Using dual labeling of thymidine analogs to investigate cell cycle dynamics in the regenerating salamander limb

Abstract

For almost two centuries, scientists have observed that most forms of regeneration fail to regrow in the absence of nerves, but at the cellular level, how the nerves regulate this phenomenon is still unknown. Understanding how cell cycle kinetics change when the nerve supply is cut would help us understand what role nerves play during regeneration. We used dual labeling of cells using thymidine analogs, 5‐bromo‐2′‐deoxyuridine (BrdU) and 5‐ethynyl‐2‐deoxyuridine (EdU), to quantify the changes in the cell cycle in the denervated regenerating limb of axolotl salamanders. Eight larval axolotls were denervated in their left limbs 12 days after amputation and injected with BrdU and EdU at 24 hour intervals. 72 hours after denervation, the limbs were collected and stained for DNA synthesis (S), mitosis (M), and cell death markers. The duration of S phase of the cell cycle shortened in the actively proliferating cells of denervated regenerating limb almost by half compared to those of the innervated regenerating limb. The total cell cycle lengths differed by about 10%, which suggests that the growth phases (G1 and G2) may have increased in length in the denervated limb. When the nerve supply was cut, less cells returned to cell cycle. Our results suggest that the nerves may regulate the duration of S phase. Future analysis of upregulated and downregulated proteins such as transcription factors during cell proliferation in denervated limb blastema may suggest what pathways are causing the observed changes in the cell cycle.Support or Funding InformationThis project was supported by Summer Scholars Independent Research Fellowship from the Office of Undergraduate Research and Fellowships and NSF grants 1558017 and 1656429.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.