THE LIMB FIELD MESODERM DETERMINES INITIAL LIMB BUD ANTEROPOSTERIOR ASYMMETRY AND BUDDING INDEPENDENT OF SONIC HEDGEHOG OR APICAL ECTODERMAL GENE EXPRESSIONS

We have analyzed the pattern of expression of several genes implicated in limb initiation and outgrowth using limbless chicken embryos. We d emonstrate that the expressions of the apical ridge associated genes, Fgf-8, Fgf-4, Bmp-2 and Bmp-4, are undetectable in limbless limb bud e ctoderm; however, FGF2 protein is present in the limb bud ectoderm. Sh h expression is undetectable in limbless limb bud mesoderm. Neverthele ss, limbless limb bud mesoderm shows polarization manifested by the as ymmetric expression of Hoxd-11, -12 and -13, Wnt-5a and Bmp-1 genes. T he posterior limbless limb bud mesoderm, although not actually express ing Shh, is competent to express it if supplied with exogenous FGF or transplanted to a normal apical ridge environment, providing further e vidence of mesodermal asymmetry. Exogenous FGF applied to limbless lim b buds permits further growth and determination of recognizable skelet al elements, without the development of an apical ridge. However, the cells competent to express Shh do so at reduced levels; nevertheless, Bmp-2 is then rapidly expressed in the posterior limbless mesoderm. li mbless limb buds appear as bi-dorsal structures, as the entire limb bu d ectoderm expresses Wnt-7a, a marker for dorsal limb bud ectoderm; th e ectoderm fails to express En-1, a marker of ventral ectoderm. As exp ected, C-Lmx1,which is downstream of Wnt-7a, is expressed in the entir e limbless limb bud mesoderm. We conclude that anteroposterior polarit y is established in the initial limb bud prior to Shh expression, apic al ridge gene expression or dorsal-ventral asymmetry. We propose that the initial pattern of gene expressions in the emergent limb bud is es tablished by axial influences on the limb field. These permit the bud to emerge with asymmetric gene expression before Shh and the apical ri dge appear. We report that expression of Fgf-8 by the limb ectoderm is not required for the initiation of the limb bud. The gene expressions in the pre-ridge limb bud mesoderm, as in the limb bud itself, are un stable without stimulation from the apical ridge and the polarizing re gion (Shh) after budding is initiated. We propose that the defect in l imbless limb buds is the lack of a dorsal-ventral interface in the lim b bud ectoderm where the apical ridge induction signal would be receiv ed and an apical ridge formed. These observations provide evidence for the hypothesis that the dorsal-ventral ectoderm interface is a precon dition for apical ridge formation.