Type XII collagen, fibroblast growth factor-1, and fibroblast growth factor-4 in newt limb and tail regeneration

Urodele (salamander and newt) limb/tail regeneration is a postembryonic form of pattern regulation that is featured by formation at the amputation level of a blastema, which is an accumulation of dedifferentiated stump tissue cells (blastema cells) covered by a wound epithelium (WE). Blastema cell proliferation requires mitogenic influences from both nerves and the WE. It also requires remodeling of the existing extracellular matrix to provide an appropriate extracellular environment for cell adhesion, migration, proliferation, and pattern formation. However, we do not yet know the functions of nerves, the WE and the remodeled extracellular matrix in limb/tail regeneration. The specific goals of the present study were to (1) determine if FGF1 is a nerve-derived blastema cell mitogen, (2) clone newt FGF4 and determine if FGF4 is a WE-derived blastema cell mitogen, and (3) clone and characterize the newt MT2 antigen, an extracellular matrix protein that is upregulated in regenerating limb/tail blastema. The results show that: (1) FGF1 in the newt is expressed by both peripheral nerves and limb blastema cells. However, the blastema FGF1 expression is negatively regulated by exogenous growth factors, including FGF1 itself, suggesting that nerve-derived FGF1 could modulate blastema FGF1 expression but its depletion due to denervation may not be the cause for denervation-induced regeneration inhibition; (2) FGF4-specific degenerate primers can PCR-amplify a full length cDNA from newt blastema RNA extract. The cDNA is most likely newt FGF4 because it is 71% homologous to chicken FGF4 at the amino acid level. However, the transcript level in the blastema is low and in situ hybridization failed to localize the cell types that transcribe this gene, thus the source and biological function of the gene product remain to be tested, (3) monoclonal antibody MT2 identifies type XII collagen (col XII) as shown by newt blastema cDNA library screening. In situ hybridization data indicate that col XII gene is expressed and developmentally regulated in regenerating limb/tail but not in developing, undifferentiated limb buds. Such a result suggests that col XII is specifically involved in remodeling of the mature matrix to provide a necessary and unique extracellular environment for blastema formation.