Cellular events associated with lung branching morphogenesis including the deposition of collagen type IV

In this study mouse lung development was examined using an in vitro model system. The culture system permitted examination of a morphogenic process that eventually led to the formation of presumptive alveoli (terminal sacs). The observations included changes in epithelial cell morphology (transition from a columnar to a spindle shape), and evidence for motile activity on the part of primitive airway epithelial cells. The importance of Type IV collagen to the cellular events associated with branching morphogenesis was investigated by immunolocalization. In addition, we assessed the similarity of normal lung development to in vitro development by comparing cultured lungs with equivalent stages of embryonic and fetal mouse lungs. The results show that cultured embryonic lung explants proceed along a morphogenic pathway that parallels normal lung development; that primitive pulmonary epithelial cells engage in motile activity and transiently acquire an extended cell shape both in vitro and in vivo; that, as suggested by others, the pattern of late branching morphogenesis is not dichotomous, but irregular; and that short wisplike fibers of Type IV collagen are present in developing embryonic and fetal lung mesenchyme. Taken together, the results show that early and late lung branching patterns differ significantly, and suggest that later stages of lung branching involve distinct epithelial cell shape transitions. The immunofluorescence data suggest that fibrous Type IV collagen may be the extracellular matrix scaffold within which early epithelial cells accomplish lung branching morphogenesis.     

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     

Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme

Mammalian lung develops as an evagination of ventral gut endoderm into the underlying mesenchyme. Iterative epithelial branching, regulated by the surrounding mesenchyme, generates an elaborate network of airways from the initial lung bud. Fibroblast growth factors (FGFs) often mediate epithelial-mesenchymal interactions and mesenchymal Fgf10 is essential for epithelial branching in the developing lung. However, no FGF has been shown to regulate lung mesenchyme. In embryonic lung, Fgf9 is detected in airway epithelium and visceral pleura at E10.5, but is restricted to the pleura by E12.5. We report that mice homozygous for a targeted disruption of Fgf9 exhibit lung hypoplasia and early postnatal death. Fgf9(-/-) lungs exhibit reduced mesenchyme and decreased branching of airways, but show significant distal airspace formation and pneumocyte differentiation. Our results suggest that Fgf9 affects lung size by stimulating mesenchymal proliferation. The reduction in the amount of mesenchyme in Fgf9(-/-) lungs limits expression of mesenchymal Fgf10. We suggest a model whereby FGF9 signaling from the epithelium and reciprocal FGF10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis.     

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.     

VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis

Vascular endothelial growth factor-A (VEGF-A) signaling directs both vasculogenesis and angiogenesis. However, the role of VEGF-A ligand signaling in the regulation of epithelial-mesenchymal interactions during early mouse lung morphogenesis remains incompletely characterized. Fetal liver kinase-1 (Flk-1) is a VEGF cognate receptor (VEGF-R2) expressed in the embryonic lung mesenchyme. VEGF-A, expressed in the epithelium, is a high affinity ligand for Flk-1. We have used both gain and loss of function approaches to investigate the role of this VEGF-A signaling pathway during lung morphogenesis. Herein, we demonstrate that exogenous VEGF 164, one of the 3 isoforms generated by alternative splicing of the Vegf-A gene, stimulates mouse embryonic lung branching morphogenesis in culture and increases the index of proliferation in both epithelium and mesenchyme. In addition, it induces differential gene and protein expression among several key lung morphogenetic genes, including up-regulation of BMP-4 and Sp-c expression as well as an increase in Flk-1-positive mesenchymal cells. Conversely, embryonic lung culture with an antisense oligodeoxynucleotide (ODN) to the Flk-1 receptor led to reduced epithelial branching, decreased epithelial and mesenchymal proliferation index as well as downregulating BMP-4 expression. These results demonstrate that the VEGF pathway is involved in driving epithelial to endothelial crosstalk in embryonic mouse lung morphogenesis.     

Protein phosphatase inhibitors arrest cell cycle and reduce branching morphogenesis in fetal rat lung cultures

Protein phosphatase 2A (PP2A) is a key signal transduction intermediate in the regulation of cellular proliferation and differentiation in vitro. However, the role of PP2A in the context of a developing organ is unknown. To explore the role of PP2A in the regulation of lung development, we studied the effect of PP2A inhibition on new airway branching, induction of apoptosis, DNA synthesis, and expression of epithelial marker genes in whole organ explant cultures of embryonic (E14) rat lung. Microdissected lung primordia were cultured in medium containing one of either two PP2A inhibitors, okadaic acid (OA, 0-9 nM) or cantharidin (Can, 0-3,600 nM), or with the PP2B inhibitor deltamethrin (Del, 0-10 microM) as a control for a PP2A-specific effect for 48 h. PP2A inhibition with OA and Can significantly inhibited airway branching and overall lung growth. PP2B inhibition with Del did not affect lung growth or new airway development. Histologically, both PP2A- and PP2B-inhibited explants were similar to controls. Increased apoptosis was not the mechanism of decreased lung growth and new airway branching inasmuch as OA-treated explant sections subjected to the terminal deoxynucleotidyltransferase dUTP nick end labeling reaction demonstrated a decrease in apoptosis. However, PP2A inhibition with OA increased DNA content and 5-bromo-2'-deoxyuridine uptake that correlated with a G(2)/M cell cycle arrest. PP2A inhibition also resulted in altered differentiation of the respiratory epithelium as evidenced by decreased mRNA levels of the early epithelial marker surfactant protein C. These findings suggest that inhibition of protein phosphatases with OA and Can halted mesenchymal cell cycle progression and reduced branching morphogenesis in fetal rat lung explant culture.     

Mesodermal deletion of transforming growth factor-beta receptor II disrupts lung epithelial morphogenesis: cross-talk between TGF-beta and Sonic hedgehog pathways

In vertebrates, Sonic hedgehog (Shh) and transforming growth factor-beta (TGF-beta) signaling pathways occur in an overlapping manner in many morphogenetic processes. In vitro data indicate that the two pathways may interact. Whether such interactions occur during embryonic development remains unknown. Using embryonic lung morphogenesis as a model, we generated transgenic mice in which exon 2 of the TbetaRII gene, which encodes the type II TGF-beta receptor, was deleted via a mesodermal-specific Cre. Mesodermal-specific deletion of TbetaRII (TbetaRII(Delta/Delta)) resulted in embryonic lethality. The lungs showed abnormalities in both number and shape of cartilage in trachea and bronchi. In the lung parenchyma, where epithelial-mesenchymal interactions are critical for normal development, deletion of mesenchymal TbetaRII caused abnormalities in epithelial morphogenesis. Failure in normal epithelial branching morphogenesis in the TbetaRII(Delta/Delta) lungs caused cystic airway malformations. Interruption of the TbetaRII locus in the lung mesenchyme increased mRNA for Patched and Gli-1, two downstream targets of Shh signaling, without alterations in Shh ligand levels produced in the epithelium. Therefore, we conclude that TbetaRII-mediated signaling in the lung mesenchyme modulates transduction of Shh signaling that originates from the epithelium. To our knowledge, this is the first in vivo evidence for a reciprocal and novel mode of cross-communication between Shh and TGF-beta pathways during embryonic development.     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

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.     

Expression of Spred and Sprouty in developing rat lung

Sproutys and Sprouty-related proteins, Spred-1 and -2, are known inhibitors of fibroblast growth factor (FGF) signaling, which plays key role in lung branching morphogenesis and the development of other tissues. The present study demonstrates that Spreds are expressed in a variety of rat embryonic tissues (brain, intestine, heart, skin) including the lung. In the embryonic lung, Spreds and Sproutys are expressed during the early stages of branching morphogenesis, but their expression profiles are both distinct and overlapping. Spreds are predominantly expressed in mesenchymal cells in contrast to Sproutys, which are abundantly expressed in epithelial cells. Spred expression is especially strong in the regions of new bud formation both in the peripheral mesenchyme as well as in the epithelium. The peripheral region also expresses FGF-10 in the mesenchymal cells and FGF-9 in the mesothelial cells. The expression profiles suggest that Spreds, Sproutys and FGF-9/FGF-10 are part of epithelial-mesenchymal interactions, which are essential for the development and maintenance of normal lung branching pattern.     

Expression of Spred and Sprouty in developing rat lung

Sproutys and Sprouty-related proteins, Spred-1 and -2, are known inhibitors of fibroblast growth factor (FGF) signaling, which plays key role in lung branching morphogenesis and the development of other tissues. The present study demonstrates that Spreds are expressed in a variety of rat embryonic tissues (brain, intestine, heart, skin) including the lung. In the embryonic lung, Spreds and Sproutys are expressed during the early stages of branching morphogenesis, but their expression profiles are both distinct and overlapping. Spreds are predominantly expressed in mesenchymal cells in contrast to Sproutys, which are abundantly expressed in epithelial cells. Spred expression is especially strong in the regions of new bud formation both in the peripheral mesenchyme as well as in the epithelium. The peripheral region also expresses FGF-10 in the mesenchymal cells and FGF-9 in the mesothelial cells. The expression profiles suggest that Spreds, Sproutys and FGF-9/FGF-10 are part of epithelial-mesenchymal interactions, which are essential for the development and maintenance of normal lung branching pattern.     

The role of integrin α8β1 in fetal lung morphogenesis and injury

Prenatal inflammation prevents normal lung morphogenesis and leads to bronchopulmonary dysplasia (BPD), a common complication of preterm birth. We previously demonstrated in a bacterial endotoxin mouse model of BPD that disrupting fibronectin localization in the fetal lung mesenchyme causes arrested saccular airway branching. In this study we show that expression of the fibronectin receptor, integrin α8β1 is decreased in the lung mesenchyme in the same inflammation model suggesting it is required for normal lung development. We verified a role for integrin α8β1 in lung development using integrin α8-null mice, which develop fusion of the medial and caudal lobes as well as abnormalities in airway division. We further show in vivo and in vitro that α8-null fetal lung mesenchymal cells fail to form stable adhesions and have increased migration. Thus we propose that integrin α8β1 plays a critical role in lung morphogenesis by regulating mesenchymal cell adhesion and migration. Furthermore, our data suggest that disruption of the interactions between extracellular matrix and integrin α8β1 may contribute to the pathogenesis of BPD.     

Emerging pulmonary vasculature lacks fate specification

Lung morphogenesis requires precise coordination between branching morphogenesis and vascularization to generate distal airways capable of supporting respiration at the cell-cell interface. The specific origins and types of blood vessels that initially form in the lung, however, remain obscure. Herein, we definitively show that during the early phases of lung development [i.e., embryonic day (E) 11.5], functional vessels, replete with blood flow, are restricted to the mesenchyme, distal to the epithelium. However, by day E14.5, and in response to epithelial-derived VEGF signals, functional vessels extend from the mesenchyme to the epithelial interface. Moreover, these vessels reside adjacent to multipotent mesenchymal stromal cells that likely play a regulatory role in this process. As well as and distinct from the systemic vasculature, immunostaining for EphrinB2 and EphB4 revealed that arterial and venous identity is not distinguishable in emergent pulmonary vasculature. Collectively, this study provides evidence that lung vascularization initially originates in the mesenchyme, distal to the epithelium, and that arterial-venous specification does not exist in the early lung. At a mechanistic level, we show that basilar epithelial VEGF prompts endothelial cells to move toward the epithelium where they undergo morphogenesis during the proliferative, canalicular stage. Thus our findings challenge existing notions of vascular origin and identity during development.     

Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung.

Mouse lung development begins when two lung buds sprout from the epithelium of the embryonic gut. Patterning of the airways is then accomplished by the outgrowth and repetitive branching of the two lung buds, a process called branching morphogenesis. One of the four fibroblast growth factor (FGF) receptor genes, FGFR2, is expressed in the epithelium of a number of embryonic organs including the lung buds. To block the function of FGFR2 during branching morphogenesis of the lung without affecting its function in other embryonic tissues, the human surfactant protein C promoter was used to target expression of a dominant negative FGFR2 exclusively to lung bud epithelium in transgenic mice. Newborn mice expressing the transgene were completely normal except that instead of normally developed lungs they had two undifferentiated epithelial tubes that extended from the bifurcation of the trachea down to the diaphragm, a defect that resulted in perinatal death. Thus, the dominant negative FGF receptor completely blocked airway branching and epithelial differentiation, without prohibiting outgrowth, establishing a specific role for FGFs in branching morphogenesis of the mammalian lung.     

Interaction of epithelium with mesenchyme affects global features of lung architecture: a computer model of development.

Lung airway morphogenesis is simulated in a simplified diffusing environment that simulates the mesenchyme to explore the role of morphogens in airway architecture development. Simple rules govern local branching morphogenesis. Morphogen gradients are modeled by four pairs of sources and their diffusion through the mesenchyme. Sensitivity to lobar architecture and mesenchymal morphogen are explored. Even if the model accurately represents observed patterns of local development, it could not produce realistic global patterns of lung architecture if interaction with its environment was not taken into account, implying that reciprocal interaction between airway growth and morphogens in the mesenchyme plays a critical role in producing realistic global features of lung architecture.     

Patterning parameters associated with the branching of the ureteric bud regulated by epithelial-mesenchymal interactions

The mechanisms by which the branching of epithelial tissue occurs and is regulated to generate different organ structures are not well understood. In this work, image analyses of the organ rudiments demonstrate specific epithelial branching patterns for the early lung and kidney; the lung type typically generating several side branches, whereas kidney branching was mainly dichotomous. Parameters such as the number of epithelial tips, the angle of the first branch, the position index of the first branch (PIFB) in a module, and the percentage of epithelial module type (PMT) were analysed. The branching patterns in the cultured lung and kidney, and in homotypic tissue recombinants recapitulated their early in vivo branching patterns. The parameters were applied to heterotypic tissue recombinants between lung mesenchyme and ureteric bud, and tip number, PIFB and PMT values qualified the change in ureter morphogenesis and the reprogramming of the ureteric bud with lung mesenchyme. All the values for the heterotypic recombinant between ureteric bud and lung mesenchyme were significantly different from those for kidney samples but similar to those of the lung samples. Hence, lung mesenchyme can instruct the ureteric bud to undergo aspects of early lung-type epithelial morphogenesis. Different areas of the lung mesenchyme, except the tracheal region, were sufficient to promote ureteric bud growth and branching. In conclusion, our findings provide morphogenetic parameters for monitoring epithelial development in early embryonic lung and kidney and demonstrate the use of heterotypic tissue recombinants as a model for studying tissue-specific epithelial branching during organogenesis.