Apical Epithelial Cap Research Articles

Expression patterns of Fgf-8 during development and limb regeneration of the axolotl.

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.

Apical epithelial cap morphology and fibronectin gene expression in regenerating axolotl limbs.

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate limbs. The regenerated structure is often indistinguishable from the developmentally produced original. Thus, the two processes by which the limb is produced - development and regeneration - are likely to use many conserved biochemical and developmental pathways. Some of these limb features are also likely to be conserved across vertebrate families. The apical ectodermal ridge (AER) of the developing amniote limb and the larger apical epithelial cap (AEC) of the regenerating urodele limb are both found at the limb's distalmost tip and have been suggested to be functionally similar even though their morphology is quite different. Both structures are necessary for limb outgrowth. However, the AEC is uniformly smooth and thickly covers the entire limb-tip, unlike the AER, which is a protruding ridge covering only the dorsoventral boundary. Previous data from our laboratory suggest the multilayered AEC may be subdivided into separate functional compartments. We used hematoxylin and eosin (H+E) staining as well as in situ hybridization to examine the basal layer of the AEC, the layer that lies immediately over the distal limb mesenchyme. In late-stage regenerates, this basal layer expresses fibronectin (FN) message very strongly in a stripe of cells along the dorso-ventral boundary. H+E staining also reveals the unique shape of basal cells in this area. The stripe of cells in the basal AEC also contains the notch/groove structure previously seen in avian and reptilian AERs. In addition, AEC expression of FN message in the cells around the groove correlates with previous amniote AER localization of FN protein inside the groove. The structural and biochemical analyses presented here suggest that there is a specialized ridge-like compartment in the basal AEC in late-stage regenerates. The data also suggest that this compartment may be homologous to the AER of the developing amniote limb. Thus, the external differences between amniote limb development and urodele limb regeneration may be outweighed by internal similarities, which enable both processes to produce morphologically complete limbs. In addition, we propose that this basal layer of the AEC is uniquely responsible for AEC functions in regeneration, such as secreting molecules to promote mesenchymal cell cycling and dictating the direction of limb outgrowth. Finally, we include here a clarification of existing nomenclature to facilitate further discussion of the AEC and its basal layer.

Expression of the 9G1 antigen in the apical cap of axolotl regenerates requires nerves and mesenchyme

Monoclonal antibody 9G1 (mAb 9G1) is reactive to the wound epithelium of axolotl larvae and therefore provided the opportunity to examine the interaction between the wound epithelium, nerves, and blastemal mesenchyme during axolotl limb regeneration. In unamputated limbs, mAb 9G1 is reactive to most or all cells of the dermis, skeletal elements, blood vessels, and nerves, to a few unidentified cells in muscle, and to none in epidermis. During regeneration of axolotl limbs, mAb 9G1 reacts strongly to an intracellular antigen of the blastemal mesenchyme and of the distal-most portion of the wound epithelium, the so-called apical epithelial cap (AEC). Because this thickened wound epithelium of regenerating amphibian limbs has been suggested as functioning in a manner similar to the apical ectodermal ridge (AER) of embryonic limb buds, it was of interest to further examine the reactivity of mAb 9G1 during various stages of regeneration. Whether mAb 9G1 reactivity in the AEC depended on mesenchyme and/or nerves was also tested. Monoclonal antibody 9G1 reactivity appears in the AEC of regenerating limbs prior to outgrowth of the blastema and persists throughout blastemal stages. Apical epithelial cap reactivity to mAb 9G1 is nerve dependent during early stages of blastema development and becomes nerve-independent at later stages. When epithelium-free blastemal mesenchyme is grafted onto injured flank musculature, ectopic limb regeneration occurs and the AEC derived from flank epidermis exhibits mAb 9G1 reactivity. These results show that a mAb 9G1 reactive AEC is characteristic of regenerating limbs and that expression of the 9G1 antigen by the AEC is dependent upon underlying blastemal mesenchyme and nerves.

The fine structure of nerves in the epidermis of regenerating salamander limbs

Intraepidermal nerves in the apical epithelial cap of the regenerating larval salamander limb were found to be entirely extracellular in their course. Bulbous enlargements of the nerves are intimately related to the epidermal cells. These nerve bulbs, which contain numerous mitochondria, small granules and vesicles, are similar in their general morphology to nerve terminals elsewhere in The body. The plasma membranes of nerve bulb and epidermal cell are separated by a space of about 200 Å and in these regions of close apposition, the plasma membranes of both nerve and epidermal cell appear very dense and an osmiophilic material is concentrated beneath the epidermal cell membrane. The morphology of the nerve bulb and nerve fibers is discussed in relation to other studies on the fine structure of nerves and the concept of a trophic action by nerves in regenerating epidermis is emphasized.

The influence of the nerve in regeneration of the amphibian extremity.

The influence of the nerve in regeneration of the amphibian extremity.