Weak magnetic fields modulate superoxide to control planarian regeneration

Reactive oxygen species (ROS) signaling regulates cell behaviors and tissue growth in development, regeneration, and cancer. Commonly, ROS are modulated pharmacologically, which while effective comes with potential complications such as off-target effects and lack of drug tolerance. Thus, additional non-invasive therapeutic methods are necessary. Recent advances have highlighted the use of weak magnetic fields (WMFs, <1 mT) as one promising approach. We previously showed that 200 μT WMFs inhibit ROS formation and block planarian regeneration. However, WMF research in different model systems at various field strengths have produced a range of results that do not fit common dose response curves, making it unclear if WMF effects are predictable. Here, we test hypotheses based on spin state theory and the radical pair mechanism, which outlines how magnetic fields can alter the formation of radical pairs by changing electron spin states. This mechanism suggests that across a broad range of field strengths (0–900 μT) some WMF exposures should be able to inhibit while others promote ROS formation in a binary fashion. Our data reveal that WMFs can be used for directed manipulation of stem cell proliferation, differentiation, and tissue growth in predictable ways for both loss and gain of function during regenerative growth. Furthermore, we examine two of the most common ROS signaling effectors, hydrogen peroxide and superoxide, to begin the identification and elucidation of the specific molecular targets by which WMFs affect tissue growth. Together, our data reveal that the cellular effects of WMF exposure are highly dependent on ROS, and we identify superoxide as a specific ROS being modulated. Altogether, these data highlight the possibilities of using WMF exposures to control ROS signaling in vivo and represent an exciting new area of research.


Introduction
Reactive oxygen species (ROS) are a group of oxygen-containing molecules with varying reactivity. Intracellular ROS are typically derived from molecular oxygen (O 2 ) and include hydrogen peroxide (H 2 [10][11][12][13][14][15]. ROS signaling plays a role in cardiomyocyte differentiation, promotes transient stem cell proliferation in mouse skin, and is required for regenerative outgrowth in a myriad of animal model systems [14][15][16][17]. Maintenance of stem cell populations requires careful control of ROS levels, which can direct them to remain quiescent, proliferate, or differentiate depending on concentration [18]. ROS signaling plays an equally complex role during tumorigenesis. The upregulation of ROS scavengers (antioxidants) is a hallmark of many cancers, functioning to allow tumorigenic cells to bypass apoptosis; however, tumor progression can be later promoted by increased ROS levels, and ROS scavenging has been found to prevent the development and progression of many cancers in cell culture [19][20][21]. The data highlight the importance of ROS manipulation as a therapeutic target in interventions where tight control of proliferating cells and tissue growth (such as in regenerative medicine and cancer treatments) is required [22]. Currently, many of the standard molecular-genetic (pharmacological) approaches to manipulating ROS come with potential therapeutic complications such as drug toxicity. To bypass these issues, research has turned to the use of nanoparticles for targeted delivery; but these efforts have been hampered in part due to patient heterogeneity that interferes with successful distribution and/or function [23,24]. Thus, the identification of additional methods to alter ROS levels is warranted for improved care and experimental approaches alike. Recent advances in our understanding of how biological systems interact with electromagnetic radiation suggest there is potential for finding such new approaches to manipulating ROS in vivo by using weak magnetic fields (WMFs, <1 mT), a form of non-ionizing radiation.
A predominant theory for understanding the biological effects of WMF exposures centers on the radical pair mechanism, which has been reviewed in detail [25][26][27][28][29][30]. Briefly, theoretical modeling ( Figure 1A) suggests that WMFs can modulate radical pairs through changes in the angular momentum of lone electrons (spin state theory). Parent molecules can both dissociate into radical pairs and recombine at given rates. For recombination to occur, the unpaired electrons on the radical pairs must have opposing valence spins. These antiparallel spin states (singlet state) allow for rapid recombination. However, if the spin states are parallel (triplet state), then recombination cannot occur, and radical pairs diffuse away from one another. Modeling indicates some WMF strengths should promote the singlet state and recombination (thereby reducing ROS), while other strengths should promote the triplet state and diffusion (increasing ROS). Overall, these data suggest that in a field-strength dependent manner WMFs might be used for the directed manipulation of ROS.
However, the extant data on biological effects from WMFs often appears incongruent or contradictory. Exposure to WMFs has been shown to alter apoptosis, necrosis, and proliferation differently depending on tissue type in rat skeletal muscle versus renal cells [31]. Mouse embryonic stem cells exposed to 400 μT WMFs had increased levels of ROS and stimulated growth factors [32]. Fibrosarcoma cells exposed to only 0.2-2 μT WMFs also increased ROS levels, while conversely exposure to WMFs less than 3 μT reduced cell survival of mouse skeletal muscle [33,34]. A recent study in planarians suggested that even at the same field strength, changes in frequency can lead to either inhibition, activation, or have no effect on regeneration [35]. These studies indicate that precise WMF exposures may hold the potential to be used as a novel therapeutic tool to control cell behaviors and alter tissue growth. But for a tool to be useful, it must be capable of inducing predictable effects on cell processes.
Unfortunately, the lack of consistency in the methods and tissues/models used for studying WMF effects on tissue growth, combined with the absence of typical pharmacological dose response curves associated with WMF exposures, has made the practical usefulness of WMFs as a tool to manipulate growth unclear.
Previously, we established an animal model system for studying effects from WMF exposures on new tissue growth using the highly regenerative, free-living planarian flatworm Schmidtea mediterranea. In this study, we use this model to test several hypotheses based on the radical pair mechanism. Overall, we hypothesize that specific field strengths will predictably alter ROS signaling, suggesting WMFs can be used for the directed manipulation of stem cell behavior in vivo. Our first hypothesis is that WMF effects, as per the radical pair mechanism, occur largely through the modulation of radical pairs. This leads to the testable prediction that at different field strengths WMFs will produce opposite effects on ROS levels, resulting in a binary switch from decreased tissue growth to increased tissue growth. A second hypothesis we also test is that the cellular signaling downstream of ROS that controls stem cell proliferation is mediated by changes in H 2 O 2 , a product of O 2 metabolism and a common second messenger in ROS signaling ( Figure 1B). These experiments aim to assess the potential for WMFs as a therapy and begin to dissect the mechanisms by which WMFs control stem cell-mediated tissue growth.
Planarians are a powerful model for investigating tissue growth mechanisms, as they can regenerate all tissues including the brain due in part to a massive population of pluripotent adult stem cells [36]. After a major injury, this stem cell population responds with increased proliferation and migration to the wound site, resulting in a blastema-undifferentiated new tissue comprised of stem cell progeny [37,38]. Pharmacological inhibition of ROS blocks planarian regeneration, while activation of ROS signaling has been shown to rescue blastema formation [39,40]. Previously, our own data demonstrated that in planarians ROS signaling is upregulated after injury and induces changes in gene expression that regulate the stem cell proliferation and differentiation required for blastema formation ( Figure 1C), all of which were inhibited by exposure to 200 μT WMFs [15]. These experiments also indicated that at a different field strength (e.g., 500 μT) tissue growth was instead increased, leading to our current hypothesis that these field strengths are predictably altering growth via changes in ROS signaling. Our current experiments reveal that exposure to different WMF strengths can be used to manipulate ROS signaling and stem cell behaviors in a predictable non-linear fashion to either inhibit or activate tissue growth. Furthermore, our data suggest that WMFs alter O 2 − and not H 2 O 2 to modulate ROS signaling, providing direction for future studies.
Frontiers in Physics frontiersin.org 04 We experimentally controlled magnetic field exposure during planarian regeneration using a custom μ-metal enclosure (MagShield Box) to block external fields combined with Helmholtz coils to produce uniform magnetic fields at specific strengths ( Figure 2). To test the hypothesis that different WMFs will produce opposite effects on new tissue growth that occur largely through modulation of radical formation, we examined both ROS accumulation and blastema formation following exposure to a controlled range of WMFs from 0 μT to 900 μT, in 100 μT increments ( Figure 3). Controls were exposed to an Earth-normal 45 μT WMF, similar in strength to the geomagnetic field (which ranges from 25-65 μT). Planarian trunk fragments were created by transverse amputation just above and below the pharynx (removing both the head and tail) and regeneration was assessed at the anterior wound site. Trunk fragments were exposed to specific WMF strengths within 5 min of amputation and thereafter until analysis. The radical pair mechanism predicts that we should see some field strengths that increase as well as those that decrease ROS levels and regenerative growth.
ROS accumulation was assessed at 1 h after injury, when it has been shown that ROS is upregulated at the wound site [15]. To visualize ROS levels in live regenerates, we used a general oxidative stress indicator dye (CM-H 2 DCFDA) that fluoresces upon ROS activity ( Figure 3A). This allowed for the quantification of signal intensities and the statistical comparison of ROS accumulation at each field strength ( Figure 3B). Our results show that compared to 45 μT controls, exposure to 200 μT WMFs prevented injury-induced ROS accumulation, while exposure to 400, 500, and 900 μT WMF exposures all caused significant increases in ROS levels. The greatest WMF effects were seen at 200 μT for inhibition and 500 μT for increased ROS accumulation.
Subsequent new tissue growth was assessed at 3 days after amputation, when blastema formation is considered complete [41]. The blastema is easily recognizable at this stage as white tissue at the wound site, since pigmentation has not yet occurred ( Figure 3C). To account for any differences in worm size, blastema size was calculated as a percentage of total regenerate size ( Figure 3D). We found that 100-400 μT exposures decreased blastema size, whereas at both 500 and 900 μT we observed the formation of larger than normal blastemas. Similar to our ROS findings, the greatest WMF effects on new tissue growth were seen at 200 μT for inhibition and 500 μT for increased blastema size. Our results demonstrate that WMFs can either increase or decrease both wound site ROS levels and tissue growth in a field strengthdependent manner.
These data suggest that a threshold potential exists to modulate tissue growth through WMF manipulation of ROS formation. Furthermore, they support 1) the hypothesis that WMF effects are consistent with our theoretical model, and 2) our hypothesis that WMF effects result mainly from the manipulation of ROS signaling. If correct, then we can predict equal and opposite changes in events mediated by ROS signaling, which in planarians includes control of adult stem cell behaviors after injury. Therefore, we next examined the effects of 200 and 500 μT WMFs (as representative of our observed effects) on ROS signaling and the resulting behaviors of stem cells during regeneration (Figure 4). At 3 days after amputation, we investigated the expression of the chaperone heat shock protein 70 (Hsp70) ( Figure 4A, top panels), which is involved in stress responses and cell survival [42,43]. In planarians, blastema-associated Hsp70 expression requires injury-induced ROS, and in turn Hsp70 upregulation is required for ROS-mediated stem cell responses during regeneration [15,44]. Therefore, at the same time point we also looked at the stem cell population using the general stem cell marker Piwi-1, as well as the late stem cell progeny marker Agat-1 ( Figure 4A, middle panels). Our data showed that as predicted, as compared to controls, 200 μT WMFs caused a significant reduction in the expression of all three genes at the wound site, while 500 μT WMFs significantly increased expression ( Figure 4B). These data demonstrate that WMF exposure can be used to directly inhibit or activate ROS signaling, depending on field strength.
Furthermore, we investigated WMF effects on proliferation at 3 h after injury ( Figure 4A, bottom panels). In planarians, stem  Frontiers in Physics frontiersin.org 07 cells have been found to be the only actively dividing cell population. Thus, we examined stem cell proliferation by looking at the presence of phosphorylated Histone H3 (pH3), which labels mitotically active cells. We found that at 200 μT there were significantly fewer mitotic cells, while at 500 μT there was a significant increase in the number of mitotic cells ( Figure 4B). These results are consistent with our prediction that WMFs could both inhibit the activation of stem cell proliferation following injury as well as increase the proliferative response.
Together, our data indicate that exposure to WMFs produces non-stochastic changes that are predictable based on our theoretical principles ( Figure 1A), which suggest that different field strengths have opposing effects. Furthermore, the data provide strong evidence that WMF effects on proliferation and tissue growth are consistent with the manipulation of ROS. These results support further investigation into the potential use of WMFs as a tool to alter stem cell activity.
Therefore, we next sought to examine the effects of WMF exposures on these specific species ( Figure 5). We hypothesized that WMFs modulate ROS signaling by influencing the formation of H 2 O 2 , since it has been well demonstrated as an ROS mediator of traditional signaling pathways.
To test this, we exposed regenerating planarians to 200 and 500 μT WMFs (with 45 μT controls) as before, and then examined the levels of H 2 O 2 using the species-specific fluorescent reporter dye peroxy orange 1. Since with our general ROS indicator dye ( Figure 3) we observed a peak at 1 h after injury, we chose that time point to examine H 2 O 2 levels at the wound site ( Figure 5A). However, there were no significant changes in the amount of H 2 O 2 at either 200 or 500 μT ( Figure 5B). In case there was a time delay in WMF effects specifically on H 2 O 2 , we also tested for effects at 2 h after injury but did not observe any significant changes ( Figures 5A, B).   39,40,47]. Therefore, we further investigated the possible differential roles for H 2 O 2 and O 2 − in mediating the effects of WMFs on planarian regeneration ( Figure  6). The general flavoenzyme inhibitor diphenyleneiodonium chloride (DPI) is often used as a pharmacological NADPH oxidase inhibitor [48,49]. To confirm a role for H 2 O 2 during regeneration, we examined the ability of exogenous H 2 O 2 (which is cell permeable and readily diffuses across the plasma membrane) to rescue tissue growth following general ROS inhibition by DPI ( Figures  6A-C). We pre-exposed animals to either 10 μM DPI or its vehicle control dimethyl sulfoxide (DMSO), amputated to produce trunk fragments, then allowed fragments to regenerate without drug exposure. At 3 days after amputation, blastema formation was significantly inhibited, while the addition of 400 μM H 2 O 2 after amputation was able to rescue/overcome this chemical block of ROS ( Figure 6C).
We repeated this H 2 O 2 rescue assay but following inhibition of ROS by 200 μT WMF exposure, and without any pre-exposure before amputation ( Figures 6D-F). Unlike chemical ROS inhibition, we found that WMF inhibition of blastema formation at 3 days could not be rescued by the addition of H 2 O 2 ( Figure 6F). To further support these findings, we also analyzed the effects of exogenous H 2 O 2 on O 2 − levels without experimentally controlled WMF exposure ( Figures 6G-I). Adding H 2 O 2 alone, even with adding a 24 h pre-treatment, failed to significantly affect injury-induced O 2 − levels at the wound site at 2 h after injury ( Figure 6I). As our results reveal that exposure to WMFs was able to alter the injury-induced accumulation of O 2 − at this same time point, the data suggest that WMF effects Frontiers in Physics frontiersin.org 08

Discussion
The study of ROS across various developmental, regenerative, and disease model systems has resulted in an explosion of data revealing the importance of this highly reactive group of oxygen-containing molecular products. In searching for ways to exert control over the vast array of cellular functions that ROS influences, researchers have turned to exploring multiple modalities. Exposures to moderate and strong magnetic fields are known to affect radicals and biological processes [52]. However, the research on WMFs (including ours) indicates that field strengths below 1 mT have important biological implications as well. While the potential of WMF exposure as a non-invasive means to control stem cell activity and cell proliferation is exciting, enthusiasm for being able to translate this potential into real-world approaches is dampened by the need to address gaps in our fundamental understanding of the mechanisms involved.
The work presented here aims to begin addressing these gaps by testing several simple, but critical, current hypotheses in the field. The first was that WMF effects, while not following the conventional dose response curves of pharmacological treatments, can be predicted based on theoretical models and therefore represent a potential tool for the directed manipulation of cell proliferation and tissue growth. The second hypothesis followed from the first, given our predictions were based on the radical pair mechanism: that the effects of WMFs during tissue growth are due largely to modulation of ROS signaling. This mechanism predicts that at different field strengths WMFs will produce opposite effects on ROS levels, resulting in a non-linear (binary) switch from decreased tissue growth to increased tissue growth. If supported, this could help explain why the data reported in the literature for effects from WMFs can often appear contradictory. Not only are the effects likely context dependent (as are most treatments) but vary by field strength.
In addition, WMF effects would also be determined in part by the different outcomes associated with individual threshold levels for free radicals such as ROS and reactive nitrogen species (RNS;  another class of molecules involved in cell signaling), which have both been implicated in a wide array of biological systems [53,54].
Our data demonstrate that consistent with the radical pair mechanism, the effects of WMFs across a range of field strengths can be predicted by the known outcomes of ROS signaling at given threshold levels. Thus, unlike many molecular-genetic tools, WMFs can be used to direct biological outcomes for both loss-and gain-of-function depending on the field strength used. Our data show that exposure to 500 μT WMFs increased ROS accumulation, resulting in upregulated gene expression, increased proliferation, and expansion of stem cell and progeny cell populations-all of which result in increased tissue growth. And (as further predicted by our theoretical model) exposure to 200 μT resulted in the opposite effect, blocking stem cell-mediated new growth as a result of inhibiting ROS accumulation after injury.
WMFs have been shown to alter ROS levels and cell behaviors in vitro under context-specific circumstances, and these effects are often attributed to the radical pair mechanism [55]. For example, WMF strengths ranging from 0 to 600 μT were shown to either inhibit or promote growth and ROS levels in fibrocarcinoma cell culture depending on field strength [56]. Both RNS and ROS signaling are important regulators of stem cells, proliferation, cell migration, and tissue growth, where they can act as extracellular chemical cues as well as intracellular second messengers [57,58]. For example, in bone marrow stem cells it was found that the addition of exogenous H 2 O 2 prevented proliferation and differentiation [59], while an earlier study on RNS signaling showed that NO plays a critical role in cell differentiation [60].
During regeneration specifically, many studies (including in axolotl, zebrafish, Xenopus, and planarians) have identified ROS signaling as necessary to drive regenerative outgrowth [11,39,61,62]. Others have shown that ROS is able to rescue pharmacologically inhibited regeneration, including a study in zebrafish that found exogenous H 2 O 2 was sufficient to rescue heart regeneration [63]. For the present work, we hypothesized that WMF effects on stem cells were mediated by H 2 O 2 specifically. There is ample evidence that H 2 O 2 signaling plays an active role in planarian regeneration. H 2 O 2 is upregulated at all wound sites within the first hour [40,47]  ONOO-signaling is known to be upstream of cell fate decisions; in neural stem and progenitor cell populations ONOO-has been shown to regulate stem cell renewal and proliferation [69,70] Figure 8B). Furthermore, our data demonstrate that these peaks are temporally distinct, with peak H 2 O 2 levels occurring at 1 h after injury and peak levels of both O 2 − and ONOO − occur subsequently at 2 h after injury ( Figure 8C).   /ONOO − signaling functions independently at later time points (for example, as a propagation signal to maintain growth). This is a future direction that we will be investigating.
Although the studies presented here did not address the role of cell migration on WMF effects during tissue growth, it is interesting to note that the migration of planarian stem cells and their progeny to the wound site and into the forming blastema does occur [72,73]. While wound closure is typically completed by 1 h post injury, migration to the wound site is known to occur later and be sustained during blastema formation. ROS in general and superoxide specifically have been shown to promote cell migration in multiple other contexts [74][75][76], which suggests the possibility that in planarians WMF effects might potentially include changes in ROS-mediated cell migration. However, since superoxide regulation of cell migration has commonly been shown to occur via SOD-induced increases in H 2 O 2 signaling [77,78], and since RNS signaling has been shown to be a negative regulator of cell migration [79,80], this area of inquiry would require a great deal more investigation.
RNS have emerged as vital components of the wound healing process, which occurs prior to and is closely tied to tissue regeneration in many species [81,82]. For example, NO has been shown to enhance wound healing in diabetic chronic wounds by accelerating cell proliferation and migration after injury [83], and as such NO donors are promising candidates for use in hydrogels to treat wounds [84]. However, like ROS, both too much and too little RNS can be harmful. And while both ROS and RNS have been shown to play roles during cell proliferation and new tissue growth, the mechanisms of RNS signaling during regeneration are much less well understood [85,86]. Although a recent study has demonstrated a role for NO during zebrafish fin regeneration [87], the role of RNS in the regenerative process is still largely uncharacterized and its role during planarian regeneration is currently unknown. Given the potential, based on the radical pair mechanism, for WMF interactions with RNS signaling during tissue growth, this is a promising area for further studies.
Moving forward, elucidation of the underling mechanisms governing the behavior of quantum phenomena in biological systems will be vital. Mounting evidence on the effects of WMFs highlight the possibilities for exposures to elicit control over disease states via ROS. In cancer research, ROS are of increasing interest as a therapeutic target and data suggest tumor cells may be more sensitive to minor changes in ROS levels than other cell types [88,89]. In the immune system, upregulation of ROS is essential to host defenses against bacterial infection, where neutrophils release high levels of ROS at the site of infection [90]. Furthermore, autoimmune diseases, such as multiple sclerosis, are associated with significantly increased ROS levels, which are thought to participate in provoking the autoimmune response [91]. Therefore, research into the mechanisms that govern the effects of WMFs on biological systems holds the potential to unlock new and innovative therapies in areas of regenerative medicine, cancer research, and more.

Animal care and amputations
The asexual clonal line of Schmidtea mediterranea (CIW4) was maintained in the dark at 18 C. Planarians were kept in ultrapure Type 1 water with Instant Ocean salts at 0.5 g/L (worm water). Animals were fed every third week with liver paste processed from a whole calf liver (antibiotic and hormone free) obtained from Creekstone Farms (Arkansas City, KS). Liver paste was never frozen or thawed more than once before feedings. Worms 2-5 mm in length were used for all experiments and worms were starved at least 1 week before use. Amputations were done as previously described [92] with a dissecting microscope on a custom-made cooling Peltier plate. Trunk fragments were produced via transverse amputation just anterior and posterior to the pharynx, with cuts made at a 90 degree angle to the sagittal plane for consistency in wounding. All untreated controls were held according to field standards in a biological oxygen demand incubator (VWR) at 18 C in the dark.

Magnetic field exposures
Experimentally-controlled static WMF exposures were done with custom-built triaxial Helmholtz coils in a μ-metal enclosure (MagShield box) to block external magnetic fields as previously described [15]. Direct electric current to Helmholtz coils was supplied by DC power sources (Mastech HY3005D-3) and was fed through both x and y axis coils to produce a uniform magnetic field. The MagShield box was kept in a temperaturecontrolled room (20 C). Animals were placed in either 35 or 60 mm Petri dishes in worm water (or in specific media as described in individual assays) in the center of each Helmholtz coil. Magnetic field exposures were performed in the dark always with one coil set at 45 μT (Earth normal average for the geomagnetic field) separated by a μ-metal partition from the other side, where a second coil was set at indicated experimental field strengths from 0 to 900 μT. Before and at the end of each experiment field strengths were confirmed using either a gauss or mG m (AlphaLab models GM1-HS or MGM). Unless otherwise specified, all planarians were exposed Frontiers in Physics frontiersin.org 15 to WMFs within 5 min of amputation and then continuously until scoring and imaging at the indicated time. For Figure 3D: total experimental replicates for blastema growth assays were n ≥ 1, with total biological replicates for each condition as follows: 45 μT, n = 164; 0 μT, n = 19; 100 μT, n = 28; 200 μT, n = 25; 300 μT, n = 18; 400 μT, n = 18; 500 μT, n = 17; 600 μT, n = 16; 700 μT, n = 14; 800 μT, n = 11; 900 μT, n = 18.

Image collection
A Zeiss V20 Fluorescence Stereomicroscope with an AxioCam MRc or MRm camera and ZEN (lite) software was used for image collection. All live images were taken while regenerates were moving (fully extended) to prevent skewing blastema size/signal intensity due to scrunching. For blastema size, animals were imaged in 100 mm Petri dishes with worm water. For live dyes, animals were imaged in 35 mm FluoroDishes (WPI FD35-100) with 25 mm round no. 1.5 coverslips (WPI 503508). For the general ROS dye CM-H 2 DCFDA, heat maps were generated using the standard rainbow lookup table (LUT) to visualize signal intensity. For each assay, samples were imaged at the same magnification and exposure levels to prevent confounding variables during comparisons (i.e., acquisition conditions were kept constant across an experiment between control/treated and/or all different time points). Photoshop (Adobe) was used to orient and scale images (and improve clarity for morphology only). No data was added or subtracted. Original images available by request.

Quantification and statistical analyses
The magnetic lasso tool in Photoshop (Adobe) was used to generate total pixel counts of the anterior blastema (white tissues) and total regenerate (entire worm including blastema). To account for any variation in worm size, blastema was calculated as percent of total body size: (blastema size/body size) x 100. The magnetic lasso tool was also used to measure gray mean values (signal intensity) of fluorescent dyes at the anterior blastema. To account for any variation in dye loading, signal intensity was calculated as the difference between signal at the blastema versus signal from the middle of the regenerate (the pharyngeal region): blastemapharyngeal region. Cell counts of pH3+ were done using the RTNC plugin tool with ImageJ. Number of mitotic cells was expressed as cells per mm 2 of the entire regenerate, with total area measured using the magnetic lasso tool (as before). Significance: either two-tailed Student's t-test with unequal variance (Microsoft Excel or GraphPad Prism 9); or one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (GraphPad Prism 7).

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
Author contributions LK, AV, and WB contributed to conception and design of the study. LK performed the experiments and statistical analyses, with the exception of the in situ hybridization experiments and analyses (which were performed by AV). LK wrote the first draft of manuscript. LK and WB contributed to manuscript revision and figure preparation. All authors read and approved the submitted version.

Funding
Funding was provided by grants to WB from the National Science Foundation (EAGER 1644384 and CAREER 1652312) and Western Michigan University (Presidential Innovation Professorship).