Recently, roles of Delta-like 4 (Dll4)-Notch signaling in angiogenesis have been demonstrated by a series of reports (Ridgway et al., 2006;Hellstrom et al., 2007;Siekmann and Lawson, 2007;Suchting et al., 2007). Murine retina heterozygous for a null mutation of the Dll4 gene showed excessive branching and this was recapitulated by administering a -secretase inhibitor, Development 138, 4763-4776 (2011Development 138, 4763-4776 ( ) doi:10.1242 SUMMARYAngiogenesis is a complex process, which is accomplished by reiteration of modules such as sprouting, elongation and bifurcation, that configures branching vascular networks. However, details of the individual and collective behaviors of vascular endothelial cells (ECs) during angiogenic morphogenesis remain largely unknown. Herein, we established a time-lapse imaging and computer-assisted analysis system that quantitatively characterizes behaviors in sprouting angiogenesis. Surprisingly, ECs moved backwards and forwards, overtaking each other even at the tip, showing an unknown mode of collective cell movement with dynamic 'cell-mixing'. Mosaic analysis, which enabled us to monitor the behavior of individual cells in a multicellular structure, confirmed the 'cell-mixing' phenomenon of ECs that occurs at the whole-cell level. Furthermore, an in vivo EC-tracking analysis revealed evidence of cell-mixing and overtaking at the tip in developing murine retinal vessels. In parametrical analysis, VEGF enhanced tip cell behavior and directed EC migration at the stalk during branch elongation. These movements were counter-regulated by EC-EC interplay via -secretase-dependent Dll4-Notch signaling, and might be promoted by EC-mural cell interplay. Finally, multiple regression analysis showed that these molecule-mediated tip cell behaviors and directed EC migration contributed to effective branch elongation. Taken together, our findings provide new insights into the individual and collective EC movements driving angiogenic morphogenesis. The methodology used for this analysis might serve to bridge the gap in our understanding between individual cell behavior and branching morphogenesis.

H. Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ.

No abstract

Sirtuins are a phylogenetically conserved NAD + -dependent protein deacetylase/ADP-ribosyltransferase family implicated in diverse biological processes. Several family members localize to mitochondria, the function of which is thought to determine the developmental potential of preimplantation embryos. We have therefore characterized the role of sirtuins in mouse preimplantation development under in vitro culture conditions. All sirtuin members were expressed in eggs, and their expression gradually decreased until the blastocyst stage. Treatment with sirtuin inhibitors resulted in increased intracellular ROS levels and decreased blastocyst formation. These effects were recapitulated by siRNA-induced knockdown of Sirt3, which is involved in mitochondrial energy metabolism, and in Sirt3 -/-embryos. The antioxidant N-acetyl-L-cysteine and lowoxygen conditions rescued these adverse effects. When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.

Protein kinase B (PKB)/Akt reportedly plays a role in the survival and/or proliferation of cells. We identified a novel protein, which binds to PKB, using a yeast twohybrid screening system. This association was demonstrated not only in vivo by overexpressing both proteins or by coimmunoprecipitation of the endogenous proteins, but also in vitro using glutathione S-transferase fusion proteins. Importantly, this protein specifically associates with the C terminus of PKB but not with other AGC kinases and enhances PKB phosphorylation and kinase activation without growth factor stimulation. Thus, we termed this Akt-specific binding protein APE (Akt-phosphorylation enhancer). Since APEinduced phosphorylation of PKB did not occur in cells treated with wortmannin or LY294002, APE itself is not a kinase but seems to enhance or prolong the phosphoinositide 3-kinase-dependent phosphorylation of PKB. In cells in which APE was suppressed by small interfering RNA, DNA synthesis was significantly reduced with suppression of PKB phosphorylation, suggesting a synergistic role of APE in PKB-induced proliferation. On the other hand, in cells overexpressing both PKB and APE, despite markedly increased basal phosphorylation of PKB, both DNA rereplication and subsequent Chk2 phosphorylation and apoptosis were seen, suggesting the involvement of APE in the regulation of cell cycling replication licensing. Taking these observations together, APE appears to be a novel regulator of PKB phosphorylation. Furthermore, the interaction between APE and PKB, possibly dependent on the expression levels of both proteins, may be a novel molecular mechanism leading to proliferation and/or apoptosis.The serine/threonine protein kinase PKB 1 (also called Akt) is thought to be a key mediator of signal transduction. Upon growth factor stimulation, a family of lipid kinases known as class 1 phosphoinositide 3-kinases (PI 3-kinases) is recruited to the plasma membrane. PI 3-kinases phosphorylate phosphatidylinositol 4,5-bisphosphate at the D-3 position of the inositol ring, converting it to phosphatidylinositol 3,4,5-trisphosphate. Following the activation of PI 3-kinase, PKBs are recruited to the plasma membrane through direct contact of the pleckstrin homology (PH) domain with phosphatidylinositol 3,4,5-trisphosphate and are phosphorylated at Thr 308 by PDK1 and at Ser 473 by PDK2, a kinase of which the molecular structure has not yet been identified (1, 2). AGC kinases other than PKB are also known to be regulated by PI 3-kinase, and PKB acts downstream from PI 3-kinase to regulate numerous biological processes, such as proliferation, antiapoptosis, cell growth, and glucose metabolism (1, 2).PKB has a wide range of substrates, including GSK-3, FKHR (FoxO1), FKHR-L1 (FoxO3), AFX (FoxO4), and eNOS, all of which have the consensus motif RXRXX(S/T) (3, 4). Protein kinases do not generally form stable complexes with their substrates, although PKB has been shown to exist in a stable complex with several of its substrates including MDM2, p21 Cip1 /WAF1, an...

AMP-activated protein kinase (AMPK) activation reportedly suppresses transcriptional activity of the cAMP-responsive element (CRE) in the phosphoenolpyruvate carboxykinase C (PEPCK-C) promoter and reduces hepatic PEPCK-C expression. Although a previous study found TORC2 phosphorylation to be involved in the suppression of AMPK-mediated CRE transcriptional activity, we herein present evidence that glycogen synthase kinase 3␤ (GSK3␤) phosphorylation induced by AMPK also plays an important role. We initially found that injecting fasted mice with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) markedly increased Ser-9 phosphorylation of hepatic GSK3␤ within 15 min. Stimulation with AICAR or the GSK3␤ inhibitor SB-415286 strongly inhibited CRE-containing promoter activity in HepG2 cells. Using the Gal4-based transactivation assay system, the transcriptional activity of cAMP-response element-binding protein (CREB) was suppressed by both AICAR and SB415286, whereas that of TORC2 was repressed significantly by AICAR but very slightly by SB415286. These results show inactivation of GSK3␤ to directly inhibit CREB but not TORC2. Importantly, the AICAR-induced suppression of PEPCK-C expression was shown to be blunted by overexpression of GSK3␤(S9G) but not wild-type GSK3␤. In addition, AICAR stimulation decreased, whereas Compound C (AMPK inhibitor) increased CREB phosphorylation (Ser-129) in HepG2 cells. The time-courses of decreased CREB phosphorylation (Ser-129) and increased GSK3␤ phosphorylation were very similar. Furthermore, AMPK-mediated GSK3␤ phosphorylation was inhibited by an Akt-specific inhibitor in HepG2 cells, suggesting involvement of the Akt pathway. In summary, phosphorylation (Ser-9) of GSK3␤ is very likely to be critical for AMPK-mediated PEPCK-C gene suppression. Reduced CREB phosphorylation (Ser-129) associated with inactivation of GSK3␤ by Ser-9 phosphorylation may be the major mechanism underlying PEPCK-C gene suppression by AMPK-activating agents such as biguanide.

DNA methylation at cytosine residues in CpG dinucleotides is a component of epigenetic marks crucial to mammalian development. In preimplantation stage embryos, a large part of genomic DNA is extensively demethylated, whereas the methylation patterns are faithfully maintained in certain regions. To date, no enzymes responsible for the maintenance of DNA methylation during preimplantation development have been identified except for the oocyte form of DNA (cytosine-5)-methyltransferase 1 (Dnmt1o) at the 8-cell stage. Herein, we demonstrate that the somatic form of Dnmt1 (Dnmt1s) is present in association with chromatin in MII-stage oocytes as well as in the nucleus throughout preimplantation development. At the early one-cell stage, Dnmt1s is asymmetrically localized in the maternal pronuclei. Thereafter, Dnmt1s is recruited to the paternal genome during pronuclear maturation. During the first two cell cycles after fertilization, Dnmt1s is exported from the nucleus in the G2 phase in a CRM1/exportin-dependent manner. Antibody microinjection and small interfering RNA-mediated knock-down decreases methylated CpG dinucleotides in repetitive intracisternal A-type particle (IAP) sequences and the imprinted gene H19. These results indicate that Dnmt1s is responsible for the maintenance methylation of particular genomic regions whose methylation patterns must be faithfully maintained during preimplantation development.

The calpains are a family of Ca 2؉ -dependent cysteine proteases implicated in various biological processes. In this family, calpain 6 (Capn6) is unique in that it lacks the active-site cysteine residues requisite for protease activity. During the search for genes downstream of the endothelin 1 (ET-1) signaling in pharyngeal-arch development, we identified Capn6. After confirming that the expression of Capn6 in pharyngeal arches is downregulated in ET-1-null embryos by in situ hybridization, we investigated its function. In Capn6-transfected cells, cytokinesis was retarded and was often aborted to yield multinucleated cells. Capn6 overexpression also caused the formation of microtubule bundles rich in acetylated ␣-tubulin and resistant to the depolymerizing activity of nocodazole. Green fluorescent protein-Capn6 overexpression, immunostaining for endogenous Capn6, and biochemical analysis demonstrated interaction between Capn6 and microtubules, which appeared to be mainly mediated by domain III. Furthermore, RNA interference-mediated Capn6 inactivation caused microtubule instability with a loss of acetylated ␣-tubulin and induced actin reorganization, resulting in lamellipodium formation with membrane ruffling. Taken together, these results indicate that Capn6 is a microtubule-stabilizing protein expressed in embryonic tissues that may be involved in the regulation of microtubule dynamics and cytoskeletal organization.