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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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

FGF signaling is essential for mammary gland development, yet the mechanisms by which different members of the FGF family control stem cell function and epithelial morphogenesis in this tissue are not well understood. Here, we have examined the requirement of Fgfr2 in mouse mammary gland morphogenesis using a postnatal organ regeneration model. We found that tissue regeneration from basal stem cells is a multistep event, including luminal differentiation and subsequent epithelial branching morphogenesis. Basal cells lacking Fgfr2 did not generate an epithelial network owing to a failure in luminal differentiation. Moreover, Fgfr2 null epithelium was unable to undergo ductal branch initiation and elongation due to a deficiency in directional migration. We identified FGF10 and FGF2 as stromal ligands that control distinct aspects of mammary ductal branching. FGF10 regulates branch initiation, which depends on directional epithelial migration. By contrast, FGF2 controls ductal elongation, requiring cell proliferation and epithelial expansion. Together, our data highlight a pleiotropic role of Fgfr2 in stem cell differentiation and branch initiation, and reveal that different FGF ligands regulate distinct aspects of epithelial behavior., (© 2014. Published by The Company of Biologists Ltd.)

Chitosan is a widely used biocompatible biomaterial in the tissue regeneration, but its utility and application in the tissue morphogenesis of salivary gland remains unclear. The study aimed to explore the effects of chitosan on the epithelial morphogenesis of submandibular gland (SMG). With chitosan, the branching morphogenesis of the whole SMG explant was facilitated, and the morphogenetic-promoting effects of mesenchymal tissue on SMG were further enhanced. Furthermore, chitosan was competent to induce recombined SMG epithelium to form branches in the serum-free condition independently. In the presence of chitosan, the morphogenetic efficacy of mesenchyme-derived growth factors responsible for epithelial morphogenesis including fibroblast growth factors 7, fibroblast growth factor 10 and hepatocyte growth factor increased. The specific epithelial phenotype induced by individual growth factor, which was required for the accomplishment of salivary epithelial morphogenesis, was promoted by chitosan. Moreover, the proliferative and the chemotactic properties of these growth factors towards the SMG epithelia were also reinforced by chitosan. Therefore, in orchestrating and intensifying the essential mesenchyme-derived growth factors, chitosan is versatile in mediating SMG epithelium to form a predetermined phenotype more efficiently and comprehensively. This study suggested that chitosan is a morphogenetic-regulating biomaterial for salivary tissue, which might be useful for the future salivary gland investigation and regeneration.

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 Cgamma1 (PLCgamma1) and phosphatidylinositol-3 kinase (PI3K) in cultured E14 SMG. However, FGF7 and FGF10 stimulated phosphorylation of both PLCgamma1 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 (PLCgamma1 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 PLCgamma1 and partly via ERK-1/2, but that FGF10 stimulates stalk elongation mainly via PLCgamma1.

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.

Branching morphogenesis (BM) of the chorionic villous tree is a crucial component of early placental formation. Fibroblast growth factors (FGFs), their receptor tyrosine kinase (RTK) and negative regulators like Sprouty (Spry) proteins are pivotal factors in the development of diverse branching organ systems. The aim of this study was to examine the effect of FGF10 and Sprouty 2 on BM of the chorionic villi in vitro. Villous explants of first trimester placentas were cultured and their outgrowths were monitored. The effect of FGF10 was tested on matrigel migration/invasion assay, collagenolytic activity of single cell trophoblasts and on villous explants outgrowths. siRNA of Spry2 was used to reduce its expression and to investigate the role of Sprouty 2 in villous explants outgrowths. Quantitative RT-PCR and immunohistochemistry were performed to determine Sprouty 2 and HLA-G (a marker of invasion) expression. FGF 10 stimulated by 8-fold the migration/invasion of single cell trophoblast enhanced their collagenolytic activity. Reduction of Spry2 expression in villous explants showed a marked increase in villous outgrowths. This was accompanied by enhanced staining for HLA-G and by the reduction of Spry2 expression that was confirmed by immunohistochemistry and by quantitative RT-PCR. We conclude that trophoblast outgrowth and invasion (part of placental villi sprouting) at the fetal maternal interface is in part under delicate control of FGF 10 and Sprouty 2. FGF 10 promotes invasion and outgrowth of trophoblasts. In addition, it increases Spry2 expression, which attenuates trophoblast sprouting.

Recent evidence has shown that retinoic acid (RA) signalling is required for early pancreatic development in zebrafish and frog but its role in later development in mammals is less clear cut. In the present study, we determined the effects of RA on the differentiation of the mouse embryonic pancreas. Addition of all-trans retinoic acid (atRA) to embryonic pancreatic cultures induced a number of changes. Branching morphogenesis and exocrine differentiation were suppressed and there was premature formation of endocrine cell clusters (although the total area of beta cells was not different in control and atRA-treated buds). We investigated the mechanism of these changes and found that the premature formation of beta cells was associated with the early expression of high-level Pdx1 in the endocrine cell clusters. In contrast, the suppressive effect of RA on exocrine differentiation may be due to a combination of two mechanisms (i) up-regulation of the extracellular matrix component laminin and (ii) enhancement of apoptosis. We also demonstrate that addition of fibroblast growth factor (FGF)-10 is able to partially prevent apoptosis and rescue exocrine differentiation and branching morphogenesis in atRA-treated cultures but not in mice lacking the FGF receptor 2-IIIb, suggesting the effects of FGF-10 are mediated through this receptor.