Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum

Abstract

The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning–candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

Advances in Decoding Axolotl Limb Regeneration

“…The axolotl generation time, just under one year, is still much shorter than many other salamanders. In the past eleven years, both transgenesis [4] and genome editing [5–8] have been successfully performed in axolotls in several laboratories. Retroviral infection, which results in genomic integration, has also been demonstrated in embryos and in larval and adult limbs [9, 10].…”

“…Two classical loss-of-function axolotl pigmentation mutants have recently been mapped, white ( d/d ,) and albino ( a/a ,) [8]. For both, linkage mapping was used to identify a small interval containing a handful of genes, and existing data from other systems for the genes within these intervals was used to narrow to a single candidate.…”

“…Sequencing revealed predicted loss-of-function mutations in the mutant stocks, and both loci were rescued using transgenesis. This study revealed that white stocks harbor a homozygous mutation in endothelin 3 , while albino stocks are mutant for tyrosinase [8]. Though the cloning of these mutants were major efforts, as the genetic linkage map is improved for axolotl, and genome editing and rescue strategies become more routine, the function of other genes will likely be illuminated with these gold-standard techniques.…”

Cooption and Specialization of Endothelin Signaling Pathways Drove Elaboration of the Neural Crest in Early Vertebrates

“…To test if lamprey Ednra and Ednrb also have dedicated ligands, we mutated ednA, ednC, and ednE, the only edns expressed in tissue-specific patterns during lamprey development. Targeting 44 , confirming that F0 mutagenesis can generate near-null edn phenotypes a high frequencies.…”

Integrative Analysis of Axolotl Gene Expression Data From Regenerative and Wound Healing Limb Tissues

“…Multiple studies have been successful in applying transgenesis and genome editing techniques on these animals [26][27][28][29][30]. While the genome of the axolotl is a simple diploid with 14 pairs of chromosomes, its enormous, highly repetitive sequence and long introns [31] have been the major hurdles towards obtaining a full genome assembly due to the inability of acquiring sufficient read-length and the absence of an improved methodology of genome assembly [22].…”