development. Mesenchymal expression of Hoxb-5 was almost completely absent in lungs treated with 100 nM T3 . Nkx2.1 expression remained present in cuboidal epithelial cell nuclei but the intensity of staining was diminished and more intermittent compared to untreated and 2 nM T3 treated lungs. The T3 -associated changes in morphologic development of embryonic lungs after 3 and 7 days in culture

were further evaluated and confirmed by morphometric analysis of lung tissue sections. These findings are described below. Morphometry Morphometric analysis of lung sections confirmed the changes in mesenchymal cell density, progressive

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development of distal airway epithelium and the dilated and abnormal saccular-like airway structures seen in the T3 -treated lungs (Fig. 4). These effects of T3 were seen after both 3 and 7 days of culture. As compared to untreated lungs, T3 -treated lungs showed a significant increase in cuboidal and flattened epithelia and decrease in mesenchyme (p < 0:05, untreated lungs verses all T3 treatments after 3 and 7 days of culture) and increase in airway space (p < 0:05, untreated control lungs verses T3 100 nM treatment after 3 days and T3 10 and 100 nM treatment after 7 days). These findings increased with progressively higher doses of T3 . The effect of dose on increased airway space and decreased mesenchyme were more prominent after treatment for 7 days compared to 3 days of treatment.

PCNA immunostaining PCNA immunostaining was performed to determine if T3 -induced changes in lung structural development were secondary to changes in epithelial and/or mesenchymal cell proliferation. PCNA, also known as cyclin, has been identified as polymerase d-associated protein. PCNA is a useful marker of cell proliferation as it is synthesized in early G1 and S phase of the cell cycle [25,31,32]. As the greatest changes in lung morphological development were seen in the cultured Gd13.5 embryonic lungs treated for 7 days, PCNA immunostaining was performed in this group of experiments. The overall histology in the lung sections evaluated for PCNA immunostaining was consistent with the histology seen and described above. In vivo Gd13.5 embryonic mouse lungs (Fig. 5B) showed more mesenchymal and epithelial cell PCNA immunostaining compared to untreated control lungs (Fig. 5C) and T3 treated lungs cultured for 7 days (Figs. 5D–F). PCNA immunostaining in untreated control lungs (Fig. 5C) was seen mostly in scattered epithelial cells and mesenchymal cell staining was minimal. As compared to untreated control lungs (Fig. 5C), epithelial cell PCNA immunostaining appeared increased in the T3 treated lungs (Figs. 5D–F), with most prominent epithelial cell immunostaining seen in the 2 nM T3 (Fig. 5D) and 10 nM T3 (Fig. 5E) treated lungs. In the absence of the antibody to PCNA, no specific staining was seen (Fig. 5A). SP-C immunostaining

Fig. 4. Morphometry of Gd13.5 embryonic mouse lungs cultured for 3 days (top panel) or 7 days (bottom panel) without T3 (control) or with added T3 . As compared to untreated control lungs, T3 -treated lungs after 3 and 7 days of culture, showed a statistically significant decrease in mesenchyme and increase in cuboidal and flattened epithelia ( p < 0:05, untreated control lungs verses all T3 treatments). Airway space was significantly increased after 3 and 7 days of culture at the higher doses of T3 treatment (# p < 0:05, untreated control lungs verses T3 100 nM, 3 day culture and T3 10 and 100 nM, 7 day culture). These effects were dose- and time-dependent. (ANOVA with Bonferroni posttest, N ¼ 4–8 observations/condition, mean  SEM).

To further study epithelial cell maturation and changes in distal airway development with T3 treatment, SP-C immunostaining was performed in consecutive sections from the same lungs used for evaluation of Nkx2.1 and Hoxb-5 immunostaining. Representative sections for 3 day cultures are shown in Fig. 6. In untreated control lungs, SP-C protein was localized to both columnar and cuboidal epithelia of developing airways where it was strongly positive (Fig. 6A). At low dose (2 nM) T3 columnar and cuboidal epithelia remained positive for SP-C protein with intensity similar to controls. At the higher dose of T3 the intensity of staining was diminished in these cells (Fig. 6, compare regions identified by arrowheads in (A)–(C)). The epithelia of the widened distal airways and the early primitive saccules in the T3 -treated lungs stained positive for SP-C (Figs. 6B and C). Furthermore, these distal airways that stain positive for SP-C correspond to the distal regions of lung that show profound T3 -dependent changes in lung remodeling and structural development, including the development of terminal sac-like structures with flattened epithelia and intervening septa. These sections, similar to Fig. 2, also show the thinning of intervening mesenchyme with increasing dose of T3

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(compare Fig. 2C with Fig. 6B and Fig. 2D with Fig. 6C). In the absence of SP-C antibody no staining was seen with the HRP reaction (data not shown). Fig. 7 shows SP-C immunostaining in consecutive lung sections from the same 7 day cultures of untreated and T3 -treated lungs shown in Fig. 3. SP-C immunolocalization in fetal lungs cultured for 7 days followed a similar pattern to that seen for Nkx2.1 protein localization. In untreated lungs, distal cuboidal cells stain positive for SP-C (Fig. 7A). With increasing dose of T3 , the cuboidal epithelial cells of the distal airways stained positive for SP-C in regions corresponding to high expression of Nkx2.1 protein (Fig. 7B as compared to Fig. 3B). At the highest dose of T3 , the cuboidal epithelia of the immensely dilated airways stained strongly positive for SP-C protein. Western blot analysis Fig. 8 shows representative Western blot densitometric analysis for Hoxb-5, Nkx2.1, and SP-C. Western blot densitometric analysis showed a T3 dose-dependent decrease in Hoxb-5 protein levels (Fig. 8, top panel). This effect was similar after 3 or 7 days of T3 treatment. At the highest dose of T3 studied, densitometry confirmed that Hoxb-5 protein levels were significantly less than in untreated lungs and were comparable to that seen in Gd18.5 and neonatal lung tissue (ANOVA with p < 0:003). In Gd13.5 embryonic lungs cultured for 3 days, Nkx2.1 protein levels (Fig. 8, middle panel) in untreated lungs were similar to levels from in vivo neonatal lungs. After 3 days of treatment, Nkx2.1 levels followed a similar trend to that seen for Hoxb-5, decreasing with increasing T3 dose. However, after 7 days of treatment with T3 , this trend of decreasing Nkx2.1 protein levels was not observed. Western blot densitometric analysis of SP-C protein levels (Fig. 8, bottom panel) showed that after 3 days of culture, SP-C protein levels were not different between untreated and T3 -treated lungs. Levels remained similar to those seen in Gd13.5 and Gd15.5 lung tissue. After 7 days of culture, SP-C protein levels in untreated lungs and lungs treated with the low (2 nM) and medium (10 nM) doses of T3 were similar to levels in Gd18.5 and neonatal lungs. However after treatment with the highest dose of T3 (100 nM), there was a trend toward decreased SP-C protein levels.

Discussion Pulmonary tissue levels of T3 during the stages of mouse lung development examined in our study are unknown but the necessity of endogenous thyroid hormone to fetal lung growth and development is well described [33–35]. It is not possible to assess the amount of T3

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absorbed by the cultured lungs from the culture medium, but the lower dose of T3 (2 nM) used to treat the cultured fetal mouse lungs approach levels seen in human fetal lung and is within range of circulating endogenous levels of T3 in the fetus [29,36–38]. The higher doses of T3 in our study have frequently been used to examine the in vitro effect of T3 on the biochemical and structural development of the lung [18,28,39–41]. In our previous study, we evaluated the effect of T3 doses less than 2 nM and found no significant difference between a lower dose of 0.2 nM T3 and untreated lungs [18]. Therefore, in this study, this lower dose was not evaluated. In our recent study, we showed that exogenous T3 treatment during the early pseudoglandular period prematurely advanced epithelial cell maturation and airway remodeling at the expense of decreased new branch formation [18]. In the present study, we have extended these observations to examine the effects of T3 beginning at a later point in lung development during the late pseudoglandular period as branching morphogenesis is completed and structural remodeling is continued into the canalicular and terminal sac periods. We have shown that the effect of T3 on distal airway development was more prominent when treatment was started during the late pseudoglandular period. T3 treatment remained developmentally specific being both dose- and time-dependent with the different doses of exogenous T3 having selective effects on distal airway structural formation and lung maturation that were dependent on the timing of the initiation of treatment and duration of treatment. While the whole lung organ culture undergoes cellular and structural maturation, it does not precisely mimic the timing or process of in vivo lung development. For example, these lungs lack perfusion, hence some aspects of vascular remodeling may be impaired. Similarly, the lack of fetal breathing may alter some processes in structural remodeling. Such considerations must be kept in mind when interpreting studies using this model. Nevertheless, it is clear that lungs in vitro develop into the saccular stage and exhibit structural and biochemical characteristics representative of this stage [16,19,25]. T3 is known to exert its effects by binding to nuclear receptors. Activated T3 receptors work in concert with other nuclear receptors of the steroid–thyroid–retinoid receptor family to control transcription of down stream genes [6,42]. Nuclear receptors and receptor binding for T3 are known to increase during the late pseudoglandular period as compared to the earlier point in lung development examined in our first study which may have resulted in a similar effect on distal airway morphology at a lower dose of T3 [6,18,43,44]. Alternatively, other work from our group shows that exogenous T3 treatment of cultured Gd19 embryonic mouse lungs down regulates thyroid and glucocorticoid nuclear receptor proteins from the steroid–thyroid–retinoid family [45]. The interactions of T3 with these and other

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Fig. 5. PCNA immunostaining in Gd13.5 embryonic lungs cultured for 7 days. Bar ¼ 25 lM. (A) Normal Gd13.5 fetal mouse lung immunostaining control without PCNA antibody. (B) Normal Gd13.5 fetal mouse lung immunostaining control with PCNA antibody. Gd13.5 fetal mouse lung sections from lungs cultured for 7 days as (C) untreated control lung; (D) with 2 nM T3 (T3 L); (E) with T3 10 nM (T3 M); and (F) with T3 100 nM (T3 H) and immunostained with PCNA antibody. In T3 treated lungs (D–F) compared to untreated control lungs (C) cultured for 7 days, the progressive dilation of airway lumens (asterisks) with increasing T3 dose was accompanied by increased in PCNA staining of epithelial cells (arrows).

Fig. 6. SP-C immunostaining in Gd13.5 embryonic mouse lungs cultured for 3 days. Bar ¼ 25 lM. (A) Untreated lung; (B) 2 nM T3 (low dose); (C) 100 nM (high dose). Untreated lung (A) showed SP-C staining (brown) in cuboidal epithelia (arrowhead) of distal airways. T3 -treated lungs showed a dose-dependent development of widened distal airways and early terminal saccules (asterisk). These early saccules are lined by flattened epithelia and cuboidal epithelia (arrowheads) that stain strongly positive for SP-C. No brown staining was seen in absence of SP-C antibody (data not shown).

Fig. 7. SP-C immunostaining in Gd13.5 embryonic lung cultured for 7 days. Bar ¼ 25 lM. (A) Untreated lung; (B) 2 nM T3 ; (C) 100 nM T3 . Untreated embryonic lung (A) showed SP-C staining (brown) in distal cuboidal and flattened epithelial cells (arrowheads). In lungs treated with 2 nM T3 (B) there was increased development of distal airways and early saccules (asterisk) with flattened epithelia and low cuboidal epithelia (arrowheads) which stain intensively for SP-C. After treatment with the highest dose of T3 (100 nM) (C), most airways are dilated (asterisk) and lined by cuboidal epithelia (arrowheads) that stain positive for SP-C.

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Fig. 8. Western blot densitometry analysis of Hoxb-5, Nkx2.1, and SPC of in vivo lungs from Gd13.5, Gd15.5, Gd18.5 and neonatal mice and Gd13.5 embryonic lungs cultured for 3 or 7 days as untreated control (Con) or with added T3 (T3L ¼ 2 nM, T3M ¼ 10 nM, T3Hi ¼ 100 nM). Top panel: Hoxb-5 protein levels decreased significantly with high dose T3 treatment in both 3 and 7 day cultures to levels comparable to neonatal lung. ( P < 0:003, Control versus high dose T3 at 3 and 7 days, N ¼ 8–10 blots from separate experiments, mean  SEM). Middle panel: After 3 days of culture, there was a trend toward decreased Nkx2.1 protein levels with increasing T3 dose. After 7 days of culture, this trend in Nkx2.1 levels was not seen in control treated lungs verses T3 -treated lungs. (N ¼ 5–6 blots from separate experiments, mean  SEM). Bottom panel: SP-C protein levels were not significantly different in control and T3 -treated lungs after 3 days of culture, whereas, after 7 days of culture, there was a trend towards decreased levels with increasing dose of T3 . (N ¼ 3 blots from separate, mean  SEM).

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regulatory pathways within the lung may have led to the T3 effects on airway development that we observed in our current study and suggests that down regulation of T3 nuclear receptors by exogenous T3 treatment may have altered downstream receptor hormone pathways involved in structural and functional maturation of the distal airways [8,16,42,45,46]. These possible mechanisms for the effect of T3 on the morphologic development of the lung are consistent with the observed effects of higher T3 doses in Gd13.5 fetal lungs in our current study. At these higher doses, the further changes in distal airway morphology indicated that at this time in lung development the distal airway epithelia had the developmental ability to respond further to T3 . The accelerated formation of distal airway structures seen after 3 days of treatment with the high dose of T3 was again seen when lungs were treated for 7 days with the low dose of T3 . This indicated that the developmentally specific effect of T3 dose and length of time in culture persisted when Gd13.5 lungs were treated for 7 days. These apparent changes in morphology were confirmed by morphometric analysis. After 3 and 7 days of culture, there was significant T3 -dependent increases in airway space and in cuboidal and flattened epithelia and a decrease in mesenchyme. Others have described similar effects of T3 or T4 on late-gestation lung morphology and/or morphometry [14,47]. Chan et al. [14] gave a single in vivo T3 dose to fetal lambs, followed by premature delivery two weeks later. This single dose resulted in improved lung compliance and gas exchange that was not related to changes in surfactant production, suggesting that T3 had changed lung structure. Similarly, Kikkawa et al. [47] showed that intraamniotic treatment of fetal rabbits with T4 followed by preterm delivery led to increased lung biochemical and structural lung maturation. The lung histology of the T4 treated lungs in this study [47] is very similar to what we observed in Gd13.5 embryonic mouse lungs treated for 3 days with 100 nM T3 or for 7 days with 2 nM T3 . Other in vivo and in vitro studies also correlate with our study showing that T3 accelerates thinning of mesenchyme and the development of saccular structures and presumptive early alveolar septa, potentially leading to improved gas exchanging surface area [9,48]. Although studies in animals show potential beneficial effects of T3 on lung maturation, clinical studies in humans have been controversial suggesting that exogenous T3 during critical periods of lung development may be deleterious [15,49]. The effects of T3 on lung development in animal and human studies are summarized in Table 1. Our current in vitro study begins to investigate possible reasons for the conflicting outcome of infants treated with regimens that alter the thyroid hormone axis to advance lung development prior to preterm birth. The potential deleterious effects of increased exogenous T3 levels in human fetuses make the abnormal lung

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Table 1 Effects of changes in thyroid hormone axis on fetal lung maturation Effect

Species

Increased surfactant production Decreased or no effect on surfactant proteins (Sp) Altered lung function/airway morphology Altered nuclear hormone receptors Increased RDS and/or neonatal chronic lung disease (CLD)

Human and rat fetal lung [8,16,28] Fetal rat lung [39–41] Fetal lamb, rabbit, rat, and mouse [4,9,11,14,18,47–49] Fetal mouse lung [45] Human neonate [13,15]

This table summarizes in vivo and in vitro treatments with thyroid releasing hormone, T3 or T4 in human and animal studies. Other studies can be found within studies referenced above.

development we noted in Gd13.5 mouse lungs treated for 7 days with the higher doses of T3 particularly important. These findings suggest that the effects of these higher doses of T3 at this point in lung morphogenesis are deleterious to distal airway formation. The marked dilation of airway lumens with T3 treatment has not been reported by others. The abnormal luminal development as well as the profound thinning of mesenchymal tissue seen in the lungs treated for 7 days with the high dose of T3 and confirmed by morphometry may have resulted from T3 -induced alterations in the coordinated interactions of mesenchyme and epithelia needed for distal airway development [50]. As evidenced by the PCNA immunostaining in the Gd13.5 embryonic lungs cultured for 7 days, this change in the mesenchymal and epithelial component of the lung may be mediated in part by changes in cell proliferation of the epithelial component of the lung. The change in the epithelial component of the lung may have altered subsequent epithelial-mesenchymal cell communication and thus cellular maturation and differentiation. To gain insight into potential T3 -induced changes in epithelial and mesenchymal cell maturation and cellular interactions we used double immunostaining to simultaneously evaluate the spatial and cellular expression of Nkx2.1 and Hoxb-5 proteins [18]. The effect of T3 on the developmental expression patterns of Hoxb-5 and Nkx2.1 correlated with the morphologic changes in the mesenchyme and epithelia. Both increasing dose of T3 and increasing time in culture were associated with progressive changes in the regional and cellular expression patterns of these proteins, with the pattern of expression advancing to that seen in neonatal lung [20,22]. The cellular and quantitative expression of Hoxb-5 protein represent an accelerated developmental pattern compared to what we have reported for neonatal mouse and human lung tissue [20,23]. Hoxb-5 protein is normally expressed in mesenchyme at high levels during branching morphogenesis until the onset of the canalicular period of lung development (Gd16.5–Gd17.5).

The ability of T3 to alter this pattern resulting in down regulation of Hoxb-5 suggests that branching morphogenesis may not be appropriately completed before the onset of the abnormal distal airway development induced by exogenous T3 treatment. Alternatively, the decrease in Hoxb-5 expression may have resulted from the ability of T3 treatment to alter the mesenchymal component of the lungs in culture thereby decreasing the number of cells that could express Hoxb-5. The T3 -induced changes in the cellular expression pattern of Nkx2.1 may have impacted on the role of Nkx2.1 in cellular differentiation of the lung epithelia. Nkx2.1 is critical for transcriptional regulation of the murine SP-C gene [51]. As T3 altered the developmental expression pattern of Nkx2.1 in our study, the ability of Nkx2.1 to modulate the expression pattern of SP-C may have also changed contributing to altered expression of SP-C. The levels of Nkx2.1 and SP-C proteins in T3 treated lungs seen on Western blot were not statistically different than the levels of these proteins in untreated lungs. However, the ability of T3 treatment to alter the developmental cellular expression patterns for Nkx2.1 and SP-C proteins as seen by immunocytochemistry make the changes in the level of these proteins worth noting. Thus, although T3 prematurely and abnormally advanced distal airway structural development, it may have inhibited functional differentiation of these abnormally developed distal airway-like structures. Additionally, the decrease in mesenchymal tissue that was visualized by microscopy and quantified by morphometry suggest that this altered functional differentiation of the distal airway may be secondary to altered mesenchymal–epithelial interactions necessary for appropriate developmental expression of Nkx2.1 and SP-C. The down regulation of mesenchymal expression of Hoxb-5 in the presence of exogenous T3 may be one mechanism involved in these altered mesenchymal–epithelial cell interactions [52]. We conclude that exogenous T3 treatment starting in the late pseudoglandular period of mouse lung development prematurely and abnormally accelerated the developmental program of the lung by effects on lung structural development. This accelerated distal airway structural development changed the cellular composition of the lung leading to abnormal cell fate and functional differentiation of the distal airway epithelium. The altered cell composition may have led to changes in epithelial–mesenchymal cell communication and subsequent abnormal and contrasting biochemical changes within the developing lung.

Acknowledgments This work was supported by American Lung Association Research Grant RG-060-N, National Institute of

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Health HD38419, National Institute of Health HL37930, New England Medical Center Research Grant, March of Dimes Grant H6 FY98-0608, American Lung Association Career Investigator Award (to Dr. Mala Chinoy) and American Heart Association Grant-in-Aid. We extend special thanks to Dr. Michael Beers and Dr. Jeffrey Whitsett for the generous contribution of their SP-C antibodies. The helpful assistance of Erdene Haltiwanger, Lucia Pham, Sam Sit, and Wilson Lee are greatly appreciated.

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