The Mexican axolotl (Ambystoma mexicanum) is a well-established tetrapod model for regeneration and developmental studies. Interestingly enough, axolotls do not metamorphose naturally; instead, only with hormone or environmental stimuli can they enter the metamorphosis stage. During this process, many tissues and organs undergo remodeling and functional changes, resulting in a reduced regeneration ability. The construction of the axolotl single-cell landscape during neotenic and metamorphic stages can open up an avenue for the understanding of the molecular mechanisms of their regenerative and metamorphic developmental process.

The research team led by Prof. GUO Guoji at the Zhejiang University School of Medicine developed a single-cell combinatorial hybridization sequencing (CH-seq) method as a new cost-effective protocol for high-throughput single-cell sequencing. Their research findings were published in the journal Nature Communications on July 22.

Using CH-seq, the research team profiled profile over 1 million cells from 19 tissues in neotenic adult axolotls, metamorphosed adult axolotls and larval stage axolotls. The adult axolotl cell landscape, which was constructed to detect transcripts fixed in dissociated cells, covered nearly 500 subtypes of cells. In addition, they created a whole-organism single-cell transcriptome landscape from adult and larval axolotls spanning Day 30 to Day 70 post-fertilization.

The research team also identified the variable expression patterns of different keratin and mucin genes in skin, tails and limbs between neotenic and metamorphosed axolotls. The upregulation of these genes was directly related to the phenotypic changes in axolotls evolving to terrestrial living environments. The increase in fibronectins and endogenous prostaglandins in the gastrointestinal tract of metamorphosed axolotls revealed the homeostatic regulatory function of the digestive metabolic system during tissue remodeling. Compared with other species, upregulation of conserved gene features during limb development, such as A2m and Matn1, may correlate with the neotenic development state in axolotls and naked mole-rats. Experimentally induced metamorphosis in axolotls could somehow reduce their regeneration ability and lifespan. Moreover, the research team found the enhanced differentiation of musculoskeletal lineages within the locomotor system and that of epithelial lineages within the respiratory system during metamorphosis in axolotls.

These resources could facilitate future exploration of neoteny, regeneration, aging and cancer research in axolotls as well as cross-species comparison.