Ferretti P, Brockes J P, Brown R
Ludwig Institute for Cancer Research UCL/Middlesex Branch, London, UK.
Development. 1991 Feb;111(2):497-507. doi: 10.1242/dev.111.2.497.
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 (NvKII) 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, NvKII mRNA is expressed both in proximal and distal blastemas, although at higher levels distally, indicating that this keratin is regeneration associated. The NvKII 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 NvKII mRNA is markedly down-regulated both in normal and regenerating limbs following intraperitoneal injection with retinoic acid, a putative endogenous morphogen in limb regeneration.
为了理解有尾两栖类肢体再生能力背后的分子机制，识别与再生相关的蛋白质并研究其调控非常重要。我们最近发现，抗细胞角蛋白单克隆抗体LP1K与蝾螈芽基细胞强烈反应，而其在正常肢体中的反应性受到限制。通过用LP1K筛选蝾螈芽基的cDNA表达文库，我们鉴定出编码一种II型角蛋白（NvKII）的cDNA克隆，该角蛋白在芽基的间充质和特化伤口上皮中均有表达。虽然该蛋白的杆状结构域高度保守，但NvKII与哺乳动物II型角蛋白在N端和C端区域的同源性明显下降。通过对各种蝾螈组织进行Northern印迹和RNA酶保护分析来分析这种角蛋白的表达，结果显示它似乎具有器官特异性，因为它仅限于正常和再生的肢体及尾巴。特别是，我们研究了这种角蛋白mRNA在正常和再生肢体中的表达。在正常肢体的近端几乎检测不到该转录本，但在远端水平较高。截肢后，NvKII mRNA在近端和远端芽基中均有表达，尽管在远端水平更高，这表明这种角蛋白与再生相关。在再生体的间充质和伤口上皮中均可检测到NvKII转录本，而在正常表皮中未检测到转录本。在腹腔注射视黄酸（一种肢体再生中假定的内源性形态发生素）后，正常和再生肢体中NvKII mRNA的水平均明显下调。
