Hiroyuki Nogawa

FGF-1 and FGF-7 Induce Distinct Patterns of Growth and Differentiation in Embryonic Lung Epithelium

Fibroblast growth factors (FGFs) and receptors (FGFRs) are expressed in the developing lung and appear to be major regulators of lung growth and differentiation. By using mesenchyme‐free lung epithelial cultures we show that FGF‐1 (aFGF) and FGF‐7 (KGF) produce different effects in the developing lung. FGF‐1 stimulates epithelial proliferation that results in bud formation (branching), while FGF‐7 promotes epithelial proliferation that leads to formation of cyst‐like structures. In addition, FGF‐7 stimulates epithelial differentiation, stimulating expression of SP‐A and SP‐B mRNA throughout the explant, and inducing formation of focal areas of highly differentiated cells. The FGF‐1 effects on differentiation are limited to induction of surfactant protein SP‐B mRNA at the tips of the explant. The FGF‐induced patterns of growth appear to correlate with the distribution of epithelial FGFRs mRNAs; FGFR‐2 IIIb (KGFR) is diffusely expressed in the day 11 lung epithelium, while FGFR‐4 appears in distal but not in proximal sites. We propose that cyst‐like structures may result from FGF‐7 binding to the uniformly distributed FGFR‐2‐IIIb. Lung bud formation may be regulated by FGF‐1 and/or other ligands binding to FGFR‐2 and a distally located FGFR, such as FGFR‐4, leading to an increasing binding and activation of FGFRs at the tips of the explant. Thus, in the embryonic lung epithelium, growth effects of FGFs appear to be dependent on location of FGFRs, while effects on differentiation are ligand‐dependent. Dev. Dyn. 208:398–405, 1997.

Bud formation precedes the appearance of differential cell proliferation during branching morphogenesis of mouse lung epithelium in vitro

Cell proliferation is an essential requirement for epithelial expansion and tubular branching; however, little is known of how these events are coupled during morphogenesis. We have previously shown that, in the absence of mesenchyme, fibroblast growth factor 1 (FGF‐1) elicits budding of the mouse lung epithelium cultured in a basement membrane matrix. Although bud formation seems to be the manifestation of a localized response of lung epithelial cells to FGF‐1, it is unclear whether budding results from induction of differential rates of cell proliferation within the epithelium. We performed continuous labeling and pulse‐chase experiments in FGF‐1‐treated mesenchyme‐free lung epithelial cultures at distinct stages of bud induction using bromodeoxyuridine (BrdU), to determine when and to what extent cell proliferation contributes to bud formation. When explants were incubated with BrdU either before bud induction (0–18 hr in culture) or at the onset of budding (24–30 hr), labeled nuclei were found distributed throughout the entire explant. In contrast, BrdU incubation after the onset of budding (30–48 hr) resulted in labeling concentrated in the budding areas, and a decrease of labeling toward the proximal region of the explant, between buds. These results demonstrate that differential rates of cell proliferation between bud and nonbud areas do not appear until when buds are almost completely formed. Thus, in the developing lung epithelium in vitro, bud outgrowth is not triggered by induction of localized cell proliferation. Dev. Dyn. 1998;213:228–235.

EGF‐dependent lobule formation and FGF7‐dependent stalk elongation in branching morphogenesis of mouse salivary epithelium in vitro

When supplemented with appropriate growth factors, salivary gland epithelial explants isolated from mouse embryos undergo branching morphogenesis in vitro in the absence of mesenchyme. Epidermal growth factor (EGF) induces lobule formation, while fibroblast growth factor 7 (FGF7) promotes stalk elongation. A mixture of EGF and FGF7 produces an intermediate morphology, which resembles the branching pattern of salivary epithelium observed in vivo. To investigate how lobule formation and stalk elongation are related to the pattern of epithelial cell proliferation induced by EGF and FGF7, we performed a bromodeoxyuridine labeling study in whole‐mount preparations. During the initial steps of lobule formation in EGF cultures, cleft and non‐cleft regions had similar proliferative activity. However, once clefts had fully deepened, cells with low proliferative activity appeared at the bottom of the clefts. In contrast, during stalk elongation in FGF7 cultures, distal regions of the explants always showed higher proliferative activity than proximal regions. These results suggest that stalk elongation, but not cleft formation, may result from differential cell proliferation. Dev Dyn 1999;215:148–154.

Significant role of laminin-1 in branching morphogenesis of mouse salivary epithelium cultured in basement membrane matrix.

Mouse submandibular epithelium shows branching morphogenesis in mesenchyme‐free conditions when covered with a basement membrane matrix (Matrigel) in medium supplemented with epidermal growth factor. In the present study, the role of laminin‐1 (LN1), a major glycoprotein of Matrigel, in this culture system was defined. When the epithelium was cultured in a LN1‐nidogen gel, the epithelium showed much branching, comparable to that observed with Matrigel. By electron microscopy, only a felt‐like matrix was formed on the epithelial surface in the LN1‐nidogen gel cultures, while an organized basal lamina structure was formed on the epithelial surface in direct or transfilter recombination cultures with mesenchyme. Next, the epithelium covered with Matrigel was cultured in medium containing either biologically active peptides from LN1, IKVAV‐including peptide (2097–2108), AG10 (2183–2194), AG32 (2370–2381) or AG73 (2719–2730) from the α1 chain, or YIGSR‐including peptide (926–933) from the β1 chain. Only AG73 (RKRLQVQLSIRT from the α1 chain carboxyl‐terminal globular domain) inhibited the epithelial branching in Matrigel. These results suggest that LN1‐nidogen can support the branching morphogenesis of submandibular epithelium even if LN1‐nidogen is not assembled into an intact basal lamina, and that the AG73 sequence is an important site on LN1, which interacts with submandibular epithelial cells.

Induction of bud formation of embryonic mouse tracheal epithelium by fibroblast growth factor plus transferrin in mesenchyme-free culture

Embryonic mouse tracheal epithelium, which branches in an epithelial–mesenchymal recombination culture with bronchial mesenchyme, was cultured under mesenchyme‐free conditions. When embedded in a basement‐membrane–like matrix and cultured in a serum‐free medium supplemented with fibroblast growth factor 1 (FGF1), the tracheal epithelium did not branch, whereas the bronchial epithelium underwent branching morphogenesis. When the medium was enriched with transferrin (Tf), bud formation was induced in the tracheal epithelium and some buds branched secondarily. FGF7 and FGF10, in cooperation with Tf, induced tracheal bud formation to the same extent as FGF1, although the optimum concentrations differed. A bromodeoxyuridine‐labeling study comparing cultures with and without Tf showed no Tf‐specific amplification of cell proliferation. A whole‐mount in situ hybridization study of the expression of Bmp4 and Shh genes in explants of mesenchyme‐free culture revealed that both genes were ubiquitously expressed and that expression did not correlate with bud formation. This expression pattern was different from the distally localized expression pattern observed in normal lung rudiments and in extratracheal buds induced by the recombined bronchial mesenchyme. These results suggest that both bronchial and tracheal bud formations were initiated without localized exposure of the epithelium to FGFs and were not accompanied by localized expression of Bmp4 and Shh in the epithelium.

Lysophosphatidic acid cooperates with EGF in inducing branching morphogenesis of embryonic mouse salivary epithelium

Epithelial morphogenesis is supported by diffusible growth factors and by nondiffusible cell substrata, such as laminin and fibronectin. When embedded in a laminin‐rich basement‐membrane substratum, embryonic mouse submandibular epithelium undergoes cell proliferation and branching morphogenesis in response to epidermal growth factor (EGF) in mesenchyme‐free culture but not in serum‐free medium. In this study, we sought to identify the biologically active factor in serum. As this factor was heat‐stable and trypsin‐resistant, the lipid fraction was analyzed. Horse serum was fractionated by ethanol extraction, Folch partition with chloroform–methanol–water, and high‐performance liquid chromatography, and we tested the branch‐inducing activity of each fraction. We also analyzed the partially purified fraction with a mass spectrometer, indicating that the active fraction largely consisted of lysophosphatidyl‐hexose. Finally we identified the molecule as lysophosphatidic acid (LPA), because, whereas lysophosphatidyl‐inositol had only a slight branch‐inducing activity, its relevant LPA fully substituted for serum and induced branching morphogenesis in cooperation with EGF. LPA receptor genes were expressed in submandibular epithelial cells. DNA‐synthesizing cells were abundant only when cultured in the presence of both EGF and LPA, but not either singly. Developmental Dynamics 235:403–410, 2006.

FGF alters epithelial competence for EGF at the initiation of branching morphogenesis of mouse submandibular gland.

Embryonic day 13 mouse submandibular gland (E13‐SMG) rudiments with two to four clefts have been commonly used in culture experiments to show that growth factors, such as epidermal growth factor (EGF) ‐family and fibroblast growth factor (FGF) ‐family ligands, are involved in branching morphogenesis. In the present study, we focused on E12 rudiments and attempted to elucidate the roles of EGF‐ and FGF‐family ligands in SMG development from E12 to E13. In mesenchyme‐free, Matrigel‐embedded cultures, EGF + lysophosphatidic acid (LPA) induced branching in E13 epithelium, whereas E12 epithelium remained spherical and no branching occurred under the same culture conditions; however, both E12 and E13 epithelia elongated in response to FGF10. Reverse transcriptase‐polymerase chain reaction studies showed that the expression of ErbB1 among four EGF receptors and Lpa3 among three LPA receptors was lower in E12 than in E13 epithelia. Fgf10, Fgf7, and their major receptor Fgfr2b were highly and equally expressed in E12 and E13 rudiments. After 24 hr of mesenchyme‐free culture with FGF10 or FGF7, E12 epithelium was primed to initiate branching morphogenesis in response to EGF + LPA coincident with ErbB1 and Lpa3 up‐regulation. These results suggest that the EGF‐family ligand–receptor system is undeveloped at E12 and that it becomes primed on E13 by the FGF ligand–receptor system to play an important role in the induction of branching morphogenesis. Developmental Dynamics 238:315–323, 2009.

FGF7 signals are relayed to autocrine EGF family growth factors to induce branching morphogenesis of mouse salivary epithelium

Background: The Matrigel‐embedded epithelium of the mouse submandibular gland undergoes branching morphogenesis when cultured in medium supplemented with fibroblast growth factor 7 (FGF7) and lysophosphatidic acid (LPA), whereas it elongates a stalk with limited branching in medium with only FGF7. Because LPA is a well‐known activator of epidermal growth factor (EGF) signaling, we hypothesized the involvement of autocrine EGF family growth factors in the branching morphogenesis. Results: Reverse transcriptase polymerase chain reaction studies showed that three members, Tgfa, Hbegf,and Nrg1 of the EGF family were expressed in the epithelium cultured with FGF7 + LPA as well as in the epithelium freshly isolated from the rudiments. All the growth factors induced extensive branching morphogenesis in the Matrigel‐embedded epithelium in the presence of LPA. Tyrphostin AG112, an inhibitor of EGF signaling, severely impaired branching morphogenesis induced by FGF7 + LPA without exogenous addition of EGF family growth factors to the culture medium. The shaking cultures, which were expected to decrease the concentration of autocrine growth factors near the epithelium by promoting their diffusion, significantly reduced branching morphogenesis induced by FGF7 + LPA. Conclusions: Autocrine EGF family growth factors are involved in epithelial branching morphogenesis induced by FGF7 + LPA. Developmental Dynamics 243:552–559, 2014.

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.

Autocrine Growth Factors Are Involved in Branching Morphogenesis of Mouse Lung Epithelium

The current model for branching morphogenesis of mouse lung proposes that the epithelium bifurcates as cells pursue separate sources of fibroblast growth factor (FGF) 10, secreted from mesenchymal tissue through interactions with epithelial tissue. If so, it may be assumed that the lung epithelium will grow into a uniform, expanding ball (without branching) when uniformly exposed to a constant concentration of FGF10. To test this hypothesis, we cultured Matrigel-embedded lung epithelium explants in FGF10-supplemented medium while shaking the culture dishes. Shaking cultures with FGF10 resulted in inferior epithelial branching compared to control cultures at rest. However, this effect was unexpectedly accompanied by poor growth rather than by ball-like expansion. When using FGF1, epithelial cultures grew and branched similarly well under either culture condition. Thus, we hypothesized that FGF10 signaling must be mediated by autocrine FGFs, such as FGF1, which might easily diffuse through the culture medium in the shaking culture. Reverse transcription-polymerase chain reaction analyses showed that FGF9 as well as FGF1 were expressed in the epithelium in vivo and in FGF10-stimulated epithelium in vitro, and FGF9 induced epithelial branching at a much lower concentration than FGF10. These results suggest that FGF1 and FGF9 may mediate FGF10 signaling and induce branching in the lung epithelium via autocrine signaling.