Abstract
The Mexican axolotl (Ambystoma mexicanum) provides an excellent model for studying heart development because it carries a simple recessive cardiac lethal mutation that results in a failure of the mutant embryonic myocardium to contract. In cardiac mutant axolotls, the hearts do not beat, apparently due to an absence of organized myofibrils. The mutant hearts can be rescued by coculturing them with normal anterior endoderm/mesoderm tissue, by a medium conditioned with normal anterior endoderm/mesoderm, or by an RNA isolated from the conditioned medium. We have previously isolated a single cDNA clone from a library prepared with total RNA from conditioned medium; this 166-nt-long in vitro synthesized RNA, directed by the unique cDNA clone (Clone #4), has the ability to correct the heart defect and promote myofibrillogenesis in mutant hearts. The criteria for rescue include contraction of mutant hearts throughout their lengths, an increase in sarcomeric tropomyosin arrays as shown by immunofluorescent confocal microscopy, and the ultrastructural appearance of organized sarcomeric myofibrils. More recently, we carried out RT-PCR with total RNAs extracted from normal or mutant axolotl embryos. A point mutation (G-T) was found in Clone #4 RNA derived from the mutant embryos at stages 20 and 30 as compared to normal axolotls at the same stages. Furthermore, we searched for the full-length Clone #4 gene in an axolotl genomic library. Primer extension yielded a product of —500 by at the 5’ end of the Clone #4 gene. The nucleotide sequence of the extended Clone #4 (Ext-Clone #4) was determined and was found to be unique because there was no significant homology with other known sequences available from the gene databases. Interestingly, T7 sense RNA from the Ext-Clone #4 (-500-nt RNA) showed a higher efficiency in rescuing mutant hearts than the T7 RNA from original Clone #4 (166-nt RNA). Our working hypothesis is that the bioactive RNA is a regulatory RNA that may directly or indirectly upregulate tropomyosin production in the axolotl heart.