The essential role of connective-tissue cells during axolotl limb regeneration

Damián García-García, Dunja Knapp, Minjoo Kim, Katelyn Jamwal, Heath Fuqua, Ryan P. Seaman, Riley E. Grindle, Sergej Nowoshilow, Maria Novatchkova, Fred W. Kolling, Joel H. Graber, Prayag Murawala

Posted on: 26 June 2025

Preprint posted on 2 April 2025

Axolotl limb regeneration decoded! Connective-tissue cells aren’t just participants – they are essential architects of new limbs. Remove them, and regeneration stalls!

Selected by Dhanuush Balakannan

Categories: cell biology, developmental biology

Background and central hypothesis

Salamander species like axolotls possess the exceptional ability to regenerate complex structures like limbs. This happens through the formation of a blastema, a mass of progenitor cells that arise from various tissues at the site of amputation. It has long been believed that connective-tissue (CT) cells are essential for both blastema formation and the regeneration process. However, this has not been functionally tested. This preprint addresses this gap by directly examining the role of CT cells in blastema formation and regeneration. The hypothesis: CT cells are not only the predominant contributors to the blastema, but are also essential for regenerating fully functional limbs in axolotls.

Key Findings

Which cell types make up the majority of the blastema?

Spatial transcriptomics analysis revealed that CT cells predominate in the blastema. At 14 days post-amputation, they made up 75% of the blastema cell population. This suggests that CT cells might be the main cellular driver of axolotl limb regeneration.

Does the cellular composition of the blastema change temporally?

The preprint authors observed that during the early stages of blastema formation, immune cells such as the macrophages predominated, but their numbers declined as CT cells took over. Muscle cells, nerve cells, and blood vessels remained at low levels throughout the early phases of regeneration. This indicates that the immune cells resolve inflammation and clear debris early on, while CT cells may orchestrate the tissue regeneration that follows.

Figure : CT-ablated limbs exhibited a smaller limb-to-body ratio compared to controls during regeneration from 0 to 60 dpa. Two distinct phenotypes emerged: delayed regeneration (fully patterned but smaller limbs) and truncated development (defective zeugopod with ≤2 digits). This image has been made available with permission of the authors.

Are these CT cells necessary for limb regeneration?

Genetic ablation of CT cells using nitroreductase/metronidazole (NTR/MTZ) caused delayed or truncated limb regeneration. This implies that CT cells are necessary for blastema formation and limb regeneration.

What molecular programs do CT cells execute during regeneration?

Microarray and spatial transcriptomics profiling revealed distinct waves of gene expression in CT cells during regeneration, including early upregulation of genes involved in chemotaxis and pH regulation, followed by activation of differentiation and limb morphogenesis programs at later stages.

Significance

The authors have implemented a novel strategy, for the first time, to study the role of a specific cell type in axolotl limb regeneration via the NTR-MTZ system. This strategy enables inducible, tissue-specific ablation in axolotls. The authors also provide an open web-based platform for CT cell gene expression data. This enables the wider research community to study the molecular players involved during CT cell-based limb regeneration. The study also opens up avenues to examine the interaction between the immune system and connective tissue cell types during the early stages of regeneration for successful regeneration.

Why I highlight this work?

As a researcher with active interest in limb regeneration, my scientific questions have been what type of cells contribute to the restoration of the limb upon amputation rather than the molecular details. The study established a method, similar to what has been done in other species like zebrafish, to ablate the cells to confirm their role in regeneration – which I find fascinating. Moreover, the lab has been a great resource to other salamander labs in terms of data availability. Their resources could be useful to several others interested in salamander work and thus I was curious to highlight this prePrint.

Questions for the authors

Question 1 : Did regeneration resume from surviving CT cells after partial ablation, or was it permanently stalled? Is there a threshold (minimum number of cells) required for regeneration?

Question 2 : According to your data, the blastema population temporally changes from immune cell to CT cell dominance. What do you think about this transition in terms of cross-talk between the immune system and the CT cells? How is this transition happening?

Question 3 : Would you consider performing single-cell multi-omics (ATAC-seq) to further resolve the regulatory landscape of CT cells during regeneration?

Tags: ablation, axolotl, blastema, limb regeneration, regeneration

doi: https://doi.org/10.1242/prelights.40933

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Author's response to questions

Damián García-García shared

Question 1 : Did regeneration resume from surviving CT cells after partial ablation, or was it permanently stalled? Is there a threshold (minimum number of cells) required for regeneration?

Response : When we performed the CT-cell ablation at 14 dpa (corresponding to the detected peak of CT-cells in the blastema), we found two different outcomes: 1) A fully patterned but smaller limb than the control condition, and 2) A truncated limb with incomplete zeugopod with a maximum of two digits. Most of the observed results corresponded to the second phenotype at 60 dpa. However, we also evaluated the regeneration process at 120 dpa to push our understanding of the ablation experiments. At this point, phenotype number 1 could recover from CT-cell ablation, whereas phenotype number 2 remained as a truncated limb (Data not shown). These results indicate that the ablation is permanently stalled in most of the limbs treated at 14 dpa.

An interesting experiment is needed for the second question: We should be able to graft mature, sorted Prrx1+ cells in a truncated limb background in a quantified control manner. This will tell us the threshold of the number of cells needed to promote limb regeneration. However, we didn’t perform these experiments, and I would find it interesting and valuable to continue in that direction.

Response : Immune cell infiltration during the first stages of tissue regeneration is a common phenomenon occurring in different regenerative processes. Resident macrophages are known to promote the recruitment of other cells, like neutrophils or monocytes, to initiate inflammation and debris clearance. However, our microarray data found a cluster of genes associated with cellular chemotaxis within CT cells at 1 dpa. With this, we hypothesize that CT-cells also promote immune cell recruitment after amputation. Following this explanation, we also found a cluster associated with pH regulation at 3 and 5 dpa. Phagocytic processes from the immune cells may cause this association with the CT-cells, suggesting a stronger communication between CT-cells and immune cells in the first stages of limb regeneration than we thought. We hypothesize that immune cells could select the CT-cells that will undergo de-differentiation, migrate, and form the blastema. However, we still need more experiments to prove this.

Question 3 : Would you consider performing single-cell multi-omics (ATAC-seq) to further resolve the regulatory landscape of CT cells during regeneration?

Response : Exploring the regulatory landscape of connective tissue cells during blastema formation through scATAC-seq is compelling. In previous studies, genome-wide chromatin profiling in mature and regenerative CT cells has identified active regeneration-specific regulatory elements prior to the cell dedifferentiation program (Kawaguchi, A et al.2024). Although that data gives us an idea of the regulatory landscape of CT cells, complementing these experiments with scATAC-seq will reveal the heterogeneity in regulatory mechanisms across different CT-cell subpopulations, allowing the identification of cell-specific regulatory elements with more detail.

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