The role of Engrailed in establishing the dorsoventral axis of the chick limb.

Expression and mutation analyses in mice suggest that the homeobox-containing gene Engrailed (En) plays a role in dorsoventral patterning of the limb. During the initial stages of limb bud outgrowth, En-1 mRNA and protein are uniformly distributed throughout the ventral limb bud ectoderm. Limbs of En-1(-/-) mice display a double dorsal phenotype suggesting that normal expression of En-1 in the ventral ectoderm is required to establish and/or maintain ventral limb characteristics. Loss of En-1 function also results in ventral expansion of the apical ectodermal ridge (AER), suggesting that En-1 is also required for proper formation of the AER. To further investigate the role En plays in dorsoventral patterning and AER formation, we have used the replication competent retroviral vector, RCAS, to mis-express mouse En-1 in the early chick limb bud. We show that ectopic En-1 expression in dorsal ectoderm is sufficient to repress the endogenous expression of the dorsal ectodermal marker Wnt7a, with a resultant decrease in Lmx1 expression in underlying dorsal mesenchyme. Furthermore, the AER is disrupted morphologically and the expression patterns of the AER signalling molecules Fgf-8 and Fgf-4 are altered. Consistent with recent evidence that there is a reciprocal interaction between signalling molecules in the dorsal ectoderm, AER, and zone of polarising activity (ZPA), loss of Wnt7a, Fgf-8 and Fgf-4 expression leads to a decrease in expression of the signalling molecule Shh in the ZPA. These results strongly support the idea that, in its normal domain of expression, En-1 represses Wnt7a-mediated dorsal differentiation by limiting the expression of Wnt7a to the dorsal ectoderm. Furthermore, our results provide additional evidence that En-1 is involved in AER formation and suggest that En-1 may act to define ventral ectodermal identity.