Keratin 8 and 18 expression in mesenchymal progenitor cells of regenerating limbs is associated with cell proliferation and differentiation.

Keratins are considered markers of epithelial differentiation. In lower vertebrates, however, immunoreactivity for keratin 8 and 18 has been reported in nonepithelial cells, particularly in mesenchymal progenitor cells of regenerating complex body structures. To confirm that such reactivity does indeed reflect keratin expression and to investigate their possible role in regeneration, we have isolated clones coding for the newt homologues of keratin 8 and 18 (NvK8 and NvK18, respectively) and studied their distribution and changes in their expression following experimental manipulations. Analysis of NvK8 and NvK18 transcripts confirms that K8 and K18 are expressed in the blastemal cells of regenerating newt limbs and that their expression is first observed 3-5 days after amputation, when the blastemal cells start to proliferate under the influence of the nerve, whose presence is essential for regeneration to proceed. In contrast, no induction of these keratins is observed following amputation of a larval limb at a stage when organogenesis is proceeding in a nerve-independent manner. To establish whether there is a causal relationship between keratin expression and cell proliferation in the adult limb blastema, we have investigated whether their expression is nerve-dependent and whether suppression of their expression in cultured blastemal cells affects cell division and differentiation. Analysis of keratins in denervated limbs demonstrates that the nerve is not necessary to induce their expression. However, treatment of cultured blastemal cells with K8 and K18 anti-sense oligonucleotides significantly decreases DNA synthesis and induces changes in cell morphology, suggesting that expression of these keratins during regeneration may be necessary for the maintenance of the undifferentiated and proliferative state of blastemal cells.