Zhang C, Meng F, Huang X P, Zajdel R, Lemanski S L, Foster D, Erginel-Unaltuna N, Dube D K, Lemanski L F
Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL 33431, USA.
Tissue Cell. 2004 Feb;36(1):71-81. doi: 10.1016/j.tice.2003.10.003.
Recessive mutant gene c in the axolotl results in a failure of affected embryos to develop contracting hearts. This abnormality can be corrected by treating the mutant heart with RNA isolated from normal anterior endoderm or from endoderm conditioned medium. A cDNA library was constructed from the total conditioned medium RNA using a random priming technique in a pcDNAII vector. We have previously identified a clone (designated as N1) from the constructed axolotl cDNA library, which has a unique nucleotide sequence. We have also discovered that the N1 gene product is related to heart development in the Mexican axolotl [Cell Mol. Biol. Res. 41 (1995) 117]. In the present studies, we further investigate the role of N1 on heartbeating and heart development in axolotls. N1 mRNA expression has been determined by using semi-quantitative RT-PCR with specifically designed primers. Normal embryonic hearts (at stages 30-31) have been transfected with anti-sense oligonucleotides against N1 to determine if downregulation of N1 gene expression has any effect on normal heart development. Our results show that cardiac N1 mRNA expression is partially blocked in the hearts transfected with anti-sense nucleotides and the downregulation of N1 gene expression results in a decrease of heartbeating in normal embryos, although the hearts remain alive as indicated by calcium spike movement throughout the hearts. Confocal microscopy data indicate some myofibril disorganization in the hearts transfected with the anti-sense N1 oligonucleotides. Interestingly, we also find that N1 gene expression is significantly decreased in the mutant axolotl hearts. Our results suggest that N1 is a novel gene in Mexican axolotls and it probably plays an important role in myofibrillogenesis and in the initiation of heartbeating during heart development.
美西螈中的隐性突变基因c会导致受影响的胚胎无法发育出收缩的心脏。通过用从正常前端内胚层或内胚层条件培养基中分离的RNA处理突变心脏，这种异常可以得到纠正。使用随机引物技术在pcDNAII载体中从总的条件培养基RNA构建了一个cDNA文库。我们之前从构建的美西螈cDNA文库中鉴定出一个克隆（命名为N1），它具有独特的核苷酸序列。我们还发现N1基因产物与墨西哥美西螈的心脏发育有关[细胞与分子生物学研究41(1995)117]。在本研究中，我们进一步研究N1在美西螈心跳和心脏发育中的作用。通过使用专门设计的引物进行半定量RT-PCR来确定N1 mRNA的表达。用针对N1的反义寡核苷酸转染正常胚胎心脏（30-31期），以确定N1基因表达的下调是否对正常心脏发育有任何影响。我们的结果表明，在用反义核苷酸转染的心脏中，心脏N1 mRNA表达部分受阻，N1基因表达的下调导致正常胚胎心跳减少，尽管通过整个心脏的钙峰移动表明心脏仍然存活。共聚焦显微镜数据表明，在用反义N1寡核苷酸转染的心脏中存在一些肌原纤维紊乱。有趣的是，我们还发现突变美西螈心脏中的N1基因表达显著降低。我们的结果表明，N1是墨西哥美西螈中的一个新基因，它可能在肌原纤维形成和心脏发育过程中心跳的起始中起重要作用。
