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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.

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 Ser473 by Western analysis in the presence of PI 3-kinase inhibitors. The major lipid product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP3), 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 PIP3 pathway.

In vitro fabricated biological tissue would be a valuable tool to screen newly synthesized drugs or understand the tissue development process. Several studies have attempted to fabricate biological tissue in vitro. However, controlling the growth and morphology of the fabricated tissue remains a challenge. Therefore, new techniques are required to modulate tissue growth. RGD (arginine-glycine-aspartic acid), which is an integrin-binding domain of fibronectin, has been found to enhance cell adhesion and survival; it has been used to modify substrates for in vitro cell culture studies or used as tissue engineering scaffolds. In addition, this study shows novel functions of the RGD peptide, which enhances tissue growth and modulates tissue morphology in vitro. When an isolated submandibular gland (SMG) was cultured on an RGD-modified alginate hydrogel sheet, SMG growth including bud expansion and cleft formation was dramatically enhanced. Furthermore, we prepared small RGD-modified alginate beads and placed them on the growing SMG tissue. These RGD-modified beads successfully induced cleft formation at the bead position, guiding the desired SMG morphology. Thus, this RGD-modified material might be a promising tool to modulate tissue growth and morphology in vitro for biological tissue fabrication.

In this unit, the authors describe a new technique, T7-SAGE, in which a high-fidelity T7 amplification step is combined with SAGE analysis. In order to avoid extra PCR or other forms of amplification, the authors incorporate only two cycles of T7-based RNA amplification as the initial step. This T7-based amplification step has high accuracy. In addition, T7 RNA polymerase has high processivity and functions effectively even when broad stretches of nucleotides are being amplified. Although no protocol that includes an amplification step can claim to permit determination of absolute transcript number, since slight changes in estimated transcript frequency are always possible, T7 procedures appear to be the safest to date. This new T7-SAGE procedure should facilitate application of SAGE for gene-expression profiling using minimal quantities of starting material, such as from embryonic tissues and microdissected cells from histological sections of tissues.

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 Ca(2+) channel blockers, diltiazem and omega-conotoxin, inhibited MSH-induced neurite outgrowth and MAPK phosphorylation in a Ca(2+)-dependent manner. A transient elevation in intracellular Ca(2+) level was observed upon MSH stimulation. These findings suggest that extracellular Ca(2+) influx, followed by activation of the Ras-MAPK signaling pathway, is required for MSH induced PC12 cell neurite outgrowth.

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.

(IQ 196.5-869.7), 73% of subjects had at least one PD 4mm in the 3rd molar region at follow-up vs. 54% below the median, and 58% of subjects had at least one PD 4mm in the non-3rd molar region at follow-up vs. 39%. Baseline levels of “orange” or “red” cluster pathogens significantly predicted follow-up 3rd molar region PD 4mm, but not non-3rd molar PD 4mm. Conclusion: The presence of risk markers for oral inflammation were associated with worsening 3rd molar and non-3rd molar periodontal pathology.

Zirconium(IV) reacts with an excess of bromopyrogallol red to form a violet complex. The constant absorbance of a solution containing the complex is obtained in the pH range from 5.0 to 5.4 at 670 mμ. About 25 min is required to complete the chelate formation in a boiling water bath. The complex thus formed is stable for at least 3 hr. The color system obeys Beer’s law in the range from 4 to 35 μg zirconium in 25 ml. The sensitivity of the reaction is 0.002 μg per cm2 for an absorbance of 0.001. In composition the complex has a metal-to-ligand ratio of 1:2. The molar absorptivity and the apparent formation constant of the complex are calculated to be 7.01×104 at 670 mμ and 1.11×109 at pH 5.2 respectively. The effect of diverse ions on the determination is examined.