Determining cDNA Sequences for Targeted Axolotl Salamander Genes Using Assembled RNA-Seq Reads

Mammalian articular cartilage and associated skeletal structures undergo morphological and functional changes during the neonatal period in association with the transition from a non-weight bearing environment in utero to increased levels of physical activity and ambulation after birth. Axolotl salamanders (Ambystoma mexicanum) remain aquatic in their natural state, during which biomechanical forces on their diarthrodial joints are reduced relative to similar salamanders living on land. However, these amphibians can be induced to metamorphose into a weight-bearing terrestrial stage. In some respects, this aquatic to terrestrial transition of axolotl salamanders through metamorphosis may model developmental skeletal changes relevant to the prenatal to postnatal parameters associated with birth in mammals. To evaluate this possibility, morphological and gene expression comparisons of skeletal tissues were made between aquatic and terrestrial axolotls that were the same age and genetically full siblings. Unfortunately, a reference genome for Ambystoma mexicanum or a closely related species is not yet available. Therefore, contigs assembled from axolotl RNA-seq reads were analyzed for homology to Genbank sequences for human and African clawed frog (Xenopus laevis). Complete axolotl cDNAs for targeted genes of interest were then generated using end-point PCR. These sequences will be used to study differential patterns of gene expression in the experimental salamander samples.