Regulation of Branching Morphogenesis in Fetal Mouse Submandibular Gland by Signaling Pathways Activated by Growth Factors and α6 Integrin

The submandibular gland (SMG) of the fetal mouse is useful for understanding the epithelial-mesenchymal interactions required for branching morphogenesis (BrM), which entails cleft formation and stalk elongation. We found that specific signaling pathways activated by growth factors or integrins regulate the processes of cleft formation and stalk elongation. Western blot analysis showed that epidermal growth factor (EGF) strongly stimulated the phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and weakly stimulated the phosphorylation of phospholipase Cγ1 (PLCγ1) and phosphatidylinositol-3 kinase (PI3K) in cultured E14 SMG. Meanwhile, fibroblast growth factor (FGF) 7 and FGF10 stimulated the phosphorylation of both PLCγ1 and PI3K, but elicited only minimal phosphorylation of ERK-1/2. Morphological studies revealed that EGF induced cleft formation of epithelial endpieces of mesenchyme-free SMG and that FGF7 stimulated both cleft formation and stalk elongation, whereas FGF10 induced only stalk elongation. U0126 (a MEK inhibitor) completely blocked cleft formation, while U73122 (a PLCγ1 inhibitor) suppressed stalk elongation. These results suggest that EGF stimulates cleft formation by activation of ERK1/2 and that FGF7 stimulates both cleft formation and stalk elongation by activation of PLCγ1 and partial activation of ERK1/2, and that FGF10 stimulates stalk elongation mainly by activation of PLCγ1. GoH3, a neutralizing antibody against the α6 integrin subunit, caused a slight increase over basal levels of ERK1/2 phosphorylation in cultured E14 SMG, but it had no discernible effect on EGF-induced phosphorylation. Based on these new findings regarding the effects of α6 integrin on growth factor receptor signals, we discuss the regulation of salivary gland development.