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

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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.