Molecular interactions between the ZPA and the AER in the developing vertebrate limb

The development of a vertebrate embryo is a complex process marked by several morphogenetic events, which create a highly reproducible pattern. The vertebrate limb has emerged as a model for studying pattern formation in the embryo mainly because limb manipulations do not affect embryo survival. Within the developing limb, experimental manipulation of the embryo resulted in the identification of the classical signaling centers known as the Zone of Polarizing Activity (ZPA) and the Apical Ectodermal Ridge (AER).;The molecular signals required for function of the ZPA and AER have been identified. They are Sonic hedgehog (Shh) from the ZPA and Fibroblast Growth Factors (Fgfs) from the AER. The functions of each of these molecules are now beginning to be understood. Analysis of Shh and hedgehog (Hh) signaling target genes has shown that Hh activation in the limb bud mesoderm is required for normal limb development. It has been stated that Hh signaling in the limb bud ectoderm cannot occur because components of the Hh signaling pathway and Hh target genes have not been found in this tissue. Contrary to previous reports, we have identified SHH protein and targets of Hh signaling present in the limb bud ectoderm including the apex of the bud. To directly test whether ectodermal Hh signaling was required for normal limb patterning we removed Smoothened (Smo), an essential component of the Hh signaling pathway, from the AER. Loss of functional Hh signaling in the AER resulted in disruption of the normal digit pattern and formation of additional cartilaginous condensations. These data indicate that contrary to previous accounts, the Hh signaling pathway is present and required in the developing limb AER for normal autopod development.;The formation of supernumerary digits, or polydactyly, is a common congenital malformation. In addition to our studies investigating the role of hedghog signaling in the AER, we characterized a spontaneous chicken mutant, known as Dorking. The hindlimbs of Dorkings form a preaxial supernumerary digit. During the early stages of limb development ectopic expression of several genes, including Sonic Hedgehog (Shh) and Fibroblast Growth Factor 4 (Fgf4), was found in Dorking hindlimbs. In addition to ectopic gene expression, a decrease in cell death in the anterior of the developing Dorking hindlimb was observed. Further molecular investigation revealed that ectopic Fgf4 expression was initiated and maintained independent of ectopic Shh. Inhibition of Fgf signaling but not hedgehog signaling was capable of restoring the normal anterior domain of cell death in Dorking hindlimbs. Our data indicate that in Dorking chickens, preaxial polydactyly is initiated independent of Shh. The sum of all findings is discussed within the context of establishing appropriate gene expression patterns, cell number and digit number in the developing limb.