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Common and diverse mechanisms of pattern formation of animals

动物模式形成的常见和多样化机制

基本信息

批准号：
14GS0319
负责人：
TOSHIHIKO Ogura
金额：
$ 199.35万
依托单位：
Tohoku University (2003-2006)Nara Institute of Science and Technology (2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Creative Scientific Research
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2006
项目状态：
已结题

项目摘要

1) Co-evolution of the Tbx2/3/4/5 gene complex and morphology of vertebrate limb and heart. We mis -expressed Tbx5 or Tbx4 gene in flank regions between wing and leg beds of chick embryos. Unexpectedly, such mis -expression induced complete wing and leg buds by tbx5 and tbx4, respectively. Compatible with these observations, over-expression of the dominant negative forms of Tbx5 and Tbx4 in the prospective wing and leg fields resulted in a complete loss of wing and leg structures, respectively. These data indicate that Tbx5 and Tbx4 is the initiator of wing and leg bud formation, lying at the most, upstream of the putative signaling pathway of lib formation.We also found that Tbx5 is specifically expressed in the left ventricle of developing mouse and chick hearts, implying that Tbx5 might be involved in specification of left and right ventricles. To confirm this possibility, we mis-expressed Tbx5 in chick hearts. When Tbx5 was expressed in the whole ventricle, ventricular septum was n … More ot formed. When Tbx5 was expressed in the right ventricle, position of septum was shifted. These indicate that Tbx5 specifies the identity of left ventricle, and that the ventricular septum was formed at the boundary of Tbx5 expression. In addition, we identified Tbx20 was a right ventricle-specific Tbx gene.In the developing limb buds, Tbx2 and Tbx3 are expressed in posterior part of limb bud, with Tbx2 in the most posterior part and Tbx3 in a more anterior, suggesting that these genes might be involved in specification of posterior digits. To confirm this, we mis -expressed these genes in chick limb buds. As expected, misexpression of these genes induces posteriorization of digit identity. Suppression of these genes induces anterior transformation, with a typical phenotype that resembles that of human ulnar-mammary syndrome.2) Cerebellum development and Irx2. We cloned 6 chick Irx genes and found that one of them, Irx2, is specifically expressed in the rhombic lip, from which cerebellum develops. When Irx2 was over-expressed in midbrain, no morphological change was obtained, indicating that another unknown factor(s) cooperate with Irx2 to regulate cerebellar development. When Irx2 and FGF8 were co-expressed in the midbrain, complete transformation of midbrain to cerebellum was observed. Biochemical analyses revealed that Irx2 is phosphorylated by MAP kinase, which is activated by FGF8. These data indicate that Irx2 is one of critical determinant of cerebellum formation, cooperating an organizer activity of FGF8/MAP kinase.3) Mechanical stresses, transcriptional control and development. In search of co-activator(s) of Tbx5, we have identified several factors that synergistically activate ANF (atrial natriuretic factor) promoter, a direct target of Tbx5. One of them, MKL2, transactivates ANF several hundred fold. Interestingly, this activation was strongly enhanced by RhoA and non-canonical Wnt signaling. When sub-cellular localization of MKL2 was analyzed in zebrafish heart, MKL2 was found to be in nucleus in beating heart, whereas in non-beating silent heart. This indicates that localization of MKL2 was controlled mechanical stresses, such as tension and sheer stresses induced heartbeat. Compatible with this, MKL2 shuttles into nucleus in response to mechanical stretch of cultured cell. Hence, we conclude that transcriptional activity of Tbx5 is regulated by mechanical stress-dependent nucleo-cytoplasmic shuttling of MKL2. This also suggests that cardiac development is regulated by the genetic and epigenetic programs, in which physical parameters of hemodynamics play important roles. In addition to MKL2, we have identified several shuttling factors. Our preliminary data indicate that such factors regulate differentiation of mesenchymal stem cells, adipogenesis vs. myogenesis, lipid metabolism and dynamic morphogenetic cell behaviors, highlighting mechanical stresses as a critical factor that is involved in various aspects of development and homeostasis. Less

1) Tbx2/3/4/5基因复合体与脊椎动物肢体和心脏形态的共同进化。Tbx5或Tbx4基因在鸡胚翼床和腿床之间的侧腹区域表达错误。出乎意料的是，tbx5和tbx4分别诱导了完整的翅膀和腿芽。与这些观察结果相一致的是，Tbx5和Tbx4的显性阴性形式在预期的翅膀和腿区过表达分别导致翅膀和腿结构的完全丧失。这些数据表明，Tbx5和Tbx4是翅膀和腿芽形成的启动器，位于lib形成的假定信号通路的最上游。我们还发现Tbx5在发育中的小鼠和鸡的左心室特异性表达，这表明Tbx5可能参与了左右心室的规范。为了证实这种可能性，我们在小鸡心脏中错误表达了Tbx5。当Tbx5在全脑室表达时，室间隔未形成。当Tbx5在右心室表达时，室间隔位置移位。这说明Tbx5指定了左心室的身份，并且在Tbx5表达的边界处形成了室间隔。此外，我们发现Tbx20是一个右心室特异性Tbx基因。在发育中的肢芽中，Tbx2和Tbx3在肢芽后部表达，Tbx2在肢芽后部表达，Tbx3在肢芽后部表达，Tbx3在肢芽前部表达，提示这两个基因可能参与了后趾的形成。为了证实这一点，我们在鸡的肢体芽中错误地表达了这些基因。正如预期的那样，这些基因的错误表达诱导了数字身份的后显化。这些基因的抑制诱导前转化，具有与人类尺乳综合征相似的典型表型。2)小脑发育与Irx2。我们克隆了6个鸡Irx基因，发现其中一个Irx2基因在小脑发育的菱形唇上特异性表达。当Irx2在中脑过表达时，未见形态学改变，说明有未知因子与Irx2协同调节小脑发育。当Irx2和FGF8在中脑共表达时，观察到中脑向小脑的完全转化。生化分析显示Irx2被MAP激酶磷酸化，而MAP激酶被FGF8激活。这些数据表明，Irx2是小脑形成的关键决定因素之一，协同FGF8/MAP激酶的组织者活性。3)机械应力、转录调控与发育。为了寻找Tbx5的共激活因子，我们已经确定了几个协同激活ANF（心房钠素因子）启动子的因子，这是Tbx5的直接靶点。其中之一，MKL2，能几百倍地激活ANF。有趣的是，RhoA和非典型Wnt信号强烈增强了这种激活。分析MKL2在斑马鱼心脏中的亚细胞定位，发现MKL2在跳动心脏的细胞核中，而在不跳动的静止心脏中。这表明MKL2的定位受到机械应力的控制，如张力和纯粹应力诱导的心跳。与此相容的是，MKL2在培养细胞的机械拉伸下进入细胞核。因此，我们得出结论，Tbx5的转录活性受MKL2机械应力依赖的核细胞质穿梭调节。这也表明心脏发育受遗传和表观遗传程序的调控，其中血液动力学的物理参数起着重要作用。除了MKL2外，我们还确定了几个穿梭因子。我们的初步数据表明，这些因素调节间充质干细胞的分化，脂肪生成与肌肉生成，脂质代谢和动态形态发生细胞行为，强调机械应力是参与发育和稳态各个方面的关键因素。少