Toru Hayashi

Mesenchymal miR-21 regulates branching morphogenesis in murine submandibular gland in vitro

Branching morphogenesis in murine submandibular glands (SMG) is regulated by growth factors, extracellular matrix (ECM) and many biological processes through interactions between the epithelium and the mesenchyme. MicroRNAs (miRNAs) are a set of small, non-protein-coding RNAs that regulate gene expression at the post-transcriptional level. We hypothesized that branching morphogenesis is partly regulated by miRNAs. Forty-four miRNAs and novel miRNA candidates were detected in SMG at embryonic day 13 by a cloning method combined with Argonaute-2 immunoprecipitation. MicroRNA21 (miR-21) expression in the mesenchyme was up-regulated and accelerated by epidermal growth factor, which is known to enhance branching morphogenesis in vitro. Down-regulation of miR-21 in the mesenchyme by locked nucleic acids was associated with a decrease in the number of epithelial buds. Relative quantification of candidates for target genes of miR-21 indicated that two messenger RNAs (for Reck and Pdcd4) were down-regulated in the mesenchyme, where miR-21 expression levels were up-regulated. These results suggest that branching morphogenesis is regulated by miR-21 through gene expression related to ECM degradation in the mesenchyme.

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 Cγ1 (PLCγ1) and phosphatidylinositol‐3 kinase (PI3K) in cultured E14 SMG. However, FGF7 and FGF10 stimulated phosphorylation of both PLCγ1 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 (PLCγ1 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 PLCγ1 and partly via ERK‐1/2, but that FGF10 stimulates stalk elongation mainly via PLCγ1.

Molecular Analysis of Deep-Sea Hydrothermal Vent Aerobic Methanotrophs by Targeting Genes of 16S rRNA and Particulate Methane Monooxygenase

Molecular diversity of deep-sea hydrothermal vent aerobic methanotrophs was studied using both 16S ribosomalDNA and pmoA encoding the subunit A of particulate methane monooxygenase (pMOA). Hydrothermal vent plume and chimney samples were collected from back-arc vent at Mid-Okinawa Trough (MOT), Japan, and the Trans-Atlantic Geotraverse (TAG) site along Mid-Atlantic Ridge, respectively. The target genes were amplified by polymerase chain reaction from the bulk DNA using specific primers and cloned. Fifty clones from each clone library were directly sequenced. The 16S rDNA sequences were grouped into 3 operational taxonomic units (OTUs), 2 from MOT and 1 from TAG. Two OTUs (1 MOT and 1 TAG) were located within the branch of type I methanotrophic ?-Proteobacteria. Another MOT OTU formed a unique phylogenetic lineage related to type I methanotrophs. Direct sequencing of 50 clones each from the MOT and TAG samples yielded 17 and 4 operational pmoA units (OPUs), respectively. The phylogenetic tree based on the pMOA amino acid sequences deduced from OPUs formed diverse phylogenetic lineages within the branch of type I methanotrophs, except for the OPU MOT-pmoA-8 related to type X methanotrophs. The deduced pMOA topologies were similar to those of all known pMOA, which may suggest that the pmoA gene is conserved through evolution. Neither the 16S rDNA nor pmoA molecular analysis could detect type II methanotrophs, which suggests the absence of type II methanotrophs in the collected vent samples.

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.

Shh/Ptch and EGF/ErbB cooperatively regulate branching morphogenesis of fetal mouse submandibular glands.

The hedgehog family includes Sonic hedgehog (Shh), Desert hedgehog, and Indian hedgehog, which are well known as a morphogens that play many important roles during development of numerous organs such as the tongue, pancreas, kidney, cartilage, teeth and salivary glands (SMG). In Shh null mice, abnormal development of the salivary gland is seen after embryonic day 14 (E14). Shh also induced lobule formation and lumen formation in acini-like structures in cultured E14 SMG. In this study, we investigated the relationship between Shh and epidermal growth factor (EGF)/ErbB signaling in developing fetal mouse SMG. Administration of Shh to cultured E13 SMG stimulated branching morphogenesis (BrM) and induced synthesis of mRNAs for EGF ligands and receptors of the ErbB family. Shh also stimulated activation of ErbB signaling system such as ERK1/2. AG1478, a specific inhibitor of ErbB receptors, completely suppressed BrM and activation of EGF/ErbB/ERK1/2 cascade in E13 SMGs cultured with Shh. The expressions of mRNA for Egf in mesenchyme and mRNA for Erbb1, Erbb2 and Erbb3 in epithelium of E13 SMG were specifically induced by administration of Shh. These results show that Shh stimulates BrM of fetal mouse SMG, at least in part, through activation of the EGF/ErbB/ERK1/2 signaling system.

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

Abstract 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.

Regulation of Expression of Sprouty Isoforms by EGF, FGF7 or FGF10 in Fetal Mouse Submandibular Glands

Abstract Branching morphogenesis of the fetal mouse submandibular gland (SMG) is regulated by signaling through the ErbB and FGF families of tyrosine kinase receptors, whose members activate the ERK-1/2 pathway. The four Sprouty (Spry) proteins are inhibitory modulators of ERK-1/2. There is little information on their expression during pre- and postnatal development of the SMG. Qualitative RT-PCR detected mRNAs for Spry1 , 2 , and 4 from embryonic day 13 (E13) through postnatal day 7 (P7), but only trace amounts of Spry1 and 2 in adult SMGs. More sensitive quantitative RT-PCR revealed that transcripts for all four Spry isoforms are expressed, and each shows individual patterns of variation across fetal and early postnatal stages, and that there are very low levels of Spry1 and 2 , but no Spry3 and 4 , in adult glands. EGF, FGF7 and FGF10 upregulate expression of mRNA for Spry1 , but only FGF7 upregulates Spry2 mRNA. EGF strongly induces an activating phosphorylation of all four Spry isoforms, but both FGFs do so only minimally. Quantitative RT-PCR of samples collected by laser capture microdissection showed that transcripts for Spry1 are confined to the epithelium of E13 SMG rudiments. The isoform-specific temporal variation in the patterns of expression of Spry1, 2, 3 and 4 suggests a potentially important role for these negative modulators of growth-factor driven ras/ERK-1/2 signaling at stages when the SMG is most actively undergoing branching morphogenesis.