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

Shh和FGF4的表达是通过正反馈诱导在肢体芽中维持的，如将Shh蛋白应用于前肢芽的外胚层FGF4表达。我们在这里已经证明FGF4蛋白也可以诱导Shh和Hoxd13在后肢芽中表达，而不是在前肢芽中表达，导致位置值的重新规范。Western blotting证实，正常表达于顶端外胚层脊（AER）的Wnt10a过表达，通过β -catenin在细胞部分的积累，导致FGF8在AER外的外胚层表达扩增。用PI-PLC和抗epha4抗体抑制细胞分选表明在肢体芽位置依赖性软骨形成过程中参与了ephrin-A和EphA的相互作用。ephrin-A2在近端肢芽强烈表达，其过表达可诱导远端指骨并指和分叉，而EphA4在远端则强烈表达。过表达抑制wnt的frp3（Frzb1）或显性阴性形式的LEF1维持未分化的肢体软骨细胞，而错误表达Wnt8或构成活性形式的LEF1促进软骨细胞成熟和肥大，导致软骨钙化。Wnt5a、Wnt11、Wnt7a和Wnt3a在发育中的肢体芽中表现出区域特异性表达。这些wnt与RCAS的过表达影响肢体芽中成肌细胞的增殖和分化。Wnt5a促进慢肌纤维分化，抑制快肌纤维分化。Wnt11的显性阴性形式对肌纤维型分化有类似的影响，而野生型Wnt11则相反。wnt结合蛋白strp2在成肌细胞祖细胞中表达，错表达时对肌肉分化有抑制作用。

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.