Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43^−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43^−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43^−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.     

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.     

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity^1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood ⁴. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo⁵. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-111^6,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands⁸. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.     

Extracellular regulated kinase5 is expressed in fetal mouse submandibular glands and is phosphorylated in response to epidermal growth factor and other ligands of the ErbB family of receptors

Growth factors and their receptors regulate development of many organs through activation of multiple intracellular signaling cascades including a mitogen‐activated protein kinase (MAPK). Extracellular regulated kinases (ERK)1/2, classic MAPK family members, are expressed in fetal mouse submandibular glands (SMG), and stimulate branching morphogenesis. ERK5, also called big mitogen‐activated protein kinase 1, was recently found as a new member of MAPK super family, and its biological roles are still largely unknown. In this study, we investigated the expression and function of ERK5 in developing fetal mouse SMGs. Western blotting analysis showed that the expression pattern of ERK5 was different from the pattern of ERK1/2 in developing fetal SMGs. Both ERK1/2 and ERK5 were phosphorylated after exposure to ligands of the ErbB family of receptor tyrosine kinases (RTKs). Phosphorylation of ERK1/2 was strongly induced by epidermal growth factor (EGF) in SMG rudiments at embryonic day 14 (E14), E16 and E18. However, ERK5 phosphorylation induced by EGF was clearly observed at E14 and E16, but not at E18. Branching morphogenesis of cultured E13 SMG rudiments was strongly suppressed by administration of U0126, an inhibitor for ERK1/2 activation, whereas the phosphorylation of ERK5 was not inhibited by U0126. BIX02188, a specific inhibitor for ERK5 activation, also inhibited branching morphogenesis in cultured SMG rudiments. These results show that EGF‐responsive ERK5 is expressed in developing fetal mouse SMG, and suggest that both ERK1/2 and ERK5 signaling cascades might play an important role in the regulation of branching morphogenesis.     

Signaling pathways activated by epidermal growth factor receptor or fibroblast growth factor receptor differentially regulate branching morphogenesis in fetal mouse submandibular glands

Although growth factor signaling is required for embryonic development of organs, individual signaling mechanisms regulating these organotypic processes are just beginning to be defined. We compared signaling activated in fetal mouse submandibular glands (SMGs) by three growth factors, epidermal growth factor (EGF), fibroblast growth factor (FGF) 7, or FGF10, and correlated it with specific events of branching morphogenesis. Immunoblotting showed that EGF strongly stimulated phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and weakly stimulated phosphorylation of phospholipase C γ 1 (PLC γ 1) and phosphatidylinositol-3 kinase (PI3K) in cultured E14 SMG. However, FGF7 and FGF10 stimulated phosphorylation of both PLC γ 1 and PI3K, but elicited only minimal phosphorylation of ERK-1/2. Morphological study of mesenchyme-free SMG epithelium cultured in Matrigel revealed that EGF induced cleft formation of endpieces, that FGF7 stimulated both cleft formation and stalk elongation, but that FGF10 induced only stalk elongation. In mesenchyme-free SMG epithelium cultured with EGF, FGF7 and FGF10, U0126 (MEK inhibitor) completely blocked cleft formation, whereas U73122 (PLC γ 1 inhibitor) suppressed stalk elongation. These finding suggest that EGF stimulates cleft formation and drives branch formation via ERK-1/2, and that FGF7 stimulates both cleft formation and stalk elongation via PLC γ 1 and partly via ERK-1/2, but that FGF10 stimulates stalk elongation mainly via PLC γ 1.     

Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis

The mouse submandibular gland (SMG) epithelium undergoes extensive morphogenetic branching during embryonic development as the first step in the establishment of its glandular structure. However, the specific signaling pathways required for SMG branching morphogenesis are not well understood. Using E13 mouse SMG organ cultures, we showed that inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), wortmannin and LY294002, substantially inhibited branching morphogenesis in SMG. Branching morphogenesis of epithelial rudiments denuded of mesenchyme was inhibited similarly, indicating that PI 3-kinase inhibitors act directly on the epithelium. Immunostaining and Western analysis demonstrated that the p85 isoform of PI 3-kinase is expressed in epithelium at levels higher than in the mesenchyme. A target of PI 3-kinase, Akt/protein kinase B (PKB), showed decreased phosphorylation at Ser(473) by Western analysis in the presence of PI 3-kinase inhibitors. The major lipid product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), was added exogenously to SMG via a membrane-transporting carrier in the presence of PI 3-kinase inhibitors and was found to stimulate cleft formation, the first step of branching morphogenesis. Together, these data indicate that PI 3-kinase plays a role in the regulation of epithelial branching morphogenesis in mouse SMG acting through a PIP(3) pathway.     

Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis

Experimental evidence is rapidly emerging that the coupling of positive regulatory signals with the induction of negative feedback modulators is a mechanism of fine regulation in development. Studies in Drosophila and chick have shown that members of the SPROUTY family are inducible negative regulators of growth factors that act through tyrosine kinase receptors. We and others have shown that Fibroblast Growth Factor 10 (FGF10) is a key positive regulator of lung branching morphogenesis. Herein, we provide direct evidence that mSprouty2 is dynamically expressed in the peripheral endoderm in embryonic lung and is downregulated in the clefts between new branches at E12.5. We found that mSprouty2 was expressed in a domain restricted in time and space, adjacent to that of Fgf10 in the peripheral mesenchyme. By E14.5, Fgf10 expression was restricted to a narrow domain of mesenchyme along the extreme edges of the individual lung lobes, whereas mSprouty2 was most highly expressed in the subjacent epithelial terminal buds. FGF10 beads upregulated the expression of mSprouty2 in adjacent epithelium in embryonic lung explant culture. Lung cultures treated with exogenous FGF10 showed greater branching and higher levels of mSpry2 mRNA. Conversely, Fgf10 antisense oligonucleotides reduced branching and decreased mSpry2 mRNA levels. However, treatment with exogenous FGF10 or antisense Fgf10 did not change Shh and FgfR2 mRNA levels in the lungs. We investigated Sprouty2 function during lung development by two different but complementary approaches. The targeted overexpression of mSprouty2 in the peripheral lung epithelium in vivo, using the Surfactant Protein C promoter, resulted in a low level of branching, lung lobe edges abnormal in appearance and the inhibition of epithelial proliferation. Transient high-level overexpression of mSpry2 throughout the pulmonary epithelium by intra-tracheal adenovirus microinjection also resulted in a low level of branching. These results indicate for the first time that mSPROUTY2 functions as a negative regulator of embryonic lung morphogenesis and growth.     

Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium

Mouse salivary epithelium cannot undergo branching morphogenesis in the absence of the surrounding mesenchyme. To clarify the nature of the mesenchymal influence on the epithelium, we have investigated the culture conditions in which the epithelium could normally branch in the absence of mesenchymal cells. Combination of basement-membrane-like substratum (Matrigel) and epidermal growth factor (EGF) could substitute for the mesenchyme, the epithelium showing typical branching morphogenesis. Transforming growth factor alpha had the same effect as EGF. Matrigel plus basic fibroblast growth factor or transforming growth factor beta 1 and collagen gel plus EGF were not sufficient to support the branching of the epithelium. These results clearly reveal that the role of mesenchyme in salivary morphogenesis is both to provide the epithelium with an appropriate substratum and to accelerate growth of the epithelium.     

Laminin alpha5 is necessary for submandibular gland epithelial morphogenesis and influences FGFR expression through beta1 integrin signaling

Laminin alpha chains have unique spatiotemporal expression patterns during development and defining their function is necessary to understand the regulation of epithelial morphogenesis. We investigated the function of laminin alpha5 in mouse submandibular glands (SMGs). Lama5(-/-) SMGs have a striking phenotype: epithelial clefting is delayed, although proliferation occurs; there is decreased FGFR1b and FGFR2b, but no difference in Lama1 expression; later in development, epithelial cell organization and lumen formation are disrupted. In wild-type SMGs alpha5 and alpha1 are present in epithelial clefts but as branching begins alpha5 expression increases while alpha1 decreases. Lama5 siRNA decreased branching, p42 MAPK phosphorylation, and FGFR expression, and branching was rescued by FGF10. FGFR siRNA decreased Lama5 suggesting that FGFR signaling provides positive feedback for Lama5 expression. Anti-beta1 integrin antibodies decreased FGFR and Lama5 expression, suggesting that beta1 integrin signaling provides positive feedback for Lama5 and FGFR expression. Interestingly, the Itga3(-/-):Itga6(-/-) SMGs have a similar phenotype to Lama5(-/-). Our findings suggest that laminin alpha5 controls SMG epithelial morphogenesis through beta1 integrin signaling by regulating FGFR expression, which also reciprocally regulates the expression of Lama5. These data link changes in basement membrane composition during branching morphogenesis with FGFR expression and signaling.     

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.     

Platelet-derived growth factor potentiates phorbol ester-induced neuronal differentiation of human neuroblastoma cells.

Previous studies have shown that platelet-derived growth factor (PDGF) and PDGF receptors are expressed in the mammalian central nervous system and that primary cultured neuroblasts from rat hindbrain have functional PDGF beta-receptors. Here, it is shown that cultured human neuroblastoma cells express PDGF alpha- and beta-receptors, but not PDGF-A and PDGF-B chain mRNA. In contrast to alpha-receptor expression, beta-receptor expression appears to be associated with a mature neuronal phenotype. Under serum-free growth conditions, PDGF-AA and -BB induce a trophic and weak mitogenic response in SH-SY5Y neuroblastoma cells, showing that the PDGF receptors in these cells are functional. In combination with 12-O-tetradecanoylphorbol-13-acetate, all three PDGF isoforms induce sympathetic neuronal differentiation of the SH-SY5Y cells, as shown by morphology and by increased expression of the genes coding for growth-associated protein 43 and neuropeptide tyrosine, respectively. This indicates a potential role for PDGF in the development of sympathetic neurons in particular and of the nervous system in general.     

TNF signalling in tooth development

Mammalian tooth development has served as an excellent model system to investigate the intricate, interactive mechanisms of patterning, morphogenesis and cytodifferentiation during organogenesis. Teeth develop from interactions between epithelium and neural crest-derived (ecto)mesenchyme that are largely mediated by ligand-receptor signalling. It is well-established that signalling molecules of the Bmp, Fgf, Wnt and Hedgehog families, are involved at multiple stages of tooth development. Recently, however, a specific role for molecules belonging to the TNF-family of ligands in tooth morphogenesis has been identified, suggesting that this pathway, acting to activate NF-kappaB, has played an important role in the development and evolution of tooth number and shape.     

Calcium and growth responses of hyperresponsive airway smooth muscle to different isoforms of platelet-derived growth factor (PDGF)

Airway smooth muscle (ASM) mass is likely to be an important determinant of airway responsiveness. Highly inbred Fisher rats model innate hyperresponsiveness, and also have more ASM in vivo than control Lewis rats. Platelet derived growth factor (PDGF) is an important endogenous growth factor for ASM, and partially purified PDGF-AB causes enhanced growth of Fisher rat ASM cells, compared to Lewis cells. The aim of the present study was to determine the mitogenic effects of all three recombinant PDGF isoforms on ASM cells, and investigate the mechanisms of enhanced Fisher ASM growth responses. The potential mechanisms assessed include PDGF receptor expression and activation (tyrosine phosphorylation), and intracellular calcium (Ca2+) responses to PDGF isoforms. Fisher ASM cells had a greater mitogenic response to PDGF-AB and -AA, and a greater Ca2+ response to -BB than Lewis ASM cells. A Ca2+ response was not necessary for a mitogenic response, and the effects of PDGF isoforms on Ca2+ were not associated with their effects on growth. Therefore, we suggest that enhanced Fisher mitogenic response to PDGF-AA and -AB is not mediated by differences in Ca2+ signalling. Western analysis of the PDGF receptor indicated a similar expression of beta-PDGF receptor in ASM cells from the two rat strains, but a greater expression of alpha-PDGF receptor in Fisher cells; however, phosphorylation of the PDGF receptor following growth stimulation did not differ between strains. This suggests a role for post-receptor signals, in addition to enhanced receptor expression, in the enhanced growth response of Fisher ASM cells to PDGF-AA and -AB.     

Involvement of heparin-binding EGF-like growth factor and its processing by metalloproteinases in early epithelial morphogenesis of the submandibular gland

In the present study, the role of a member of the epidermal growth factor (EGF) family, heparin-binding EGF-like growth factor (HB-EGF), in organ development was investigated by using developing mouse submandibular gland (SMG), in which the EGF receptor signaling and heparan sulfate chains have been implicated. HB-EGF mRNA was detected in developing SMG by RT-PCR analysis and was expressed mainly in epithelium and weakly in mesenchyme of the embryonic SMG. Epithelial morphogenesis was inhibited by a synthetic peptide corresponding to the heparin-binding domain of HB-EGF and by anti-HB-EGF neutralizing antibody. An in vitro assay using an EGF receptor ligand-dependent cell line, EP170.7 cells, allowed us to detect the growth factor activity in SMG-conditioned media, which was significantly reduced by anti-HB-EGF antibody. Furthermore, treatment of SMG rudiments with the hydroxamate-based metalloproteinase inhibitor OSU8-1, which inhibits processing of EGFR ligands including HB-EGF, markedly diminished the growth factor activity in conditioned media and resulted in almost complete inhibition of SMG morphogenesis. The inhibitory effects on morphogenesis were reversed, though partially, by adding the soluble form of HB-EGF. Our results provide the first evidence that HB-EGF is a crucial regulator of epithelial morphogenesis during organ development, highlighting the importance of its processing by metalloproteinases.     

Specific heparan sulfate structures modulate FGF10-mediated submandibular gland epithelial morphogenesis and differentiation

FGF10, a heparan sulfate (HS)-binding growth factor, is required for branching morphogenesis of mouse submandibular glands (SMGs). HS increases the affinity of FGF10 for FGFR2b, which forms an FGF10.FGFR2b.HS ternary signaling complex, and results in diverse biological outcomes, including proliferation and epithelial morphogenesis. Defining the HS structures involved in specific FGF10-mediated events is critical to understand how HS modulates growth factor signaling in specific developmental contexts. We used HS-deficient BaF3/FGFR2b cells, which require exogenous HS to proliferate, to investigate the HS requirements for FGF10-mediated proliferation and primary SMG epithelia to investigate the structural requirements of HS for FGF10-mediated epithelial morphogenesis. In BaF3/FGFR2b cells, heparin with at least 10 saccharides and 6-O-, 2-O-, and N-sulfates were required for maximal proliferation. During FGF10-mediated SMG epithelial morphogenesis, HS increased proliferation and end bud expansion. Defined heparin decasaccharide libraries showed that 2-O-sulfation with either an N-or 6-O-sulfate induced end bud expansion, whereas decasaccharides with 6-O-sulfation alone induced duct elongation. End bud expansion resulted from increased FGFR1b signaling, with increased FGFR1b, Fgf1, and Spry1 as well as increased Aqp5 expression, a marker of end bud differentiation. Duct elongation was associated with expression of Cp2L1, a marker of developing ducts. Collectively, these findings show that the size and sulfate patterns of HS modulate specific FGF10-mediated events, such as proliferation, duct elongation, end bud expansion, and differentiation, and provide mechanistic insight as to how the developmental localization of specific HS structures in tissues influences FGF10-mediated morphogenesis and differentiation.     

Expression of Fgf10 and Fgf receptors during development of the embryonic chicken stomach

Fibroblast growth factor 10 (FGF10) is involved in numerous different aspects of embryonic development and especially in active epithelial-mesenchymal interactions during morphogenesis of many organs as a mesenchymal regulator by activating its receptors (FGFR1b and FGFR2b) expressed in the epithelial tissue. FGFR2b is also activated by FGF7 although FGF7 does not bind to FGFR1b. To provide basic data to analyze function of FGFs in the developing gut, here we cloned Fgf7 and studied expression patterns of Fgf7, Fgf10 and Fgfr1-4 during the development of chicken stomach (glandular stomach; proventriculus and muscular stomach; gizzard). Fgf10 is expressed both in the proventricular and gizzard mesenchyme while Fgf7 is expressed only in gizzard mesenchyme. Fgfr1-4 are expressed both in the epithelium and mesenchyme with a different spatial expression patterns. Furthermore, RT-PCR analysis reveals that Fgfr1b and Fgfr2b are expressed only in epithelia of both organs.