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.

Fig. 1

Method used to apply electric fields to newt forelimbs. (A) Anesthetized newts were gently strapped to an IC-Spacing perfboard with Stretch Magic elastic cord (restraints). Restraints were applied to the neck, trunk, and both forelimbs. Newts were submerged in PBS and electrodes were placed parallel to the stylopodium so that the electrodes did not touch the skin. (B) An enlarged dorsal view of panel A showing that the electrodes were spaced 3 mm apart to prevent contact with the skin. The electric field was applied in pulses as described in the Materials and methods section. For EGFP expression, the expression construct was injected as shown just prior to applying the electric field. Injections were not performed in experiments designed to determine the effect of electric fields on intact limbs.

Fig. 2

Cell cycle reentry and general histolysis in newt forelimbs following application of an electric field. Newts were injected intraperitoneally with BrdU approximately 12 hours before collecting the limbs for examination. (A) An intact nonelectroporated newt limb control. Note that DNA synthesis (represented by brown nuclei that have incorporated BrdU) only occurred in the epidermal cells. (B, D, F, and H) Newt limbs were amputated and allowed to regenerate for the specified time period. Note that general histolysis of the tissues and cell cycle reentry began on day 5 postamputation and increased through regeneration days 14 to 21. (C, E, G, and I) Electric fields were applied to intact newt limbs as described in the text and limbs were examined at the specified time periods. Note that general histolysis and cell cycle reentry began on day 5 and increased through days 7 to 14. By day 21, myofibers were beginning to form again, while cells residing between the myofibers as well as periosteal cells continue to synthesize DNA. Arrows show examples of nuclei that were actively synthesizing DNA (B, C, I). b, bone; p, periosteal cells; m, muscle; d, dermis; e, epidermis. The scale bar in panel I is for all panels.

Fig. 3

Application of an electric field induces cell cycle reentry and general histolysis in newt hindlimbs and tails. (A and D) Intact, nonelectroporated hindlimb or tail control, respectively. (B and E) Hindlimb and tail, respectively, 14 days postamputation. (C and F) Hindlimb or tail, respectively, 14 days following application of an electric field. Note that cell cycle reentry and histolysis of internal appendage cells occurred following either amputation or application of an electric field. The scale bar in panel F is for all panels.

Fig. 4

Regulation of muscle differentiation and blastemal markers following the application of an electric field to newt forelimbs. (A) Immunofluorescence assay on intact nonelectroporated control limbs demonstrated high expression of 12/101 in myofibers (red fluorescence). Tissue counterstained with DAPI to show nuclei (blue fluorescence). (B) Limb regenerate 10 days postamputation. Note the reduction in 12/101 fluorescence in cells proximal to the apical epithelial cap (AEC). The fluorescing cells also appear to be fragmenting to form smaller cells (arrows). (C-F) Intact electroporated limbs at 7, 10, 14, and 21 days following the application of an electric field. Note the marked reduction of 12/101 expression and that some cells continue to express the 12/101 antigen during myofiber cleavage and histolysis (C-E). By day 21, myofibers were starting to re-form and there was a concomitant increase in 12/101 expression (F). (G) Immunofluorescence assay of an intact nonelectroporated control limb demonstrated no expression of tenascin (MT1 epitope, red fluorescence) in the muscle tissues (arrow), a result consistent with previous studies (Onda et al., 1990; Onda et al., 1991). However, other tissues, such as the epidermis, tendons, and periosteum express tenascin in intact limbs and presumably the positive signal in the upper right hand corner of the panel represents such tissues (arrowhead). (H) Limb regenerate 14 days postamputation. Note the increase in MT1 fluorescence in the late dedifferentiation/early blastemal tissues (arrow). (I) An intact electroporated limb 14 days following the application of an electric field. Note the increase in MT1 fluorescence in the dedifferentiating myofibers (arrow).

Fig. 5

A time course for complete regeneration of tissue structure following application of an electric field. Hematoxylin and eosin-stained tissue sections from newt forelimbs were taken at weekly intervals following the application of an electric field. (A and B) Note that histolysis of muscle and dermal tissues (including skin glands) was prevalent 7 and 14 days post electrical stimulation. (C and D) By days 21 and 28, myofibers and skin glands were starting to re-form. (E) By day 35, the histology of the tissues was often indistinguishable from the intact, nonelectroporated control (F). Scale bar shown in panel F is for all panels.

Fig. 6

Application of low level electric fields causes little cell death in newt forelimbs. TUNEL assays were performed to assess level of apoptosis following application of an electric field. Brown nuclei represent cells positive for the TUNEL assay, while nonapoptotic nuclei are blue. (A) Positive control for apoptosis. Tissue section was treated with DNase I to create strand breaks before performing the TUNEL assay. (B) Intact, nonelectroporated control newt limb. Very few, if any, cells were TUNEL positive. (C) Limb regenerate 11 days postamputation. Only a few cells were TUNEL positive. (D) Intact newt limb 11 days following application of an electric field. Very few, if any, cells were TUNEL positive. (E) High powered examination of hematoxylin and eosin-stained tissue sections of newt limbs 7 days following the application of an electric field revealed no evidence of necrosis. However, evidence for cell survival and growth were present in the tissues undergoing histolysis. Black arrow denotes a mitotic cell in late anaphase or early telophase. White arrow points to a healthy mononucleated cell with bright-staining cytoplasm. Scale bar in panel D is for panels A-D.

Fig. 7

Dedifferentiation correlates with electric field strengths sufficient to cause electroporation of cell membranes. Newt limbs were injected with an EGFP expression construct followed by the application of electrical pulses at varying electric field strengths. EGFP expression was monitored 7 days following application of the electric field and cell cycle reentry and histolysis were examined. (A and B) No electrical pulses were given following injection. No EGFP expression or cell cycle reentry was observed. (C and D) Five 20 V (Electric field strength = 67 V/cm), 100 msec pulses were delivered to the newt forelimb. Low levels of EGFP expression were observed and a few internal cells, mostly periosteal cells (black arrow) but also some muscle cells (white arrows), have reentered the cell cycle. Very little histolysis was observed. (E and F) Five 50 V (Electric field strength = 167 V/cm), 100 msec pulses were delivered to the newt forelimb. Expression of EGFP was abundant and both cell cycle reentry and histolysis of the tissues were observed. Scale bar in panel E is for panels A, C, and E. Scale bar in panel F is for panels B, D, and F.

Fig. 8

Spatial expression patterns of upregulated genes are similar following amputation or electroporation. RNA in situ hybridization of amputated and electroporated forelimb using riboprobes directed towards three MMP genes revealed that these genes were expressed in similar tissues following amputation or electroporation. Arrows point to areas of MMP expression: blue arrows, epithelium; black arrows, periosteum; red arrows, muscle; green arrows, endosteum. MMP denote MMP expression in the epidermis, black arrows point to MMP expression in the periosteum denote areas of MMP expression. (A-C) MMP3/10b antisense probe hybridized to a 1-day limb regenerate (A), an intact limb 1 day postelectroporation (B), and intact, nonelectroporated control limb (C). Expression was observed in the basal layer of the AEC or epidermis and in the muscle tissues of the stimulated limbs, but not the control. (D-F) nCol antisense probe hybridized to a 5 day limb regenerate (D), an intact limb 1 day postelectroporation (E), and intact, nonelectroporated control limb (F). Expression was observed in the basal layer of the AEC or epidermis and periosteal cells of the stimulated tissues, but not in the control. (G-I) MMP9 antisense probe hybridized to a 1 day limb regenerate (G), an intact limb 1 day postelectroporation (H), and intact, nonelectroporated control limb (I). Expression was observed in the basal layer of the AEC or epidermis, periosteal cells, and endosteal cells but not the control. Control sense probes did not hybridize to tissue sections taken from the same limbs (data not shown). Scale bar shown in panel I is for all panels.