Secreted inhibitors drive the loss of regeneration competence in Xenopus limbs

Background and results

 

Many amphibian species can regenerate lost appendages after amputation, whereas amniotes (such as humans and chicks) cannot. A well known example are Salamanders, which preserve regeneration competency to the adult stage. Xenopus leavis is not able to regenerate lost limbs at the adult stage after amputation but can do so at the embryonic stage. The progressive loss of regeneration competency is associated with the formation of apical-epithelial-cap (AEC) cells. AEC cells are a layer of cells that localise distally to secrete mitotic factors (e.g. FGF) to promote blastema cell proliferation and patterning of the re-growing limb. However, how AEC is formed, and why it is only formed in early but not late stages of  Xenopus development remains unclear.

 

To understand the mechanisms behind AEC cell formation, Aztekin et al performed single-cell RNA sequencing in developing and regenerative hindlimb tissues, at different stages of the developing Xenopus tadpoles, and in different regions along the proximal to distal axis of the limb. From a total of 42,348 cells, the authors identified 60 putative cell types/states, including the AEC cell population in amputated samples. Transcriptomic analysis on the AEC cell population suggests that their gene signature is highly similar to the AER cells, the layer of ectodermal cells that, during normal limb development, promote limb bud outgrowth and distal patterning. Both AER and AEC cells express genes for ligands of the FGF, BMP, WNT and TGF-b signalling pathways but the levels of expression diverge slightly. This result thus confirms the previous notion that the AEC cells are analogous to the AER.

 

To investigate the mechanisms causing AEC formation, the authors deployed an in vitro explant culture assay. Here, they dissected the stylopod, or zeugopod and stylopod segments of the limb and cultured them in growth medium. They observed that the proximal and distal end of the explants show distinct developmental trajectories such that the distal site forms AEC, whereas the proximal end undergoes chondrogenesis. This positional effect is in accord with the expected outcomes in vivo. Pharmacological treatments suggested that AEC formation receives various signalling inputs, including FGF, BMP, Wnt, Notch, and TGFB pathways. Interestingly, EdU tracing analyses indicated that only 40% of the AEC cells were formed by cell division, suggesting that de-differentiation might also be involved in AEC formation. To further corroborate this, with single-cell RNA-sequencing analysis the authors identified a putative differentiation trajectory from basal epidermal cells to the AEC cells, suggesting that amputation might trigger signalling cues to convert some epidermal cells into AEC.

 

But what regulates the transition between regeneration competent to regeneration incompetent states? The authors first wanted to understand whether secreted factors can account for this transition. The authors performed co-culture experiments with both regeneration competent and regeneration incompetent tissues. Remarkably, regeneration incompetent tissues blocked the regeneration of competent tissues. Consistently, applying the medium from regeneration incompetent tissues overturned the regeneration competent state. These experiments hint that the appearance of regeneration inhibitors accounts for the loss of regeneration potential.

 

The loss of regeneration capacity coincides with the increase in the chondrogenic lineage. The authors, therefore, asked whether the factors secreted by chondrogenic lineage cells can negatively affect regeneration. The chondrogenic cell population expresses high levels of Noggin transcripts. Significantly, the addition of Noggin protein to the culture medium of regeneration competent tissues blocked AEC formation, and consequently the regeneration process. On the other hand, neutralizing Noggin proteins by means of antibodies enhanced the formation of AEC. Hence, Noggin secretion by the chondrogenic lineage can negatively affect the regeneration of limbs. The chondrogenesis progression shows a positional gradient from proximal to distal axis. By modulating FGF signalling activities, the authors showed that the FGF pathway negatively regulates chondrogenesis and the expression of Noggin. Therefore, chondrogenic lineage and AEC mutually inhibit each other to establish the distal to proximal gradient of regeneration capacity. To further support this idea, the authors co-administrated Noggin antibodies and FGF10 proteins, which were able to induce ectopic AEC in the proximal amputation site.

 

In conclusion, the results of this manuscript suggest that enhanced chondrogenesis contributes to the loss of regeneration ability during Xenopus limb development.

 

What I like about this paper?

 

Regeneration is an evolutionary trait that is present in marine species but not in amniotes. This work asks the question of how and why the regeneration potential of lost limbs can be absent. In doing so, the authors combined cutting-edge technologies with classical embryology methods. What I particularly liked about this work is the good balance that the authors have managed in using these two types of approaches. The knowledge from scRNA-seq results are able to instruct the experiments, and the experiments can corroborate the sequencing analysis. In summary, the authors were able to to make solid conclusions and provided useful information to the regeneration field.

 

Questions to the authors:

 

1. To what extent do the authors think that the chondrogenesis process can account for the regeneration competent to incompetent transition?
2. Are there any other chondrogenic factors besides Noggin that are likely to affect regeneration potential?
3. Could the chondrogenesis mechanism explain the eternal regeneration potential in Salamanders?

 

 

Figure. The development of the chondrogenic lineage contributes to the transition between regeneration competent to regeneration incompetent states in the Xenopus limb. In particular, chondrogenic cells secrets BMP modulators including Noggin that antagonise the AEC formation upon limb amputation.

 

doi: https://doi.org/10.1242/prelights.24915

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