A newt type II keratin restricted to normal and regenerating limbs and tails is responsive to retinoic acid

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Abstract
In order to understand the molecular mechanisms underlying the regenerative ability of the urodele limb, it is important to identify regeneration-associated proteins and to study their regulation. We have recently shown that the anti-cytokeratin monoclonal antibody LP1K reacts strongly with newt blastemal cells, while its reactivity is restricted in normal limbs. By screening a cDNA expression library from the newt blastema with LP1K, we have identified cDNA clones coding for a type II keratin (NvKH) expressed both in the mesenchyme and the specialized wound epithelium of the blastema. While the rod domain of the protein is highly conserved, the homology between NvKII and mammalian type II keratins drops markedly at the N- and C-terminal regions. The expression of this keratin was analysed by Northern blotting and RNAase protection analysis of various newt tissues, and appears to be organ specific, since it is restricted to normal and regenerating limbs and tails. In particular, we have investigated the expression of this keratin mRNA in normal and regenerating limbs. The transcript is barely detectable in the proximal portion of the normal limb, but its level is high in the distal one. After amputation, NvKH mRNA is expressed both in proximal and distal blastemas, although at higher levels distally, indicating that this keratin is regeneration associated. The NvKH transcript is detectable both in mesenchyme and in the wound epithelium of the regenerate, while no transcript is detectable in normal epidermis. The level of NvKH mRNA is markedly down-regulated both in normal and regenerating limbs following intraperitoneal injection with retinoic acid, a putative endogenous morphogen in limb regeneration.