Creation of chimeric mutant axolotls: a model to study early embryonic heart development in Mexican axolotls

The Mexican axolotl (Ambystoma mexicanum) provides an excellent model for s tudying heart development since it carries a cardiac lethal mutation in gen e c that results in failure of contraction of mutant embryonic myocardium. In cardiac mutant axolotls (c/c) the hearts do not beat, apparently because of an absence of organized myofibrils. To date, there has been no way to a nalyze the genotypes of embryos from heterozygous spawnings (+/cx+/c) until stage 35 when the normal (+/c or +/+) embryos first begin to have beating hearts; mutant (c/c) embryos fail to develop normal heartbeats. In the pres ent study, we created chimeric axolotls by using microsurgical techniques. The general approach was to transect tailbud embryos and join the anterior and posterior halves of two different individuals. The chimeric axolotl is composed of a normal head and heart region (+/+), permitting survival and a mutant body containing mutant gonads (c/c) that permits the production of c/c mutant offspring: 100% c/c offspring were obtained by mating c/c chimer as (c/cxc/c). The mutant phenotypes were confirmed by the absence of beatin g hearts and death at stage 41 in 100% of the embryos. Examination of the m utant hearts with electron microscopy and comfocal microscopy after immunof luorescent staining for tropomyosin showed identical images to those descri bed previously in naturally-occurring c/c mutant axolotls (i.e., lacking or ganized sarcomeric myofibrils). These "c/c chimeric" axolotls provide a use ful and unique way to investigate early embryonic heart development in card iac mutant Mexican axolotls.