Time-lapse observation of branching morphogenesis of the lung bud epithelium in mesenchyme-free culture and its relationship with the localization of actin filaments

It has been shown that branching morphogenesis of the lung bud is mediated by epithelial-mesenchymal interaction via such molecules as FGF10, BMP4 and Shh. However, a recent study showed that the isolated lung epithelium still undergoes branching morphogenesis in vitro even in the absence of mesenchyme (Nogawa and Ito, 1995). In the present study, we observed in vitro the dynamic movement of the isolated lung epithelium of the fetal mouse using time-lapse recording, and investigatedthe roles of actinfilaments in branching of the lung bud. First, time-lapse observation of the initial phase of lung branching morphogenesis revealed that at the sites of cleft formation, the epithelial surface was retracted inward from its original position. From this observation we assumed that there should be some structures which exert a physical force on the epithelium, and the localization and arrangement of actin fibers in the cultured lung epithelium were examined at various stages of branching morphogenesis. At the prebudding (6 h) and onset-budding (24 h) stages, no specific localization of actin filaments was observed in the lung bud epithelium, but at the postbudding stage (48 h) they were localized densely in the cells at the tip of the branched lung epithelium. The cell density was not different between the tip and cleft regions of the lung bud epithelium. When cultured with FGF-soaked beads, an actin-rich region was induced at the tip of the lung bud which was growing toward an FGF-soaked bead. These results indicate that actin fibers do not play a significant part in cleft formation but can be secondarily induced by FGF in the surrounding matrix and play some roles at later shaping of the branch in lung morphogenesis.     

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.     

Heparan sulfate-FGF10 interactions during lung morphogenesis

Signaling by fibroblast growth factor 10 (FGF10) through FGFR2b is essential for lung development. Heparan sulfates (HS) are major modulators of growth factor binding and signaling present on cell surfaces and extracellular matrices of all tissues. Although recent studies provide evidence that HS are required for FGF-directed tracheal morphogenesis in Drosophila, little is known about the HS role in FGF10-mediated bud formation in the vertebrate lung. Here, we mapped HS expression in the early lung and we investigated how HS interactions with FGF10-FGFR2b influence lung morphogenesis. Our data show that a specific set of HS low in O-sulfates is dynamically expressed in the lung mesenchyme at the sites of prospective budding near Fgf10-expressing areas. In turn, highly sulfated HS are present in basement membranes of branching epithelial tubules. We show that disrupting endogenous gradients of HS or altering HS sulfation in embryonic lung culture systems prevents FGF10 from inducing local responses and markedly alters lung pattern formation and gene expression. Experiments with selectively sulfated heparins indicate that O-sulfated groups in HS are critical for FGF10 signaling activation in the epithelium during lung bud formation, and that the effect of FGF10 in pattern is in part determined by regional distribution of O-sulfated HS. Moreover, we describe expression of a HS 6-O-sulfotransferase preferentially at the tips of branching tubules. Our data suggest that the ability of FGF10 to induce local budding is critically influenced by developmentally regulated regional patterns of HS sulfation.     

Phosphoinositide 3-Kinase Alpha-Dependent Regulation of Branching Morphogenesis in Murine Embryonic Lung: Evidence for a Role in Determining Morphogenic Properties of FGF7

Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling.     

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.     

Chondroitin sulfate proteoglycans are required for lung growth and morphogenesis in vitro

Proteoglycans (PGs) have been shown to play a key role in the development of many tissues. We have investigated the role of sulfated PGs in early rat lung development by treating cultured tissues with 30 mM sodium chlorate, a global inhibitor of PG sulfation. Chlorate treatment disrupted growth and branching of embryonic day 13 lung explants. Isolated lung epithelium (LgE) migrated toward and invaded lung mesenchyme (LgM), and chlorate irreversibly suppressed this response. Chlorate also inhibited migration of LgE toward beads soaked in FGF10. Chlorate severely decreased branching morphogenesis in tissue recombinants consisting of LgM plus either LgE or tracheal epithelium (TrE) and decreased expression of surfactant protein C gene (SP-C). Chlorate also reduced bone morphogenetic protein-4 expression in cultured tips and recombinants but had no effect on the expression of clara cell 10-kDa protein (CC10), sonic hedgehog (Shh), FGF10, and FGF receptor 2IIIb. Chlorate reduced the growth of LgE in mesenchyme-free culture but did not affect SP-C expression. In contrast, chlorate inhibited both rudiment growth and the induction of SP-C in mesenchyme-free cultured TrE. Treatment of lung tips and tissue recombinants with chondroitinase ABC abolished branching morphogenesis. Chondroitinase also suppressed growth of TrE in mesenchyme-free culture. Chondroitinase treatment, however, had no effect on the induction of SP-C expression in any of these cultures. These results demonstrate the overall importance of sulfated PGs to normal lung development and demonstrate a dynamic role for chondroitin sulfate PGs in embryonic lung growth and morphogenesis.     

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.     

FGF-10 induces SP-C and Bmp4 and regulates proximal-distal patterning in embryonic tracheal epithelium

The induction, growth, and differentiation of epithelial lung buds are regulated by the interaction of signals between the lung epithelium and its surrounding mesenchyme. Fibroblast growth factor-10 (FGF-10), which is expressed in the mesenchyme near the distal tips, and bone morphogenetic protein 4 (BMP4), which is expressed in the most distal regions of the epithelium, are important molecules in lung morphogenesis. In the present study, we used two in vitro systems to examine the induction, growth, and differentiation of lung epithelium. Transfilter cultures were used to determine the effect of diffusible factors from the distal lung mesenchyme (LgM) on epithelial branching, and FGF-10 bead cultures were used to ascertain the effect of a high local concentration of a single diffusible molecule on the epithelium. Embryonic tracheal epithelium (TrE) was induced to grow in both culture systems and to express the distal epithelial marker surfactant protein C at the tips nearest the diffusible protein source. TrE cultured on the opposite side of a filter to LgM branched in a pattern resembling intact lungs, whereas TrE cultured in apposition to an FGF-10 bead resembled a single elongating epithelial bud. Examination of the role of BMP4 on lung bud morphogenesis revealed that BMP4 signaling suppressed expression of the proximal epithelial genes Ccsp and Foxj1 in both types of culture and upregulated the expression of Sprouty 2 in TrE cultured with an FGF-10 bead. Antagonizing BMP signaling with Noggin, however, increased expression of both Ccsp and Foxj1.     

Modelling in vitro lung branching morphogenesis during development

It has been shown experimentally that lung epithelial explants have an ability to undergo branching morphogenesis without mesenchyme. However, the mechanisms of this phenomenon remain to be elucidated. In the present study, we construct a mathematical model that can reproduce the dynamics of in vitro branching morphogenesis. We show that the system is essentially governed by three variables--c(0) which is the initial fibroblast growth factor (FGF) concentration, D which is the diffusion coefficient of FGF, and beta which describes the mechanical strength of the cytoskeleton. It is confirmed by numerical simulations that this model can reproduce the experimentally obtained patterns qualitatively. Finally, we experimentally verify two predictions from the model: effects of very high FGF concentration and effects of small mechanical contributions of the cytoskeleton. The theoretical predictions match well with the experimental results.     

FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis

Branching morphogenesis of mouse submandibular glands is regulated by multiple growth factors. Here, we report that ex vivo branching of intact submandibular glands decreases when either FGFR2 expression is downregulated or soluble recombinant FGFR2b competes out the endogenous growth factors. However, a combination of neutralizing antibodies to FGF1, FGF7 and FGF10 is required to inhibit branching in the intact gland, suggesting that multiple FGF isoforms are required for branching. Exogenous FGFs added to submandibular epithelial rudiments cultured without mesenchyme induce distinct morphologies. FGF7 induces epithelial budding, whereas FGF10 induces duct elongation, and both are inhibited by FGFR or ERK1/2 signaling inhibitors. However, a PI3-kinase inhibitor also decreases FGF7-mediated epithelial budding, suggesting that multiple signaling pathways exist. We immunolocalized FGF receptors and analyzed changes in FGFR, FGF and MMP gene expression to identify the mechanisms of FGF-mediated morphogenesis. FGFR1b and FGFR2b are present throughout the epithelium, although FGFR1b is more highly expressed around the periphery of the buds and the duct tips. FGF7 signaling increases FGFR1b and FGF1 expression, and MMP2 activity, when compared with FGF10, resulting in increased cell proliferation and expansion of the epithelial bud, whereas FGF10 stimulates localized proliferation at the tip of the duct. FGF7- and FGF10-mediated morphogenesis is inhibited by an MMP inhibitor and a neutralizing antibody to FGF1, suggesting that both FGF1 and MMPs are essential downstream mediators of epithelial morphogenesis. Taken together, our data suggests that FGFR2b signaling involves a regulatory network of FGFR1b/FGF1/MMP2 expression that mediates budding and duct elongation during branching morphogenesis.     

Iroquois genes influence proximo-distal morphogenesis during rat lung development

Lung development is a highly regulated process directed by mesenchymal-epithelial interactions, which coordinate the temporal and spatial expression of multiple regulatory factors required for proper lung formation. The Iroquois homeobox (Irx) genes have been implicated in the patterning and specification of several Drosophila and vertebrate organs, including the heart. Herein, we investigated whether the Irx genes play a role in lung morphogenesis. We found that Irx1-3 and Irx5 expression was confined to the branching lung epithelium, whereas Irx4 was not expressed in the developing lung. Antisense knockdown of all pulmonary Irx genes together dramatically decreased distal branching morphogenesis and increased distention of the proximal tubules in vitro, which was accompanied by a reduction in surfactant protein C-positive epithelial cells and an increase in beta-tubulin IV and Clara cell secretory protein positive epithelial structures. Transmission electron microscopy confirmed the proximal phenotype of the epithelial structures. Furthermore, antisense Irx knockdown resulted in loss of lung mesenchyme and abnormal smooth muscle cell formation. Expression of fibroblast growth factors (FGF) 1, 7, and 10, FGF receptor 2, bone morphogenetic protein 4, and Sonic hedgehog (Shh) were not altered in lung explants treated with antisense Irx oligonucleotides. All four Irx genes were expressed in Shh- and Gli(2)-deficient murine lungs. Collectively, these results suggest that Irx genes are involved in the regulation of proximo-distal morphogenesis of the developing lung but are likely not linked to the FGF, BMP, or Shh signaling pathways.     

FGF8 dose-dependent regulation of embryonic submandibular salivary gland morphogenesis

FGF8 has been shown to play important morphoregulatory roles during embryonic development. The observation that craniofacial, cardiovascular, pharyngeal, and neural phenotypes vary with Fgf8 gene dosage suggests that FGF8 signaling induces differences in downstream responses in a dose-dependent manner. In this study, we investigated if FGF8 plays a dose-dependent regulatory role during embryonic submandibular salivary gland (SMG) morphogenesis. We evaluated SMG phenotypes of Fgf8 hypomorphic mice, which have decreased Fgf8 gene function throughout embryogenesis. We also evaluated SMG phenotypes of Fgf8 conditional mutants in which Fgf8 function has been completely ablated in its expression domain in the first pharyngeal arch ectoderm from the time of arch formation. Fgf8 hypomorphs have hypoplastic SMGs, whereas conditional mutant SMGs exhibit ontogenic arrest followed by involution and are absent by E18.5. SMG aplasia in Fgf8 ectoderm conditional mutants indicates that FGF8 signaling is essential for the morphogenesis and survival of Pseudoglandular Stage and older SMGs. Equally important, the presence of an initial SMG bud in Fgf8 conditional mutants indicates that initial bud formation is FGF8 independent. Mice heterozygous for either the Fgf8 null allele (Fgf8(+/N)) or the hypomorphic allele (Fgf8(+/H)) have SMGs that are indistinguishable from wild-type (Fgf8(+/+)) mice which suggest that there is not only an FGF8 dose-dependent phenotypic response, but a nonlinear, threshold-like, epistatic response as well. We also found that enhanced FGF8 signaling induced, and abrogated FGF8 signaling decreased, SMG branching morphogenesis in vitro. Furthermore, since FGF10 and Shh expression is modulated by Fgf8 levels, we postulated that exogenous FGF10, Shh, or FGF10 + Shh peptide supplementation in vitro would largely "rescue" the abnormal SMG phenotype associated with decreased FGF8 signaling. This is as expected, though there is no synergistic effect with FGF10 + Shh peptide supplementation. These in vitro experiments model the principle that mutations have different effects in the context of different epigenotypes.     

Potential role of RGD-binding integrins in mammalian lung branching morphogenesis.

Cell-matrix interactions are generally considered critical for normal lung development. This is particularly likely to be true during the glandular stage, when the primitive airways are formed through a process termed branching morphogenesis. Integrins, transmembrane receptors that bind to extracellular matrices, are likely to mediate important interactions between embryonic cells and their matrices during branching morphogenesis. In this report, we examine the role of integrin receptors in this process. Immunohistochemical studies revealed that the integrins VLA 3, VLA 5 and integrin receptors to vitronectin are expressed in the epithelium and/or mesenchyme during the glandular stage of murine lung development. To correlate expression with function, an in vitro model of murine lung branching morphogenesis was utilized to examine branching in the presence of inhibitors of ligand binding to integrin receptors. One such reagent, a hexapeptide containing the RGD (Arg-Gly-Asp) sequence, diminished branching and resulted in an abnormal morphology, whereas a control peptide RGESP (Arg-Gly-Glu-Ser-Pro) had no effect. These findings suggest a critical role for cell-matrix interactions mediated via integrin receptors in early stages of mammalian lung development.     

Heparanase cleavage of perlecan heparan sulfate modulates FGF10 activity during ex vivo submandibular gland branching morphogenesis

Heparan sulfate proteoglycans are essential for biological processes regulated by fibroblast growth factors (FGFs). Heparan sulfate (HS) regulates the activity of FGFs by acting as a coreceptor at the cell surface, enhancing FGF-FGFR affinity, and being a storage reservoir for FGFs in the extracellular matrix (ECM). Here we demonstrate a critical role for heparanase during mouse submandibular gland (SMG) branching morphogenesis. Heparanase, an endoglycosidase, colocalized with perlecan in the basement membrane and in epithelial clefts of SMGs. Inhibition of heparanase activity in organ culture decreased branching morphogenesis, and this inhibition was rescued specifically by FGF10 and not by other FGFs. By contrast, exogenous heparanase increased SMG branching and MAPK signaling and, surprisingly, when isolated epithelia were cultured in a three-dimensional ECM with FGF10, it increased the number of lateral branches and end buds. In a solid-phase binding assay, an FGF10-FGFR2b complex was released from the ECM by heparanase. In addition, surface plasmon resonance (SPR) analysis showed that FGF10 and the FGF10-FGFR2b complex bound to purified perlecan HS and could be released by heparanase. We used the FGF10-FGFR2b complex as a probe for HS in SMGs, and it colocalized with perlecan in the basement membrane and partly colocalized with syndecan 1 in the epithelium, and binding was reduced by treatment with heparanase. In summary, our results show heparanase releases FGF10 from perlecan HS in the basement membrane, increasing MAPK signaling, epithelial clefting, and lateral branch formation, which results in increased branching morphogenesis.     

Semaphorin3a inhibits ureteric bud branching morphogenesis

Class 3 semaphorins are guidance proteins involved in axon pathfinding, vascular patterning and lung branching morphogenesis in the developing mouse embryo. Semaphorin3a (Sema3a) is expressed in renal epithelia throughout kidney development, including podocytes and ureteric bud cells. However, the role of Sema3a in ureteric bud branching is unknown. Here we demonstrate that Sema3a plays a role in patterning the ureteric bud tree in both metanephric organ cultures and Sema3a mutant mice. In vitro ureteric bud injection with Sema3a antisense morpholino resulted in increased branching, whereas recombinant SEMA3A inhibited ureteric bud branching and decreased the number of developing glomeruli. Additional studies revealed that SEMA3A effects on ureteric bud branching involve downregulation of glial cell-line derived neurotrophic factor (GDNF) signaling, competition with vascular endothelial growth factor A (VEGF-A) and decreased activity of Akt survival pathways. Deletion of Sema3a in mice is associated with increased ureteric bud branching, confirming its inhibitory role in vivo. Collectively, these data suggest that Sema3a is an endogenous antagonist of ureteric bud branching and hence, plays a role in patterning the renal collecting system as a negative regulator.