Cellular electroporation induces dedifferentiation in intact newt limbs

Abstract

Newts have the remarkable ability to regenerate lost appendages including their forelimbs, hindlimbs, and tails. Following amputation of an appendage, the wound is rapidly closed by the migration of epithelial cells from the proximal epidermis. Internal cells just proximal to the amputation plane begin to dedifferentiate to form a pool of proliferating progenitor cells known as the regeneration blastema. We show that dedifferentiation of internal appendage cells can be initiated in the absence of amputation by applying an electric field sufficient to induce cellular electroporation, but not necrosis or apoptosis. The time course for dedifferentiation following electroporation is similar to that observed following amputation with evidence of dedifferentiation beginning at about 5 days postelectroporation and continuing for 2 to 3 weeks. Microarray analyses, real-time RT-PCR, and in situ hybridization show that changes in early gene expression are similar following amputation or electroporation. We conclude that the application of an electric field sufficient to induce transient electroporation of cell membranes induces a dedifferentiation response that is virtually indistinguishable from the response that occurs following amputation of newt appendages. This discovery allows dedifferentiation to be studied in the absence of wound healing and may aid in identifying genes required for cellular plasticity.

<i>In Vivo</i> Electroporation Induces Cell Cycle Reentry of Myonuclei in Rat Skeletal Muscle

“…In addition, the mouse myotubes that responded to the newt regeneration extract were cleaved into smaller myotubes or proliferative mononucleated cells, indicating that even the myofibers that have both the nuclei reentering the cell cycle and remaining in the quiescent state may be able to dedifferentiate after electroporation. While the electroporation to newt limbs induces cell cycle reentry of nuclei within skeletal muscle tissues, regardless of the presence of plasmid DNA [3], the present study showed that plasmid DNA is essential to the cell cycle reentry of nuclei within myofibers after electroporation. This indicates that the presence or the expression of plasmid DNA can influence the cell cycle reentry of myonuclei of mammals.…”

“…Atkinson et al showed that electroporation to newt appendages and tail induces cell cycle reentry of nuclei within skeletal muscle tissue, and raised the possibility of dedifferentiation of myofibers [3]. In the regenerative process following amputation of newt appendages, not only the myonuclei but also the nuclei of activated satellite cells, which are separated from myofibers by sarcolemma [9], reenter the cell cycle [7,30].…”

“…Besides these uses for gene transfer, a recent study showed that electroporation to newt appendages can induce the dedifferentiation response that is indistinguishable from the response after amputation [3]. In the present study, we examined whether mammalian myofibers can reenter the cell cycle after electroporation.…”

Looking proximally and distally: 100 years of limb regeneration and beyond

“…Proteomic analysis suggests that reduced metabolic activity, up-regulation of the unfolded protein response (UPR), and differential regulation of apoptotic pathways may largely prevent apoptosis of limb cells (Rao et al, 2009), which is known to be minimal during blastema formation (Mescher et al, 2000;Atkinson et al, 2006). Studies on cultured chondrocytes, b-cells, and Muller glia cells of the retina suggest that cells dedifferentiate as part of a mechanism to combat apoptotic cell stress (for discussion, see Rao et al, 2009).…”

Pseudotyped retroviruses for infecting axolotl in vivo and in vitro

“…Transgenesis is possible (Sobkow et al, 2006) but is relatively inefficient, suffers from the long generation time of the axolotl, and has only recently been coupled with any induction system (Whited et al, 2012) or tissue-specific promoters (Monaghan and Maden, 2012). Electroporation is currently the predominant means with which to mis-express genetic elements in salamanders (Berg et al, 2010;Echeverri and Tanaka, 2002;Echeverri and Tanaka, 2003;Echeverri and Tanaka, 2005;Kumar et al, 2007;Mercader et al, 2005), yet this method has drawbacks, such as dilution of plasmid with cell division, and the procedure itself has been shown to cause some degree of cellular dedifferentiation in newts (Atkinson et al, 2006) akin to the dedifferentiation that is a hallmark of limb regeneration.…”