Strain-triggered mechanical feedback in self-organizing optic-cup morphogenesis.

Organogenesis is a self-organizing process of multiple cells in three-dimensional (3D) space, where macroscopic tissue deformations are robustly regulated by multicellular autonomy. It is clear that this robust regulation requires cells to sense and modulate 3D tissue formation across different scales, but its underlying mechanisms are still unclear. To address this question, we developed a versatile computational model of 3D multicellular dynamics at single-cell resolution and combined it with the 3D culture system of pluripotent stem cell-derived optic-cup organoid. The complementary approach enabled quantitative prediction of morphogenesis and its corresponding verification and elucidated that the macroscopic 3D tissue deformation is fed back to individual cellular force generations via mechanosensing. We hereby conclude that mechanical force plays a key role as a feedback regulator to establish the robustness of organogenesis.

Deletion of thyrotropin receptor residue Asp403 in a hyperfunctioning thyroid nodule provides insight into the role of the ectodomain in ligand-induced receptor activation.

Somatic mutations of the TSH receptor (TSHR) gene are the main cause of autonomously functioning thyroid nodules. Except for mutations in ectodomain residue S281, all of the numerous reported activating mutations are in the TSHR membrane-spanning region. Here, we describe a patient with a toxic adenoma with a novel heterozygous somatic mutation caused by deletion of ectodomain residue Asp403 (Del-D403). Subsequent in vitro functional studies of the Del-D403 TSHR mutation demonstrated greatly increased ligand-independent constitutive activity, 8-fold above that of the wild-type TSHR. TSH stimulation had little further effect, indicating that the mutation produced near maximal activation of the receptor. In summary, we report only the second TSHR ectodomain activating mutation (and the first ectodomain deletion mutation) responsible for development of a thyroid toxic adenoma. Because Del-D403 causes near maximal activation, our finding provides novel insight into TSHR structure and function; residue D403 is more likely to be involved in the ligand-mediated activating pathway than in the ectodomain inverse agonist property.

The effects of seasonal and interannual variability of oceanic structure in the western Pacific North Equatorial Current on larval transport of the Japanese eel Anguilla japonica.

As the North Equatorial Current (NEC)-bifurcation is known to be related to El Niño-Southern Oscillation (ENSO) events, the influence of the position of the NEC bifurcation on transport success of the larval Japanese eel Anguilla japonica was investigated. Using a Lagrangian modelling approach, larval transport was simulated and the relative influence of El Niño and La Niña events and the NEC-bifurcation position on the success of particle transport analysed. The number of particles transported from the NEC to the Kuroshio tended to be lowest during El Niño years, and differences between La Niña and regular years were small. The transport success observed in simulations showed some relationships to annual A. japonica glass eel recruitment to Tanegashima Island over 1993 to 2001, but not in 2002. The study shows that particle tracking simulations can be used to improve knowledge of the oceanic migration of A. japonica but further studies are required, including comparisons with the effects on larval survival of fluctuations in temperature and food availability.

Directed differentiation of neural and sensory tissues from embryonic stem cells in vitro.

We have recently identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells from mouse embryonic stem (ES) cells. Particularly, midbrain TH+ dopaminergic neurons are generated efficiently in this system. These dopaminergic neurons are transplantable and survive well in the 6-OHDA-treated mouse striatum. SDIA induces co-cultured ES cells to differentiate into rostral central nervous system (CNS) tissues containing both ventral and dorsal cells. While early exposure of SDIA-treated ES cells to BMP4 suppresses neural differentiation and promotes epidermogenesis, late BMP4 exposure after the 4th day of co-culture causes differentiation of neural crest cells and dorsal-most CNS cells, with autonomic system and sensory lineages induced preferentially by high and low BMP4 concentrations, respectively. In contrast, Sonic Hedgehog (Shh) suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons and HNF3beta+ floor plate cells with axonal guidance activities. Thus, SDIA-treated ES cells generate naïve precursors that have the competence of differentiating into the "full" dorsal-ventral range of neuroectodermal derivatives in response to patterning signals. I also discuss the role of SDIA and the mode of rostral-caudal specification of neuralized ES cells. In addition, I would like to discuss them in the light of control of in vitro neural production for the use in regenerative medicine for parkinsonism and retinal degeneration.

International prognostic scoring system and TP53 mutations are independent prognostic indicators for patients with myelodysplastic syndrome.

We applied the International Prognostic Scoring System (IPSS) to our series of 118 patients with myelodysplastic syndrome (MDS) to determine its validity, and also used univariate and multivariate analyses to evaluate the prognostic significance of TP53 configurations. Sixteen patients with the mutation had a strikingly worse prognosis and the multivariate analysis demonstrated that this alteration was the most significant factor. The prognostic comparison between patients with and without the mutation within each IPSS subgroup showed a significant difference in the intermediate subgroups. A combination of clinical manifestations and genetic configurations provided us with more accurate prognostic information in MDS patients.

Requirement of FoxD3-class signaling for neural crest determination in Xenopus.

Fox factors (winged-helix transcription factors) play important roles in early embryonic patterning. We show here that FoxD3 (Forkhead 6) regulates neural crest determination in Xenopus embryos. Expression of FoxD3 in the presumptive neural crest region starts at the late gastrula stage in a manner similar to that of Slug, and overlaps with that of Zic-r1. When overexpressed in the embryo and in ectodermal explants, FoxD3 induces expression of neural crest markers. Attenuation of FoxD3-related signaling by a dominant-negative FoxD3 construct (FoxD3delN) inhibits neural crest differentiation in vivo without suppressing the CNS marker Sox2. Interestingly, these loss-of-function phenotypes are reversed by coinjecting SLUG: In animal cap explants, neural crest differentiation induced by Slug and Wnt3a is also inhibited by FoxD3delN but not by a dominant-negative form of XBF2. Loss-of-function studies using dominant-negative forms of FoxD3 and Slug indicate that Slug induction by Zic factors requires FoxD3-related signaling, and that FoxD3 and Slug have different requirements in inducing downstream neural crest markers. These data demonstrate that FoxD3 (or its closely related factor) is an essential upstream regulator of neural crest determination.

Roles of Sox factors in neural determination: conserved signaling in evolution?

Neural differentiation in amphibian embryos is initiated by the neural inducers emanating from the Spemann-Mangold organizer. The fate of uncommitted ectoderm is determined by graded BMP activity along the dorsal-ventral axis. Several transcriptional regulators acting in early neural differentiation have been identified, including Sox, Zic, Pou, HLH and Fox factors. In this paper, I review recent molecular studies on neural determination, focusing mainly on Sox factors. I also discuss the possible conservation of regulatory factors in neural differentiation, comparing Xenopus and Drosophila counterparts.

Regulation of neural determination by evolutionarily conserved signals: anti-BMP factors and what next?

The evolutionary conservation of Chordin/bone morphogenetic protein (BMP) signaling supports the hypothesis of dorsal-ventral axis inversion of vertebrates and invertebrates, and implies that the invention of a central nervous system occurred only once during animal evolution. This hypothesis is further strengthened by recent findings of the conservation of downstream genes and modifier genes of neural induction. On the other hand, in contrast with such gross conservation, recent data suggest that the requirement for some signals in neural determination may differ even within the vertebrate subphylum.

Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.

We have identified a stromal cell-derived inducing activity (SDIA) that promotes neural differentiation of mouse ES cells. SDIA accumulates on the surface of PA6 stromal cells and induces efficient neuronal differentiation of cocultured ES cells in serum-free conditions without use of either retinoic acid or embryoid bodies. BMP4, which acts as an antineuralizing morphogen in Xenopus, suppresses SDIA-induced neuralization and promotes epidermal differentiation. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained from SDIA-treated ES cells. When transplanted, SDIA-induced dopaminergic neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase expression. Neural induction by SDIA provides a new powerful tool for both basic neuroscience research and therapeutic applications.

Relationship between breast milk feeding and atopic dermatitis in children.

OBJECTIVE: To determine whether or not the breast milk feeding has a role in the prevalence of atopic dermatitis among children. METHODS: The target population of the study was all children participating in health check-up program for 3-year-old children in 60 municipalities locating 10 selected prefectures during designated 2 months between October and December 1997. Using a questionnaire, information on nutrition in infants (breast milk only, bottled milk only, or mixed), parity, mothers' age at birth, and a history of atopic dermatitis was obtained. Besides, data on potential confounding factors were obtained. RESULTS: Questionnaires from 3856 children (81.6% of those who were to participate in the programs, and 96.4% of children who participated them) were analyzed. After the adjustment for all potential confounding factors using unconditional logistic models, the risk of atopic dermatitis was slightly higher among children with breast milk (odds ratio [OR] = 1.16 with 95% confidence interval [CI] 0.96-1.40). Mothers' age at birth (OR for those who were more than 30 years or older in comparison with those who were younger than 30 years = 1.15; 95% CI, 0.96-1.37) and those with second or later parity orders (OR = 1.14, 95% CI; 0.95-1.35) showed odds ratios that were higher than unity without statistical significance. CONCLUSION: Breast milk elevates the risk of atopic dermatitis slightly without statistical significance; the risk may be, however, higher in children in second or later parity orders.

Xenopus kielin: A dorsalizing factor containing multiple chordin-type repeats secreted from the embryonic midline.

The midline tissues are important inductive centers of early vertebrate embryos. By signal peptide selection screening, we isolated a secreted factor, Kielin, which contains multiple cys-rich repeats similar to those in chordin (Chd). Expression of Kielin starts at midgastrula stages in the notochord and is detected in the floor plate of neurula embryos. Kielin is induced in mesoderm and in ectoderm by nodal-related genes. Chd is sufficient to activate Kielin expression in mesoderm whereas Shh or HNF-3beta in addition to Chd is required for induction in ectoderm. Kielin has a distinct biological activity from that of Chd. Injection of Kielin mRNA causes dorsalization of ventral marginal zone explants and expansion of MyoD expression in neurula embryos. Unlike Chd, Kielin does not efficiently induce neural differentiation of animal cap ectoderm, suggesting that the activity of Kielin is not simply caused by BMP4 blockade. Kielin is a signaling molecule that mediates inductive activities of the embryonic midline.

[Qualitative study of municipalities' maternal and child health promotion planning].

From 1997, basic maternal and child health services have been provided through municipalities. The ministry of Health and Welfare has requested all municipalities to publish a MCH promotion plan. We conducted a qualitative analysis of important contents of the plans using an evaluation index of original dichotomous variables. Out of 3,256 municipalities, 2,873 developed the plans for MCH promotion. Most of the plans present descriptions of the significance of MCH promotion planning, goals of the plans, problems of current MCH statistics and services, and plans of providing services. The proportion was low for plans which have needs assessment of target population, indicators of evaluation of plans, description of the relationship between objectives and services, importance of objectives, action plans, specific chapter or evaluation of the plan, monitoring of the plan, and plans for informing public of the MCH plan. Therefore, the MCH promotion plans have weaknesses in evaluation and action. The MCH promotion plans of middle sized cities were more likely to have these important contents. We will continue to analyze the processes for producing of excellent MCH promotion plans to extract universal promoting factors for producing MCH promotion plans.

Xenopus Xenf: an early endodermal nuclear factor that is regulated in a pathway distinct from Sox17 and Mix-related gene pathways.

We report a novel zygotic gene encoding a Xenopus endodermal nuclear factor, Xenf. Expression of Xenf starts at the late blastula stages and is decreased after gastrulation. Xenf shows no structural homology to any known proteins. When GFP-tagged Xenf is overexpressed in Xenopus cells, Xenf protein is localized to the nucleus, associating closely with the chromosomes. In animal cap assays, Xenf expression is strongly activated by mRNA injection of Vg1 and VegT, maternal vegetal genes that can induce endodermal differentiation. In contrast, Xenf is not induced by endoderm-inducing zygotic transcription factors such as Sox17 and Mix-related genes. In turn, Xenf does not activate expression of Sox17, Mixer or Milk. Thus, Xenf is regulated by maternal vegetal positional information in a parallel manner to Sox17 and Mix-related gene pathways.

Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm.

From early stages of development, Sox2-class transcription factors (Sox1, Sox2 and Sox3) are expressed in neural tissues and sensory epithelia. In this report, we show that Sox2 function is required for neural differentiation of early Xenopus ectoderm. Microinjection of dominant-negative forms of Sox2 (dnSox2) mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Expression of dnSox2 in developing embryos suppresses expression of N-CAM and regional neural markers. We have analyzed temporal requirement of Sox2-mediated signaling by using an inducible dnSox2 construct fused to the ligand-binding domain of the glucocorticoid receptor. Attenuation of Sox2 function both from the late blastula stage and from the late gastrula stage onwards causes an inhibition of neural differentiation in animal caps and in whole embryos. Additionally, dnSox2-injected cells that fail to differentiate into neural tissues are not able to adopt epidermal cell fate. These data suggest that Sox2-class genes are essential for early neuroectoderm cells to consolidate their neural identity during secondary steps of neural differentiation.

Myelodysplastic syndrome with clonal eosinophilia accompanied by eosinophilic pulmonary interstitial infiltration.

We report a case of de novo myelodysplastic syndrome with clonal eosinophilia (MDS-Eo) and eosinophilic pulmonary interstitial infiltration, confirmed by autopsy. Cytogenetic study using Giemsa banding identified 47,XY,+1,der(1;7)(q10;p10),+8 in the marrow cells. Simple Giemsa staining revealed the same chromosomal aberration in metaphase spreads with eosinophilic granules, indicating the clonal proliferation of eosinophils. To our knowledge, our case is the 6th reported case of MDS-Eo with cytogenetically confirmed clonal eosinophilia, and the first autopsy of MDS-Eo. A review of the literature combined with our findings suggests that this type of chromosomal aberration might be involved in the as yet unknown pathogenesis of MDS-Eo.

Genotype of glutathione S-transferase and other genetic configurations in myelodysplasia.

We examined polymorphisms of glutathione S-transferase (GST) genes in 159 Japanese patients with myelodysplasia and compared the incidence with that in 43 normal individuals to clarify their pathogenetic significance in myelodysplasia. In individuals with the GSTT1 null genotype, the odds ratios for disease risk were elevated to 2.65 (95%CI; 1.27-5.52) in de novo MDS, 4.62 (1.48-14.4) in therapy-related AML, and 2.94 (1.07-8.07) in AML with triliniage dysplasia. Other representative polymorphisms of GSTs had a similar incidence among patients with myelodysplasia, and those of the controls and other hematological disorders. To further investigate the genetic pathway of myelodysplasia, the association between GST genotype and karyotype or configurations of TP53 and NRAS was evaluated, but no relationship was noted. These results suggest that the GSTT1 null genotype may play a role in an increased risk of myelodysplasia unrelated to other mechanisms of myelodysplasia, such as chromosomal alterations or mutation of TP53 or NRAS.

Distinct genetic involvement of the TP53 gene in therapy-related leukemia and myelodysplasia with chromosomal losses of Nos 5 and/or 7 and its possible relationship to replication error phenotype.

We examined chromosomes and molecular aberrations in 21 patients with therapy-related leukemias (t-AML) or myelodysplastic syndromes (t-MDS). All patients showed abnormal karyotypes, and chromosomal losses of No. 5 and/or No. 7 (-5/5q- and/or -7/7q-) were identified in 12 patients. Among these 12, six patients (50%) harbored a TP53 mutation, and two of five examined showed microsatellite instability, suggesting replication error (RER+) phenotype. Meanwhile, among the other nine patients without -5/5q- and/or -7/7q-, none harbored a TP53 mutation, and none of five examined showed RER+ phenotype. Thus, TP53 mutations and RER+ phenotype were preferentially associated with specific chromosomal losses in t-AML/MDS. We then screened for mutational events in representative DNA mismatch repair genes; exons 5-7 and 12-15 of the hMSH2 gene and exon 9 of hMLH1. Notably, two unrelated patients showing RER+ phenotype had an identical missense alteration at codon 419 of hMSH2 in their marrow cells and fibroblasts, which were not found in 120 DNA samples from healthy volunteers or patients with other hematological disorders. Consequently, this study revealed a possible relationship of RER+ phenotype accompanying an hMSH2 alteration to the development of therapy-related AML/MDS in association with TP53 mutations and specific chromosomal losses, and suggests that some patients may be predisposed to myelodysplasia after chemotherapy for their primary tumor.

[Syngeneic peripheral blood stem cell transplantation for chronic myelogenous leukemia associated with Klinefelter's syndrome].

We report on a patient with Klinefelter's syndrome who underwent successful syngeneic peripheral blood stem cell transplantation (PBSCT) for chronic myelogenous leukemia (CML). A-46-year-old man was given a diagnosis of chronic phase CML in May 1994 on the basis of findings of leukocytosis (54,000/microliter) and bone marrow chromosomal abnormalities [47, XXY, t(9; 22; 14) (q34; q11; q24)]. Hydroxyurea and interferon alpha were administered. In August 1996, a syngeneic transplant was performed following myeloablative therapy, using peripheral blood stem cells collected from the patient's identical twin brother, who had been pretreated with rhG-CSF. Following transplantation (4.0 x 10(6) CD34+ cells/kg) and the subsequent administration of rhG-CSF, the patient rapidly achieved normal tri-lineage hematopoiesis. A post-transplant chromosomal analysis of the patient's bone marrow cells detected the 47, XXY karyotype. Although the major BCR-ABL gene had been detected in bone marrow by RT-PCR methods prior to the syngeneic PBSCT (August 1996), it was not detected after PBSCT (January 1997). In March 1998, interphase fluorescence in situ hibridization (FISH) procedures disclosed XXY signal patterns in peripheral blood lymphocyte samples from the patient and donor, at frequencies of 96% and 97%, respectively. Both the patient and donor had high levels of serum FSH and LH and low levels of serum testosterone.