Fig. 6. A biophysical model of the mechanisms controlling tail regeneration. (A-F) Schematics of the physiological events occurring in tails under different conditions. The intact tail (A,A') is generally hyperpolarized, with a distributed pattern of depolarized cells. The regenerating tail exhibits a bud that is first depolarized (B) but is then repolarized by the normal expression of the V-ATPase or induced expression of the PMA H⁺-pump (B'); by 24 hpa, a depolarized cell group has appeared in the center of the trunk just anterior to the bud (the `shoulder region'). (C) V-ATPase-inhibited, or PTX-treated, tails are fully depolarized and are not able to repolarize the bud by 24 hpa (C'). (D,D') Refractory tails are likewise unable to repolarize the bud and do not possess a depolarized cell group in the shoulder region. (E) Thus, the tail possesses two separate biophysical components: a transepithelial potential normally driven by proton-extruder expression at the edge [likely to result in an electric field (curved lines) that may guide axons into the bud], and a population of cells in the shoulder region that becomes depolarized in tails capable of regenerating. (F) The significance of the shoulder region is unknown, but it may be a region of highly active morphogenesis, as evidenced by the disorganization of the mature melanocytes in this region. (G) A step-wise model of tail regeneration consisting of physiological, gene expression and morphogenetic modules. Amputation triggers a cassette of ion transporter expression in existing cells, with V-ATPase expressed as early as 6 hpa and inducing KCNK1 (12-24 hpa). This results in a particular pattern of relative hyper- and depolarization in the regeneration bud and shoulder cells, respectively (characterized at 24 hpa in Fig. 3). A key parameter here is the physiological condition of bud cells (membrane voltage); when hyperpolarized by the activity of a H⁺ pump, whether naturally or through judicious misexpression of specific transporters, this leads to depolarization in a rostral cell group (24 hpa), an upregulation of mitosis (48 hpa), and subsequent axonal outgrowth (48-72 hpa), ultimately resulting in the regeneration of the complete tail. Refractory-stage larvae cannot regenerate owing to a failure to repolarize the bud and depolarize shoulder region cells. Ectopically-induced H⁺ flux can rescue upstream steps and initiate the program of regeneration, thus representing a tractable initiation point for therapeutic approaches.