Enduring questions in regenerative biology and the search for answers

Modern scientific research generally reflects Thomas Kuhn’s notion of normal science, whereby technological advances enable researchers to pursue incremental confirmations of existing theory¹. This approach does, on occasion, produce unexpected insight into outstanding problems, but its fidelity to conventional frameworks discourages the sort of creative experimentation and custom tool-building that produces paradigm-shifting results. Moreover, deploying new technology just because it is available can create the illusion of advancement, i.e., posing questions that have already been answered with less-advanced methods but are proffered as unresolved in order to apply the latest sophisticated (and usually expensive) technology. This is often done with good intentions, e.g., the hope of discovering something new with more sensitive detection or detail, but ultimately does not actually remove the most critical barriers that must be surmounted to move a field forward². Furthermore, this kind of “modern science” incentivizes specialization and, in doing so, focuses effort away from bigger questions that require interdisciplinary efforts and have the potential to advance multiple fields.

In an attempt to think beyond “normal science,” we wrote this perspective piece to synthesize and share ideas discussed at a Banbury Center workshop on enduring questions in regenerative biology (Box 1). As a group broadly representative of the regeneration field, we reflected on the significant progress made in the past four decades and discussed the types of ambitious community-led projects that we need to pursue to uncover answers to new and enduring questions. There was a strong feeling that by doing this collectively and with the input of experts from other disciplines, regeneration researchers would be better prepared to harness existing or new technologies and design experiments that can begin to address these questions. When scientific collectives assemble to examine major problems, it motivates collaborative efforts across research groups and disciplines. Knowledge creation in one field often spurs progress in related areas, generating benefits for science far beyond the original goals.

Despite the varied expertise among workshop participants and wide-ranging discussions about how regeneration occurs among diverse species, we found broad agreement on identifying several enduring, fundamental questions where scientists should direct their efforts. Importantly, our task was to identify common problems that are not overly reductionist or specific to a particular organism. In essence, we focused on the forest to identify major driving questions while at the same time considering why some trees remain undescribed, hidden, or unknown. We found common ground on the notion that regeneration remains vastly understudied, in part because the most commonly used model organisms do not have robust regenerative capacities. Unsurprisingly, there was no strong impetus for creating new technologies to specifically study regeneration; the enduring technological hurdle in our field is the application of specific transgenic tools to highly regenerative research models. Thus, the perspective put forth here represents a synthesis of focused discussions that aim to stimulate an exchange of ideas and future collaborations between experts in many fields, including regeneration.

Despite general agreement for defining regeneration (see Box 2), what remains ill-defined is the set of component processes that comprise regeneration from induction to resolution. While it is clear that regeneration is induced by significant tissue loss or wounding, when and how regeneration-specific processes can be distinguished from those that occur during wound healing and fibrotic repair remain unresolved. Historically, many researchers studied tissue repair mechanisms holistically across diverse species despite varied healing outcomes, i.e., fibrotic or regenerative³. However, modern (1980s-present) wound/tissue repair research has largely been siloed from epimorphic regeneration research because studies on wound repair rely heavily on mice, rats, and humans (i.e., non-regenerative species in which wound healing normally generates scar tissue). Similarly, research into epimorphic regeneration has relied on a few highly regenerative invertebrate and vertebrate models (e.g., Hydra, planarians, zebrafish, salamanders). As regeneration research expands its scope to include new research organisms that can be genetically manipulated and maintained in a laboratory setting⁴ or that enable comparisons of regenerative success and failure between closely related and divergent species, our definition of “regenerative capacity” needs re-evaluation. For instance, does a species’ “regenerative capacity” signify the inheritance of a single, albeit complex, trait or a combination of separate, interwoven processes? If the latter, are all component processes required for successful regeneration, or might some tissues/species omit one or more? Conversely, do all non-regenerative organisms diverge at the same stage of this progression? Ultimately, refining the definition of regenerative capacity returns us to a fundamental question: what are the component events that comprise regeneration? Defining these components allows one to determine the extent to which they share similarities with or are distinct from processes that occur during fibrotic repair. Thus, we identified a set of fundamental processes that occur across most regenerative species (summarized in Table 1).

After defining component processes common to regeneration, we considered how these events might vary across an ever-broadening set of organisms. For instance, complex tissue regeneration may be evolutionarily constrained such that the entire regenerative response, including all component processes, is canalized with low variation between species for any specific process or set of interrelated processes (Fig. 1a, b). This could be true even if regenerative ability has evolved multiple times in different lineages. Conversely, regeneration could have arisen via convergent evolution (i.e., homoplasy) and variability among component processes may be large across the entire process set or for most of the processes (Fig. 1c). Alternatively, variation across species could be relatively large for some processes yet low for others, i.e., high conservation of specific processes (Fig. 1d). Defining the processes outlined in Table 1 as component parts of regeneration provides a framework to study each one across organisms and facilitates the generation of testable hypotheses to ask what is lacking (or modified) in non-regenerative organisms. For example, one testable hypothesis is that events one through five outlined in Table 1 constitute a general wound response that occurs in all animals, regardless of subsequent steps, with regeneration requiring a distinct mechanism that transitions tissues into regenerative healing (steps six through nine, Table 1). Another hypothesis is that specific events occur during the early wounding response to trigger regenerative healing.

In line with the second hypothesis, one enduring question is whether a particular injury signal predicts the final healing outcome. For example, is there a unique trigger for wound healing versus regeneration⁵? Many investigators have advanced the hypothesis that particular immune cells and their products are specifically required for regeneration, independent of their role in regulating and resolving inflammation⁶. For instance, studies in several adult regeneration models suggest blocking immune cell infiltration (e.g., monocytes and macrophages) or depleting specific immune cell subtypes (e.g., macrophages) prevents normal wound healing and the transition to regenerative healing^7,8,9,10,11. In contrast, removing similar immune cell types when trying to stimulate regeneration can enhance the response (microglia)¹². However, it remains an open question if immune phenotypes exist that regulate and promote regeneration-specific processes. The hypothesis that regeneration-promoting immune cell states exist could be tested by comparing immune system responses across different types of injuries in the same species, i.e., where one injury induces regeneration and the other does not (e.g., lizard tails vs. limbs). This hypothesis could also be tested by comparing the immune response in different aged animals or closely related species in which identical tissues heal via regeneration and fibrotic repair, respectively. Comparing the regenerative response in divergent regenerative species may also offer insight into whether components of the immune response are permissive or instructive relative to regeneration.

Component processes and the transitions between them are also critical to define because they establish a framework for generating specific datasets (i.e., cell type and time-point specific) that can capture cell state changes to compare across species. Specifically, changes in chromatin state or genomic architecture, which impact gene regulatory networks (GRNs) and their outputs, may be broadly conserved at these cellular and temporal transitions¹³. Importantly, cell state changes accompanying reactivation of developmental genes can provide signposts for exploring chromatin states associated with activation or repression of gene expression and thus provide insight into both activation and constraint of regenerative ability¹⁴. These types of comparisons also lay the groundwork for evaluating cell-type evolution models as they relate to the presence or absence of regenerative capacity^15,16. Lastly, breaking regeneration into component processes provides a framework for comparing cellular transitions as they occur during regeneration and embryonic development (Boxes 3 and 4).

Cells detect and respond to injury regardless of the healing outcome (regeneration or scarring), which raises another outstanding question: to what degree do different healing trajectories overlap (Fig. 2a–d)? For example, the ERK/MAPK signaling pathway is rapidly induced upon tissue injury in both non-regenerating and regenerating species and inhibition of its activation impairs wound healing and regeneration^{5,17,18,19,20,21}. What remains unclear is whether the requirement of ERK/MAPK signaling in regeneration directly results from its role in wound healing or if this pleiotropic pathway induces multiple downstream mechanisms that are separately required for multiple regeneration steps (Table 1). Similarly, are there inductive molecules with the dual capacity to promote regeneration and antagonize fibrosis?²² In broader terms, is it possible to identify a set of conserved cellular and molecular mechanisms that initiate regeneration and thus define the “beginning” of regeneration? As discussed above, it remains unclear whether the early events of wound healing are common across most species and contexts with the unique regenerative response initiated later, or if the different healing trajectories (regeneration vs. scarring) are established during the healing process (Fig. 2c, d). If the latter, how can we discover molecular signals and cell states specific to regeneration?

a, b Examples of variation in regenerative ability across species. a Tissue healing is quite different between closely related (~18 mya) Acomys cahirinus (spiny mouse) and Mus musculus (laboratory mouse; Adobe Stock Image used with educational license) where Acomys exhibit complex tissue regeneration in the ear pinna and identical injuries heal with scar tissue (and no regeneration) in Mus. b While two flatworm species Schmidtea mediterranea (orange) and Dendrocoelum lacteum (gray) are capable of head regeneration, D. lacteum exhibits poor head regeneration from posterior fragments (S. mediterranea has near limitless regenerative ability from any fragment). c Comparing regeneration and fibrotic repair, the early events that occur prior to new tissue formation could be similar between species, only to diverge as mechanisms specific to regeneration or fibrotic repair are activated. In the example presented, this divergence occurs immediately prior to activation of a developmental state (i.e., blastema formation), although under this hypothesis it could occur later. d An alternative is that regenerative and fibrotic healing are evolutionarily distinct and thus upon injury two different healing trajectories, and their mechanistic underpinnings, are expressed.

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To distinguish between regenerative responses and more general repair processes, it can be useful to identify events that occur during both regeneration and embryonic development. As embryogenesis unfolds in a temporal sequence beginning from fertilization, tissues, and organs arise at precise positions and prescribed times based on a specific developmental plan. Although it is also difficult to ascribe an initiating event in organ development, developmental biologists largely agree that the formation of a tissue anlagen or primordium is associated with precursor cells that are competent to receive inductive signals. In response to inductive signals (cell-autonomous or non-cell-autonomous), precursor cells launch a developmental program and self-organize into tissues comprising a diverse array of differentiated cell types. Thus, it may be appropriate to consider the beginning of regeneration per se as the point when injury-activated cells accumulate at the injury site and adopt a development-like state (Table 1). This would align the beginning of regeneration with anlagen formation and distinguish wound healing from events specific to regeneration (Fig. 2c).

In support of this proposition, data from salamanders suggests that injury-induced cell accumulation is necessary but insufficient for regeneration (reviewed in ref. ²³). Across countless experiments, researchers performed surgical interventions to study how nerve-secreted signals regulate the regenerative response in ambystomid salamanders and newt limbs^24,25. These results show that wound healing and proliferative cell accumulation occur after denervating a limb, but aggregated cells fail to progress to morphogenesis. In further support of this idea, inhibiting certain signaling pathways (e.g., Wnt and Hedgehog signaling) allows damaged tissue to repair, but regeneration does not occur^26,27,28. Together, these findings support the concept that the ability to accumulate proliferative cells is a necessary but insufficient feature that distinguishes wound healing processes from regeneration. Instead, it suggests the transition to regeneration is characterized by proliferative cells acquiring the ability to undergo patterning, differentiation, and growth, a state often referred to as blastema formation²⁹. However, data linking signaling pathways and nerves to regenerative competence also points to the existence of other essential cellular and molecular events that are required for regeneration to begin in a robust and recognizable way^26,27,28. Thus, while the blastema is a common element of what may represent an evolutionarily conserved regenerative feature, it also highlights the critical need to determine which specific cellular and molecular characteristics of the local cellular environment are essential for regeneration to proceed.

The discussion of molecular signals that initiate regeneration evokes broader questions about what initiates the start of regeneration. For example, how is the amount of loss or damage requiring regeneration detected? Are specific regenerative programs only initiated after significant cell loss? If so, what are the mechanisms that activate them? There are many unknowns regarding injury-sensing mechanisms. Are there particular molecules or physiological changes communicating the amount or extent of tissue damage? Does the loss of cell-cell contacts act additively or in parallel to chemical sensing mechanisms? Does the new interaction of cells from different anatomical positions stimulate the regenerative program^30,31,32? Overall, the common theme that emerges from these questions is the need to expand data collection on regenerative ability within and across species, particularly by adding data from more examples of failed or intermediate regenerative success. More comprehensive data will allow researchers to better identify cellular, molecular, and functional commonalities in successful regenerative processes versus unsuccessful ones.

Indisputably, the most desirable outcome of a regeneration process is to regain tissue shape and function. Thus, understanding and defining when and how a regenerative process restores a functional state is equally important to determining how it starts. Most descriptions of tissue or whole-body regeneration indicate that repair processes shift to a remodeling and growth/scaling phase after the initial expansion of cellular material and patterning³³. But exactly how regeneration restores form and function is not clearly defined. For example, as cells accumulate and proliferate, how do they appropriately regulate pathways that terminate cell cycle progression and how is this balanced with tissue scaling? Work on the Hippo pathway has demonstrated that its signals can be modulated to regulate growth during development³⁴. A similar use of this pathway likely controls growth during regeneration^34,35. However, precisely clarifying when regenerative healing and tissue restoration shift to a growth phase and identifying the molecular mechanisms that regulate this transition is a major objective for our field.

One possibility is that the end of regeneration recapitulates the patterning and growth of embryonic organs, where growth ultimately becomes regulated at the organismal level. For example, regeneration in molting animals (e.g., crustaceans) produces a miniature facsimile of the original appendage, which is only capable of additional growth upon subsequent molts^36,37,38. In zebrafish, caudal fin regeneration is generally consistent with the overall size of the animal, but genetic mutants do exist in which regenerating fins lose their allometry and produce dramatically overgrown fins³⁹. Nonetheless, regeneration generally restores missing tissues, their structures, and their functions, suggesting that although the precise mechanisms underlying the integration of new and existing tissue may vary across species, they achieve a common output.

In trying to understand why regeneration is absent in some animal species or tissue types, one hypothesis is that regeneration initiators are missing or not sufficiently activated, while another suggests that some animals lack the capacity to create restorative cell states. Another hypothesis is that mechanisms that normally terminate phases of regenerative healing have been co-opted to inhibit regeneration completely. For example, the evolution of “molecular brakes” in some animals may have rendered certain tissues regeneration-incompetent (e.g., heart muscle and the auditory epithelium)⁴⁰. The acquisition of molecular breaks is also observed within an animal’s lifetime: neonatal mammals can regenerate a subset of tissues, including cardiac muscle⁴¹. Notably, cardiac muscle has been observed to regenerate in newborn mice for approximately one week before this ability is lost⁴². Could this apparent loss of regenerative ability as the animal ages result from inhibitory mechanisms that halt a specific component process? Comparisons across regenerative versus non-regenerative species/tissues are likely to assist in exploring this hypothesis²⁹. In addition, a greater emphasis on pinpointing mechanisms that terminate or inhibit regenerative processes in cases where regeneration appears to be interrupted or halted after successful initiation may have profound implications for understanding how reduced regenerative capacity evolved in certain animal lineages.

Notably, unbridled overgrowth of regenerating tissue is rarely observed, supporting the hypothesis that regenerative species have mechanisms that provide tight control over proliferation and morphogenesis. Furthermore, reports of tumors in highly regenerative species are uncommon in the literature, although not unknown (reviewed in^43,44). Neoplasms can be induced in newts⁴⁵ and zebrafish (reviewed in⁴⁶), and epidermal neoplasms occur with some frequency in captive salamander colonies (see “De-Mystifying Salamander Cancers” Facebook group). In spite of these observations, the idea that highly regenerative organisms live tumor-free and are resistant to developing cancer remains a widely held belief^43,47. Interestingly, epimorphic regeneration often requires highly conserved pathways that are associated with cancer, and disrupting tumor suppressor genes such as Hippo, p53, or PTEN in planarians and zebrafish can occasionally lead to the formation of tumor-like structures^48,49,50,51. Thus, while the incidence of significant, bona fide tumorigenesis in highly regenerative animals appears low, it seems likely that pathways commonly dysregulated in human cancer are tightly controlled during regenerative healing^52,53,54,55. Alternatively, the expansion of tumor suppressor gene families in mammals may correlate with their reduced ability to regenerate⁵⁴. For example, the human ARF protein produced as an alternately spliced gene product from the p16 locus appears to be absent from the genomes of many regenerative species⁵⁴. In addition, injecting newt myotubes with a plasmid encoding the human tumor suppressor p16^INK4a blocks de-differentiation and cell cycle re-entry⁵⁶. Therefore, tumor suppressor genes may represent another layer of complexity that negatively regulates regenerative responses in mammals. Unraveling the functional relationship between tumor suppression, cell cycle control, and regeneration will require either in vivo genome editing or the introduction of genetically edited cells. However, these tools are currently limited to only a few regenerative organisms.

Understanding the mechanisms by which organisms set and restore size and shape is a formidable challenge in development and regeneration. Evidence from divergent organisms indicates that patterning and growth during regeneration depend on the activation of major components of developmental GRNs and signaling pathways. For example, a conserved role for Wnt signaling in re-establishing polarity during regeneration is supported by work in many regeneration systems, including Hydra, acoels, planarians, and amphibians^13,27,57,58. Despite redeploying conserved developmental pathways, these processes need to operate on very different timescales and, in some cases, across orders of magnitude in scale, which remains difficult to reconcile with current knowledge about patterning in fields of cells.

In one illustration of how organisms can cope with scaling problems, Drosophila embryos balance cell proliferation and apoptosis to regulate proportion during development. Specifically, Bicoid protein is distributed in a gradient across the developing embryos and organizes anterior development^59,60. Yet classic experiments revealed that increases in Bicoid dosage levels, which expand the anterior fate map of the embryo and should give rise to patterning defects, can produce normal larvae⁶⁰. This restoration of proper proportion is driven by increased cell death in the expanded anterior regions, suggesting that the embryos have mechanisms for sensing and adjusting the relative proportion of anterior fated cells⁶¹. Cell death has also been implicated as a key process required in Hydra, planarian, and mammalian liver regeneration⁶². But how do cells detect when there are too few or too many cells and adjust the rate of proliferation or cell death? How do they maintain the correct ratio of specific cell types, particularly in a replacement tissue that must match the scale of the existing animal? Clearly, these are complex, challenging problems that are relevant to both development and regeneration, making experiments in one context informative for the other.

Besides balancing cell proliferation and death to achieve a proper number of building blocks, injured tissues must specify the fates of new cells and rearrange them appropriately to establish tissue size, proportion, and function. One historical view of how this is accomplished is that cells carry information in a Cartesian code to interpret positional details, and this code is deployed in response to morphogen gradients for pattern formation⁶³. A molecular version of this theory is that cells have different chromatin and transcriptomic states based on their developmental history and can both emit and respond to signals in their local environment. Therefore, overall patterning can vary depending on the type or strength of the signals present and the state of cells interpreting the morphogenic cues. A striking example of the importance of this type of injury-induced communication is the dysregulation of canonical Wnt signaling in planarians, which disrupts anteroposterior polarity and leads to the regeneration of ectopic heads or tails⁵⁷. In addition, the Wnt-signaling pathway also plays a role in regulating the proportion of new tissues, as disrupting the striatin-interacting phosphatase and kinase (STRIPAK) complex increases worm length by expanding the posterior wnt1 signaling center and dysregulating axial scaling⁶⁴.

Important regeneration and growth signals are likely not restricted to secreted ligands and well-known signaling pathways. They likely involve ECM components, biomechanical inputs, redox state fluctuations, and changes in metabolic states^65,66,67,68. Ion sensing has also been linked to organ size and regeneration⁶⁹. For example, the zebrafish mutant longfin exhibits fin overgrowth due to ectopic expression of the ion channel Kcnh2a³⁹. Thus, ion sensing and regulation may be a genetically encoded and tunable mechanism for “reading” positional information and producing the correct amount of growth. However, we do not yet know what controls the strength of different types of signals in different contexts and how the regulatory pathways in development might differ from those activated during regeneration. Additionally, although particular signaling pathways may be conserved across contexts, the exact cellular and molecular mechanisms in which they are deployed may differ depending on the size, proportion, and types of tissue being replaced. To this end, embryos typically develop on a much smaller scale than the replacement tissue that is produced during restorative regeneration, so while embryonic pathways may be re-used during regeneration, there are likely key mechanistic differences in how they are employed during the latter.

One rapidly expanding area of interest in regeneration biology is the application of genomic tools to understand how gene expression is regulated over time, in different injury and tissue contexts, and between species. Many recent studies have focused on identifying regulatory elements, particularly enhancers, that are potentially unique or specifically activated after injury in regenerative organisms/tissues (see Box 5). These efforts are rational extensions of studies showing that enhancers are sites of dynamic genome interactions with gene promoters during cell fate transitions in various developmental contexts and organisms^{70,71,72,73,74}. Further, the genetic tools and cell culture systems used routinely in Drosophila and ex vivo mammalian cells to dissect regulatory mechanisms are not yet optimized in most highly regenerative organisms. Thus, it is reasonable to use paradigms emerging from traditional models as the basis for targeted experiments in regenerative organisms as a starting point for uncovering gene regulatory modules that function during regeneration. However, given that the genome regulatory mechanisms that drive regenerative processes and transitions may be different from those operating in non-regenerative species, it is also critical for our field to expand our hypotheses and approaches beyond those that emerge from studies in common animal models. To ensure that we identify the key regulatory mechanisms, including potentially novel ones, that control essential regenerative processes, we must invest the resources needed to customize problem-specific tools in regenerative models. For example, there are chemical biology tools (e.g., degrons, azide-labeled non-canonical amino acids) that are not commonly used in our fields but would allow us to address specific, mechanistic questions arising from decades of observational and functional studies.

Any individual group studying genome regulation during regeneration will likely find that evaluating all possible mechanisms of genome regulation is a formidable challenge. Thus, innovation and impactful discovery in this area will particularly benefit from coordinated interaction and collaboration across research groups so that experimental design and genomic data collection can be readily comparable. In addition, a broad view of genomic regulation across multiple regenerative species will allow us to identify conserved mechanisms, even if the specific genetic modules they activate or silence are different¹³ (Fig. 3). Whether there is greater conservation of upstream genome regulatory mechanisms, the specific proteins orchestrating them, or the genetic modules they target is not likely to have a single or simple answer (Fig. 3). However, we can better distinguish which molecular mechanisms are functionally significant during regeneration by focusing on how specific genetic modules that are known to be activated and essential for regeneration are regulated in regenerative versus non-regenerative contexts.

a In this model, specific gene modules (e.g., coding genes and enhancers) are conserved among various species but regulated differently after injury. This model represents situations where specific genes (e.g., gene X) are present in multiple species, but only activated after injury in regenerative species. It also reflects the possibility that injury-induced genes are activated by different mechanisms in different regenerative species. b In this model, injury induces similar genome regulatory mechanisms (e.g., changes in histone modifications and the expression of pioneering transcription factors) but they have different functional outcomes due to evolutionary differences in the presence and arrangement of specific genetic modules (e.g., the enhancer is only present at this locus in regenerative organisms).

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For example, in the regeneration-competent zebrafish retina, expression of transcription factor ascl1 is induced upon retinal injury and required for retinal regeneration^75,76. Interestingly, Ascl1 is conserved in mice and required for their retinal development^77,78, but Ascl1 expression is not induced after injury of the non-regenerative mouse retina^79,80. This observation inspired experiments in which Asl1 was ectopically expressed in explanted adult mouse retinas⁸¹. However, although Ascl1 functions like a pioneer factor in some ways (e.g., inducing transcription of some relevant target genes), ectopic Ascl1 expression alone was insufficient to induce functional de-differentiation, proliferation, or redifferentiation in injured mouse retinas⁸¹. Yet, importantly, further experiments found that treatment of mouse retinas with ectopic Ascl1 expression in combination with a histone deacetylase (HDAC) inhibitor (which broadly increases genome accessibility) is significantly more successful in activating productive de-differentiation, proliferation, and redifferentiation of mouse cells⁸². These results are exciting, as they suggest it is indeed possible to reactivate existing genes and induce regeneration in non-regenerative tissues⁸³. Nevertheless, many unanswered questions remain: are HDACs facilitating Ascl1 binding to the genome, and if so, at which loci? Which of these newly bound Ascl1 loci are functionally important, and for which steps in the regenerative processes? Why is the induced regeneration of mouse retinas still less robust than the endogenous regeneration process in zebrafish? What regulatory mechanisms or targeted gene expression programs are missing (or inhibitory)? Experiments addressing these specific questions have the potential to both further our understanding of retinal regeneration and uncover widely conserved molecular mechanisms that regulate essential genetic modules in other regenerative contexts.

From a broader perspective, there are many interesting and unanswered questions regarding the role of genome structure and function in regenerative processes. For example, is it relevant that many regenerative organisms, including vertebrates (e.g., axolotls) and invertebrates (e.g., planarians), have large genomes containing significant amounts of repetitive sequence? What do the repetitive sequences signify? Are these sequences serving as regulatory platforms for transcription factor binding, or do they play more instructive roles in regulating cellular plasticity? Interestingly, studies in mammalian stem cells and early embryos suggest that transient activation of repetitive regions plays a major role in regulating gene expression and chromatin state at critical developmental transitions^84,85,86. Are similar mechanisms operating during cell state transitions after an injury? As with the other major knowledge gaps in our field, more comparative studies across species and developmental contexts will be needed to unravel the answers to these questions.

One of the oldest and most enduring questions identified by regenerative and developmental biologists is why regenerative ability is unevenly distributed among metazoans⁸⁷. Why, with over two centuries of regeneration research behind us and major technological advances in molecular biology, next-generation sequencing, and genetic engineering occurring at an ever-quickening pace, does this problem continue to challenge our field? First, evolutionary problems that span large phylogenetic distances are notoriously difficult to address experimentally, and debates about the adaptive nature of regenerative ability remain unresolved. Second, tackling this question necessitates expanding the diversity of species used in regeneration studies⁸⁸, which faces significant financial and practical barriers. Understandably, many scientists prefer to take advantage of the extensive toolkits available in a small subset of genetic model organisms and shy away from the risks associated with learning or developing a new model system. Of course, the decision to study a new model or species should be driven by scientific questions.

In our discussions at the Banbury workshop, there was broad consensus that comparative studies are essential to identifying the possible cell states, mechanisms, and functions critical for those processes outlined in Table 1. Unfortunately, experimental workflows designed for one species are rarely practical across multiple species due to various confounding factors (e.g., regeneration timing, anatomical differences, genome quality disparities, the need for species-specific expertise, etc.). Instead, the field should invest in collectively designing experimental pipelines that can be deployed across species in individual laboratories with high fidelity. Such pipelines would use existing technologies to generate data that may not be significant in any species but could synergize to generate and address many testable hypotheses. Multi-species studies can provide a platform to test the widely held position that a core regenerative program is conserved among metazoans, and its unequal distribution reflects loss and re-emergence in distant lineages. As the number of species used in regeneration research grows within taxonomic groups and across increasingly distant lineages, it will also provide an opportunity to rigorously examine if the alternative hypothesis may be true: that regeneration has independently evolved in numerous lineages. If the latter hypothesis was shown to be true, it would radically expand potential avenues to explore in regenerative therapies.

Adding to the complexity of discovering the mechanistic basis for interspecific differences in regenerative ability, cellular changes associated with aging (e.g., mutations, metabolism, epigenetic states) are gaining recognition as complementary problems with solutions that may help unlock the potential to stimulate regeneration in humans. Suggestively, animals with the capacity for whole-body regeneration, like Hydra and planarians, appear to be negligibly senescent⁸⁹, animals with indeterminate growth have high regenerative ability⁹⁰, and recent work in spiny mice suggests connective tissue cells from these regenerative mammals are highly resistant to stress-induced cellular senescence⁹¹. Moreover, long-lived animals and those with post-metamorphic life stages often lose or have diminished regenerative capacities in adulthood compared to their fetal, neonatal, or juvenile life stages⁹². While the cellular and molecular mechanisms underlying this loss remain poorly understood, possibilities include changes in how cells sense injury signals, cell-autonomous features that prevent cell activation or cell state alternations, or a decline in homeostatic cellular and tissue renewal (Table 1 and Box 2). Together, it is increasingly clear that major advances in dissecting the molecular logic of regeneration (and reconstructing it in humans) cannot occur by studying a handful of organisms. Instead, expanding the collective effort of many research labs across an increasing diversity of organisms can provide answers to some enduring questions in animal biology while potentially unlocking new breakthroughs in human regenerative medicine. Excellent examples of such efforts include comparative studies contrasting species of salamanders, fish, or flatworms^{8,93,94,95,96}, which revealed cellular and molecular insights.

Of course, science at the scale we suggest requires coordination across organisms, labs, and institutions, not to mention a financial strategy to support such activities. Additionally, there is a need for investigators to invest in multiple approaches to rigorously test conclusions on which subsequent studies are based. Rigor and reproducibility, once a cornerstone of the scientific method, have taken a distant backseat to novelty⁹⁷. Potentially insightful work demonstrated in one model system, or species is strengthened, not weakened, through repeated experimental testing by multiple groups and through testing in other regenerative organisms⁹⁸. We must ask this of our field. Cross-species study reproduction and hypothesis testing provide another dimension related to the conservation and canalization of regenerative mechanisms. To understand the fundamental principles of regeneration, we urge our peers and other scientists to revisit some of the most enduring questions in our field.