NOVEL REGULATORY INTERACTIONS REVEALED BY STUDIES OF MURINE LIMB PATTERN IN WNT-7A AND EN-1 MUTANTS

Classical embryological experiments have demonstrated that dorsal-vent ral patterning of the vertebrate limb is dependent upon ectodermal sig nals, One such factor is Wnt-7a, a member of the Wnt family of secrete d proteins, which is expressed in the dorsal ectoderm, Loss of Wnt-7a results in the appearance of ventral characteristics in the dorsal hal f of the distal limb, Conversely, En-1, a homeodomain transcription fa ctor, is expressed exclusively in the ventral ectoderm, where it repre sses Wnt-7a. Eu-1 mutants have dorsal characteristics in the ventral h alf of the distal limb, Experiments in the chick suggest that the dors alizing activity of Wnt-7a in the mesenchyme is mediated through the r egulation of the LIM-homeodomain transcription factor Lmx-1, Here we h ave examined the relationship between Wnt-7a, En-1 and Lmx-1b, a mouse homolog of chick Lmx-1, in patterning the mammalian limb, We find tha t Wnt-7a is required for Lmx-1b expression in distal limb mesenchyme, and that Lmx-1b activation in the ventral mesenchyme of En-1 mutants r equires Wnt-7a. Consistent with Lmx-1b playing a primary role in dorsa lization of the limb, we find a direct correlation between regions of the anterior distal limb in which Lmx-1b is misregulated during limb d evelopment and the localization of dorsal-ventral patterning defects i n Tnt-7a and En-1 mutant adults, Thus, ectopic Wnt-7a expression and L mx-1b activation underlie the dorsalized En-1 phenotype, although our analysis also reveals a Wnt-7a-independent activity for En-1 in the re pression of pigmentation in the ventral epidermis, Finally, we demonst rate that ectopic expression of Wnt-7a in the ventral limb ectoderm of En-1 mutants results in the formation of a second, ventral apical ect odermal ridge (AER) at the junction between Wnt-7a-expressing and none xpressing ectoderm, Unlike the normal AER, ectopic AER formation is de pendent upon Wnt-7a activity, indicating that distinct genetic mechani sms may be involved in primary and secondary AER formation.