| Budget Amount *help |
¥311,480,000 (Direct Cost: ¥256,580,000、Indirect Cost: ¥54,900,000)
Fiscal Year 2006: ¥59,930,000 (Direct Cost: ¥46,100,000、Indirect Cost: ¥13,830,000)
Fiscal Year 2005: ¥59,410,000 (Direct Cost: ¥45,700,000、Indirect Cost: ¥13,710,000)
Fiscal Year 2004: ¥59,020,000 (Direct Cost: ¥45,400,000、Indirect Cost: ¥13,620,000)
Fiscal Year 2003: ¥59,540,000 (Direct Cost: ¥45,800,000、Indirect Cost: ¥13,740,000)
Fiscal Year 2002: ¥73,580,000 (Direct Cost: ¥73,580,000)
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| Research Abstract |
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
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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
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