Takayoshi Sakai

Fibronectin requirement in branching morphogenesis

Many organs, including salivary glands, lung and kidney, are formed during embryonic development by epithelial branching. In branching morphogenesis, repetitive epithelial cleft and bud formation create the complex three-dimensional branching structures characteristic of many organs. Although the mechanisms are poorly understood, one might involve the site-specific accumulation of some regulatory protein. Here we show that the extracellular matrix protein fibronectin is essential for cleft formation during the initiation of epithelial branching. Fibronectin messenger RNA and fibrils appeared transiently and focally in forming cleft regions of submandibular salivary-gland epithelia, accompanied by an adjacent loss of cadherin localization. Decreasing the fibronectin concentration by using small interfering RNA and inhibition by anti-fibronectin or anti-integrin antibodies blocked cleft formation and branching. Exogenous fibronectin accelerated cleft formation and branching. Similar effects of fibronectin suppression and augmentation were observed in developing lung and kidney. Mechanistic studies revealed that fibrillar fibronectin can induce cell–matrix adhesions on cultured human salivary epithelial cells with a local loss of cadherins at cell–cell junctions. Thus, fibronectin expression is required for cleft formation in branching morphogenesis associated with the conversion of cell–cell adhesions to cell–matrix adhesions.

Btbd7 Regulates Epithelial Cell Dynamics and Branching Morphogenesis

Epithelial Cleft Formation The internal architecture of many embryonic organs is established by repetitive branching of epithelia. Epithelial clefts and outgrowths generate this internal branching of glands and other organs. Onodera et al. (p. 562) identify a gene, Btbd7, as a regulator of epithelial dynamics and cleft formation, linking the extracellular matrix with morphogenesis. Btbd7 is induced by a matrix protein at sites of cleft progression and induces a transcription factor and suppresses cell adhesion. The resulting local cell separation and motility contribute to transient tissue gaps that contribute to clefts that help form branched organs. A regulatory gene suppresses cell adhesion and enhances cell motility to help form branched organs. During embryonic development, many organs form by extensive branching of epithelia through the formation of clefts and buds. In cleft formation, buds are delineated by the conversion of epithelial cell-cell adhesions to cell-matrix adhesions, but the mechanisms of cleft formation are not clear. We have identified Btbd7 as a dynamic regulator of branching morphogenesis. Btbd7 provides a mechanistic link between the extracellular matrix and cleft propagation through its highly focal expression leading to local regulation of Snail2 (Slug), E-cadherin, and epithelial cell motility. Inhibition experiments show that Btbd7 is required for branching of embryonic mammalian salivary glands and lungs. Hence, Btbd7 is a regulatory gene that promotes epithelial tissue remodeling and formation of branched organs.

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.

Mouse Semaphorin H Induces PC12 Cell Neurite Outgrowth Activating Ras-Mitogen-activated Protein Kinase Signaling Pathway via Ca2+ Influx

We recently showed that mouse semaphorin H (MSH), a secreted semaphorin molecule, acts as a chemorepulsive factor on sensory neurites. In this study, we found for the first time that MSH induces neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of Ras-mitogen-activated protein kinase (MAPK) signaling pathways between MSH and nerve growth factor (NGF) revealed that these pathways are crucial for MSH action as well as NGF. K-252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks), did not inhibit the action of MSH, suggesting that MSH action occurs via a different receptor than NGF. L- and N-types of voltage-dependent Ca2+ channel blockers, diltiazem and ω-conotoxin, inhibited MSH-induced neurite outgrowth and MAPK phosphorylation in a Ca2+-dependent manner. A transient elevation in intracellular Ca2+ level was observed upon MSH stimulation. These findings suggest that extracellular Ca2+ influx, followed by activation of the Ras-MAPK signaling pathway, is required for MSH induced PC12 cell neurite outgrowth.

DEVELOPMENTAL LOCALIZATION OF SEMAPHORIN H MESSENGER RNA ACTING AS A COLLAPSING FACTOR ON SENSORY AXONS IN THE MOUSE BRAIN

Semaphorins/collapsins, a family of genes with a semaphorin domain conserved from insects through to mammals, are believed to be involved in axon guidance during neuronal development. We report the expression patterns of mouse semaphorin messenger RNAs. Among secreted semaphorins, mouse semaphorin H is structurally most similar to semaphorin III/D, the first semaphorin identified as a collapsing factor for sensory axons. However, its expression patterns apparently differ from those of semaphorin III/D. The messenger RNAs are distributed in the brain widely but unevenly during development, in particular, in the main olfactory bulb, hippocampus and pontine nucleus. In the trunk, the expression level is high in mesodermal tissues surrounding the dorsal root ganglia, while it is low in the spinal cord. Moreover, we examined whether this molecule has activity to collapse growth cones of sensory neurons, as well as semaphorin III/D. Mouse semaphorin H collapsed growth cones of sensory neurons of the dorsal root ganglion in a dose-dependent manner, and anti-neuropilin antibodies inhibited this activity. Taken together, these results suggest that mouse semaphorin H can function as a chemorepellent to guide sensory peripheral nerves, most likely via neuropilin as a receptor.

Mouse semaphorin H inhibits neurite outgrowth from sensory neurons

Mouse semaphorin H (M-semaH) was structurally similar to semaphorin III/D, a mammalian homologue of collapsin 1 which was identified as a collapsing factor for sensory nerves. In this study we investigated the expression patterns of M-semaH mRNA and the protein binding sites in the trunk of mouse embryos. M-semaH mRNA was expressed in the mesenchymal tissues surrounding each dorsal root ganglia. These tissues include the caudal sclerotome and perinotochordal mesenchyme, which were thought to express factors repulsive to axons. M-semaH binding was detected on the spinal nerves. We further investigated, using in vitro co-culture assay, whether M-semaH acted as a chemorepulsive molecule on sensory axons. The results suggested that M-semaH was a candidate for a chemorepellent expressed in the mesenchyme surrounding the sensory ganglia, which is involved in the axonal guidance mechanism of sensory nerves in the trunk.

Neurotrophic effect of Semaphorin 4D in PC12 cells

Semaphorins provide crucial attractive and repulsive cues involved in axon guidance during neural development. Out of them, Semaphorin 4D (Sema4D) is enriched in the nervous and immune tissues, and acts as proliferative and survival factors of peripheral lymphocytes in the immune system, but is poorly understood in the nervous system. By using PC12 cells which are well known to differentiate into neural cells in response to nerve growth factor (NGF), we found that soluble forms of Sema4D had neurotrophic effects which were inhibited by neutralizing antibodies to Sema4D. Sema4D strikingly potentiated neurite outgrowth in the presence of 50 ng/ml NGF and increased sensitivity to NGF. Cells responded to very low concentrations of NGF in the presence of 1 nM Sema4D. Activation of following signal proteins, protein kinase C (PKC), L-type of voltage-dependent Ca(2+) channel, and phosphatidylinositol (PI) 3-kinase mediated neurotrophic neurite-outgrowth action of Sema4D. These findings suggest a new function of Sema4D as a neurotrophic signal in PC12 cells.

Effects of human fibroblasts on invasiveness of oral cancer cells in vitro: Isolation of a chemotactic factor from human fibroblasts

Oral fibroblasts stimulated invasion of oral‐carcinoma cells into the collagen matrix. The mechanisms of the fibroblast‐induced stimulation of invasiveness was further investigated by examining cell motility and proteolytic activity of tumor cells, using mainly an adenoid‐cystic‐carcinoma cell line (ACCS) and normal fibroblasts from gingival tissues. Conditioned medium from the fibroblasts grown in serum‐free medium was fractionated on a Superdex 200 pg column, and Peak 1 eluted at 200 to 300 kDa and Peak 2 eluted at 50 to 100 kDa were found to contain different specific activity. Treatment of ACCS cells with Peak 1 resulted in an increase in the production of proteolytic enzymes. Peak 2 stimulated both chemotaxis and chemokinesis of ACCS cells. A chemotactic factor was purified from the heparin‐unbound fraction of Peak 2 by anion exchange and hydrophobic chromatography, and was named “fibroblastderived motility factor (FDMF)”. At 1 μg/ml, FDMF stimulated chemotaxis of ACCS cells by 4‐fold compared with unstimulated controls. Characterization of the physicochemical properties of FDMF suggested that it might be different from any known motility factors. Exposure of ACCS cells to FDMF resulted in reduced amounts of actin stress fiber in the cytoplasm and induction of tyrosine phosphorylation of several cellular proteins detectable 30 to 60 min after treatment. These FDMF‐induced changes were blocked by pre‐treatment either with genistein or with pertussis toxin. These findings suggest that FDMF may be a novel protein which stimulates cell motility via a signaling pathway mediated by a pertussis‐toxin‐sensitive G protein and tyrosine phosphorylation.

Regenerating Salivary Glands in the Microenvironment of Induced Pluripotent Stem Cells.

This report describes our initial attempt to regenerate salivary glands using induced pluripotent stem (iPS) cells in vivo and in vitro. Glandular tissues that were similar to the adult submandibular glands (SMGs) and sublingual glands could be partially produced by the transplantation of iPS cells into mouse salivary glands. However, the tumorigenicity of iPS cells has not been resolved yet. It is well known that stem cells affect their microenvironment, known as a stem cell niche. We focused on the niche and the interaction between iPS cells and salivary gland cells in our study on salivary gland regeneration. Coculture of embryonic SMG cells and iPS cells have better-developed epithelial structures and fewer undifferentiated specific markers than monoculture of embryonic SMG cells in vitro. These results suggest that iPS cells have a potential ability to accelerate differentiation for salivary gland development and regeneration.

Embryonic Organ Culture

This unit provides detailed protocols for dissecting embryonic organs and performing organ culture to study questions in developmental biology. Procedures are described here for dissecting organs such as kidney, lung, and salivary gland. The unit also contains commentary including troubleshooting for embryonic organ culture. Curr. Protoc. Cell Biol. 41:19.8.1‐19.8.8.