SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and Plasticity
Thyroid hormone affects embryonic mouse lung branching morphogenesis and cellular differentiation

Kwanchai Archavachotikul1, Teriggi J. Ciccone1, Mala R. Chinoy2, Heber C. Nielsen1, and Maryann V. Volpe1

1 Department of Pediatrics, Division of Newborn Medicine, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111; and 2 Lung Development Research Program, Department of Surgery, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although thyroid hormone (T3) influences epithelial cell differentiation during late fetal lung development, its effects on early lung morphogenesis are unknown. We hypothesized that T3 would alter embryonic lung airway branching and temporal-spatial differentiation of the lung epithelium and mesenchyme. Gestational day 11.5 embryonic mouse lungs were cultured for 72 h in BGJb serum-free medium without or with added T3 (0.2, 2.0, 10.0, or 100 nM). Evaluation of terminal bud counts showed a dose- and time-dependent decrease in branching morphogenesis. Cell proliferation was also significantly decreased with higher doses of T3. Morphometric analysis of lung histology showed that T3 caused a dose-dependent decrease in mesenchyme and increase in cuboidal epithelia and airway space. Immunocytochemistry showed that with T3 treatment, Nkx2.1 and surfactant protein SP-C proteins became progressively localized to cuboidal epithelial cells and mesenchymal expression of Hoxb5 was reduced, a pattern resembling late fetal lung development. We conclude that exogenous T3 treatment during early lung development accelerated epithelial and mesenchymal cell differentiation at the expense of premature reduction in new branch formation and lung growth.

triiodothyronine; lung development; Nkx2.1; Hoxb5; surfactant protein-C


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DURING FETAL DEVELOPMENT, lung morphology and differentiation of the airway lining epithelium change with advancing gestation. In the mouse, lung morphogenesis starts at gestational day 9.5 (Gd 9.5) with the evagination of the lung primordium from the ventral foregut to form the prospective trachea and two lung buds followed by the dichotomous branching of each lung bud through the process of branching morphogenesis. Differentiation of this lung primordium into the bronchial and pulmonary acinar systems begins during this period. The restriction of cell types and in some cases loss of cell-specific markers herald changes in the stage of bronchiolar versus distal epithelial cell fate and differentiation (48, 58). These processes of airway branching and cytodifferentiation require a close interaction between endodermal epithelium and adjacent mesenchyme and are affected by specific factors and hormonal systems active in the developing embryo and fetus (1, 22, 24, 35, 38). In particular, in rodent lung organ cultures and human lung explant cultures, triiodothyronine (T3), the active form of thyroid hormone, influences late fetal lung development by accelerating epithelial cell differentiation leading to surfactant phospholipid synthesis and by regulating lung structural development and alveolar septation (9, 11, 26, 38). Little is known about the cellular expression pattern of T3 receptors during fetal lung development, but they are present in rodent and human lung tissue during the pseudoglandular period and increase in quantity and binding affinity as lung development progresses (6, 13, 18, 20, 23). Although there are relatively low levels of T3 and its receptors in early fetal life, maternal hypo- or hyperthyroidism leads to characteristic changes in lung growth, morphology, and function (6, 10, 17, 32). T3 exerts its effects by binding to specific nuclear receptors with subsequent binding of either T3-receptor monomers, of homodimer complexes, or of heterodimers with retinoic acid receptors (RXRs) to specific response elements in downstream genes regulated by T3 (28, 40). Although much is understood concerning T3-mediated molecular and cellular interactions and its potential role in late lung development, the effect of T3 on the progression of airway branching morphogenesis and early lung cellular differentiation and the specific developmentally regulated factors that control these processes have not been studied.

There are several mesenchyme-specific and epithelial cell-specific proteins whose expression patterns at different stages of lung development reflect epithelial differentiation and airway morphogenesis. Hox proteins are highly conserved, mesenchymally derived transcription factors that control cell fate and organ-specific morphogenesis in the developing embryo (34). In particular, Hoxb5 is one of the Hox proteins that are strongly expressed in the mesenchyme of the developing lung, where it helps regulate branching morphogenesis and patterning of the conducting airways (2, 31, 52, 55, 56). Inhibition of Hoxb5 in developing embryonic mouse lung leads to inhibition of branching morphogenesis (55). The expression of Hoxb5 protein is developmentally regulated, decreasing with advancing gestation and the onset of alveolar formation. Factors that enhance branching morphogenesis upregulate Hoxb5, whereas factors that accelerate lung maturation downregulate Hoxb5 (12, 53, 55). It is not known if thyroid hormone regulates Hoxb5 or other Hox genes.

Nkx2.1 (also known as thyroid transcription factor 1, TTF1, and TITF1) is an epithelial cell-specific homeodomain transcription factor expressed in the developing thyroid gland, brain, and lung. Although Nkx2.1 regulates thyroid-specific gene transcription, the interaction of T3 and Nkx2.1 during lung development has not been studied. In the lung, Nkx2.1 is expressed in epithelial cells of the lung primordium, where it regulates branching morphogenesis during early lung development. The expression of Nkx2.1 becomes progressively localized to distal epithelium as lung development proceeds, regulating epithelial differentiation in early and late lung development (21, 29, 59, 60). Similar to Nkx2.1, surfactant protein C (SP-C) is also produced by pulmonary epithelial cells and is detected throughout the epithelium during early embryonic mouse lung development. SP-C follows changes in cellular and spatial expression that are similar to Nkx2.1. SP-C becomes restricted to distal epithelium of developing saccules, reflecting the progression of epithelial differentiation during late lung development (60). Nkx2.1 directly regulates expression of SP-C as well as expression of surfactant proteins, SP-A, SP-B, and the Clara cell secretory protein genes in the developing lung (4, 5, 21). It is not known if Nkx2.1 is directly regulated by Hox genes, but recent evidence suggests that expression of Hox genes in lung mesenchyme may modulate epithelial expression of Nkx2.1 (2). Recently, with the use of cotransfection assays in NIH3T3 cells or HeLa cells, a Hox gene has been shown to regulate in vitro expression of Nkx2.1 (27).

To explore the effects of T3 on early lung development, we studied the impact of T3 treatment on embryonic lung morphogenesis and on the temporal-spatial differentiation of the respiratory epithelium and mesenchyme. We hypothesized that exogenous T3 treatment would interfere with airway branching and cellular differentiation of the embryonic lung. We explored this hypothesis by studying the effects of T3 on airway branching morphogenesis, cell proliferation, lung morphometry, and on the developmentally regulated and cell-specific expression of Hoxb5, Nkx2.1, and SP-C proteins.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. The animal study protocol was approved by the Institutional Animal Research Committee. Principles of laboratory animal care (National Institutes of Health publication 86-23, revised 1985) were followed.

Timed pregnant Swiss-Webster mice (Taconic Farms, Germantown, NY) were killed by CO2 inhalation at Gd 11.5. (Gd 0.5 is defined as the morning of identification of the vaginal plug; term is Gd 19.) This day of gestation was chosen because it represents the early pseudoglandular stage of lung development in the mouse, during the period of active branching morphogenesis, before the onset of regional changes in cell fate and epithelial cell differentiation.

Reagents. Mouse monoclonal antibody to Nkx2.1 was purchased from Neomarkers (Fremont, CA). Hoxb5 rabbit polyclonal antibody was previously developed in our laboratory (52). Rabbit polyclonal antibody to SP-C was a gift from Dr. Michael Beers, University of Pennsylvania. T3 was purchased from Sigma (St. Louis, MO), and [3H]thymidine (specific activity of 50 Ci/mM) was purchased from ICN Pharmaceuticals (Irvine, CA). Immunostaining reagents were purchased from Vector Laboratories (Burlingame, CA). All other reagents were from Sigma unless otherwise specified.

Embryonic lung culture. Embryonic lung cultures were prepared as we and others have previously described (35, 42, 55, 57). After maternal death, sterile laparotomy was performed and the uterus was removed with embryos intact and placed in ice-cold Hanks' balanced salt solution. Under a dissecting microscope (×20 magnification), using microdissection forceps, we removed the fetuses from the uterus and dissected lungs free of surrounding structures. Embryonic lungs were then placed in 70-mm culture dishes on GVWP membranes (Millipore, Bedford, MA) at the air-liquid interface in serum-free and hormone-free (BGJb) medium (Gibco BRL) containing 0.2 mg ascorbic acid, 50 µg streptomycin, and 50 units penicillin per milliliter of culture medium and cultured for 72 h as control or with added T3. Untreated control lungs received no additional treatment. A stock solution of 0.1 and 1 µM T3 in BGJb medium was prepared and added to cultures to produce experimental conditions of 0.2, 2, 10, or 100 nM T3. These doses of T3 represent doses within the probable range of T3 levels and receptor binding capacity in embryonic mouse lung. T3 levels in human embryonic lung are 0.5 nM, and the nuclear binding coefficients of T3 receptors in rodent embryonic lung have been shown to be 0.5 nM. A similar range of T3 concentrations has also been used in cultured fetal lung and showed a dose-response effect on surfactant phospholipid synthesis (13, 25, 26, 36). Cultured lungs were maintained at 37°C in 95% room air-5% CO2 for the 72 h of culture. Culture medium and treatments were changed daily. After 72 h, lungs were processed as described below.

Evaluation of branching morphogenesis. Branching morphogenesis was evaluated as we have previously described (35, 42, 55). Briefly, embryonic lungs in culture were evaluated at time zero and at 24-h intervals in culture using an inverted Diaphot microscope at ×4 magnification. For each lung in which the right and left lung could be clearly identified and were structurally intact, the number of terminal buds in the left lung of each explant was recorded at 0, 24, 48, and 72 h and compared between untreated control and treatment conditions.

Evaluation of cell proliferation. The incorporation of radiolabeled thymidine into DNA was used to assess the affect of T3 on changes in cell proliferation. Embryonic lungs were incubated with [3H]thymidine (1 µCi/ml) for the last 24 h of culture. Embryonic lungs for each culture condition were harvested into phosphate-buffered saline (PBS), pH 7.4, and homogenized by hand with Dounce glass homogenizing tubes. Each homogenized sample was evaluated for [3H]thymidine levels and DNA content using a modified procedure from our previous studies (41, 42). Each homogenate was subjected to DNA precipitation with three cycles of precipitation with 0.4 M perchloric acid followed by resuspension in 0.5 ml of PBS, pH 7.4. An equal aliquot of each sample was removed for determination of total DNA. Liquid scintillation cocktail was then added to an equal volume of each sample, and total disintegrations per minute (dpm) were determined in a scintillation counter. Total disintegrations per minute per nanomole of DNA were calculated for each culture condition.

Nkx2.1 and Hoxb5 immunostaining. At 72 h, representative lungs from each culture condition (2-3 lungs/condition) were processed for Nkx2.1 and Hoxb5 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 optimum cutting temperature frozen specimen medium (Miles, Elkhart, IN) and sectioned (6 µm) 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, and 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. Nonspecific binding sites were blocked by sequential incubations with avidin (15 min) and 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). Endogenous peroxidase activity was eliminated by washing in a hydrogen peroxide and methanol (1:4) solution for 30 min. Specific peroxidase activity was then detected by incubation with diaminobenzidine (DAB) reagent for 5 min. We then prepared the same sections to detect Hoxb5 protein localization by washing them 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 Hoxb5 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 (30 min). Hoxb5 protein sites were then detected with alkaline phosphatase substrate (Vector Blue, 30 min). Sections were counterstained with nuclear fast red, dehydrated through a graded alcohol series (95, 100%), and cleared with Histoclear (National Diagnostics, Atlanta, GA). Coverslips were applied, and sections were analyzed under an inverted light microscope.

SP-C immunostaining. In separate lung sections from the same lungs in which Nkx2.1 and Hoxb5 were studied, the spatial and cellular expression pattern of SP-C protein was evaluated using an HRP detection protocol modified from Beers et al. (3). 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 with methanol-H2O2 incubation followed by blocking of nonspecific sites 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 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, and cleared with Histoclear (National Diagnostics, Atlanta, GA). Coverslips were applied, and sections were analyzed using an inverted light microscope.

Morphometry. Lung sections from 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 µm) and were representative of the overall morphology in the untreated and treated lungs. Criteria for columnar, cuboidal, or flattened epithelial cells and mesenchymal cells and air space were predefined and based on published descriptions of lung histology (48). In each lung section, the percentage of grid points intersecting columnar or cuboidal epithelium, airway space or mesenchyme was statistically compared in the untreated and T3-treated lungs. Analyses were performed by the same observer without knowledge of results from untreated and treated lungs until the evaluations were completed.

Statistical analysis. For evaluation of branching morphogenesis, mean terminal bud counts for each culture condition at each time interval were compared for effects of T3 and length of time in culture using Kruskal-Wallis nonparametric ANOVA with Dunnett's multiple comparison test, with a value of P <=  0.05 considered significant. For evaluation of cell proliferation, the ratio of [3H]thymidine (dpm) to total DNA (nM) in untreated control and T3-treated specimens was statistically compared by Kruskal-Wallis nonparametric ANOVA followed by Dunnett's multiple comparison test with level of P <=  0.05 taken as significant. Morphometric data were compared by ANOVA with Bonferroni's multiple comparison test.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Branching morphogenesis. Representative lungs after 24, 48, and 72 h of culture are shown in Fig. 1. Untreated control lungs exhibited the normal progression of monochotomous and dichotomous airway branching that we and others have previously described (35, 42, 55, 57). Visually, lungs treated with 0.2 and 2 nM T3 also had a similar branching pattern to untreated lungs but developed fewer branches over the 72 h of culture (see Figs. 1 and 2). Although the decreased airway branching seen with T3 treatment occurred throughout the lung without a prominent effect on particular airway generations, the terminal airway generations in the lungs treated with 10 and 100 nM T3 appeared dilated, and lungs appeared decreased in size. The visual appearance of fewer airway generations in T3-treated lungs was confirmed by objective evaluation of terminal bud counts in the left lung of each explant (Fig. 2). After 72 h of culture, terminal bud counts were decreased in a dose-dependent fashion by 7% (0.2 nM T3), 23% (2 nM T3), 28% (10 nM T3), and 25% (100 nM T3) compared with untreated lungs. Kruskal-Wallis nonparametric ANOVA demonstrated significant effects of both T3 dose (P < 0.0001) and time in culture (P < 0.0001) on terminal bud counts. Multiple comparison testing was performed on data at 48 and 72 h to identify individual differences. Compared with untreated lungs, the effects of 2-, 10-, and 100-nM doses of T3 on branching were statistically significant after both 48 and 72 h (Fig. 2).


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Fig. 1.   Representative photographs of gestational day (Gd) 11.5 cultured embryonic mouse lungs. Untreated lung (control) photographed after 24 (A), 48 (B), and 72 h (C) of culture; lung treated with 0.2 nM thyroid hormone triiodothyronine (T3) after 24 (D), 48 (E), and 72 h (F); lung treated with 2 nM T3 after 24 (G), 48 (H), and 72 h (I); lung treated with 10 nM T3 after 24 (J), 48 (K), and 72 h (L) of culture; and lung treated with 100 nM T3 after 24 (M), 48 (N), and 72 h (O) of culture. Bar = 500 µm, with same magnification for all lungs. Photographs represent individual lungs with the same lung being photographed at 24, 48, and 72 h of culture for each culture condition. At time zero and after 24 h of culture, untreated control lungs (A) and T3-treated lungs (D, G, J, M) had similar numbers of terminal branches. After 48 and 72 h of culture with the higher doses of T3 treatment, the development of airway branching was progressively decreased and overall lung size appeared diminished.



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Fig. 2.   Gd 11.5 cultured embryonic mouse lungs. Effect of T3 on left lung terminal bud counts. Lungs in all treatment conditions showed an increase in terminal bud counts with time in culture. Nonparametric ANOVA showed that both time in culture (P < 0.0001) and T3 treatment (P < 0.0001) affected terminal bud counts. Compared with untreated control lungs, terminal bud counts in 0.2 nM T3-treated lungs were decreased by 5% after 48 h and 7% after 72 h. Dunnett's multiple comparison testing showed that this decrease in terminal bud counts became significant in 2, 10, and 100 nM T3-treated lungs both after 48 and 72 h compared with untreated control lungs cultured for the same period of time. Bars represent means ± SE; n = 11-76 individual lungs from 7 experiments. *P < 0.01 for no T3 condition vs. 2-, 10-, 100-nM conditions at 48 and 72 h.

Cell proliferation. The incorporation of thymidine into DNA in the last 24 h of culture was evaluated as an index of whole lung cell proliferation and lung growth in untreated control lungs compared with T3-treated lungs. The effect of T3 on thymidine incorporation into DNA is shown for untreated control lungs and lungs treated with 2, 10, and 100 nM T3, as these doses showed the most profound changes in terminal bud counts and lung histology (Fig. 3). At these doses of T3, thymidine incorporation into DNA was decreased compared with untreated lungs. With 100 nM T3 treatment, thymidine incorporation into DNA was decreased by 50% in T3-treated lungs compared with untreated control (P < 0.05, mean ± SE, 1,366 ± 329 dpm/nM DNA in 100 nM T3-treated lungs; 2,872 ± 340 dpm/nM DNA in untreated control lungs).


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Fig. 3.   Gd 11.5 embryonic mouse lungs cultured for 72 h. Effect of T3 on [3H]thymidine incorporation into DNA. Gd 11.5 embryonic mouse lungs treated with T3 for 72 h had decreased [3H]thymidine incorporation into DNA. Bars represent means ± SE of 3-5 observations. Kruskal-Wallis ANOVA with Dunnett's multiple comparison test showed P < 0.05 for 100-nM T3 treatment vs. no T3 condition (*).

Immunostaining. The effect of T3 treatment on changes in epithelial and mesenchymal cell morphological maturation was studied using immunohistochemistry. The simultaneous spatial and cellular expression of Nkx2.1 and Hoxb5 protein was evaluated using double immunostaining methods to allow simultaneous evaluation of Nkx2.1 and Hoxb5 protein expression in the context of histological characterization of airway epithelial morphology and airway branching patterns. Nkx2.1 and Hoxb5 are homeodomain transcription factor proteins that exhibit characteristic changes in spatial and cellular expression with advancing lung development and maturation, with Nkx2.1 being specific for epithelial cells and Hoxb5 specific for mesenchymal cells.

Untreated lungs (Fig. 4, A-C) and lungs treated with 0.2 nM T3 (Fig. 4D) showed branching airway patterns characteristic of the early to midpseudoglandular period of lung development. Airways were lined by proximal columnar and distal cuboidal cells. Nkx2.1 protein expression was prominent in the nuclei of both these cell types throughout the lung. There was no apparent difference in the cellular expression of Nkx2.1 protein between the columnar and cuboidal epithelial cells (Fig. 4, B and C, untreated lung; Fig. 4D, 0.2 nM-treated lung). Mesenchyme that surrounded these airways was fairly dense, and mesenchymal cell nuclei were diffusely positive for Hoxb5 protein (Fig. 4, A-D). In contrast to untreated lungs, lungs treated with the higher doses of T3 (Fig. 4, F and G, 100 nM T3; Fig. 4H, 10 nM T3) had progressive changes in airway morphology and in the spatial and cellular localization Nkx2.1 and Hoxb5 proteins. Representative lung sections from lungs treated with 10 nM (Fig. 4H) and 100 nM T3 (Fig. 4, F and G) demonstrate a progressively more disorganized pattern of airway branches with abnormal or incompletely developed airway subdivisions. Some airway lumens appeared small, with a disorganized epithelial lining, whereas, especially at the highest dose of T3 studied, some distal airways had the appearance suggestive of early saccules with dilated lumens lined by cuboidal epithelium (Fig. 4, E-G). The mesenchyme also appeared progressively less dense around these abnormally developed airways. With increasing doses of T3, Nkx2.1 was more intermittently expressed in columnar epithelial cells but remained prominent and more distally located in cuboidal epithelial cells, beginning at the columnar-cuboidal cell transitional zone (Fig. 4H, 10 nM T3; Fig. 4, F and G, 100 nM T3). Similarly, regional expression of Hoxb5 protein also changed with increasing doses of T3 treatment. Hoxb5 became progressively localized to peripheral mesenchymal cells adjacent to airways with columnar and cuboidal epithelia, with an apparent decrease in Hoxb5-positive nuclei in the mesenchyme around airways that lacked Nkx2.1 protein expression (Fig. 4, F-H). In the absence of either primary antibody, no specific staining was seen for Nkx2.1 (Fig. 4A) or Hoxb5 protein (Fig. 4E). In the absence of both primary antibodies, no staining was seen with the HRP or alkaline phosphatase reactions (data not shown).


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Fig. 4.   Gd 11.5 embryonic mouse lungs cultured for 72 h. Double label immunostaining for Nkx2.1 protein (brown nuclei) and Hoxb5 protein (blue nuclei). Coronal lung sections from untreated lung (A-C), 0.2 nM T3-treated lung (D), 100 nM T3-treated lungs (E-G), and 10 nM T3-treated lung (H). A (untreated lung) and E (100 nM T3 treatment): immunostaining controls with normal serum in place of primary antibody to Nkx2.1 (A) and primary antibody to Hoxb5 (E), respectively. Bar = 100 µm in A, B, and F. Bar = 50 µm in C-E, G, and H. * in A: mesenchyme surrounding trachea and mainstem bronchus, which is known not to express Hoxb5 protein (6, 49, 52). In untreated lung (B and C) and in lung treated with 0.2 nM T3 (D), Nkx2.1 protein was strongly expressed in both columnar (arrowhead) and cuboidal (arrows) epithelial cell nuclei, and Hoxb5 protein was strongly expressed in most mesenchymal cell nuclei. Immunostaining demonstrated T3 dose-related changes [compare 0.2 nM T3 (D), 10 nM T3 (H), and 100 nM T3 (E-G)] in the regional and cellular expression of Nkx2.1 and Hoxb5 proteins. With increasing T3 treatment (H, E, F, G), Nkx2.1 expression became progressively intermittent in columnar epithelium (arrowhead), and after 100-nM T3 treatment Nkx2.1 was predominantly localized to distal cuboidal epithelium (arrows). Hoxb5 protein became progressively restricted to distal mesenchymal cells around columnar and cuboidal epithelia with increasing dose of T3 (H, E, F, G).

The immunohistology of SP-C protein expression was studied as a marker gene to gain additional insight into changes in epithelial cell fate with T3 treatment. Changes in the cellular expression of SP-C was used to further identify proximal versus distal airway morphological development, because, as the lung enters the later stages of morphogenesis, SP-C expression is extinguished in proximal epithelia and becomes more highly expressed in progenitor epithelial cells of distal airways (58). Immunostaining for SP-C in the untreated control lungs was seen in the apical and basal cytoplasm of many columnar and cuboidal epithelial cells, reflecting the immature stage of epithelial differentiation in which the respiratory epithelia do not exhibit complete restricted gene expression (Fig. 5B). With increasing doses of T3, the pattern of SP-C protein expression became characteristic of a more terminally differentiated lung and followed that seen with Nkx2.1 protein expression. SP-C decreased in more proximal columnar epithelia and became restricted to more distal low columnar and cuboidal epithelial cells of distal airways (Fig. 5, C and D).


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Fig. 5.   Gd 11.5 embryonic mouse lungs cultured for 72 h. Immunostaining for surfactant protein (SP)-C protein. Representative coronal lung sections from untreated lung (A), immunostaining control, with normal rabbit serum in place of primary antibody; SP-C immunostaining in untreated lung (B); 10 nM T3-treated lung (C); and 100 nM T3-treated lung (D). Bar = 25 µm. In untreated lung (B), SP-C immunostaining (brown cytoplasm) was present in basal and apical regions of many columnar epithelial cells (arrowheads) and also in some cuboidal epithelial cells (arrows). The pattern of SP-C immunolocalization in the T3-treated lungs (C, D) was more restricted. T3-treated lungs (C, D) had decreased and more intermittent staining of tall columnar epithelial cells (arrowhead), especially in basal regions of these cells. There continued to be specific staining of low columnar epithelia (open arrow) but with progressive localization of SP-C protein to distal cuboidal epithelial cells of the more distally located airways (arrows).

Morphometry. The apparent changes in mesenchyme and airway histology seen on lung sections were confirmed objectively by morphometric analysis (Fig. 6). Point-count evaluations identified dose-dependent changes in volume density of mesenchyme, cuboidal epithelium, and air space (ANOVA, P < 0.05). Multiple comparison testing was performed to identify individual differences. This testing showed that, compared with untreated controls, significant increases in cuboidal epithelia were observed in all T3-treated groups (P < 0.01), and significant decreases in mesenchyme and increased airway space occurred in 100 nM T3-treated lungs (P < 0.05).


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Fig. 6.   Morphometry of Gd 11.5 embryonic mouse lungs cultured for 72 h without T3 or with added T3 at 0.2-, 2-, 10-, or 100-nM concentrations. T3 treatment caused dose-dependent changes in lung morphology as evidenced by an increase in cuboidal epithelia (P < 0.0001), decrease in mesenchyme (P < 0.02), and increase in airway space with increasing T3 dose (ANOVA). Bonferroni's multiple comparison posttest identified significant individual differences for cuboidal epithelia in all T3-treated groups vs. untreated lungs (*P < 0.01) and for mesenchyme and airway in untreated lungs vs. 100 nM T3-treated lungs (*P < 0.05). Bars represent means ± SE; n = 6-8 lung sections per treatment condition from 2-3 lungs per condition.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

During the entire period of gestational development, the fetal lung parenchyma undergoes significant structural and cytomorphological changes that assure precise control of regional and cell-specific growth and maturation (30, 50). During the early stages of lung development, the processes of growth and differentiation of the future air-conducting structures predominate, whereas the development of terminal airways and the gas exchange apparatus become prominent late in fetal life and postnatally (50). Thyroid hormone is one factor that affects late gestational and postnatal lung structural development and late epithelial differentiation. In late-gestation lung, thyroid hormone upregulates surfactant phospholipid synthesis and downregulates the production of antioxidant enzymes (superoxide dismutase and catalase) and expression of surfactant protein gene expression (43, 47). Thyroid hormone may also be a physiological regulator of septation and the size of the gas-exchanging apparatus late in gestation and early postnatal life (9, 38). However, the effects of exogenous thyroid hormone on early lung morphogenesis and cell differentiation have not been studied.

In the human, fetal serum T3 is low but detectable in fetal tissues as early as the end of the first trimester. It has been suggested that local tissue conversion to biologically active T3 may occur in fetal tissues. T3 nuclear receptors, binding of T3, and increasing binding capacity are all present in human fetal lung tissue at 12-19 wk of gestation and in other species at comparable times (6, 13, 18, 23). Thus low levels of endogenous T3 may play a role in lung morphogenesis during the early pseudoglandular period of lung development. Evidence of lung hypoplasia or altered lung growth and differentiation has been reported in infants after maternal hypothyroidism and after experimentally induced maternal hypothyroidism. Conversely, maternal hyperthyroidism and exogenous maternal thyroid releasing hormone have been associated with concerning fetal outcome (10, 14, 16, 17). This indicates that the precise balance of exposure to thyroid hormone during fetal lung development is important. Little is known about the cellular expression pattern of T3 receptors in the developing lung. However, the effect of T3 on lung surfactant phospholipid synthesis is mediated by direct action on epithelial cells consistent with the expression of T3 receptors on adult rat type 2 cells (37, 45). RXR-alpha and -beta that dimerize with T3 receptors are ubiquitously expressed in developing lung (7). The effect of T3 on the activity of T3 receptors and RXR heterodimers may further alter the response of lung epithelia and mesenchyme to T3.

Costa et al. (13) reported that tissue T3 concentration in the human embryonic lung is 0.5 nM. Our study used concentrations in a range spanning these levels to identify dose-dependent effects. Additionally, based on T3 receptor binding studies in human and rabbit embryonic lung, the nuclear binding coefficient of T3 receptors in embryonic lung tissue is also within the range of the T3 doses used in our study (23, 36). Concentrations of T3 in culture media are not necessarily reflective of tissue concentrations, such that true tissue concentration may be significantly lower if delivery is limited or higher if T3 becomes concentrated in the cells. Nevertheless, we observed significant effects of T3 with doses at or above this measured physiological level. Similar doses were shown in other in vitro studies to affect the biochemical and structural development of the lung in late fetal and postnatal life. (11, 19, 25, 26, 51). Thus the dose effect of T3 that we have observed in early lung development is comparable to previous studies in late lung development in which doses <1 nM had little or no effect and doses of 1-100 nM produced progressive changes in lung maturation, with a maximum effect at 10 nM (11, 23, 26). In our studies, these higher doses of T3 showed the most prominent effect on cellular proliferation, changes in overall lung and airway morphology, lung morphometry, and on the progression of cellular differentiation.

We found that airway branching morphogenesis was significantly decreased in treated lungs after 48 and 72 h. This response suggests that T3 effects on branching morphogenesis were related in part to the overall decreased cell proliferation that we observed in the T3-treated lungs. The lungs continued to branch and grow even at the highest dose of T3, and thymidine incorporation did not decrease significantly between doses of 2 and 100 nM. This indicates that our results are unlikely due to a nonspecific effect but more likely due to altered mesenchymal and epithelial cell proliferation in the face of decreased branching morphogenesis. Active airway branching morphogenesis is associated with changes in mesenchymal and epithelial cell proliferation and lung growth as well as associated regional changes in apoptosis, which allow for the remodeling of airways and surrounding mesenchyme as new airway branches form (33, 35, 42). This overall decreased cell proliferation we demonstrated in the T3-treated lungs is consistent with a T3-mediated change in the growth potential of branching airway epithelium and of surrounding mesenchyme that is normally present during the early pseudoglandular period of lung development. High-dose T3-treated lungs also appeared to have dilated terminal airways lined by cuboidal epithelium resembling early terminal saccules, suggesting that distal airways that would normally still be involved in branching morphogenesis were being stimulated to undergo terminal differentiation. These apparent gross morphological changes in the airway epithelia and airway space were confirmed objectively by morphometric analysis of lung sections, which showed an increase in cuboidal epithelia and airway space and a decrease in mesenchyme with increasing T3 doses. These morphological and proliferative changes seen in the T3-treated lungs in our study appear to herald the effects of T3 seen in later lung development, including the thinning of mesenchyme and the formation of thinned walled saccules. These processes result in improved lung compliance and gas exchange at term gestation but, at this early gestation, occur at the expense of decreased airway branching that, in vivo, may ultimately decrease the overall surface area of the lung available for gas exchange (8, 30, 38, 51).

Although statistical analysis confirmed a dose-dependent effect of T3 on branching, the differences at doses beyond 2 nM were small, which raises the question of what is the nature of the effect of T3 on branching. The pattern of a "dose response" in terms of airway branching likely depends on the mechanism by which altered branching occurs. Changes in the amount of instructive proteins may show graded responses producing the traditional appearance of a steadily changing dose-response effect. However, alteration of the phenotype of morphogenetic cells may produce an effect that is closer to a quantum "on-off" response, in which branching morphogenesis is either normal or reduced. Increasing doses of the agent likely would not produce further changes in branching because the mechanism involved is a simple on-off switch. Examples of such a simple on-off effect on branching morphogenesis have been reported, in which once an effective dose was reached, increasing doses did not produce further large changes in bud formation (8, 35, 44, 49). However, in the same system, the cell-specific expression of specific proteins, which are markers of stages in cell differentiation, might show more obvious progressive changes with increasing doses of the agent, as documented by our studies of expression of Hoxb5, Nkx2.1, and SP-C.

The decreased branching morphogenesis that we observed might also be secondary to T3-associated changes in the regional expression of Nkx2.1. Functional knockouts of Nkx2.1 expression exhibit reduced branching morphogenesis in vitro (39). The impairment of branching morphogenesis induced by T3 was also associated with a restriction of epithelial phenotypes, further supporting the notion that T3 treatment altered the growth potential of less-differentiated cells by committing them to a more-differentiated phenotype. We observed that with increasing doses of T3, Nkx2.1 expression became progressively extinguished in columnar epithelia and more localized to the cuboidal epithelial cells of distal airways, representing an accelerated change in the natural course of Nkx2.1 expression (29, 39, 60). During in vivo lung development, Nkx2.1 expression becomes extinguished in proximal pulmonary epithelium (trachea and primary bronchial epithelium) and becomes confined to distal cuboidal epithelial cells as branching morphogenesis is completed and epithelial differentiation proceeds (60). This T3-associated change in Nkx2.1 expression may have altered the branching potential of the lung during the phase of active branching morphogenesis and suggests that the T3-treated lungs exhibited advanced cellular differentiation compared with the untreated control lungs.

The effect of T3 on the cellular and regional expression pattern of Hoxb5 protein was consistent with the above findings. In the lungs treated with T3, Hoxb5 became progressively localized to peripheral mesenchyme in subepithelial regions, with an overall reduction in the apparent number of mesenchymal cells expressing Hoxb5 protein. The decreased mesenchymal expression of Hoxb5 may also be related to the overall decrease in the mesenchymal component of the T3-treated lungs that we confirmed with the morphometric evaluations. These changes in lung mesenchyme and Hoxb5 expression are normally seen in later lung development, during the canalicular period after the completion of branching morphogenesis when distal airways are progressing toward alveolar development (12, 52). As branching morphogenesis is completed, toward the end of the pseudoglandular period (Gd 15.5-16.5), Hoxb5 becomes restricted to subepithelial mesenchyme around developing bronchioles, suggesting that after branching morphogenesis is completed the restricted expression pattern of Hoxb5 may be involved in the further maturation of bronchioles and surrounding mesenchyme. Interestingly, we have also shown that antisense inhibition of Hoxb5 expression in embryonic mouse lung culture also causes reduced airway branching morphogenesis (55). In our present study, T3 accelerated this developmental program of Hoxb5 such that the cellular expression of Hoxb5 was prematurely decreased at a point in lung development when branching morphogenesis is still active and Hoxb5 expression is normally quite high and diffusely expressed throughout the lung mesenchyme. This ability of T3 to cause a premature reduction and change in the cellular expression pattern of Hoxb5 may be one mechanism by which T3 altered branching morphogenesis. Airways in the T3-treated lungs that lacked epithelial expression of Nkx2.1 appeared to have a regional decrease in Hoxb5-positive mesenchymal cells underlying these airways. These findings and the known role of Hoxb5 and Nkx2.1 during normal branching morphogenesis (39, 55) suggest an association or interaction of mesenchymal expression of Hoxb5 and epithelial expression of Nkx2.1 during normal branching morphogenesis that was altered by T3 treatment. It is not known if Nkx2.1 expression in epithelial cells is regulated by Hoxb5 expression in subepithelial mesenchyme. However, recent evidence suggests that Nkx2.1 may be regulated by Hox genes and that mesenchymal expression of Hox genes in lung development mediates epithelial cell-specific expression of Nkx2.1 (2, 27).

Regional changes in SP-C expression in the T3-treated lungs also support the conclusion that T3 mediated changes in epithelial cell fate. In embryonic lung during the early pseudoglandular period, SP-C expression is seen in both proximal and distal airways (58). In T3-treated lungs, SP-C protein expression became more localized to distal cuboidal epithelial cells, mimicking a later period of lung development. This change in the spatial and cellular expression of SP-C followed the change in expression pattern seen for Nkx2.1 in the T3-treated lungs. This finding might be expected since Nkx2.1 regulates the transcription of surfactant protein genes SP-A, SP-B, and SP-C (4, 5, 21). Therefore, in the T3-treated lungs, regional changes in SP-C protein expression are likely mediated by the changes in the cellular expression of Nkx2.1 protein expression. The regionalization of SP-C expression also suggests that T3 treatment advanced epithelial differentiation. Although thyroid hormone is known to increase surfactant phospholipid production in fetal lungs, thyroid hormone either decreases or has no effect on the surfactant proteins (25, 26, 43, 50). Our findings on the regional changes in SP-C protein expression are consistent with the studies of Ramadurai et al. (43), in which in vivo treatment decreased SP-C mRNA in fetal rat lung. Our data suggest that the reported changes in SP-C mRNA levels seen with T3 treatment are in part due to a decrease in the number of cells expressing SP-C.

The data from the current study also suggest a basic difference between T3 and epidermal growth factor (EGF)-induced effects on embryonic lung branching morphogenesis and epithelial differentiation. In contrast to the effects of T3 shown in this study, we previously showed that EGF treatment of Gd 10.5 embryonic mouse lungs stimulated lung branching morphogenesis, increased cell proliferation, and enhanced epithelial expression of SP-C and surfactant phospholipid in embryonic mouse lung cultures (42). Coincident with an increase in branching morphogenesis, EGF treatment of Gd 10.5 embryonic lungs increased Hoxb5 protein expression, whereas EGF- receptor-deficient mice have impaired branching morphogenesis, decreased Hoxb5 expression, and deficient alveolar development and alveolar surface area (53, 54). Interestingly, retinoic acid, another important regulator of lung morphogenesis both during airway branching morphogenesis and in alveolar development, increases EGF receptor binding but can also interact with T3 to directly downregulate transcription of the EGF receptor (15, 28). These data suggest potential cross-modulation of T3, retinoic acid, and EGF effects during different stages of lung development. EGF effects on branching morphogenesis may predominate during early lung development. As completion of branching morphogenesis occurs toward the end of the pseudoglandular period, T3 and retinoic acid regulation of distal airway development may then modulate or alter EGF receptor signal pathways to allow for proximal and distal epithelial cell differentiation. This is consistent with studies in human lung tissue showing that T3 receptors are present in human fetal lung as early as 12-13 wk of pregnancy and increase in binding capacity toward the end of airway branching morphogenesis with the onset of acinar development (23).

In summary, our data suggest that exogenous T3 treatment during the early pseudoglandular period of lung development accelerated embryonic lung cellular differentiation, resulting in prematurely advanced epithelial cell differentiation and premature reduction in new branch formation. We speculate that the effect of T3 on distal airway development and differentiation are mediated, in part, by altering the developmental clock of Nkx2.1 and Hoxb5 protein expression. Further investigations of the possible cis- and trans-related effects of T3 on Nkx2.1 and Hoxb5 will be necessary to fully evaluate the interactions of these factors in early lung morphogenesis.


    ACKNOWLEDGEMENTS

We extend special thanks to Dr. Michael Beers for the generous contribution of SP-C antibody. The helpful assistance of Erdene Haltiwanger, Lucia Pham, and Sam Sit is greatly appreciated.


    FOOTNOTES

This work was supported by American Lung Association Research Grant RG-060-N, National Institutes of Health Grants HD-38419 and HL-37930, and a New England Medical Center research grant.

Address for reprint requests and other correspondence: M. V. Volpe, Dept. of Pediatrics, Div. of Newborn Medicine, New England Medical Center, Box 44, 750 Washington St., Boston, MA 02111 (E-mail: mvolpe1{at}lifespan.org).

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.00400.2000

Received 22 November 2000; accepted in final form 18 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alescio, T, and Cassini A. Induction in vitro of tracheal buds by primary mesenchyme grafted onto tracheal epithelium. J Exp Zool 150: 83-94, 1962[ISI].

2.   Aubin, J, Lemieux M, Tremblay M, Berard J, and Jeannotte L. Early postnatal lethality in Hoxa-5 mutant mice is attributable to respiratory tract defects. Dev Biol 192: 43-445, 1997.

3.   Beers, MF, Kim CY, Dodia C, and Fisher AB. Localization, synthesis, and progression of surfactant protein SP-C in rat lung analyzed by epitope specific antipeptide antibodies. J Biol Chem 269: 20318-20328, 1994[Abstract/Free Full Text].

4.   Bohinski, RJ, Di Lauro R, and Whitsett JA. The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3 indicating common factors for organ-specific expression along the foregut axis. Mol Cell Biol 14: 5671-5681, 1994[Abstract].

5.   Bruno, MD, Bohinski RJ, Huelsman KM, and Whitsett JA. The lung-cell-specific expression of the murine surfactant protein A (SP-A) gene is mediated by interactions between the SP-a promoter and thyroid transcription factor-1. J Biol Chem 270: 6531-6536, 1995[Abstract/Free Full Text].

6.   Burrow, GN, Fisher DA, and Larsen PR. Maternal and fetal thyroid function. N Engl J Med 331: 1072-1078, 1994[Free Full Text].

7.   Cardoso, WV. Transcription factors and pattern formation in the developing lung. Am J Physiol Lung Cell Mol Physiol 269: L429-L442, 1995[Abstract/Free Full Text].

8.   Cardoso, WV, Williams MC, Mitsialis SA, Joyce-Brady M, Rishi AK, and Brody JS. Retinoic acid induces changes in the pattern of airway branching and alters epithelial cell differentiation in the developing lung in vitro. Am J Respir Cell Mol Biol 12: 464-476, 1995[Abstract].

9.   Chan, L, Miller TF, Yuxin J, Farina C, Chander A, Shaffer TH, and Wolfson MR. Antenatal triiodothyronine improves neonatal pulmonary function in preterm lambs. J Soc Gynecol Investig 5: 122-126, 1998[ISI][Medline].

10.   Church, J, Khafayan E, Chechani V, Sadiq F, Devaskar S, deMello D, and Devaskar U. Transplacental stimulation of fetal lung maturation: effect of triiodothyronine in the female and male rabbit fetus. Biol Neonate 52: 157-65, 1987[ISI][Medline].

11.   Chinoy, MR, Gonzales LW, Ballard PL, Fisher AB, and Eckenhoff RG. Elemental composition of lamellar bodies from fetal and adult human lung. Am J Respir Cell Mol Biol 13: 99-108, 1995[Abstract].

12.   Chinoy, MR, Volpe MV, Cilley RE, Zgleszewski SE, Vosatka RJ, Martin A, Nielsen HC, and Krummel TM. Growth factors and dexamethasone regulate Hoxb5 protein in cultured murine fetal lungs. Am J Physiol Lung Cell Mol Physiol 274: L610-L620, 1998[Abstract/Free Full Text].

13.   Costa, A, Arisio R, Benedetto C, Bertino E, Fabris C, Giraudi G, Marozio L, Maula V, Pagliano M, Testori O, and Zoppetti G. Thyroid hormones in tissues from human embryos and fetuses. J Endocrinol Invest 14: 559-568, 1991[ISI][Medline].

14.   Crowther CA and the ACTOBAT Study Group. Australian collaborative trial of antenatal thyrotropin-releasing hormone: adverse affects at 12 month followup. Pediatrics 99: 311-317, 1997[Abstract/Free Full Text].

15.   Dammann, CEL, and Nielsen HC. Regulation of the epidermal growth factor receptor in fetal rat lung fibroblasts during late gestation. Endocrinology 139: 1671-1677, 1998[Abstract/Free Full Text].

16.   Erenberg, A, Omori K, Menkes JH, Oh W, and Fisher DA. Growth and development of thyroidectomized ovine fetus. Pediatr Res 8: 783-789, 1974[ISI][Medline].

17.   Erenberg, A, Rhodes ML, Weinstein MM, and Kennedy RL. The effect of fetal thyroidectomy on ovine fetal lung maturation. Pediatr Res 13: 230-235, 1979[ISI][Medline].

18.   Ferreiro, B, Bernal J, Goodyer CG, and Branchard CL. Estimation of nuclear thyroid hormone receptor saturation in human fetal brain and lung during early gestation. J Clin Endocrinol Metab 67: 853-856, 1988[Abstract].

19.   Floros, J, Gross I, Nichols KV, Veletza SV, Dynia D, Lu H, Wilson CM, and Peterec SM. Hormonal effects of the surfactant protein B (SP-B) mRNA in cultured fetal rat lung. Am J Respir Cell Mol Biol 4: 449-454, 1991[ISI][Medline].

20.   Forrest, D, Sjoberg M, and Vennstrom B. Contrasting developmental and tissue-specific expression of alpha and beta thyroid hormone receptor genes. EMBO J 9: 1519-1528, 1990[Abstract].

21.   Ghaffari, M, Zeng X, Whitsett JA, and Yan C. Nuclear localization domain of thyroid. Transcription factor-1 in respiratory epithelial cells. Biochem J 328: 757-771, 1997[ISI][Medline].

22.   Gluckman, PD, Sizonenko SV, and Bassett NS. The transition from fetus to neonate---an endocrine perspective. Acta Paediatr Suppl 428: 7-11, 1999.

23.   Gonzalez, LW, and Ballard PL. Identification and characterization of nuclear 3,5,3' triiodothyronine-binding sites in fetal human lung. J Clin Endocrinol Metab 53: 21-28, 1981[Abstract].

24.   Gross, I. Regulation of fetal lung maturation. Am J Physiol Lung Cell Mol Physiol 259: L337-L344, 1990[Abstract/Free Full Text].

25.   Gross, I, Dynia DW, Wilson CM, Ingelson LD, Gewolb IH, and Rooney SA. Glucocorticoid-thyroid hormone interactions in fetal rat lung. Pediatr Res 18: 191-196, 1984[Abstract].

26.   Gross, I, and Wilson C. Fetal lung in organ culture IV. Supra-additive hormone interactions. J Appl Physiol Respir Environ Exercise Physiol 52: 1420-1425, 1982[Abstract/Free Full Text].

27.   Guazzi, S, Lonigro R, Pintonello L, Boncinelli E, Di Lauro R, and Mavilio F. The thyroid transcription factor-1 gene is a candidate target for regulation by Hox proteins. EMBO J 13: 3339-3347, 1994[Abstract].

28.   Hudson, LG, Santon JV, Glass CK, and Gill GN. Ligand-activated thyroid hormone and retinoic acid receptors inhibit growth factor receptor promoter expression. Cell 62: 1165-1175, 1990[ISI][Medline].

29.   Ikeda, K, Clark JC, Shaw-White JR, Stahlman MT, Boutell CJ, and Whitsett JA. Gene structure and expression of human thyroid transcription factor-1 in respiratory epithelial cells. J Biol Chem 270: 8108-8114, 1995[Abstract/Free Full Text].

30.   Inselman, LS, and Mellins RB. Growth and development of the lung. J Pediatr 98: 1-15, 1981[ISI][Medline].

31.   Kappen, C. Hox genes in the lung. Am J Respir Cell Mol Biol 15: 156-162, 1996[ISI][Medline].

32.   Kikkawa, BWY, Orzalesi MM, Motoyama EK, Kaibara M, Zigas CJ, and Cook CD. The effect of thyroxine on the maturation of fetal rabbit lung. Biol Neonate 22: 161-168, 1973[ISI][Medline].

33.   Kresch, MJ, Christian C, Wu F, and Hussain N. Ontogeny of apoptosis during lung development. Pediatr Res 43: 426-431, 1998[Abstract].

34.   Krumlauf, R. Hox genes in vertebrate development. Cell 78: 191-201, 1994[ISI][Medline].

35.   Levesque, BM, Vosatka RJ, and Nielsen HC. Dihydrotestosterone stimulates branching morphogenesis, cell proliferation, and programmed cell death in mouse embryonic lung explants. Pediatr Res 47: 481-491, 2000[Abstract/Free Full Text].

36.   Lindenberg, JA, Brehier A, and Ballard PL. Triiodothyronine nuclear binding in fetal and adult rabbit lung and cultured lung cells. Endocrinology 103: 1725-1731, 1978[Abstract].

37.   Luo, M, Faure R, Tong YA, and Dussault JH. Immunocytochemical localization of the nuclear 3,5,3'-triiodothyronine receptor in the adult rat: liver, kidney, heart, lung and spleen. Acta Endocrinol 120: 451-458, 1989[ISI][Medline].

38.   Massaro, D, Teich N, and Massaro GD. Postnatal development of pulmonary alveoli: modulation in rats by thyroid hormones. Am J Physiol Regulatory Integrative Comp Physiol 250: R51-R55, 1986[ISI][Medline].

39.   Minoo, P, Hamdan H, Bu D, Warburton D, Stepanik P, and deLemos R. TTF-1 regulates lung epithelial morphogenesis. Dev Biol 172: 694-698, 1995[ISI][Medline].

40.   Motomura, K, and Brent GA. Mechanisms of thyroid hormone action. Implications for the clinical manifestation of thyrotoxicosis. Endocrinol Metab Clin North Am 27: 1-23, 1998[ISI][Medline].

41.   Nielsen, HC, Kirk WO, Sweezy N, and Torday JS. Regulation of growth and differentiation in the fetal lung. Exp Cell Res 188: 89-96, 1990[ISI][Medline].

42.   Nielsen, HC, Martin A, Volpe MV, Hatzis D, and Vosatka RJ. Growth factor control of growth and epithelial differentiation in embryonic lungs. Biochem Mol Med 60: 38-48, 1997[ISI][Medline].

43.   Ramadurai, S, Chen Y, Hatzis D, Sosenko IRS, and Nielsen HC. Differential effects in vivo of thyroid hormone on the expression of surfactant phospholipid, surfactant protein mRNA and antioxidant enzyme mRNA in fetal rat lung. Exp Lung Res 24: 641-657, 1998[ISI][Medline].

44.   Shiratori, M, Oshika EE, Ung LP, Singh G, Shinozuka H, Warburton D, Michalopoulos G, and Katyal SL. Keratinocyte growth factor and embryonic rat lung morphogenesis. Am J Respir Cell Mol Biol 15: 328-338, 1996[Abstract].

45.   Smith, BT, and Sabry K. Glucocorticoid-thyroid synergism in lung maturation: a mechanism involving epithelial-mesenchymal interaction. Proc Natl Acad Sci USA 80: 1951-1954, 1983[Abstract].

46.   Spooner, BS, and Wessels NK. Mammalian lung development: interactions in primordium formation and bronchial morphogenesis. J Exp Zool 175: 445-454, 1970[ISI][Medline].

47.   Sosenko, IRS, and Frank L. Thyroid hormone depresses antioxidant enzyme maturation in fetal rat lung. Am J Physiol Regulatory Integrative Comp Physiol 253: R592-R598, 1987[Abstract/Free Full Text].

48.   Ten Have-Opbroek, AAW Lung development in the mouse embryo. Exp Lung Res 17: 111-130, 1991[ISI][Medline].

49.   Thompson, HA, and Spooner BS. Inhibition of branching morphogenesis and alteration of glycosaminoglycan biosynthesis in salivary glands treated with beta -D-xyloside. Dev Biol 89: 417-424, 1982[ISI][Medline].

50.   Veletza, SV, Nichols KV, Gross I, Lu H, Dynia DW, and Floros J. Surfactant protein C: hormonal control of SP-C mRNA levels in vitro. Am J Physiol Lung Cell Mol Physiol 262: L684-L687, 1992[Abstract/Free Full Text].

51.   Volpe, MV, Chinoy MR, Archavachotikul K, Cheng ZH, Cilley RE, and Nielsen HC. Thyroid hormone affects airway morphogenesis and epithelial cell fate during the late pseudoglandular period of mouse lung development (Abstract). Am J Respir Crit Care Med 161: A564, 2000.

52.   Volpe, MV, Martin A, Vosatka RJ, Mazzoni CL, and Nielsen HC. Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate. Histochem Cell Biol 108: 495-504, 1997[ISI][Medline].

53.   Volpe, MV, and Nielsen HC. Hoxb-5 protein expression: regulation by epidermal growth factor and transforming growth factor beta  in embryonic lung (Abstract). Am J Respir Crit Care Med 151: A298, 1995.

54.   Volpe, MV, Sibilia M, Wagner EF, and Nielsen HC. Embryonic lung branching morphogenesis, cell proliferation, and differentiation are altered in epidermal growth factor receptor (EGF-R) -/- transgenic 129/SV × C57BL/6 mice (Abstract). Pediatr Res 39: 70A, 1996.

55.   Volpe, MV, Vosatka RJ, and Nielsen HC. Hoxb-5 control of early airway formation during branching morphogenesis in the developing mouse lung. Biochim Biophys Acta 1475: 337-345, 2000[ISI][Medline].

56.   Wall, NA, Jones CM, Hogan BLM, and Wright CVE Expression and modification of Hox 2.1 protein in mouse embryos. Mech Dev 37: 111-120, 1992[ISI][Medline].

57.   Warburton, D, Seth R, Shum L, Horcher PG, Hall FL, Werb Z, and Slavkin HC. Epigenetic role of EGF expression and signaling in embryonic mouse lung morphogenesis. Dev Biol 149: 123-133, 1992[ISI][Medline].

58.   Wert, SE, Glasser SW, Korfhagen TR, and Whitsett JA. Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol 156: 426-443, 1993[ISI][Medline].

59.   Yuan, B, Li C, Kimura S, Engelhardt RT, Smith BR, and Minoo P. Inhibition of distal lung morphogenesis in Nkx2.1 (-/-) embryos. Dev Dyn 217: 180-190, 2000[ISI][Medline].

60.   Zhou, L, Lim L, Costa RH, and Whitsett JA. Thyroid transcription factor-1, hepatocyte nuclear factor-3beta , surfactant protein B, C, and clara cell secretory protein in developing mouse lung. J Histochem Cytochem 44: 1183-1193, 1996[Abstract/Free Full Text].


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