Isolation of differentially expressed genes from wild-type and Twist mutant mouse limb buds

In the mouse, Twist is required for normal limb and craniofacial development. We show that the aristaless-like transcription factors, Alx3 and Alx4 are down-regulated in the Twist^-/- mutant and may be potential targets of Twist. By suppression subtractive hybridization we isolated 31 and 18 unique clones representing mRNAs that are putatively downregulated and upregulated respectively in Twist^-/- forelimb buds. These included genes encoding cytoskeletal components, metabolic enzymes, hemoglobin molecules, membrane transport proteins, components of transcription and translation complexes, protein modification enzymes and proteins related to cell proliferation and apoptosis. Differential expression of selected clones was validated by whole mount in situ hybridization to E10.5 wild-type and Twist^-/- embryos. We show that four novel clones are expressed in the Twist-expressing craniofacial tissues and paraxial mesoderm and downregulated in Twist^-/- embryos, raising the possibility that they are, in addition to genes of the Alx family, downstream targets of Twist.