Shimizu-Nishikawa K, Tsuji S, Yoshizato K
Regenerative Biology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, Higashihiroshima, Japan.
Dev Dyn. 2001 Jan;220(1):74-86. doi: 10.1002/1097-0177(20010101)220:1<74::AID-DVDY1090>3.0.CO;2-Q.
Formation of the blastema is a key event for limb regeneration in urodele amphibians, and skeletal muscle has been thought to be a major origin of the multipotent blastemal mesenchyme. In the present study, we used differential display to identify the genes expressed differentially in the muscle at the amputation site. We have isolated a cDNA clone that was upregulated during limb regeneration of the Japanese newt, Cynops pyrrhogaster. Deduced amino acid sequence revealed that the cloned cDNA was a newt homolog of rad (ras associated with diabetes), a gene overexpressed in skeletal muscle of Type II diabetic patients. Expression of newt rad (nrad) was not observed in unamputated normal limb muscle, increased within 4 hr after amputation, and then decreased to the level of normal muscle between 11 and 21 days after amputation. In situ hybridization showed that the transcripts of nrad were localized around most of the nuclei of skeletal muscle near the amputation site, indicating the expression of nrad in the multinucleate myotubes. This expression gradually decreased along the distal to proximal axis. No signals were observed in apical epidermal cap or blastemal mesenchyme. However, reverse transcription-PCR analysis detected a very low level of nrad expression in blastema, suggesting the carry-over of nrad expression in blastema from muscle. Administration of retinoic acid, which has been shown to cause an enhanced dedifferentiation in the regenerating limbs, increased nrad expression in more proximally located limb muscle tissues and prolonged the expression period. Thus, it was strongly suggested that the nrad expression is correlated with the dedifferentiation of myotubes of regenerating limbs. We also analyzed the expression of nrad during development. Transcripts were observed in immature oocytes, seen faintly or not seen thereafter until stage 57 when its expression increased again. These results indicated that nrad may play a role(s) in the developmental process as well as limb regeneration.
芽基的形成是有尾两栖动物肢体再生的关键事件，并且骨骼肌一直被认为是多能芽基间充质的主要来源。在本研究中，我们使用差异显示技术来鉴定在截肢部位的肌肉中差异表达的基因。我们分离出一个在日本蝾螈（Cynops pyrrhogaster）肢体再生过程中上调的cDNA克隆。推导的氨基酸序列显示，克隆的cDNA是rad（与糖尿病相关的Ras）的蝾螈同源物，该基因在II型糖尿病患者的骨骼肌中过表达。在未截肢的正常肢体肌肉中未观察到蝾螈rad（nrad）的表达，截肢后4小时内其表达增加，然后在截肢后11至21天降至正常肌肉水平。原位杂交显示，nrad的转录本定位于截肢部位附近骨骼肌的大多数细胞核周围，表明nrad在多核肌管中表达。这种表达沿远端到近端轴逐渐降低。在顶端表皮帽或芽基间充质中未观察到信号。然而，逆转录 - PCR分析检测到芽基中nrad表达水平非常低，表明芽基中nrad的表达是从肌肉延续而来的。已证明视黄酸可导致再生肢体中去分化增强，视黄酸处理增加了更靠近近端的肢体肌肉组织中nrad的表达并延长了表达期。因此，强烈提示nrad的表达与再生肢体肌管的去分化相关。我们还分析了nrad在发育过程中的表达。在未成熟卵母细胞中观察到转录本，此后直到第57阶段其表达再次增加之前，转录本表达微弱或未观察到。这些结果表明，nrad可能在发育过程以及肢体再生中发挥作用。
