Sonic hedgehog (shh) expression in developing and regenerating axolotl limbs.

Sonic hedgehog (shh) expression is detectable in the posterior mesenchyme of many developing vertebrate limbs. We have isolated an RT-PCR fragment from the axolotl, Ambystoma mexicanum, that has high identity to other vertebrate shh genes. We describe the localization of this transcript during development and regeneration and in response to tissue grafts and retinoic acid (RA) exposure in the axolotl. Even though axolotl digits show a reversed polarity of differentiation (anterior [A] to posterior [P]) when compared to other tetrapods (P to A), shh is nevertheless expressed on the posterior margin of developing and regenerating limb buds. When A cells are grafted adjacent to P cells, an ectopic domain of shh is induced. Exposure to retinoic acid (RA), a molecule known to alter pattern in all three limb axes in urodeles, results in ectopic expression of shh in anterior cells of the regeneration blastema. Prior to this induced expression in response to RA, there is an earlier response by the endogenous domain of shh, which is downregulated within the first few hours of exposure.

Expression of HoxD genes in developing and regenerating axolotl limbs.

Hox genes play a critical role in the development of the vertebrate axis and limbs, and previous studies have implicated them in the specification of positional identity, the control of growth, and the timing of differentiation. Axolotl limbs offer an opportunity to distinguish these alternatives because the sequence of skeletal differentiation is reversed along the anterior-posterior axis relative to that of other tetrapods. We report that during early limb development, expression patterns of HoxD genes in axolotls resemble those in amniotes and anuran amphibians. At later stages, the anterior boundary of Hoxd-11 expression is conserved with respect to morphological landmarks, but there is no anterior-distal expansion of the posterior domain of Hoxd-11 expression similar to that observed in mice and chicks. Since axolotls do not form an expanded paddle-like handplate prior to digit differentiation, we suggest that anterior expansion of expression in higher vertebrates is linked to the formation of the handplate, but is clearly not necessary for digit differentiation. We also show that the 5' HoxD genes are reexpressed during limb regeneration. The change in the expression pattern of Hoxd-11 during the course of regeneration is consistent with the hypothesis that the distal tip of the regenerate is specified first, followed by intercalation of intermediate levels of the pattern. Both Hoxd-8 and Hoxd-10 are expressed in non-regenerating wounds, but Hoxd-11 is specific for regeneration. It is also expressed in the posterior half of nerve-induced supernumerary outgrowths.

Nerve dependency of regeneration: the role of Distal-less and FGF signaling in amphibian limb regeneration.

Dlx-3, a homolog of Drosophila Dll, has been isolated from an axolotl blastema cDNA library, and its expression in developing and regenerating limbs characterized. The normal expression pattern, and the changes that occur during experimental treatments, indicate a correlation between Dlx-3 expression and the establishment of the outgrowth-permitting epidermis. Dlx-3 is expressed at high levels in a distal-to-proximal gradient in the epidermis of developing limb buds, and is upregulated in the apical ectodermal cap (AEC) during limb regeneration. Expression is maximal at the late bud stage of regeneration, coincident with the transition from the early phase of nerve dependency to the later phase of nerve independence. Dlx-3 expression in the epidermis is rapidly downregulated by denervation during the nerve-dependent phase and is unaffected by denervation during the nerve-independent phase. We investigated this relationship between nerves and Dlx-3 expression by implanting FGF-2 beads into regenerates that had been denervated at a nerve-dependent stage. Dlx-3 expression was maintained by FGF-2 after denervation, and regeneration progressed to completion. In addition, we detected FGF-2 protein in the AEC and in nerves, and observed that the level of expression in both tissues decreases dramatically in response to denervation. We conclude that both limb development and regeneration require a permissive epidermis, characterized by Dlx-3 and FGF expression, both of which are maintained by FGF through an autocrine loop. The transformation of the limb epidermis into a functional AEC that produces and responds to FGF autocatalytically, is presumed to be induced by FGF. Since nerves appear to be a source of this priming FGF, it is possible that a member of the FGF family of growth factors is the elusive neurotrophic factor of limb regeneration.

Distribution of biglycan and its propeptide form in rat and bovine aortic tissue.

The matrix proteoglycan biglycan was identified in bovine and rat aortic tissue by Western blot analysis and by immunohistochemistry, using polyclonal antibodies raised against peptides of the propeptide and the hypervariable region of the rat biglycan core protein. Western blot analysis of proteoglycans isolated from bovine and rat aortas by ion exchange chromatography and treated with chondroitin ABC lyase, with antibody against propeptide, demonstrated core proteins with molecular weights ranging from 43,000 to 45,000 daltons. Similar results were obtained with Western blot studies using the peptide antibody to the hypervariable region of biglycan, except the antibody did not recognize the core protein of bovine biglycan. Location of biglycan within bovine and rat aortic tissue sections by immunoperoxidase histochemistry using the antibody raised against the propeptide revealed intense intracellular staining of medial myocytes and endothelial cells but no extracellular staining. In contrast, immunohistochemistry performed with the purified antibody to the hypervariable region revealed significant extracellular staining of the adventitia proximate to the media and of the endothelial lining but no intracellular staining of rat aortic tissue, with no observable staining of bovine aortic tissue. These data demonstrate that, in contrast to cultured smooth muscle cells, biglycan containing the propeptide is not secreted and deposited within the extracellular matrix by smooth muscle cells and endothelial cells from aortic tissue.

cDNA sequence for bovine biglycan (PGI) protein core.

The nucleotide sequence of the protein core for bovine aortic smooth muscle cell biglycan was determined using recombinant DNA technology. Analysis of the deduced amino acid sequence for bovine biglycan revealed a striking homology, 94.6% and 95.7%, to human and rat biglycan, respectively. The bovine biglycan protein core has four potential O-linked and two potential N-linked glycosylation sites and is composed of 11 leucine-rich repeat units.