🦎 Scientists discover how some vertebrates regenerate their limbs

Some animals in the world possess the ability to regenerate tissues, allowing them to regrow parts of limbs or entire limbs after amputation.

In a study published in the journal PNAS, a research team from Inserm, the University of Montpellier, and the Montpellier University Hospital shows that mouse embryos cultivated in the laboratory can only initiate regeneration of anterior limb buds, such as forelimbs, during an extremely short period: between 10.5 and 12.5 days after fertilization, just as the limb buds are beginning to form. Outside this window, this ability completely disappears.


The scientists demonstrate that this phenomenon relies on a specific population of neural crest cells, known to play a key role in the development of many tissues. Their results suggest that these cells are at the heart of regeneration mechanisms in all vertebrates. The team also managed to induce regeneration after amputation by transplanting these cells into an embryo that lacked them and had lost its regenerative capacity.


This study, conducted as part of the Biotherapies and bioproduction of innovative therapies research program, marks a new step toward regenerative medicine, capable of restoring biological tissues and their functions.

Unlike lizards and salamanders whose tails can regrow, tissue regeneration is very limited in mammals. However, there are a few well-known exceptions: deer renew their antlers every year, and rabbits are capable of repairing excised tissue in their ears.

Another notable exception concerns mouse embryos. A previous study [1] showed that in the latter, regeneration of anterior limb buds (the forelimbs) is possible during the tenth day after fertilization. However, until now it was unknown how long this ability persisted and what biological and cellular mechanisms made it possible.


A new study conducted as part of the Biotherapies and bioproduction of innovative therapies research program, funded by the France 2030 investment plan, led by Inserm and the CEA, now provides answers to these questions. Published in the journal PNAS, this work was led by a research team from Inserm, the University of Montpellier, and the Montpellier University Hospital.

"Our discovery shows that mouse embryos can only initiate regeneration of anterior limb buds during an extremely narrow time window of development: between 10.5 and 12.5 days after fertilization, just as the buds are beginning to form, explains Farida Djouad, senior author of this study. Outside this period, this ability completely disappears."

Concretely, the scientists amputated the anterior limb bud of mouse embryos cultivated in the laboratory 10.5 days after fertilization, and observed that the bud began to regenerate within the following 24 hours. On the other hand, when the same experiment was performed 12.5 days after fertilization, no regeneration was observed.


The study reveals that this ability relies on a specific population of cells called neural crest cells, known to play a key role in the development of the nervous system, the facial skeleton, and many tissues.

Within three hours following amputation, these cells migrate to the injured area and participate in the formation of a blastema, a cluster of immature cells that gives rise to regrowth. "When these cells are absent, regeneration fails. But if we transplant them again, this ability seems to be restorable", emphasizes Farida Djouad.

Using DNA chips allowing the analysis of the activity of thousands of genes simultaneously, the scientists showed that the bmp4 and fgf8 genes, specific markers activated during limb formation from the embryonic stage, were lost after amputation but reactivated during regeneration, highlighting their essential role in the restoration of amputated structures.

Other genes, WNT1 and FOXD3, characteristic of neural crest cells and normally active at an even earlier stage in the embryo (between the 8th and 10th day after fertilization, when limb buds begin to form), were also reactivated. "This temporary reactivation seems to allow the cells to regain a younger, more flexible state, capable of mobilizing and participating in tissue reconstruction", continues the senior author.

Previous studies had already highlighted the role of neural crest cells during tail or limb regeneration in newts [2], as well as in the regeneration of fingertip tips in mouse embryos [3].

"Our results suggest that neural crest cells are at the heart of regeneration mechanisms in all vertebrates, from amphibians to mammals", explains Jholy De La Cruz, co-first author of the study.

These results offer a starting explanation for the loss of regenerative capacity in adult mice: neural crest cells are present, but they can no longer reactivate the genes necessary for tissue regeneration expressed in the embryonic state.

The research team now wishes to know if these mechanisms also exist in humans.

"In the long term, we hope our work will contribute to a better understanding of tissue regeneration, including in humans, and how it might one day be possible to reactivate these mechanisms for therapeutic purposes," concludes Farida Djouad.

Notes:


[1] Chan WY, Lee KK, Tam PP. Regenerative capacity of forelimb buds after amputation in mouse embryos at the early-organogenesis stage. J Exp Zool. 1991 Oct;260(1):74-83. doi: 10.1002/jez.1402600110. PMID: 1791423.

[2] Kumar A, Godwin JW, Gates PB, Garza-Garcia AA, Brockes JP. Molecular basis for the nerve dependence of limb regeneration in an adult vertebrate. Science. 2007 Nov 2;318(5851):772-7. doi: 10.1126/science.1147710. PMID: 17975060; PMCID: PMC2696928.

[3] Johnston AP, Yuzwa SA, Carr MJ, Mahmud N, Storer MA, Krause MP, Jones K, Paul S, Kaplan DR, Miller FD. Dedifferentiated Schwann Cell Precursors Secreting Paracrine Factors Are Required for Regeneration of the Mammalian Digit Tip. Cell Stem Cell. 2016 Oct 6;19(4):433-448. doi: 10.1016/j.stem.2016.06.002. Epub 2016 Jul 1. PMID: 27376984.