Signaling factor interaction affecting Shh, Wnt, and BMP expression during pattern formation in the developimg limb

信号因子相互作用影响发育肢体模式形成过程中的 Shh、Wnt 和 BMP 表达

基本信息

批准号：
13680813
负责人：
NOHNO Tsutomu
金额：
$ 2.3万
依托单位：
KAWASAKI MEDICAL SCHOOL
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Expression of Shh and FGF4 is maintained in the limb bud through positive feedback induction, as indicated by ectodermal FGF4 expression by Shh protein applied to the anterior limb bud. We have shown here that FGF4 protein can also induce Shh and Hoxd13 expression in the hindlimb bud, but not in the forelimb bud, leading to respecification of the positional value.Overexpression of Wnt10a normally expressed in the apical ectodermal ridge(AER) resulted in ectodermal expansion of FGF8 expression outside of the AER, through beta-catenin accumulation in the cellular fraction, as demonstrated by Western blotting.Inhibition of cell sorting with PI-PLC and anti-EphA4 antibody suggests involvement of ephrin-A and EphA interaction during position dependent cartilage formation in the limb bud. Overexpression of ephrin-A2 that intensely expressed in the proximal limb bud can induce syndactyly and bifurcation of the phalangeal bones distally, where EphA4 is intensely expressed.Overexpression of Wnt-inhibiting Sfrp3(Frzb1) or dominant-negative form of LEF1 maintains undifferentiated limb chondrocytes, whereas misexpression of Wnt8 or constitutive-active form of LEF1 promotes chondrocyte maturation and hypertrophy, leading to calcification of cartilage. Wnt5a, Wnt11,Wnt7a, and Wnt3a show region specific expression in the developing limb bud. Overexpression of these Wnts with RCAS affects myoblast proliferation and differentiation in the limb bud. Wnt5a promotes slow muscle fiber differentiation and inhibits fast muscle fiber differentiation. Dominant-negative form of Wnt11 has similar effect on muscle fibretype differentiation, which is reversed by wild-type Wnt11. Wnt-binding protein Sfrp2 is expressed in the myoblast progenitor cells and has inhibitory effect on muscle differentiaion upon misexpression.

通过正反馈诱导，Shh 和 FGF4 的表达在肢芽中得以维持，如应用于前肢芽的 Shh 蛋白的外胚层 FGF4 表达所表明的那样。我们在此表明，FGF4蛋白还可以诱导Shh和Hoxd13在后肢芽中表达，但不能在前肢芽中表达，从而导致位置值的重新特化。通常在顶外胚层脊（AER）中表达的Wnt10a的过度表达导致外胚层通过细胞部分中 β-连环蛋白的积累，FGF8 表达在 AER 之外扩展，如蛋白质印迹所证明的。使用 PI-PLC 和抗 EphA4 抗体进行的细胞分选表明，在肢芽位置依赖性软骨形成过程中，肝配蛋白 A 和 EphA 相互作用参与其中。在近端肢芽中强烈表达的肝配蛋白-A2 的过度表达可诱导远端指骨的并指和分叉，其中 EphA4 强烈表达。Wnt 抑制性 Sfrp3(Frzb1) 或 LEF1 的显性失活形式的过度表达可维持未分化的肢体软骨细胞，而 Wnt8 或 LEF1 的组成型活性形式的错误表达会促进软骨细胞成熟和肥大，导致软骨钙化。 Wnt5a、Wnt11、Wnt7a 和 Wnt3a 在发育中的肢芽中显示区域特异性表达。这些 Wnt 与 RCAS 的过度表达会影响肢芽中成肌细胞的增殖和分化。 Wnt5a促进慢肌纤维分化并抑制快肌纤维分化。 Wnt11 的显性失活形式对肌肉纤维类型分化具有类似的作用，而野生型 Wnt11 可逆转这种作用。 Wnt结合蛋白Sfrp2在成肌细胞祖细胞中表达，错误表达时对肌肉分化具有抑制作用。