ANALYSIS OF THE GENETIC PATHWAY LEADING TO FORMATION OF ECTOPIC APICAL ECTODERMAL RIDGES IN MOUSE ENGRAILED-1 MUTANT LIMBS

The apical ectodermal ridge (AER), a rim of thickened ectodermal cells at the interface between the dorsal and ventral domains of the limb b ud, is required for limb outgrowth and patterning. We hare previously shown that the limbs of En1 mutant mice display dorsal-ventral and pro ximal-distal abnormalities, the latter being reflected in the appearan ce of a broadened AER and formation of ectopic ventral digits. A detai led genetic analysis of wild-type, En1 and Wnt7a mutant limb buds duri ng AER development has delineated a role for En1 in normal AER formati on. Our studies support previous suggestions that AER maturation invol ves the compression of an early broad ventral domain of limb ectoderm into a narrow rim at the tip and further show that En1 plays a critica l role in the compaction phase. Loss of En1 leads to a delay in the di stal shift and stratification of cells in the ventral half of the AER. At later stages, this often leads to development of a secondary ventr al AER, which can promote formation of an ectopic digit. The second AE R forms at the juxtaposition of the ventral border of the broadened mu tant AER and the distal border of an ectopic Lmx1b expression domain. Analysis of En1/Wnt7a double mutants demonstrates that the dorsalizing gene Wnt7a is required for the formation of the ectopic AERs in En1 m utants and for ectopic expression of Lmx1b in the ventral mesenchyme, We suggest a model whereby, in En1 mutants, ectopic ventral Wnt7a and/ or Lmx1b, expression leads to the transformation of ventral cells in t he broadened AER to a more dorsal phenotype. This leads to induction o f a second zone of compaction ventrally, which in some cases goes on t o form an autonomous secondary AER.