Molecular Mechanism of Retinoic Acid-Induced Pattern Duplication During Limb Development

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Anterior-posterior pattern of the limb is known to be determined by a signal emanating from the zone of polarizing activity(ZPA) located in the posterior margin of limb bud. Retinoic acid was postulated to be a morphogen of the ZPA.However, recent findings on the mechanism of limb development argue that retinoic acid is an inducer of polarizing activity but not a direct ZPA signal. Mechanism of retinoic acid-induced limb duplication was investigated using chick limb bud, and following results were obtained.1. Expression of HoxD induced by ZPA implantation to the anterior region of the limb bud is distal to the graft and subjacent to the apical ectodermal ridge(AER). Removal of the AER in the posterior region results in the decrease in HoxD expression. Signaling factor from the AER is therefore required for the induction and maintenance of HoxD expression in addition to the ZPA factor. A msh-related homeobox gene, Msx1, is expressed in the AER and underlying distal mesenchyme of the lim … More b bud. Expression of the Msx1 gene depends on the signals from the AER, whereas mesodermal expression of Prx1, a homeobox gene related to Drosophila paired, is independent of the AER.These homeobox genes expressed specifically in the limb bud are differentially regulated by signals from the AER and ZPA.2. Several growth factors, including fibroblast growth factor(FGF) and transforming growth factor beta(TGF-beta), are presumed to be involved in limb development, since the growth factors are known to play important roles for mesoderm induction and axial determination in Xenopus emgbryogenesis.(1) Four FGF receptor genes have been identified, and are expressed in the limb bud. Among them, FGFR2 reveals ectodermal expression and centrally restricted mesodermal expression, suggesting the involvement of the FGF family in skin and bone morphogenesis. The FGF-4 gene is also expressed in the AER of the chick limb bud, as originally observed in the mouse limb bud in organ culture. Ectopic expression of the FGF-4 gene using retrovirus vector results in the supernumeral digits in the chick embryo. Thus, FGF-4 is likely to be one of the AER factors responsible for limb outgrowth.(2) Type I and II receptors for TGF-beta family are known to have characteristic intracellular Ser/Thr kinase domain. Two chicken activin receptors were identified, and the receptor genes are weakly expressed in the early limb bud. Later, the receptor genes are expressed in the prechondrium, myoblasts and ectoderm. Two novel members of the type I receptors were identified in the chick ; one for BMP-2/4 and the other for TGF-beta1. The former is weakly expressed in the prechondrium of the developing limb.(3) Hepatocyte growth factor(HGF) is known to induce secondary body axis in the chick embryo. HGF-producing MRC-5 cells were shown to induce duplicated limb when implanted to the anterior region of the leg bud at budding stages. The HGF gene is expressed in the distal mesenchyme of the limb bud underneath the AER, suggesting HGF as an AER-inducing and/or maintenance factor during limb development. Less