THE ROLE OF ENGRAILED IN ESTABLISHING THE DORSOVENTRAL AXIS OF THE CHICK LIMB

Expression and mutation analyses in mice suggest that the homeobox-con taining 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 sugg esting that normal expression of En-1 in the ventral ectoderm is requi red to establish and/or maintain ventral limb characteristics. Loss of En-1 function also results in ventral expansion of the apical ectoder mal ridge (AER), suggesting that En-1 is also required for proper form ation of the AER, To further investigate the role En plays in dorsoven tral patterning and AER formation, we have used the replication compet ent retroviral vector, RCAS, to mis-express mouse En-1 in the early ch ick limb bud. We show that ectopic En-1 expression in dorsal ectoderm is sufficient to repress the endogenous expression of the dorsal ectod ermal marker Wnt7a, with a resultant decrease in Lmx1 expression in un derlying dorsal mesenchyme. Furthermore, the AER is disrupted morpholo gically and the expression patterns of the AER signalling molecules Fg f-8 and Fgf-l 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 si gnalling molecule Shh in the ZPA. These results strongly support the i dea that, in its normal domain of expression, En-l represses Wnt7a-med iated dorsal differentiation by limiting the expression of Wnt7a to th e dorsal ectoderm. Furthermore, our results provide additional evidenc e that En-l is involved in AER formation and suggest that En-1 may act to define ventral ectodermal identity.