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It has been shown that mirror-image duplications of the zeugopodia and digits are formed when MRC-5 fibroblasts producing hepatocyte growth factor (HGF) are applied to the anterior region of the chick limb bud (Yonei et al. [1993] Dev. Biol. 160:246-253). To evaluate the role of HGF in limb development, we observed the expression pattern of the HGF gene using in situ hybridization. The HGF gene was expressed in the mesoderm of the limb bud and in the central core region of mandibular arch and maxillary processes at stages 17 to 24. When both wing and leg buds begin to extend distally, the HGF gene is expressed in the mesenchymal cells, but not in the ectodermal cells and somites. Concomitant with establishment of the apical ectodermal ridge, distal mesenchymal cells of the limb bud express the HGF gene intensely with a gradient higher in the distal region. The HGF expression is later confined to the ventral and subapical mesenchyme of the limb bud, although no signal is detectable in the apical and non-ridge ectoderm. However, signal for the c-met proto-oncogene encoding the HGF receptor is not detectable in the limb bud at stages 17 to 24. These results suggest that HGF produced in the limb mesoderm may be involved in initial induction and maintenance of the apical ectoderm during limb development.

We identified a homeobox-containing gene, Prx-1, isolated from the chick limb bud cDNA library. The homeodomain sequence is related to Drosophila paired and goose-berry and mouse Pax-3, Pax-6, and Pax-7. The deduced amino acid sequence of the Prx-1 gene product reveals the absence of a paired-box sequence and extensive similarity to mouse S8 and MHox homeodomain proteins, thus constituting a new class of homeobox gene. Using an in situ hybridization method, the Prx-1 gene is shown to be expressed predominantly in the limb bud and visceral arches. At early stages of limb development, distal mesodermal cells express the homeobox gene with an apparent gradient along the proximal-distal axis. The signal is absent in the apical and nonridge ectoderm. Removal of the apical ectodermal ridge had no apparent effect on the subsequent expression of Prx-1 in the limb mesenchyme. The Prx-1-expressing cells are later confined to the interdigital and perichondrial regions. The Prx-1 transcripts are also detectable in the mesenchyme of the visceral arches and facial primordia subjacent to the ectoderm. The Prx-1 gene is weakly expressed in somites and condensing vertebrae. No signal is detectable in neural tube and ectodermal epithelium. These results suggest that the Prx-1 homeodomain protein is involved in the differentiation of bone, muscle, and other tissues of mesodermal origin during limb development.

When a mouse zone of polarizing activity (ZPA) at the posterior margin of the limb bud was grafted at the anterior margin of the chick limb bud, we found that expression of the chick homeobox genes, Chox-4.7 and -4.8, was induced prior to the formation of chick extra digits. The induction was observed in the restricted domain close to both grafted mouse ZPA and the chick apical ectodermal ridge (AER). When the posterior half of the AER was removed, the normal expression was diminished in the posterodistal region. Thus, it is likely that at least two distinct factors, one from the ZPA and the other from the AER, provide positional information to induce cooperatively the sequential expression of the Chox-4 genes.

Expression patterns of three fibroblast growth factor receptor genes, FGFR1 (cek1), FGFR2 (cek3) and FGFR3 (cek2), were observed in limb and feather morphogenesis. Expression of FGFR1 was observed in the mesoderm of limb bud, in the mesenchyme just underneath the feather placode, and then in the anterior mesenchyme of the feather bud. While expression of FGFR2 was observed in both surface ectoderm and mesenchymal aggregates corresponding to the future bones of the limb, the mesenchyme between the feather placode, and surface ectoderm of feather buds. Expression of FGFR3 was observed rather ubiquitously over mesoderm of limb and feather buds. Differential expression of these FGF receptor genes suggested that differential roles of these receptors in epithelia-mesenchymal interactions of limb and feather morphogenesis.

Activin is known to induce axial mesoderm during early development in Xenopus embryo. Activin receptor was recently identified to be a member of transmembrane serine/threonine kinase family. We have studied the role of activin-mediated signaling in the limb morphogenesis by identifying the target cells. We isolated cDNAs encoding chicken activin receptors, cARIIA and cARIIB, and examined their expression patterns during chick embryogenesis. The cARIIA gene is expressed in the apical ectoderm of the limb bud at stage 20-21, whereas the cARIIB gene is expressed uniformly in the limb mesenchyme. Expression of the cARIIA gene is confined to dorsal and ventral mesenchyme at stage 23, and later confined to precartilaginous cells. Transcripts of the cARIIA gene are found in developing neuroepithelium of spinal cord, brain and eye, surface ectoderm differentiating to epidermis, and myoblasts differentiating to muscle. The IIB receptor gene is highly expressed in the developing brain. These results suggest that the activins and their receptors are implicated in the limb development, especially, in differentiation of muscle, skin and bone.

We have isolated and identified four chicken homeobox genes in the upstream region of the Chox-4 complex. The Chox-4g and -4f genes, at the 5' extremity of the complex, were expressed locally in the vicinity of the zone of polarizing activity (ZPA) at early stages of limb development, substantiating the involvement of the genes in anteroposterior axis determination. To confirm their function, we implanted a bead containing retinoic acid, or the ZPA itself, in the anterior margin of the limb bud, leading to formation of mirror-image duplicated digits, and observed the resultant change in gene expression. Expression of the Chox-4g and -4f genes was induced in the new digit-forming region. Those results suggest that positional information assigned by a ZPA morphogen is imprinted on cellular memory by expression of the Chox-4 genes to maintain positional signaling along the anteroposterior axis in the limb field.