The classical mouse mutant postaxial hemimelia results from a mutation in the Wnt 7a gene.

The study of spontaneous mutations has aided the understanding of developmental processes. A large collection of spontaneous or "classical" mouse mutations has been accumulated over many decades. One of the mutations causes the postaxial hemimelia (px) phenotype, which consists of limb patterning defects accompanied by Müllerian duct-associated sterility in both sexes. We were intrigued that both the limb and the Müllerian duct px phenotypes are similar to those caused by mutations in the gene encoding the Wnt 7a signaling molecule. In this paper, we investigate the nature of the px mutation. Morphological analysis and breeding experiments demonstrate that the px phenotype indeed results from a mutation in the Wnt 7a gene. Molecular analysis demonstrates that px results from a 515-bp deletion in the Wnt 7a gene. This generates an abnormal splicing event, which ultimately produces a truncated Wnt 7a protein of half the normal size. Thus, the px mutation is predicted to be a likely null allele of the Wnt 7a gene. Our results provide another interesting example of a classical mutation that disrupts an important patterning gene in development.

Novel regulatory interactions revealed by studies of murine limb pattern in Wnt-7a and En-1 mutants.

Classical embryological experiments have demonstrated that dorsal-ventral patterning of the vertebrate limb is dependent upon ectodermal signals. One such factor is Wnt-7a, a member of the Wnt family of secreted proteins, which is expressed in the dorsal ectoderm. Loss of Wnt-7a results in the appearance of ventral characteristics in the dorsal half of the distal limb. Conversely, En-1, a homeodomain transcription factor, is expressed exclusively in the ventral ectoderm, where it represses Wnt-7a. En-1 mutants have dorsal characteristics in the ventral half of the distal limb. Experiments in the chick suggest that the dorsalizing activity of Wnt-7a in the mesenchyme is mediated through the regulation of the LIM-homeodomain transcription factor Lmx-1. Here we have 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 that 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 requires Wnt-7a. Consistent with Lmx-1b playing a primary role in dorsalization of the limb, we find a direct correlation between regions of the anterior distal limb in which Lmx-lb is misregulated during limb development and the localization of dorsal-ventral patterning defects in Wnt-7a and En-1 mutant adults. Thus, ectopic Wnt-7a expression and Lmx-1b activation underlie the dorsalized En-1 phenotype, although our analysis also reveals a Wnt-7a-independent activity for En-1 in the repression of pigmentation in the ventral epidermis. Finally, we demonstrate that ectopic expression of Wnt-7a in the ventral limb ectoderm of En-1 mutants results in the formation of a second, ventral apical ectodermal ridge (AER) at the junction between Wnt-7a-expressing and nonexpressing ectoderm. Unlike the normal AER, ectopic AER formation is dependent upon Wnt-7a activity, indicating that distinct genetic mechanisms may be involved in primary and secondary AER formation.

Promoter specificity mediates the independent regulation of neighboring genes.

Although enhancers can exert their influence over great distances, their effect is generally limited to a single gene. To discern the mechanism by which this constraint can he mediated, we have studied three neighboring Drosophila genes: decapentaplegic (dpp), SLY1 homologous (Slh) and out at first (oaf). Several dpp enhancers are positioned close to Slh and oaf, and yet these genes are unaffected by the dpp elements. However, when a transposon is located within the oaf gene, the dpp enhancers activate the more distant transposon promoters while still ignoring the closer Slh and oaf start sites. To test whether this promoter specificity accounts for the regulatory autonomy normally found for the three genes, we used in vivo gene targeting to replace the oaf promoter with a dpp-compatible one in an otherwise normal chromosome. Strikingly, this chimeric gene is now activated by the dpp enhancers. Thus, the properties of the promoters themselves are sufficient to mediate the autonomous regulation of genes in this region.

Regulatory autonomy and molecular characterization of the Drosophila out at first gene.

Our previous work has shown that the expression of the Drosophila decapentaplegic (dpp) gene in imaginal disks is controlled by a 30 kb array of enhancers located 3' of the dpp coding region. Here, we describe the cloning and characterization of out at first (oaf), a gene located near this enhancer region. Transcription of oaf results in three classes of alternatively polyadenylated RNAs whose expression is developmentally regulated. All oaf transcripts contain two adjacent open reading frames separated by a single UGA stop codon. Suppression of the UGA codon during translation, as seen previously in Drosophila, could lead to the production of different proteins from the same RNA. During oogenesis, oaf RNA is expressed in nurse cells of all ages and maternally contributed to the egg. During embryonic development, zygotic transcription of the gene occurs in small clusters of cells in most or all segments at the time of germband extension and subsequently in a segmentally repeated pattern in the developing central nervous system. The gene is also expressed in the embryonic, larval and adult gonads of both sexes. We also characterize an enhancer trap line with its transposon inserted within the oaf gene and use it to generate six recessive oaf mutations. All six cause death near the beginning of the first larval instar, with two characterized lines showing nervous system defects. Last, we discuss our data in light of the observation that the enhancers controlling dpp expression in the imaginal disks have no effect on the relatively nearby oaf gene.