Researchers at Yale University have used an adapted method of haploid genetic screening, which involves creating transplantable grafts from mutated tissue containing only half of the genome, to reveal critical genes involved in limb regeneration in axolotls. Published in the journal eLife, this study highlights that by adapting established methods of genetic screening, researchers can expand on the findings of earlier studies that were limited by the methods used.
The axolotl, a type of salamander, has been at the centre of studies in regenerative medicine for years. It has the ability to regenerate not only its tail and limbs but also parts of internal organs such as the brain, heart, pancreas and kidney. “It regenerates almost anything after almost any injury that doesn’t kill it,” said Grant Parker Flowers, Professor of Cellular and Developmental Biology at Yale, and second author of this innovative paper. As such, this unassuming salamander has become a key player in the drive to understand the mechanisms of regeneration, with wider implications for human health and disease, such as the development of new therapies following tissue damage.
In the study, researchers replaced normal limb tissue in developing axolotls with limb tissue that only had half of a full set of genes; this is referred to as haploid donor tissue. They then induced targeted mutations in the donor tissue and the resulting axolotl had observable differences in the genetic makeup of one of its limbs compared to the rest. From studying the development of the grafted donor tissue and the new limb that formed, researchers were able to identify two genes, catalase and fetuin-b, from a selection of twenty-five that were suspected of being critical in axolotl limb regeneration. The same two genes were also shown to be involved in regeneration of the tail when severed.
The study highlights that the expression of these two genes must be tightly controlled, which aligns with results from previous studies involving other animal models. For example, the overexpression of catalase inhibits regeneration in zebrafish, whereas complete inhibition of catalase delays tail regeneration in frog larvae. As for fetuin-b, it is involved in maintaining structural support in the cells of mammals when expressed locally (e.g. in the liver) and then transported into the blood.
It is worth noting that research thus far has demonstrated that regenerated limbs are genetically similar to the original limbs that they had replaced. Previous studies have also shown that during limb regeneration in axolotls, cells at limb bud sites (blastema) do not become pluripotent, i.e. capable of developing into any cell type as in embryonic development. Instead, they retain a ‘memory’ of being limb cells and so proceed to follow a mechanism of development involving critical limb genes. This particular study excluded genes expected to have a role in development and focused primarily on those involved in regeneration. Yet research suggests that there may be phases of regeneration during which both developmental genes and regenerative genes are expressed simultaneously. As such, mapping the progression of gene expression during limb regeneration and the phases of overlap could be another interesting area to explore.
In the axolotl, an animal with a genome ten times larger than that of humans, the identification of genes involved in regeneration is a starting point for further translational research to determine the role of the same genes and others alike in mammalian regeneration.
This post was written by Ebony Coward and edited by Miles Martin