NEW YORK (GenomeWeb) – A new study has offered a window into the molecular processes involved in limb regeneration of the axolotl (Ambystoma mexicanum), an amphibian known for its ability to re-grow lopped off arms or legs.
The researchers used a combination of single-cell RNA sequencing and reporter-based lineage tracking to retrace the cellular origins and molecular features of blastema tissue that gathers at the site of amputation and ultimately morphs into a new limb. Their results, reported online today in Science, suggested that differentiated connective tissue types in adult axolotl limbs undergo reprogramming to flip back to more closely resemble multipotent embryonic limb bud progenitor cells after a severe, amputating injury.
"Our work provides a molecular view of individual cells that build a blastema and reconstitute a patterned limb skeleton," co-senior authors Barbara Treutlein, a Max Planck Institute for Evolutionary Anthropology researcher, and Elly Tanaka, a researcher affiliated with Vienna BioCenter and the Center for Regenerative Therapies, and their colleagues wrote.
The researchers set out to profile cell types present in axolotl limbs before injury with single-cell RNA-seq on 2,379 individual limb cells, done using high-throughput, droplet-based method from 10X Genomics. From expression marker clusters, they identified connective tissue cells as well as endothelial, epidermal, immune, muscle, and red blood cells.
Using a combination of fluorescence activated cell sorting and single-cell transcriptomics, meanwhile, the team fleshed out eight clusters under the connective tissue umbrella. These connective tissue cells were analyzed by lineage tracking with a Cre-loxP reporter system in germline transgenic axolotls, the authors explained, with exposure to tamoxifen at the limb bud stage successfully marking adult connective tissue cells and blastema cell precursors.
Likewise, with Fluidigm C1 system capture and high-coverage single-cell RNA-seq, the researchers characterized hundreds of cells collected over time from axolotls before and after forelimb loss, through to full limb regeneration. Such data made it possible to not only explore the processes, connective cell subtypes, and connective cell lineages involved in regeneration, but also the transcriptomic shifts that occurred along the way and the features that resembled typical axolotl limb development.
"This profiling has indicated that CT cells express adult phenotypes that are lost upon induction of regeneration and funnel into an embryonic limb bud-like phenotype including multipotency within the CT lineage," the researchers concluded. "The molecular reprogrammability of adult cells to cells of embryonic limb potential capable of orchestrating complex limb morphogenesis has clear implications for future prospects of regenerative engineering."