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Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.

We have investigated the effect of blending on the orientation of cylindrical domains in binary mixture of asymmetric polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) block copolymers. Thin films of PMMA-cylinder-forming block copolymer were prepared on flat substrate with silicon oxide surface. Atomic force microscope observation reveals that the PMMA cylinders orient perpendicular to the substrate by tuning the film thickness. Further, the grating substrate was used to guide the alignment of the perpendicular cylinders. As a result, an array of highly ordered, hexagonally-packed PMMA cylinders in PS matrix with domain spacing of less than 30nm has been produced.

We report here mutations affecting various aspects of liver development and function identified by multiple assays in a systematic mutagenesis screen in Medaka. The 22 identified recessive mutations assigned to 19 complementation groups fell into five phenotypic groups. Group 1, showing defective liver morphogenesis, comprises mutations in four genes, which may be involved in the regulation of growth or patterning of the gut endoderm. Group 2 comprises mutations in three genes that affect the laterality of the liver; in kendama mutants of this group, the laterality of the heart and liver is uncoupled and randomized. Group 3 includes mutations in three genes altering bile color, indicative of defects in hemoglobin-bilirubin metabolism and globin synthesis. Group 4 consists of mutations in three genes, characterized by a decrease in the accumulation of fluorescent metabolite of a phospholipase A(2) substrate, PED6, in the gall bladder. Lipid metabolism or the transport of lipid metabolites may be affected by these mutations. Mutations in Groups 3 and 4 may provide animal models for relevant human diseases. Group 5 mutations in six genes affect the formation of endoderm, endodermal rods and hepatic bud from which the liver develops. These Medaka mutations, identified by morphological and metabolite marker screens, should provide clues to understanding molecular mechanisms underlying formation of a functional liver.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.

A case of pheochromocytoma was discovered incidentally during long-term hemodialysis for chronic renal failure due to acquired cystic kidney disease. A 52-year-old male patient was examined for weight loss of 3 kg during over a period of 3 months. Abdominal computed tomography (CT) revealed a left adrenal mass (3.0 cm in size). Plasma adrenaline and noradrenaline were increased to 521 pg/ml and 1,341 pg/ml, respectively, and the metoclopramide provocative test was positive. Further, in the scintiscan using 123I-metaiodobenzylguanidine (MIBG), an accumulation of the radionuclide in the left adrenal tumor region was confirmed. The patient is currently under observation and conservative treatment due to the possible occurrence of arterial hypotension after resection of the tumor and to lesser possibility of the malignancy.

A 35-year-old man was admitted because of significant hepatic dysfunction with mild splenomegaly and intra-abdominal lymphadenopathy of unknown cause. Infectious mononucleosis was suggested by subsequently detected high fever, pharyngotonsillitis and cervical lymphadenopathy, but IgM to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) showed dual positivity. A definite diagnosis of EBV-induced infectious mononucleosis was established 3 months later on the basis of seroconversion to Epstein-Barr nuclear antigen (EBNA)-IgG positivity and reduced CMV-IgM titer with persistently negative CMV-IgG. This case highlights the initial diagnostic difficulties of EBV-induced infectious mononucleosis particularly in older patients, due to concomitant abnormal humoral immunity and unusual initial manifestations such as significant liver injury and extensive intra-abdominal lymphadenopathy.

Mice lacking the stress-signaling kinases SEK1 and MKK7 die from embryonic day 10.5 (E10.5) to E12.5 with a defect in liver formation. However, the mechanism of the liver defect has remained unknown. In this review, we first introduce a monoclonal antibody, anti-Liv2, which specifically recognizes murine hepatoblasts, for the analysis of liver development, and further, we describe the genetic interaction of sek1 with the tumor necrosis factor-α receptor 1 gene (tnfr1) and the protooncogene c-jun, which are also responsible for liver formation and cell apoptosis. The defective liver formation in sek1 -/- embryos was not protected by an additional tnfr1 mutation that rescues the embryonic lethality of mice lacking nuclear factor-kappa B (NF-κB) signaling components. There was a progressive increase in hepatoblast cell numbers in wild-type embryos from E10.5 to E12.5. In contrast, impaired hepatoblast proliferation was observed in sek1 -/- livers from E10.5, although fetal liver-specific gene expression was normal. The impaired phenotype in sek1 -/- livers was more severe than that in c-jun -/- embryos, and sek1 -/- c-jun -/- embryos died earlier before E8.5. The hepatoblast proliferation required no hematopoiesis, because liver development was not impaired in AML1 -/- mice, which lack hematopoietic functions. Stimulation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) by hepatocyte growth factor was attenuated in sek1 -/- livers. Thus, SEK1 and MKK7-mediated SAPK/JNK signaling appears to playa crucial role in hepatoblast proliferation and survival in a manner that is apparently different from that of NF-κB or c-Jun.

Stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK), belonging to the mitogen-activated protein kinase family, plays an important role in stress signaling. SAPK/JNK activation requires the phosphorylation of both Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 and MKK7 have been identified as the dual specificity kinases. In this study, we generated mkk7(-/-) mouse embryonic stem (ES) cells in addition to sek1(-/-) cells and compared the two kinases in terms of the activation and phosphorylation of JNK. Although SAPK/JNK activation by various stress signals was markedly impaired in both sek1(-/-) and mkk7(-/-) ES cells, there were striking differences in the dual phosphorylation profile. The severe impairment observed in mkk7(-/-) cells was accompanied by a loss of the Thr phosphorylation of JNK without marked reduction in its Tyr-phosphorylated level. On the other hand, Thr phosphorylation of JNK in sek1(-/-) cells was also attenuated in addition to a decreased level of its Tyr phosphorylation. Analysis in human embryonic kidney 293T cells transfected with a kinase-dead SEK1 or a Thr-Pro-Phe mutant of JNK1 revealed that SEK1-induced Tyr phosphorylation of JNK1 was followed by additional Thr phosphorylation by MKK7. Furthermore, SEK1 but not MKK7 was capable of binding to JNK1 in 293T cells. These results indicate that the Tyr and Thr residues of SAPK/JNK are sequentially phosphorylated by SEK1 and MKK7, respectively, in the stress-stimulated ES cells.

A 51-year-old man on hemodialysis was studied with Tc-99m MIBI and I-123 scintigraphy for the assessment of a parathyroid mass. Although the results for the parathyroid detection were negative, bone structure was incidentally visualized with Tc-99m MIBI. Bone scintigraphy with Tc-99m MDP and marrow scintigraphy with In-111 chloride did not indicate a high bone turnover or marrow hyperplasia.

The first successful treatment by plasma exchange of acute renal failure and disseminated intravascular coagulation, as well as liver and central nervous system damage, was achieved in two patients with severe multiorgan damage associated with extensive muscle tissue damage caused by heatstroke and/or electrical injury. Substances derived from damaged muscle tissue, such as myoglobin and enzymes, were efficiently removed by a cellulose acetate membrane filter. Improvement of consciousness, disturbance, coagulation disorder, and renal failure were observed. The disorders associated with extensive rhabdomyolysis seem to be a promising indication for this therapy. Plasma exchange with a membrane filter is useful for treatment of patients with renal failure, being readily connected on-line with conventional dialysis.

膵に存在する蛋白分解酵素の1つであるelastaseには, 動脈壁のelastinの代謝に対するとともにリポ蛋白代謝にも作用するというユニークな性質があると言われているので, 透析患者に見られる脂質代謝異常と動脈の石灰化に対する治療効果を検討した. 維持透析患者を次の2群に分け, 関連するparameterの経時的変化を観察した. すなわち, E群はelastase含有カプセル (Elaszym) を1日5,400EL単位ずつ投与した50名で, 平均年齢は50.5±13.0年であった. C群は対照とした24名で, 平均年齢は45.0±7.7歳であった. 糖尿病やX線上明らかな腎性骨異栄養症の所見を呈する患者は, 対象より除外した. 透析開始前の総コレステロール, 中性脂肪, HDL-コレステロールの他, 透析管理に必要な一般血液検査を隔週間隔で行った. 観察開始の時点では, カルシュウム代謝に関したパラメータを含め, これらの値には, 両群間で差がなかった. 1年間の観察期間にも, これらの値は僅かな変化を呈するのみであった. E群の15名, C群の11名につき, 腹部大動脈のX線像を3-6ヵ月の間隔で2年間追跡した. このE群の年齢は57.4±9.0歳, 透析期間は8.4±7.9年. C群については, それぞれ47.5±7.9歳と4.7±3.0年であった. E群はC群に比べ年齢が高く透析期間が長いなど血管障害の進展に関するリスクファクターが多いにもかかわらず, 15例中2例に石灰化像の改善が認められたが, C群には改善例はなかった. 石灰化像の進展は, C群では11例中5例に認められたが, E群では2例のみであった. 1例でelastaseカプセル服用後軽度の胸焼けを認めた他には, 特別の副作用はなかった. 以上の結果より, elastaseは透析患者の血管の石灰化病変の治療に対し有望ではないかと思われた. なお, 脂質代謝異常に関しては今回肯定的結果が得られなかったが, もっと異常の顕著な症例についての検討がさらに必要と思われた.

SAPK/JNK, which belongs to the family of mitogen-activated protein kinase (MAPK), is activated by many types of cellular stresses or extracellular signals and is involved in embryonic development, immune responses, and cell survival or apoptosis. However, the physiological roles of SAPK/JNK in the signaling of stress-induced apoptosis are still controversial. To evaluate the precise function, SAPK/JNK-inactivated mouse embryonic stem (ES) cells were generated by disrupting genes of the MAPK activators, SEK1 and MKK7. Although SAPK/JNK activation by various stresses was completely abolished in sek1(-/-) mkk7(-/-) ES cells, apoptotic responses including DNA fragmentation and caspase 3 activation still occurred normally, which displays a sharp contrast to apaf1(-/-) ES cells exhibiting profound defects in the mitochondria-dependent apoptosis. These normal apoptotic responses without SAPK/JNK activation were also observed in fibroblasts derived from sek1(-/-) mkk7(-/-) ES cells. Instead, interleukin-1 beta (IL-1 beta)-induced IL-6 gene expression was greatly suppressed in sek1(-/-) mkk7(-/-) fibroblasts. These results clearly show that SAPK/JNK activation is responsible for the inflammatory cytokine-induced gene expression but not essentially required for the mitochondria-dependent apoptosis at least in ES or fibroblast-like cells, which are prototypes of all cell lineages.