Ion channel signaling influences cellular proliferation and phagocyte activity during axolotl tail regeneration

Abstract

Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, K2.1, K2.2, L-type Ca channels and H/K ATPases) or completely (GlyR, GABAR, K1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair.

Amputation‐induced reactive oxygen species signaling is required for axolotl tail regeneration

“…For each embryo, three to eight sections were analyzed to identify anatomically matching sections between control and treated embryos. For each section, all cells staining positive for PH3 within the distal 0.5 mm 2 of the amputation plane were counted as well as cells staining positive for DAPI (Franklin et al, ). A mitotic index was calculated by dividing the number of PH3 positive cells by the number of DAPI positive cells in a common area for each tissue section.…”

“…Cell proliferation measurements were performed as described in (Franklin et al, ) with some modifications. We were interested in measuring cell proliferation within the regenerating tissue, distal to the amputation plane; these areas where used for quantification.…”

More Than Just a Bandage: Closing the Gap Between Injury and Appendage Regeneration

“…Pharmacological inhibition of NaV1.2 activity also impedes regeneration, preventing proliferation, or activation of BMP and Notch pathways (Tseng et al, 2010). Other perturbations of membrane potential (V mem ) similarly inhibit regeneration in tadpoles (Tseng and Levin, 2012) and in axolotls (Franklin et al, 2017). Numerous ion channels, including those for H + , Na + , K + , Ca ++ , and Cl - , are transcriptionally differentially expressed over the course of regeneration with variable temporal dynamics (Chang et al, 2017), suggesting that the role of ion channel activity in regeneration may be complex and interregulated with other mechanisms.…”

Salamander-like tail regeneration in the West African lungfish

“…Various pathways required for tail regeneration in Xenopus tadpoles are also necessary for salamander tail regeneration. Pharmacological inhibition of Wnt, Fgf and Tgf-b pathways [13,18], ion channels [19], or Shh signaling [18,20] block salamander tail regeneration. Pharmacological inhibition of Wnt signaling alters the expression profiles of genes associated with multiples pathways implicated in tail regeneration in Xenopus, such as Egf, Notch, and others [13].…”