The Role of Early Bioelectric Signals in the Regeneration of Planarian Anterior/Posterior Polarity.

Article The Role of Early Bioelectric Signals in the Regeneration of Planarian Anterior/Posterior Polarity Fallon Durant,1 Johanna Bischof,1 Chris Fields,1 Junji Morokuma,1 Joshua LaPalme,1 Alison Hoi,1 and Michael Levin1,* 1 Allen Discovery Center at Tufts University, Department of Biology, Tufts University, Medford, Massachusetts ABSTRACT Axial patterning during planarian regeneration relies on a transcriptional circuit that confers distinct positional information on the two ends of an amputated fragment. The earliest known elements of this system begin demarcating differences between anterior and posterior wounds by 6 h postamputation. However, it is still unknown what upstream events break the axial symmetry, allowing a mutual repressor system to establish invariant, distinct biochemical states at the anterior and posterior ends. Here, we show that bioelectric signaling at 3 h is crucial for the formation of proper anterior-posterior polarity in planaria. Briefly manipulating the endogenous bioelectric state by depolarizing the injured tissue during the first 3 h of regeneration alters gene expression by 6 h postamputation and leads to a double-headed phenotype upon regeneration despite confirmed washout of ionophores from tissue. These data reveal a primary functional role for resting membrane potential taking place within the first 3 h after injury and kick-starting the downstream pattern of events that elaborate anatomy over the following 10 days. We propose a simple model of molecular-genetic mechanisms to explain how physiological events taking place immediately after injury regulate the spatial distribution of downstream gene expression and anatomy of regenerating planaria. INTRODUCTION Regeneration requires the reconstruction of complex anatomical structures and their appropriate integration with the remaining body via precise control of scaling, position, and organ identity. Planaria are free-living flatworms that have an incredible ability to regenerate missing tissue after damage and amputation despite having a rich set of internal organs, three body axes, and a complex brain and central nervous system (1–4), all of which must be recapitulated each time they regenerate. The process by which each wound blastema in a fragment decides what anatomical structure to form has been the subject of study for over 100 years (5,6). Despite considerable progress on the genetics of stem-cell differentiation and signaling pathways controlling these decisions (7–10), many gaps remain in our understanding of how tissue fragments are able to determine which cell types and body structures are missing and at which locations they need to be recreated (11,12). This general question can be assessed most clearly in planaria by investigating the robust ability of cut fragments to establish Submitted August 14, 2018, and accepted for publication January 16, 2019. *Correspondence: Editor: Stanislav Shvartsman. https://doi.org/10.1016/j.bpj.2019.01.029 Ó 2019 Biophysical Society. 948 Biophysical Journal 116, 948–961, March 5, 2019 proper anterior-posterior (AP) axial polarity (13,14). This process includes three functional endpoints: forming the correct number of heads and tails, creating each one at the correct end with respect to the original orientation of the fragment within the host, and scaling new growth (and remaining soma) appropriately to regain proper overall proportions. The current molecular models of AP polarity establishment in planaria involve feedback loops between Wnt signaling (15) and other genetic determinants of polarity, such as the ERK signaling pathway (14). Components of the Wnt pathway, b-catenin and wnt1, both repress head formation and promote tail regeneration at posterior wounds in the regenerating planarian (16–21). Consequently, knockdown of b-catenin and wnt1 both result in the growth of ectopic heads instead of tails. Furthermore, RNAi (RNA interference) knockdown of known inhibitors of the Wnt pathway such as axin (22) and APC-1 (17) induce two-tailed phenotypes. Interestingly, most components of the Wnt pathway do not show differential expression along the AP axis early during regeneration. Wnt1, for example, is expressed at both wounds of a middle fragment (19,21,23) and thus does not explain the differential fate of the two ends. Similarly, Hedgehog Early Bioelectric Control of Polarity signaling, which may in part regulate posterior-specific induction of wnt genes (24), seems to operate along the entire nervous system rather than only posteriorly (24). Notum, another inhibitor of the Wnt pathway (25), is the only known gene with an asymmetrical transcriptional response in the first 24 h postamputation (26). Notum expression first appears at the anterior blastema 6 h after injury (26) and is required for the establishment of proper polarity (27). Notum has been shown to interact with b-catenin via negative feedback (27), but not much is known about what initially breaks the symmetry of the b-catenin-Wnt amplification loop leading to the early asymmetric expression of notum (26) and its subsequent repression of b-catenin (27). To generate the large-scale AP patterning observed in fragments of planaria, the transcriptional circuits in individual cells need spatial inputs that provide positional cues with respect to the axes of the organism. What might be the input that breaks symmetry for the b-catenin-Wnt amplification loop with respect to the two wounds in a fragment and ensures that the respective ends of the fragment acquire the correct anterior and posterior identities? In other systems, such as left-right axis establishment in vertebrates, upstream physiological signals drive transcriptional cascades that implement positional information; these pathways amplify small biophysical biases to align the differential expression of the earliest genes with the correct geometrical regions in the early embryo (28–30). Here, we investigate the hypothesis that a similar system functions during AP axis specification during planarian regeneration. One type of biophysical cue is the distribution of cell resting potentials across tissues in vivo, which feed into numerous downstream pathways during regenerative pattern control in a range of model systems (31–33). It is already known that bioelectric states are involved in planarian regenerative patterning (11), mirroring conserved roles for biophysical pathways in organ- and organism-scale patterning in vertebrate and invertebrate models (31–33). Classical gain-of-function experiments by Marsh and Beams (34–36) showed the reset of axial polarity by applying external electric fields to regenerating flatworms (37,38). More recently, imaging of endogenous bioelectric gradients (39–41) and loss-of-function strategies targeting ion channels, pumps, and gap-junction proteins have implicated bioelectrics in planarian cell cycle regulation (42), control of head shape (43), size modulation (44), and stab (...truncated)