Actively cycling cells in uninjured connective tissue are not a prerequisite for appendage regeneration

Technical Summary: Actively Cycling Cells in Uninjured Connective Tissue Are Not a Prerequisite for Appendage Regeneration

Problem Statement
A prevailing hypothesis in regenerative biology posits that the ability of vertebrates to regenerate complex tissues is linked to their growth mode. Specifically, it suggests that animals with indeterminate growth (e.g., salamanders, fish) maintain a population of actively cycling stromal cells in uninjured connective tissue, which are readily accessible for recruitment during regeneration. Conversely, animals with determinate growth (e.g., mammals) are hypothesized to lack this population, with their cells being refractory to cell cycle progression, thereby limiting regenerative capacity. This study challenges the necessity of pre-existing, actively cycling stromal cells as a prerequisite for regeneration and investigates whether tissue injury induces a systemic "primed" state in distant, uninjured tissues across species with varying regenerative abilities.

Methodology
The authors employed an EdU-BrdU pulse-chase strategy to track cell cycle dynamics in four vertebrate species representing different growth modes and regenerative capabilities:

• Indeterminate growers: Danio rerio (zebrafish) and Ambystoma mexicanum (axolotl).
• Determinate growers: Acomys dimidiatus (spiny mouse, high regenerative capacity) and Mus musculus (laboratory mouse, low regenerative capacity).

Experimental Design:

1. Pulse-Chase Protocol: Uninjured animals were exposed to EdU for 9 hours to label cells in S-phase prior to injury. Following injury (ear punch in mammals, fin/limb amputation in aquatic species), animals were exposed to BrdU for 9 hours at 15 or 63 hours post-injury (hpi).
2. Tissue Collection: Injured tissues were collected at 24 or 72 hpi to quantify:
   • EdU+ cells: Cells cycling prior to injury.
   • BrdU+ cells: Cells entering the cell cycle de novo post-injury.
   • Double-positive (EdU+/BrdU+) cells: Cells that were cycling pre-injury and continued cycling post-injury.
3. Systemic Assessment: Contralateral (uninjured) tissues (ear pinna, limb, fin) were collected to test for systemic cell cycle activation distant from the injury site.
4. Cell Type Identification: Immunohistochemistry was performed using markers for leukocytes (CD45, IBA1) and stromal cells (PDGFRa) to distinguish between infiltrating inflammatory cells and resident stromal cells in the EdU+ population.

Key Results

• Baseline Proliferation: Consistent with the growth mode hypothesis, Danio and Ambystoma possessed a small population of actively cycling stromal cells in uninjured connective tissue (~1% and 0.5%, respectively). In contrast, Acomys and Mus showed virtually no cycling stromal cells in uninjured tissue, though epithelial cells in hair follicles and epidermis remained active.
• Contribution to Regeneration:
  • In Danio, the pre-existing population of cycling stromal cells did not contribute significantly to blastema formation; regeneration was driven by cells that entered the cell cycle de novo post-injury.
  • In Acomys and Mus, despite the absence of pre-injury cycling stromal cells, stromal cells successfully re-entered the cell cycle in response to injury.
  • In mammals (Acomys and Mus), the majority of EdU+ cells observed at 24 and 72 hpi were identified as infiltrating leukocytes (CD45+ and IBA1+), rather than resident stromal cells.
• Systemic Activation: The study found no evidence of systemic cell cycle activation in contralateral tissues. In all four species, the percentage of BrdU+ cells in uninjured contralateral tissues remained negligible (<0.5%) at 24 and 72 hpi, indicating that injury does not trigger a widespread "primed" state in distant stromal or epidermal tissues within the acute timeframe studied.

Significance and Claims
The paper concludes that the presence of actively cycling stromal cells in uninjured connective tissue is not a prerequisite for complex tissue regeneration. The authors demonstrate that:

1. Cell cycle re-entry is a universal response to injury: Both highly regenerative (Acomys, Danio, Ambystoma) and poorly regenerative (Mus) species exhibit robust cell cycle re-entry in response to tissue damage, regardless of their baseline growth mode.
2. De novo activation drives regeneration: Regenerative healing relies on cells entering the cell cycle de novo after injury rather than the recruitment of a pre-existing, quiescent cycling pool.
3. No systemic priming: Contrary to some previous reports in specific stem cell niches (e.g., muscle satellite cells), the study finds no evidence of systemic cell cycle activation in stromal or epidermal tissues across the vertebrates tested.

The authors suggest that the difference in regenerative outcomes between species (e.g., Acomys vs. Mus) likely lies not in the ability to re-enter the cell cycle, but in the subsequent regulation of cell cycle progression and exit, potentially involving tumor suppressor pathways (e.g., p21, p27) that prevent prolonged proliferation and fibrosis in non-regenerative species.