Fgf‐8 is one of the key signaling molecules implicated in the initiation, outgrowth, and patterning of vertebrate limbs. However, it is not clear whether FGF‐8 plays similar role in development and regeneration of urodele limbs. We isolated a Fgf‐8 cDNA from the Mexican axolotl (Ambystoma mexicanum) through the screening of an embryo cDNA library. The cloned 1.26‐kb cDNA contained an open reading frame encoding 212 amino acid residues with 84%, 86%, and 80% amino acid identities to those of Xenopus, chick, and mouse, respectively. By using the above clone as a probe, we examined the temporal and spatial expression patterns of Fgf‐8 in developing embryos and in regenerating larval limbs. In developing embryos, Fgf‐8 was expressed in the neural fold, midbrain‐hindbrain junction, tail and limb buds, pharyngeal clefts, and primordia of maxilla and mandible. In the developing axolotl limb, Fgf‐8 began to be expressed in the prospective forelimb region at pre–limb‐bud and limb bud stages. Interestingly, strong expression was detected in the mesenchymal tissue of the limb bud before digit forming stages. In the regenerating limb, Fgf‐8 expression was noted in the basal layer of the apical epithelial cap (AEC) and the underlying thin layer of mesenchymal tissue during blastema formation stages. These data suggest that Fgf‐8 is involved in the organogenesis of various craniofacial structures, the initiation and outgrowth of limb development, and the blastema formation and outgrowth of regenerating limbs. In the developing limb of axolotl, unlike in Xenopus or in amniotes such as chick and mouse, the Fgf‐8 expression domain was localized mainly in the mesenchyme rather than epidermis. The unique expression pattern of Fgf‐8 in axolotl suggests that the regulatory mechanism of Fgf‐8 expression is different between urodeles and other higher species. The expression of Fgf‐8 in the deep layer of the AEC and the thin layer of underlying mesenchymal tissue in the regenerating limbs support the previous notion that the amphibian AEC is a functional equivalent of the AER in amniotes. © 2001 Wiley‐Liss, Inc.

We have identified and characterized a new amphibian orphan member of the nuclear receptor superfamily and termed it FOR1 (farnesoid X receptor (FXR)-like Orphan Receptor) because it shares the highest amino acid identity with the mammalian FXR. We also identi- The nuclear receptor superfamily comprises a large group of structurally related ligand-dependent transcription factors regulated by a variety of steroid and non-steroid hormones. It also includes a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (reviewed in Refs. 1-3). The nuclear receptors modulate target gene transcription by direct binding to specific DNA sequences, called hormone response elements (HRE), 1 which are generally located in the promoter of the specific target genes. In general, both classic nuclear hormone receptors and orphan nuclear hormone receptors consist of four or five different modules or domains; A/B, C, D, E, and F (1). The non-conserved N-terminal region of nuclear receptors (A/B domain) is involved in transactivation in some cases but is of unknown function or is absent in others. The DNA-binding C-domain (DBD) shows the strongest sequence similarity among different nuclear receptors and is engaged in the binding of these receptors to cognate HREs. The C-domain consists of 65-68 amino acids, among which 8 cysteine residues are absolutely conserved and form two zincbinding modules (1, 3, 4). The D-domain, called the hinge region, shows relatively low sequence similarity and contains sequences involved in HRE binding at its N terminus. The E-domain directly binds to ligands or hormones and is also involved in nuclear localization and receptor dimerization. The C terminus of the LBD contains a conserved motif that, together with other portions of the LBD, forms the binding site for transcriptional coactivators (1). The F-domain is an additional C-terminal extension found in only subset of receptors. The function of this non-conserved segment is unclear.A large number of orphan nuclear receptor genes have been discovered by several different approaches. These include 1) screening cDNA libraries with conventional receptor cDNA probes at relaxed stringency (5) or with degenerate oligonucleotides based on the conserved regions (6), 2) performing PCR with degenerate oligonucleotide PCR primers from the DBD (7, 8), 3) screening cDNA libraries using nuclear receptor ligand binding domains (LBD) or receptor interaction domains of coactivators as bait in a yeast two-hybrid system (9, 10).