How do newts regrow their limbs?

by Michaela Alden

Adult urodeles, including newts and salamanders, have unique healing abilities that allow them to regenerate lost limbs, tails, retinae, intestines, and spinal cords. After a loss of limbs or tail, the urodele’s terminally differentiated dermal tissues (regular skin cells) become “reprogrammed,” first healing into a regenerative epithelium (RE) and then transforming into a blastema, a mass of cells capable of growth and development. Blastemata are normally seen only in embryos and the early stages of development, so the ability of fully differentiated adult urodele tissue to dedifferentiate into blastemata is of great interest to researchers. This area of study has potential applications in limb and organ regeneration of other adult vertebrates, including humans, but it is still unclear how urodeles transition from the initial stages of wound healing to blastema recruitment, and from blastema recruitment to tissue replacement.

It was previously thought that the blastema is composed of truly pluripotent stem cells, but blastemata are now understood to be specific to their tissue of origin, retaining partial differentiation. The replacement of a missing structure is accomplished through the recapitulation of the signaling molecules and pathways used when that structure was originally formed, pathways “remembered” by the cells of the blastema. This means that regeneration is specific to the location of the injury: an amputation at the wrist will result in the formation of a new hand, not an entire arm. The blastema is an autonomous morphogenic entity, meaning that if a wrist blastema is removed and grafted onto another part of the body, it will still produce a hand. The search for regeneration signaling molecules expressed in amputated newt limbs is an area of investigation that contributes to our understanding of the overall process of tissue regeneration.

How do blastemal cells “know” their position on a limb? One of the proteins involved is called Prod1. This protein, attached to the surface of the blastemal cells, helps dedifferentiated cells to “remember” their positional identity and is crucial to regeneration. The amount of Prod1 attached to a cell marks its position along the limb, with proximal cells (near the body) having more Prod1 than the distal cells (at the end of the limb). Prod1 is 69 amino acids long and a member of the three-finger protein (TFP) superfamily, a category including proteins present in many metazoans.

In 2009, Garza-Garcia et al. investigated some of the properties of Prod1 in the Eastern Newt, Notophthalmus viridescens. They were searching for orthologs, which are genes in different species that originated from a single gene of a common ancestor. The first step was to resolve the structure of Prod1, which involved both visualization of the protein by heteronuclear magnetic resonance (NMR) spectroscopy and a molecular dynamics-based computer simulation based on Prod1’s amino acid sequence. The structure of Prod1 was compared to known structures of other TFPs. The researchers then paired this structure-based approach with sequence-based phylogenetic analysis, using alignment software to compare the DNA sequence coding for Prod1 with the DNA sequence of other TFPs. Their analysis showed that Prod1 is not similar to other known proteins in structure or in sequence. These results suggest that proteins like Prod1 are specific to urodeles.

Nerves are required in regeneration and are thought to play a major role, possibly even initiating the entire process. In a study published in 2007, Kumar et al. searched for molecules that interact with Prod1 and identified a candidate ligand, dubbed the newt Anterior Gradient or “nAG” protein, that is secreted by nerves. To do this, they used a yeast two-hybrid screening assay, a technique commonly used to check for protein-protein interactions. In this technique, two factors required for transcription initiation are separately attached to “bait” and “prey” proteins of interest. Successful transcription indicates that the two proteins have interacted. 

When denervated (separated from the nerve), the regenerative epithelium (RE) does not contain nAG, showing that it is supplied to the RE by nerve axons. Specifically, nAG is secreted by Schwann cells, the principle type of glial cell (support neuron). After identifying nAG as a Prod1 ligand, Kumar et al. used nAG-specific antibodies to investigate when and where nAG is expressed in the process of regeneration. They found that nAG is expressed after amputation in the nerve and, later, in the RE of the regenerating structure.

Urodele tissue regeneration will continue to be a major area of research in developmental biology because it is an unusual case of differentiated tissues being able to revert to a somewhat undifferentiated state and recapitulate their original growth. Perhaps the most interesting question raised by this research is not why adult urodeles can regenerate, but why other adult vertebrates cannot. One direction for research could involve the introduction of molecular signals like Prod1 and nAG to non-urodele embryonic tissues. With Prod1 proteins expressed since the beginning of development, perhaps it would be possible for fully differentiated adult non-urodele cells to retain the memory of their positional identity. If Prod1 and nAG could not be expressed for some reason in other vertebrates, a search for orthologs of nAG could help researchers design a pathway similar to the Prod1-nAG system.

Sources (click through to read more!)

Garza-Garcia, A, R Harris, D Esposito, PB Gates, and PC Driscoll. 2009. Solution structure and phylogenetics of Prod1, a member of the three-finger protein superfamily implicated in salamander limb regeneration. PLoS ONE 4: e7123.

Kumar, A, JW Godwin, PB Gates, AA Garza-Garcia, JP Brockes. 2007. Molecular basis for the nerve dependence of limb regeneration in an adult vertebrate. Science 318: 772-777.