Smad1 expression and function during mouse embryonic lung branching morphogenesis

Bone morphogenetic protein (BMP) 4 plays very important roles in regulating developmental processes of many organs, including lung. Smad1 is one of the BMP receptor downstream signaling proteins that transduce BMP4 ligand signaling from cell surface to nucleus. The dynamic expression patterns of Smad1 in embryonic mouse lungs were examined using immunohistochemistry. Smad1 protein was predominantly detected in peripheral airway epithelial cells of early embryonic lung tissue [embryonic day 12.5 (E12.5)], whereas Smad1 protein expression in mesenchymal cells increased during mid-late gestation. Many Smad1-positive mesenchymal cells were localized adjacent to large airway epithelial cells and endothelial cells of blood vessels, which colocalized with a molecular marker of smooth muscle cells (alpha-smooth muscle actin). The biological function of Smad1 in early lung branching morphogenesis was then studied in our established E11.5 lung explant culture model. Reduction of endogenous Smad1 expression was achieved by adding a Smad1-specific antisense DNA oligonucleotide, causing approximately 20% reduction of lung epithelial branching. Furthermore, airway epithelial cell proliferation and differentiation were also inhibited when endogenous Smad1 expression was knocked down. Therefore, these data indicate that Smad1, acting as an intracellular BMP signaling pathway component, positively regulates early mouse embryonic lung branching morphogenesis.     

Gremlin negatively modulates BMP-4 induction of embryonic mouse lung branching morphogenesis

Bone morphogenetic protein-4 (BMP-4) is a key morphogen for embryonic lung development that is expressed at high levels in the peripheral epithelium, but the mechanisms that modulate BMP-4 function in early mouse lung branching morphogenesis are unclear. Here, we studied the BMP-4 antagonist Gremlin, which is a member of the DAN family of BMP antagonists that can bind and block BMP-2/4 activity. The expression level of gremlin in embryonic mouse lungs is highest in the early embryonic pseudoglandular stage [embryonic days (E) 11.5-14.5] and is reduced during fetal lung maturation (E18.5 to postnatal day 1). In situ hybridization indicates that gremlin is diffusely expressed in peripheral lung mesenchyme and epithelium, but relatively high epithelial expression occurs in branching buds at E11.5 and in large airways after E16.5. In E11.5 lung organ culture, we found that exogenous BMP-4 dramatically enhanced peripheral lung epithelial branching morphogenesis, whereas reduction of endogenous gremlin expression with antisense oligonucleotides achieved the same gain-of-function phenotype as exogenous BMP-4, including increased epithelial cell proliferation and surfactant protein C expression. On the other hand, adenoviral overexpression of gremlin blocked the stimulatory effects of exogenous BMP-4. Therefore, our data support the hypothesis that Gremlin is a physiologically negative regulator of BMP-4 in lung branching morphogenesis.     

Canonical Wnt signaling negatively regulates branching morphogenesis of the lung and lacrimal gland

Key gene families such as FGFs and BMPs are important mediators of branching morphogenesis. To understand whether Wnt genes, and in particular, the canonical Wnt signaling pathway also function in the branching process, we have used a combination of experimental and genetic gain and loss of function approaches to perturb the levels of canonical Wnt signaling in two arborized structures, the lung and the lacrimal gland. Here, we show that the addition of Wnt3a conditioned medium or LiCl strongly represses growth and proliferation of the lung and lacrimal gland, a result that was confirmed in vivo using a dominant stable mutation of beta-catenin conditionally expressed in the lacrimal gland epithelium. In agreement with these data, knockdown of Wnt signaling with beta-catenin morpholinos results in a greater number of branches and increased cell proliferation. In addition, we show that canonical Wnt signaling is able to modulate the levels of Fgf10 and suppress BMP-induced proliferation in the lacrimal gland. Thus, canonical Wnt signaling negatively regulates branching morphogenesis providing a balance to FGFs and BMPs which positively regulate this process. This multilayered control of growth and proliferation ensures that branched structures attain the morphology required to function efficiently.     

Thyroid hormone affects embryonic mouse lung branching morphogenesis and cellular differentiation

Although thyroid hormone (T(3)) influences epithelial cell differentiation during late fetal lung development, its effects on early lung morphogenesis are unknown. We hypothesized that T(3) 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 T(3) (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 T(3). Morphometric analysis of lung histology showed that T(3) caused a dose-dependent decrease in mesenchyme and increase in cuboidal epithelia and airway space. Immunocytochemistry showed that with T(3) 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 T(3) treatment during early lung development accelerated epithelial and mesenchymal cell differentiation at the expense of premature reduction in new branch formation and lung growth.     

Smurf1 regulates the inhibitory activity of Smad7 by targeting Smad7 to the plasma membrane

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT-type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces cytoplasmic localization of Smad7. Smurf1 then associates with transforming growth factor-beta type I receptor (TbetaR-I) and enhances the turnover of this receptor. However, the mechanisms of the nuclear export and plasma membrane localization of the Smurf1.Smad7 complex have not been elucidated. We show here that Smurf1 targets Smad7 to the plasma membrane through its N-terminal conserved 2 (C2) domain. Both wild-type Smurf1 (Smurf1(WT)) and Smurf1 lacking the C2 domain (Smurf1(deltaC2)) bound to Smad7 and translocated nuclear Smad7 to the cytoplasm. However, unlike Smurf1(WT), Smurf1(deltaC2) did not move to the plasma membrane and failed to recruit Smad7 to the cell surface TbetaR-II.TbetaR-I complex. Moreover, although Smurf1(deltaC2) induced ubiquitination of Smad7, it failed to induce the ubiquitination and degradation of TbetaR-I and did not enhance the inhibitory activity of Smad7. Thus, these results suggest that the plasma membrane localization of Smad7 by Smurf1 requires the C2 domain of Smurf1 and is essential for the inhibitory effect of Smad7 in the transforming growth factor-beta signaling pathway.     

Collagen involvement in branching morphogenesis of embryonic lung and salivary gland

The possibility that extracellular collagen is involved in branching morphogenesis of mouse embryo lung and salivary glands has been explored duringin vitro organ culture. Control cultures of both rudiment types contain abundant collagen in extracellular spaces between mesenchymal cells and in the epithelial-mesenchymal interface. Branching morphogenesis of lungs and salivary glands is not perturbed by the presence of β-aminopropionitrile, implying that extracellular collagen cross-linking is not required, but is perturbed by α,α′-dipyridyl orl-azetidine-2-car?ylic acid (LACA), agents reported to interfere with collagen synthesis and secretion. Analysis of the structural and biosynthetic effects of LACA revealed a severe inhibition of collagen synthesis, as monitored by hydroxyproline synthesis, and extracellular collagen accumulation. Cell and tissue integrity was not affected, but a slight inhibition of general protein synthesis, protein accumulation, and epithelial expansion was observed. The strong correlations between collagen biosynthesis, extracellular collagen presence, and branching morphogenesis are consistent with an integral role for collagen in embryonic lung and salivary gland morphogenesis.     

Quantitation of 3D ureteric branching morphogenesis in cultured embryonic mouse kidney

The growth and branching of the epithelial ureteric tree is critical for development of the permanent kidney (metanephros). Current methods of analysis of ureteric branching are mostly qualitative. We have developed a method for measuring the length of individual branches, and thereby the total length of the ureteric tree in 3 dimensions (3D). The method involves confocal microscopy of whole-mount immunostained metanephroi and computer-based image segmentation, skeletonisation and measurement. The algorithm performs semi-automatic segmentation of a set of confocal images and skeletonisation of the resulting binary object. Length measurements and number of branch points are automatically obtained. The final representation can be reconstructed providing a fully rotating 3D perspective of the skeletonised tree. After 36 h culture of E12 mouse metanephroi, the total length of the ureteric tree was 6103 +/- 291 microm (mean +/- SD), a four-fold increase compared with metanephroi cultured for just 6 h (1522 +/- 149 microm). Ureteric duct length increased at a rate of 153 microm/h over the first 30 h period and was maximal between 18 and 24 h at 325 microm/h. The distribution of branch lengths at the six time points studied was similar, suggesting tight control of ureteric lengthening and branching. This method will be of use in analysing ureteric growth in kidneys cultured in the presence of specific molecules suspected of regulating ureteric growth. The method can also be used to analyse in vivo kidneys and to quantify branching morphogenesis in other developing organs.     

E-cadherin negatively regulates CD44-hyaluronan interaction and CD44-mediated tumor invasion and branching morphogenesis

CD44 is a principal cell-surface receptor for hyaluronan (HA). Up-regulation of CD44 is often associated with morphogenesis and tumor invasion. On the contrary, reduction of cell-cell adhesion due to down-regulation of E-cadherin is associated with the invasive and metastatic phenotype of carcinomas. In our current study, we investigated the functional relationship between CD44 and E-cadherin. We established an inverse correlation between CD44 and E-cadherin indicating that the cells expressing higher levels of E-cadherin display weaker binding affinity between CD44 and HA. By using TA3 murine mammary carcinoma (TA3) cells, which display CD44-dependent HA binding, branching morphogenesis, and invasion, we demonstrated an inverse functional relationship between CD44 and E-cadherin by transfecting exogenous E-cadherin into the cells. Our results showed that increased expression of E-cadherin in TA3 cells, but not ICAM-1, weakens the binding between CD44 and HA and blocks spreading of the cells on HA substratum and CD44-mediated branching morphogenesis and tumor cell invasion. The results reported here demonstrated for the first time that E-cadherin negatively regulated CD44-HA interaction and CD44 function and suggested that balanced function of CD44 and E-cadherin may be essential for normal epithelial cell functions, and imbalanced up-regulation of CD44 function and/or down-regulation of E-cadherin function likely contributes to tumor progression.     

Smad7 and Smad6 differentially modulate transforming growth factor beta -induced inhibition of embryonic lung morphogenesis

Transforming growth factors beta (TGF-beta) are known negative regulators of lung development, and excessive TGF-beta production has been noted in pulmonary hypoplasia associated with lung fibrosis. Inhibitory Smad7 was recently identified to antagonize TGF-beta family signaling by interfering with the activation of TGF-beta signal-transducing Smad complexes. To investigate whether Smad7 can regulate TGF-beta-induced inhibition of lung morphogenesis, ectopic overexpression of Smad7 was introduced into embryonic mouse lungs in culture using a recombinant adenovirus containing Smad7 cDNA. Although exogenous TGF-beta efficiently reduced epithelial lung branching morphogenesis in control virus-infected lung culture, TGF-beta-induced branching inhibition was abolished after epithelial transfer of the Smad7 gene into lungs in culture. Smad7 also prevented TGF-beta-mediated down-regulation of surfactant protein C gene expression, a marker of bronchial epithelial differentiation, in cultured embryonic lungs. Moreover, we found that Smad7 transgene expression blocked Smad2 phosphorylation induced by exogenous TGF-beta ligand in lung culture, indicating that Smad7 exerts its inhibitory effect on both lung growth and epithelial cell differentiation through modulation of TGF-beta pathway-restricted Smad activity. However, the above anti-TGF-beta signal transduction effects were not observed in cultured embryonic lungs with Smad6 adenoviral gene transfer, suggesting that Smad7 and Smad6 differentially regulate TGF-beta signaling in developing lungs. Our data therefore provide direct evidence that Smad7, but not Smad6, prevents TGF-beta-mediated inhibition of both lung branching morphogenesis and cytodifferentiation, establishing the mechanistic basis for Smad7 as a novel target to ameliorate aberrant TGF-beta signaling during lung development, injury, and repair.     

Hoxb-5 control of early airway formation during branching morphogenesis in the developing mouse lung

Hox proteins control structural morphogenesis, pattern formation and cell fate in the developing embryo. To determine if Hoxb-5 participates in patterning of early airway branching during lung morphogenesis, gestational day 11.5 embryonic lung cultures were treated with retinoic acid (RA) to up-regulate and antisense oligonucleotides to down-regulate Hoxb-5 protein expression. RA (10^?6 M) and Hoxb-5 antisense oligonucleotide (20 μM) treatment each significantly decreased branching morphogenesis (P<0.001), but the morphology of branching under these conditions was very different. RA-treated lungs had elongated primary branches but decreased further branching with increased Hoxb-5 immunostaining in subepithelial regions underlying these elongated airways. Western blots confirmed that Hoxb-5 protein was increased by 189±20% (mean±S.E.M., P<0.05) in RA-treated lungs compared to controls. In contrast, lungs treated with Hoxb-5 antisense oligos plus RA had foreshortened primary branches with rudimentary distal clefts resulting in decreased numbers of primary and subsequent branches. Immunohistochemistry confirmed that Hoxb-5 antisense oligos inhibited Hoxb-5 protein expression even in the presence of RA. We conclude that regional and quantitative changes in Hoxb-5 protein expression influence morphogenesis of the first airway divisions from the mainstem bronchi. RA-induced alterations in branching are mediated in part through regulated Hoxb-5 expression.     

Suppression of embryonic lung branching morphogenesis by antisense oligonucleotides against HOM/C homeobox factors

The role of HOM/C homeobox genes on rat embryonic lung branching morphogenesis was investigated using the lung bud explant culture system in an air/liquid interface. Knock down of homeobox b3 and b4 expression by antisense oligonucleotide treatment repressed airway branch formation, while antisense oligonucleotide against homeobox a3 showed no effect. Addition of antisense Hoxb3 oligonucleotide resulted in upregulation of collagen type III mRNA and fibroblast growth factor 10 mRNA, while that of the T-box regulatory factor-4 was decreased. Consequently, expression of Clara cell-specific secretory protein was decreased. These results suggest a critical role for homeobox b3 and b4 genes in lung airway branching morphogenesis.     

Identification of laminin domains involved in branching morphogenesis: effects of anti-laminin monoclonal antibodies on mouse embryonic lung development

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal anti-laminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50-100 micrograms/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.     

Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate

 Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5–15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell–cell communication, cell fate, and differentiation of conducting airway epithelia. Accepted: 5 May 1997     

Nox1 regulates apoptosis and potentially stimulates branching morphogenesis in sinusoidal endothelial cells

Tubulogenic transformation of a nontubulogenic endothelial cell line NP31 by a constitutively activated form of the Flt-1 kinase (NP31/kinase) was accompanied by an increased expression of Nox1 by sixfold over NP31. Overexpression of Nox1 in NP31 cells (NP31/Nox1) stimulated branching morphogenesis in Matrigel but surprisingly cords lacked a lumen. The branching morphogenesis by NP31/kinase and NP31/Nox1 cells was blocked either by N-acetyl-l-cysteine (NAC) or Tiron. Vascular endothelial growth factor (VEGF)-dependent sinusoidal endothelial cells (SEC) in primary culture showed fivefold increase in Nox1 expression 4 days after VEGF stimulation. Interestingly, VEGF-resistant apoptosis in SEC at day 7 was inhibited by NAC or by anti-Nox1 siRNA. These results suggest that Nox1 regulates apoptosis in SEC and can potentially stimulate branching morphogenesis in SEC-derived NP 31 cells.     

Cripto-1 enhances migration and branching morphogenesis of mouse mammary epithelial cells

Cripto-1 is an EGF-CFC protein that performs an important role during early vertebrate development and is overexpressed in several types of human cancer. In the present study mouse EpH4, NMuMG, and TAC-2 mammary epithelial cells that are negative for endogenous cripto-1 expression were transfected with the murine cripto-1 cDNA. Cripto-1-transfected cell lines exhibited functional and physiological differences from the original cell lines including enhanced anchorage-independent growth in soft agar (EpH4 cells), growth in serum-free medium, increased proliferation, and formation of branching, duct-like structures when grown in a three-dimensional collagen type I matrix. Furthermore, cripto-1-expressing cell lines showed elevated migration in vitro in Boyden chamber and wound-healing assays. These results indicate that cripto-1 can function through an autocrine pathway that enables mammary epithelial cells to undergo an epithelial to mesenchymal transition.     

Sucrose stimulates branching morphogenesis of embryonic mouse lung in vitro: a problem of osmotic balance between lumen fluid and culture medium

In organ cultures of lung rudiments from 11-day mouse embryos, it was found that addition of sucrose to the culture medium stimulated branching morphogenesis and reduced lumen distension. Two possible roles of sucrose were postulated: one as a nutrient and another as a generator of osmotic pressure inducing osmosis of water from the lumen fluid to the culture medium across a simple columnar epithelial cell layer. To assess which was the case, branching morphogenesis was investigated in lung rudiments cultured in medium in which osmotic pressure was increased by the addition of lactose or NaCl rather than sucrose: similar acceleration of branching was observed in both. In another experiment, lumen fluid of cultured lung rudiments was mechanically drained each day, and significantly stimulated branching morphogenesis was observed even when sucrose was not added to the culture medium. Heparin is known to induce abnormal lumen distension and inhibits branching morphogenesis. Heparin-induced abnormal morphogenesis was prevented either by the addition of sucrose to the culture medium or by the mechanical drainage of lumen fluid. These results suggest that lumen distension caused by the accumulation of lumen fluid disrupts lung branching morphogenesis in vitro, even when the mechanism of branching morphogenesis is intact.