Roger Downie
Froglife and University of Glasgow
Limb loss in humans is a very significant source of disability and distress. One estimate is that in the USA alone, 3.6 million people will be affected by 2050, as a result of war and other traumatic injuries, or after diabetes-related amputation (Ziegler-Graham et al. 2008). There are two possible routes to the treatment of these losses. Until recently, the most hopeful way forward was advances in prosthetic limb development, related to robotic and micro-electronic engineering progress. Encouraging regeneration of the lost limb seemed less likely, despite many years of research. This is because organ regeneration in mammals is extremely limited in nature. However, recent work based at Tufts University in the USA, using the African clawed frog Xenopus laevis, suggests that limb regeneration in mammals could be stimulated with the right interventions (Murugan et al. 2022).
The ability to repair tissue damage is very widespread in the animal kingdom. In ourselves, the most obvious example is wound healing, with a complex sequence of events following an injury: local cessation of blood flow; blood clotting and formation of a scab to prevent entry of micro-organisms; mobilisation of surrounding cells to close the wound; tissue remodelling below the skin to tidy up the damage, sometimes involving formation of scar tissue. However, some animals have the capacity to replace complete and functional organs if they are lost. In amphibians and reptiles, this capability has an odd distribution.
Some species of lizards show autotomy, the deliberate loss of part or all of the tail in response to predator attack (autotomy also happens elsewhere in the animal kingdom: starfish, stick insects etc.). In these lizards, muscles in the tail on the body side contract, severing a line of weakness built into the vertebrae, and the predator is left with the twitching end of the tail, while the rest of the lizard escapes. The tail stump then heals, and the tail regenerates, but not as before. The regenerate is mis-shapen and its skeletal core is a stiff rod of tissue, rather than a set of vertebrae. If the lizard is attacked again, autotomy can only occur in the part of the tail containing the original structures. The costs and benefits of tail autotomy in lizards are finely balanced, and this ability has been lost and evolved many times in different lineages (Clause and Capaldi 2006). Where the tail is important in balance, as in fast-running species, or in social signalling, as a sign of quality as in Uta species (Fox 1998), autotomy tends to be rare or does not occur at all. Cooper et al. (2004) showed that selection related to predation pressure could influence the occurrence of autotomy in different populations of a single lizard species.
In newts and salamanders, adults can regenerate a range of complex organs: parts of the eye, brain, and heart, as well as limbs and tail. This does not involve autotomy, and unlike the case of lizard tails, regeneration results in the re-formation of a normal, functional limb or tail. Limb regeneration follows an orderly sequence: closing of the wound is followed by the formation of a ‘blastema’- a mass of tissue with the characteristics of tissue at the distal end of an embryonic developing limb. Somehow, the blastema ‘knows’ how much of the limb has been lost, and it reforms only the missing elements in order, from proximal to distal ends. As the process progresses, nerves grow into the regenerate, and these provide signalling molecules essential for full regeneration. The ability to regenerate limbs is somewhat variable between species, with larger species and individuals generally having less regenerative capacity than smaller ones (Joven et al. 2019).
In frogs and toads, regeneration of larval tails and early-stage limb buds occurs, but limb regeneration in adults hardly at all. In some families (pipids, discoglossids, hyperoliids), a blastema forms and a partial regenerate grows from the stump, a bit like the regenerated tails of lizards, but it never approaches the size and complexity of the original limb. In bufonids and ranids, wound healing occurs, but no regeneration at all (Scadding 1981). Mammals are like adult amphibians, with no limb regeneration at all.
This is where the new results from Murugan et al. are revelatory. After some years of technique development, they now report substantial hindlimb regeneration in experimentally-amputated Xenopus laevis adults ( X. laevis has a long history as a laboratory species for biological research following its early 20th Century use in human pregnancy testing). The method involves applying a temporary ‘collar’ (called a ‘Biodome’) to the limb stump following amputation. The collar is made of silicone and silk fibres, impregnated with a solution containing a mix of five molecules known to act as signals during normal limb development (such as growth hormone and retinoic acid). It therefore provides a moist environment for the stump tissue, including limb development molecules that would not normally be present there in damaged adult Xenopus limbs. The collars were left in place for only 24 hours, then removed, and the frogs then followed for 18 months to assess how well the amputated limbs regenerated. After decades of failure in such experiments, the results were spectacular.
The regenerates formed went well beyond the mis-shapen spikes usually formed by Xenopus stumps. There was long-term growth, including formation of bones and other normal tissues, neuromuscular repair so that the limbs could move, and the formation of digit-like projections at the distal ends: the animals were able to use their repaired limbs for more or less normal movements. Murugan et al. comment that while regeneration was not perfect in their experiment, the set-up provides considerable scope for alteration of the details: the mix and concentration of the signalling molecules, the duration of their application; also, the possible addition of electrical stimulation, which has shown some positive effects in other experiments. The authors are hopeful that, if regeneration can be stimulated in Xenopus, similar methods may work for mammals, including humans.
The occurrence and distribution of organ regeneration has long intrigued biologists (Bely 2010). For example, why should complete regeneration occur in newt and salamander adults, but not at all in frogs and toads? One theory concerns the period of impairment while the limb is regenerating, which can take months. Newt locomotion can remain effective in the absence of a functional limb (in many species, the limbs are greatly reduced and even absent in some), whereas the absence of a frog hindlimb for some months would be disastrous. The argument from this is that limb regeneration in frogs would be too slow to be effective, and therefore that it does not occur. This argument can be extended to mammals: the high demand for food in warm-blooded animals means that a period of immobility while a limb regenerates would not be useful (Elder 1979). Most discussion of limb regeneration assumes that the benefit follows limb loss after predation, as in lizards, but there are very few studies investigating the ecological role of regeneration in newts and salamanders.
Finally, a comment on ethics. Those opposed to any animal experimentation will not approve of Murugan et al.’s studies. Those with a more utilitarian outlook, will hope that the discomfort and death of a hundred or so Xenopus will lead eventually to new treatments that could benefit millions of people (and even some other animals). Those who prioritise animal welfare will study the conditions under which the Xenopus lived and look to see that the experimental protocols minimised any pain and discomfort. However, there is another ethical aspect: on behalf of Tufts University, where the experiments have been done, patents for the methodology have been applied for. I find it distressing that scientists should seek to make profit from advances in medical research, especially when it has been publicly funded. The notion that, in the future, victims of war should have to pay a royalty to Tufts so that their limbs can be repaired, is distasteful. But then we are all used these days to noticing that some companies have done very well financially out of the Covid pandemic.
References
Bely, A.E. 2010. Evolution of animal regeneration: re-emergence of a field. Trends in Ecology and Evolution 25, 161-170.
Clause, A.R. and Capaldi, E.A. 2006. Caudal autotomy and regeneration in lizards. Journal of Experimental Zoology 305A, 965-973.
Cooper, W.E. et al. 2004. Ease and effectiveness of costly autotomy vary with predation intensity among lizard populations. Journal of Zoology 262, 243-255.
Elder, D. 1979. Why is regeneration capacity restricted in higher organisms? Journal of Theoretical Biology 81, 563-568.
Joven, A. et al. 2019. Model systems for regeneration: salamanders. Development 146, dev167700.
Murugan, N.J. et al. 2022. Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis. Science Advances 8 (4).
Scadding, S.R. 1981. Limb regeneration in adult amphibia. Canadian Journal of Zoology 59, 34-46.
Ziegler-Graham et al. 2008. Estimating the prevalence of limb loss in the United States, 2005-2050. Archives of Physical Medicine and Rehabilitation 89, 422-429.