Inter-organ communication during tissue regeneration Free

Regeneration is the process of tissue replacement and functional restoration following damage or loss of body parts. At its core are a myriad of coordinated cellular behaviors and molecular programs. Injury stimulates the immediate onset of inflammatory responses, during which immune cells are recruited to the wound to clear invading microbes, process damaged and dying tissues, and regulate the behaviors of other immune cells (Eming et al., 2017; Lai et al., 2019). Cells at the wound border are often stimulated to migrate and/or proliferate by cell signaling or mechanical cues (King and Newmark, 2012). These early events generate a pro-regenerative environment that contains signals and builds scaffolds conducive to morphogenesis rather than scarring (González-Rosa et al., 2017). Spared cells in the injury site are cued to activate and respond to developmental signals that promote tissue construction. These processes share some similarities with embryonic patterning but greatly differ in scale, the nature of the starting materials, and the need to functionally incorporate new cells with old. Elucidating the regulatory mechanisms of these dynamic events in tissues, such as limb stumps or the border zones of cardiac or brain infarcts, has been a primary challenge and focus of regeneration research over the past few decades. Understanding regeneration in vertebrate and invertebrate animal models, such as flatworms, Drosophila, fish, salamanders, mice and deer, can illuminate ideas and methods to enhance the repair capacity of injured human organs.

Recent studies are beginning to provide new evidence that tissue regeneration cannot be optimally achieved by individual organs in isolation. Organisms are connected circuits of organ systems, and the impact of a severe injury can extend far beyond the injured organ. Disruption of homeostasis in one organ can elicit organism-wide cellular, molecular and physiological changes in animals. In some cases, distant organs are activated as part of the compensatory mechanism for changes in functionality of an injured organ (Hartupee and Mann, 2017; Schefold et al., 2016). Additionally, nerves and circulating hormones are able to transmit signals over long distances and have been implicated in regenerative events (see below), further connecting remote organs with tissue regeneration. In this Review, we discuss key themes and examples of inter-organ communication during regeneration, including emerging evidence of distant responses to tissue regeneration, communication networks that connect intact and regenerating tissues, mediators of regeneration with long-range activity, and how differences in regenerative potential between different life stages or species may be underpinned by inter-organ communication. We do not discuss the role of circulating immune cells here, but this topic has been covered in other reviews (Aurora and Olson, 2014; Eming et al., 2017; Shanley et al., 2021; Simoes and Riley, 2022; Wynn and Vannella, 2016). Finally, we summarize the potential significance of this line of research and perspectives for mechanistically studying the collective efforts of multi-organ systems during regeneration.

Compared with regenerative programs that activate locally at a wound site, responses of tissues and organs distant from the trauma might not be as easily noticeable. However, changes are evident at many levels. Within hours of a resection injury at the apex of the zebrafish ventricle, the inner endocardial cells of the ventricle, including those hundreds of micrometers away from the injury, change their thin, elongated shape into a round and disorganized morphology and detach transiently from their underlying myofibers (Kikuchi et al., 2011). The gene encoding the retinoic acid-synthesizing enzyme Aldehyde dehydrogenase 1 family, member A2 (Aldh1a2) is induced at an organ-wide level in endocardial cells, providing a source of retinoic acid to promote cardiomyocyte proliferation during heart regeneration (Kikuchi et al., 2011). Similar organ-wide endocardial activation has been observed with many other markers, including il11a and heg (heg1) (Fang et al., 2013; Kikuchi et al., 2011). Epicardial cells surrounding the cardiac chambers also react strongly and rapidly to the distant injury with cell proliferation and gene induction (Huang et al., 2012; Kikuchi et al., 2011; Lepilina et al., 2006; Schnabel et al., 2011; Zhou et al., 2011). These organ-wide molecular and morphological events in injured hearts become restricted to the wound site in the following days (Fang et al., 2013; Kikuchi et al., 2011; Lepilina et al., 2006). Heart regeneration occurs effectively in zebrafish but not adult mammals and, intriguingly, the expression of aldh1a2 is not nearly as dynamic or widespread in injured adult mouse hearts (Kikuchi et al., 2011). This suggests the possibility that the dynamism of the response in non-muscle cells, going from immediate organ-wide to later localized activation, may be relevant to the organ's innate regenerative capacity.

Patterned cell proliferation at the wound drives events such as planarian head regeneration, Drosophila imaginal disc regeneration, and salamander limb regeneration (Abbott et al., 1981; Baguñà et al., 1989; Crucianelli et al., 2022; Kragl et al., 2009; Newmark and Sanchez Alvarado, 2000; O'Brochta and Bryant, 1987; Smith-Bolton et al., 2009; Sobkow et al., 2006; Wagner et al., 2011). Strikingly, major injuries can also activate cell cycling in body parts far from the amputation plane (Ricci and Srivastava, 2018). Planarian head amputation triggers a biphasic sequence of mitotic events, with body-wide cell cycling events preceding localized proliferation at the wound site (Fig. 1A) (Baguñà, 1976; Saló and Baguñà, 1984; Wenemoser and Reddien, 2010). The first mitotic phase is an immediate response of neoblasts to the disruption of body integrity, independent of tissue loss or the formation of a regeneration blastema (Wenemoser and Reddien, 2010), and was recently suggested to be required for proper regeneration at the injured site (Fan et al., 2023). The response to limb amputation differs in axolotls; in this case, only amputation injuries generating massive tissue loss can activate cell proliferation across the organism (in the contralateral limb, heart, liver and spinal cord). These organism-wide cell cycling events are induced concomitantly with cell proliferation triggered locally at the amputation stump (Fig. 1B) (Johnson et al., 2018).

Global cell cycle entry has not yet been reported as part of the injury responses of adult mice. Instead, studies have demonstrated that injuries such as a skeletal muscle lesion can trigger the transition of distant stem cells (such as satellite cells or fibro-adipogenic progenitor cells in the contralateral limb, and long-term hematopoietic stem cells) from a G₀ non-cycling, quiescent state into an ‘alert’ state (G_alert) (Fig. 1C) (Rodgers et al., 2014). Compared with quiescent and activated stem cells, these alert stem cells show intermediate properties of cell cycle kinetics, cell morphology, transcriptional activity and mitochondrial metabolism. Alert stem cells are also primed to respond to a local injury/stimulus more quickly and enter the cell cycle more rapidly than quiescent stem cells, boosting a future regenerative response. The mitotic response to limb amputation in axolotls has also been reported to elevate the regenerative responsiveness of the contralateral limb (Payzin-Dogru et al., 2021 preprint), but mechanisms that underly this response remain unclear. In addition, whether other axolotl organs influenced by limb amputation, such as the heart, liver and spinal cord, are also primed to respond to a second local injury is still unknown. A deeper understanding of the identities of cycling cells in distant tissues, their molecular signatures, and their fates after a round of division will help reveal the roles of these dynamic organism-wide responses.

Although cell cycling responses to major injury have been reported in planarians, axolotls and mice, the molecular mechanisms that mediate these distant responses may be different among these species. A recent study in planarians reported a model in which an Erk activity signal is elicited at the injury site and propagates through longitudinal muscles at a speed of 1 mm/h to reach neoblasts millimeters away and activate cell cycling events (Fan et al., 2023). In contrast, and consistent with the scale and anatomies of salamanders and mice, signals triggered by injury described thus far in these larger animals are transmitted by the circulatory or peripheral nervous systems (discussed below) (Payzin-Dogru et al., 2021 preprint; Rodgers et al., 2017). The extent to which the cell cycle alternations in distant tissues and their primed status following a remote injury are conserved across species is unclear, as a world of vertebrate and invertebrate species awaits similar examination. It is also unclear to what extent the severity and type of injury might influence distant stem cells to enter an alert or cycling state. In some cases, injuries might direct cell cycling in distant tissues in an opposite direction. For instance, in contrast to observations in planarians, axolotls and mice, Drosophila larvae with wing pouch ablation injuries induce cell cycling at the wound but suppress it distantly in the wing hinge and notum region of regenerating discs (Crucianelli et al., 2022; Smith-Bolton et al., 2009). In addition, it is largely unclear whether activating cell cycling or the alert state of stem cells is the major or only strategy animals adopt to prime distant tissues for regeneration. A recent study in mice identified priming of hair follicle stem cells located within a ∼7 mm distance from the wound, and it emphasized the contribution of enhanced migratory potential to their elevated ability to re-epithelialize after a second injury (Levra Levron et al., 2023).

In insects, regeneration of damaged imaginal tissue or appendages can interfere with the pupariation or molting of developing larvae (Kunkel, 1977; O'Farrell and Stock, 1954; Simpson et al., 1980). Lesions of Drosophila melanogaster imaginal tissues induced by irradiation, temperature-sensitive mutations, mechanical manipulation, or genetic ablation significantly delay metamorphosis and reduce the growth rate of uninjured imaginal tissues, effectively allowing imaginal tissues to regrow before pupariation and synchronize their size to the uninjured organs (Fig. 2) (Halme et al., 2010; Hussey et al., 1927; Poodry and Woods, 1990; Shearn et al., 1971; Simpson et al., 1980; Szabad and Bryant, 1982). Molting is similarly postponed in cockroaches that lose their metathoracic legs through autotomy (Kunkel, 1977; O'Farrell and Stock, 1954). Ecdysteroid levels in injured animals are lower than in uninjured animals, suggesting that the central structure for molting hormone secretion responds to peripheral injuries (Kunkel, 1977; O'Farrell and Stock, 1954). Indeed, injury specifically affects a group of neurons within the midbrain, inhibiting transcription of the gene encoding prothoracicotropic hormone (PTTH) (Halme et al., 2010). Suppression of Ptth synthesis delays the release of the molting hormone ecdysone from the ring gland, which subsequently slows developmental progression in regenerating animals (Fig. 2) (Halme et al., 2010; Karanja et al., 2022). This is a prototypical example of how regeneration leads to animal-wide responses through the targeted influence of a specific distant organ.

In addition to inducing organ-wide or organism-wide changes in animals, injuries are known to elicit more preferential cell activation in specific tissues and organs. A field study of white-tailed bucks more than 50 years ago demonstrated that experimentally amputating the rear leg of the deer promotes the regeneration of a severely malformed antler on the contralateral side of the injury following casting (natural shed of antlers) (Marburger et al., 1972). A similar contralateral effect is observed in roe deer, where amputation of the whole antler pedicle results in excessive branching on the unoperated side (Bubenik and Pavlansky, 1965). Severe surgical removal of a pedicle together with its underlying frontal bone induces benign bone tumors, known as exostoses, centimeters away from the pedicle bases on both sides and, in the following cycle of antler growth, a short antler grows out from the exostoses contralateral to the injury (Fig. 3A) (Bubenik and Pavlansky, 1965). More generally, it appears that the intact, contralateral side of paired organs can be prominently reactive to damage (Busse et al., 2018). These physiological and morphological changes reported in uninjured contralateral organs can emerge as quickly as seconds or as slowly as years, challenging their elucidation, especially for internal paired organs, such as brain hemispheres, lungs and kidneys of mammals.

Importantly, the distant injury responses by contralateral uninjured tissues revealed in these and other studies challenge their suitability as controls in regeneration experiments. Transcriptional and epigenetic profiling of contralateral tissues has revealed both gene expression level changes and chromatin accessibility changes in contralateral limbs compared with naïve limbs of uninjured animals (Payzin-Dogru et al., 2021 preprint; Rodgers et al., 2014). One must consider then that the tissues of naïve littermates or clutchmates, never having been injured previously, likely provide the most pristine controls for profiling and other high-resolution comparisons.

Signals elicited by injuries can be potent triggers of local regenerative programs. To initiate responses across tissue space during regeneration, animals must deliver injury messages to remote, uninjured structures. The identity, availability and transmission of these long-range signals should determine the extent to which remote organs are activated by injury (systemic versus targeted) and the duration of responses in remote organs (transient versus permanent). Identification of long-range injury signals and understanding their regulation is challenging but crucial to understand how activities in multiple organs are coordinated during regeneration.

An intuitive hypothesis is that regenerating tissues function as signaling centers, producing and secreting key molecules that act upon distant organs. Early studies of Drosophila imaginal tissue regeneration provided evidence supporting this paradigm. Complete ablation of imaginal tissues does not impact the development of injured larvae, whereas partial perturbation does (Poodry and Woods, 1990; Shearn et al., 1971; Simpson et al., 1980; Szabad and Bryant, 1982), and transplantation of bisected genital discs delays the pupariation of the uninjured host (Dewes, 1975). These findings indicated that damaged imaginal tissue is necessary and sufficient to induce animal-wide growth responses, and that this is likely achieved through the release of a negative regulator of metamorphosis during regeneration. Nearly 40 years later, two independent genetic studies revealed this factor to be a secreted insulin-like peptide hormone, Dilp8 (Ilp8), expression levels of which are positively correlated with the severity of perturbation in imaginal tissues (Colombani et al., 2012; Garelli et al., 2012). Dilp8 is synthesized and released from the epithelium of imaginal discs and diffuses to the brain through hemolymph, where it suppresses ecdysone synthesis and associated metamorphosis and growth (Fig. 2) (Colombani et al., 2012; Garelli et al., 2012).

Activation of distant tissue need not involve de novo synthesis and secretion of signaling molecules from injured tissues. For instance, injuries can directly or indirectly activate blood-borne factors with long-range signaling properties. Skeletal muscle satellite cells in mice have been proposed to adopt the G_alert state in response to hepatocyte growth factor activator (HGFA; HGFAC) (Rodgers et al., 2017). The inactive precursor of HGFA, pro-HGFA, is constitutively present in the circulatory system in uninjured animals, and is catalyzed to its active form by activated thrombin (Rodgers et al., 2017). Active HGFA circulates in the injured animals, processing resident inactive hepatocyte growth factor (HGF) in distant responding tissues into active HGF to induce the G_alert state of distant stem cells. In this scenario, the liver is the primary source for producing the inactive long-range signaling molecule pro-HGFA and the inactive form of thrombin, prothrombin. The circulatory system is not merely a transport system but also functions as a reservoir of long-range signaling molecules (Fig. 1C). Rather than acting as a source of signals, the damaged tissue uncages pre-existing signals to regulate distant responses.

The nervous system enables the coordination of organ function in response to contextual changes, for which regeneration is no exception. In contrast to routing signals through vascular highways, sensory neurons in peripheral organs convert local chemical, mechanical or thermal cues into electrical signals that rapidly traverse to the spinal cord or brain. Peripheral sensory neurons elicit one of the earliest remote responses to tissue damage by activating ion channels located at the axon terminals upon exposure to noxious stimuli, which generate action potentials to stimulate the perception of pain (Pinho-Ribeiro et al., 2017). During regeneration, both the sensory input from the injured organ and the efferent output at the target tissue have been reported to be required for distant responses. In the oriental cockroach (Blattella orientalis), resection of the nerve projecting from the thoracic ganglion to the autotomized leg, or the connection between those ganglia and the brain, eliminates the delay in molting usually caused by leg loss and affects proper regeneration (Kunkel, 1977). In axolotl, α-adrenergic signals are required for activating cell proliferation and the priming response in the contralateral side after limb amputation, and denervating either the injury side or the contralateral side of the axolotl limb immediately before amputation impedes the proliferative response in the contralateral tissue (Payzin-Dogru et al., 2021 preprint). Although the sensory arm of the peripheral nervous system is clearly involved in activating distant responses, more work is needed to explore the diversity of sensory neurons involved in injury and regeneration, how injury and regeneration are sensed differently by animals, how different regeneration-related sensations are detected by neurons, and how different subtypes of neurons coordinate signaling to distant tissues. Customized and controlled involvement of both the nervous and circulatory systems may add another layer of complexity to how different organs communicate during regeneration.

Neurons are not the only cells in a biological system that can deliver electric signals. Bioelectric signals can be generated in virtually all cells by ion flow across the plasma membrane, and these altered membrane potentials can propagate to neighboring cells through gap junctions to coordinate the bioelectric state across space (Levin, 2021). It is now clear that bioelectric signals can regulate gene expression and cellular behaviors, including proliferation, differentiation, apoptosis and migration (Atsuta et al., 2019; Benham-Pyle et al., 2021; Bi et al., 2013; Franklin et al., 2017; Hiratsuka et al., 2015; Konig et al., 2006; Lansu and Gentile, 2013; Pai et al., 2015). During regeneration, injury alters the bioelectric field of regenerating organs (Borgens et al., 1977a,b; Busse et al., 2018). Manipulating the bioelectric state of regenerating tissue by regulating ion channel activity or applying an external electric field has been reported to disrupt and even direct proper growth and patterning of a regenerating tissue in various regenerative species and contexts, suggesting an instructive role of bioelectricity (Adams et al., 2007, 2013, 2016; Borgens, 1986; Durant et al., 2019, 2017; Oviedo et al., 2010; Pai et al., 2012; Sharma and Niazi, 1990; Smith, 1974; Vandenberg et al., 2011). This has been reviewed in detail elsewhere (Harris, 2021; Levin, 2021; McLaughlin and Levin, 2018).

Recent studies in Xenopus embryos revealed long-range instructive roles of such bioelectric signals during development (Blackiston et al., 2011; Lobikin et al., 2015; Pai et al., 2020, 2015). A similar far-reaching effect was suggested by a recent study of Xenopus limb regeneration. Amputation of frog hindlimbs in their highly regenerative stage induces depolarization in the epidermis of the contralateral uninjured limb within seconds (Busse et al., 2018). Depolarization peaks at 30 mins post-amputation and then decreases sharply by 24 h post-amputation. The region of depolarization in the contralateral limb corresponds to the location of the amputation plane of the injured limb (Fig. 3B). This study presents an intriguing scenario in which bioelectric signals encode positional information about the amputation, propagate rapidly across the animal, and deliver targeted information to the designated structure. However, the exact mechanism underlying this long-range signal transmission remains to be elucidated. For example, it is unclear how positional information is converted into electric signals, how bioelectric signals are propagated in seconds to regions that are centimeters away, and how the propagation of signals is directionally restricted toward the contralateral limb. Interestingly, removal of one eye or one kidney does not induce depolarization in the contralateral partner, suggesting that this contralateral depolarization effect is specific to limb amputation (Busse et al., 2018). Analyzing bioelectric features of zebrafish pectoral fins or axolotl limbs during regeneration will elucidate whether this is an evolutionarily conserved feature across analogous regenerative structures. It will also be important to understand the cellular effects of depolarization in the contralateral limb. Previous studies demonstrated that transient changes in the bioelectric state can have prolonged or permanent effects on animals (Durant et al., 2019; Eischen-Loges et al., 2018). Thus, we speculate that there may be a biphasic change in cellular behavior: one transient response to the local bioelectric signals, which may relate to the regeneration of the injured limb, and one prolonged effect due to the ‘memory’ of this depolarization event, which may affect the regenerative response of the contralateral limb to a future injury event.

Sending signals via the circulatory system would be expected to expand the distance these signals can travel at the expense of specificity. However, evidence indicates that the extent to which systemic factors elicit responses can be highly programmed, targeting specific organs. Ostensibly, specific structures in these distant organs must detect and process these systemic signals. Identifying these discrete structures can explain how and why certain remote tissues respond to regeneration. For instance, genetic studies identified Lgr3 as the target of Dilp8 during imaginal tissue regeneration. The Drosophila relaxin receptor Lgr3 is present both in neuronal subsets of the central nervous system (CNS) and in the prothoracic gland of the ring gland (Colombani et al., 2015; Garelli et al., 2015; Jaszczak et al., 2016). In the CNS, a pair of bilateral neurons located at the pars intercerebralis (a neuroendocrine system at the anterior midline of the brain) is in contact with PTTH neurons and insulin-producing neurons and consistently respond to the Dilp8 stimulus during imaginal tissue regeneration (Colombani et al., 2015; Garelli et al., 2015; Vallejo et al., 2015). These Lgr3-expressing neurons suppress PTTH neuron activities in response to Dilp8 signals, thus decreasing ecdysone synthesis and delaying development during imaginal tissue regeneration (Colombani et al., 2012; Garelli et al., 2015; Vallejo et al., 2015). In parallel, the neuronal Dilp8-Lgr3 signaling axis also inhibits insulin-producing neurons in the brain and activates nitric oxide synthetase (NOS) in the ring gland, both of which suppress ecdysone synthesis to slow down the growth of the uninjured tissues (Fig. 2) (Jaszczak et al., 2016, 2015; Vallejo et al., 2015). In the ring gland, Dilp8-Lgr3 signaling is not required for regeneration-induced developmental delay, but instead retards the growth of intact organs by activating NOS, ultimately helping to coordinate growth between regenerating and uninjured organs (Jaszczak et al., 2016) (Fig. 2). This provides a central control mechanism for ensuring the proportionality and bilateral symmetry of organs before proceeding to the next stage of development. Studies such as these highlight the concept that responding to regeneration from distant regions can involve a relay of signals across multiple organs. Distant organs in this case are not mere signal recipients but can function as transmitters, emitting a second long-range signal to connect the regenerating tissue with its other remote effectors.

A recent study in zebrafish identified a class of DNA regulatory elements termed remote tissue regeneration enhancer elements (r-TREEs), described as enhancers that act as the detecting structures in a distant organ to initiate regeneration-responsive programs (Sun et al., 2022). Analogous DNA regulatory elements that respond locally to injury and regeneration events were previously described in the imaginal discs of Drosophila larvae and in the regenerating heart and fins of zebrafish and named TREEs by our group (Harris et al., 2016; Kang et al., 2016). TREEs are categorized by their ability to direct gene expression changes in the presence of injury, maintain this expression for the duration of regeneration, and temper it as regeneration concludes. Profiling the transcriptomic and chromatin accessibility changes in the brain during zebrafish heart regeneration revealed an enhancer, CEN, that detects corticosteroid hormone signals and instructs expression of the cebpd gene in the uninjured brain tissue (Sun et al., 2022). Interestingly, further profiling during heart regeneration of a second remote tissue, whole kidney marrow, revealed a largely distinct set of candidate r-TREEs that regulate whole kidney marrow-associated programs (Sun et al., 2022). We speculate that tissues employ distinct classes of r-TREEs to filter long-range injury signals and direct the appropriate responses, presumably via navigation of the local chromatin environment by available tissue-specific transcription factors and tissue-specific features.

Responses of distant tissues to a traumatic injury have long been recognized as part of the compensatory mechanism animals use to restore physiological homeostasis in the body. However, there is emerging evidence for an instructive role of these distant responders in regulating tissue regeneration events at the site of injury. Identifying the factors that distant structures produce to modulate tissue regeneration can lead to intriguing ideas for remote control of regenerative therapies.

In the early 20th century, many experiments were performed in newts that removed endocrine glands prior to or coincident with limb amputation. In one of the earliest works on this topic, thyroidectomy (removal of the thyroid gland) hindered limb regeneration in the adult Triton cristatus (Walter, 1910). This observation was further supported by similar studies in Triturus viridescens (Richardson, 1940; Schotté and Washburn, 1954). In 1926, Schotté surgically removed the pituitary gland (a process termed hypophysectomy) from the adult Triturus and found that amputated limbs of these hypophysectomized newts could no longer regenerate (Schotté, 1926). Later work found that removal of the pancreas inhibits the regeneration of the forelimb and tail in adult Diemictylus viridescens (Vethamany-Globus and Liversage, 1973). More recent studies with Xenopus laevis showed that the nascent brain is required for distant organ patterning and morphogenesis during development (Herrera-Rincon and Levin, 2018), and that injury to the basal hypothalamus through electrocautery microsurgery leads to limb regeneration defects in Xenopus tadpoles (Zhang et al., 2018). Although extirpation of endocrine glands is detrimental to animals in many aspects, these observations specifically demonstrate the necessity of distant organs in regulating local regeneration processes, and they suggest that signals outside the local injury environment can control tissue regeneration.

Hormones, mentioned above in descriptions of ecdysone and relaxin functions in Drosophila, constitute a class of signaling molecules that cannot be overlooked when discussing inter-organ communication. They are typically synthesized in internal endocrine glands and secreted into the blood supply, functioning by binding to their receptors in target organs. In addition to their well-established roles in metabolism, physiology, reproduction and growth, hormones can regulate tissue regeneration and repair (comprehensively reviewed by Losner et al., 2021). The administration of anterior pituitary extracts or crude growth hormone extracts can restore limb regeneration in adult hypophysectomized newts (Liversage and Fisher, 1974; Liversage and Scadding, 1969; Richardson, 1945; Wilkerson, 1963). The effective components in these extracts were later demonstrated to be growth hormone and prolactin (Connelly et al., 1968; Tassava, 1969; Wilkerson, 1963). Injection of purified growth hormone on its own or applying a combination of prolactin (which is secreted from the pituitary gland) and thyroxin (secretion of which is regulated by the hypothalamus-pituitary-thyroid axis) can effectively restore limb regeneration in adult hypophysectomized newts (Connelly et al., 1968; Tassava, 1969; Wilkerson, 1963). The application of thyroxin alone is sufficient to rescue limb regeneration in thyroidectomized newts (Richardson, 1945). These findings suggest that limb regeneration is dependent on systemic hormone signals derived from distant endocrine tissues. In the context of deer antler regeneration, systemic hormone signals initiate regeneration and also regulate tissue maturation and growth during regeneration. Casting happens when levels of circulating testosterone drop in the spring (Goss, 1968). Administration of testosterone during fall or winter inhibits casting (Goss, 1968), and castration in the fall or winter leads to early casting due to the early drop of testosterone levels in the deer (Goss, 1983). Regenerated antlers in castrated deer are less ossified, and their velvet is never shed, resulting in amorphous growth of the regenerated antlers (Goss, 1983).

The effect of hormones on tissue regeneration is highly dependent on the dose, duration and timing of their administration, and on species-dependent factors (Amram et al., 2021; Easterling et al., 2019). A recent study of cardiac regeneration in Xenopus laevis tadpoles demonstrated that either applying or depriving thyroid hormone can impair regeneration, indicating the importance of fine-tuning hormone levels (Marshall et al., 2019). Examples also exist demonstrating that transitions in tissue regenerative capacity during development are associated with a peak of hormone production or prolonged elevation of hormone levels in animals (Alberch et al., 1986; Hirose et al., 2019; Larras-Regard et al., 1981). The inability of tissue to regenerate following hormone exposure is not only limited to tissue that has undergone an injury; tissue exposed to high levels of hormones during development may also lack the ability to regenerate later in life (Grobstein, 1947), suggesting that transient exposure to hormone signals can sculpt future regenerative responses to injury.

Hormones can also function differently depending on the developmental stage. Schotté suggested long ago that pituitary hormones are necessary for limb regeneration in adult Triturus, but dispensable for similar events in larvae (Schotté, 1926; Schotté and Droin, 1965). In Drosophila, low-level ecdysone is essential for imaginal tissue regeneration at the early larval stage, whereas high levels of ecdysone trigger metamorphosis and impede imaginal disc repair at the late larval stage (Karanja et al., 2022). In these examples, development is accompanied by alternations in the external environment, internal physiology and intrinsic cellular properties. It is thus reasonable to speculate that there exist intrinsic factors that regulate how injured tissue perceives hormone signals at different developmental stages, as well as extrinsic factors that interact with hormone signals to regulate how hormones impact injured tissues during regeneration.

Vitamin D, known for its roles in maintaining calcium homeostasis and bone integrity in animals, is an extraordinarily well-studied signaling factor. A recent chemical screen using zebrafish larvae identified vitamin D as an activator of cardiomyocyte cycling (Han et al., 2019). Intriguingly, administration of vitamin D analogs to adult zebrafish also boosted cycling events in epicardial cells, endocardial cells, dermal osteoblasts, basal epithelial cells, corneal epithelial cells and retinal cells, suggesting vitamin D acts as a pan-mitogen that promotes cell proliferation to regulate tissue growth in adults (Han et al., 2019). This broad mitogenic response of vitamin D has not been reported in rodent systems. Future mechanistic studies in zebrafish will hopefully elucidate how a systemic regulator with a broad mitogenic effect regulates the growth of injured organs specifically during tissue regeneration. Dissecting the downstream effectors of vitamin D signaling across tissue types in adult zebrafish and rodents could provide insights into the species-specific roles of vitamin D and how vitamin D functions diverged during evolution.

Parabiosis is a procedure whereby the circulatory systems of two mice are surgically joined together, enabling sharing of hematopoietic cells and factors that circulate in the bloodstream. It was introduced by Paul Bert in 1865 (Bert, 1865) and has found many recent applications across fields of biology, with one study demonstrating that parabiosis between a young mouse and an aged mouse enhances the regenerative capacities of skeletal muscle satellite cells and hepatocytes in the aged mouse, and decreases the regenerative potential of satellite cells and proliferation of hepatocytes in the young mouse (Conboy et al., 2005). This finding established two milestones for the field: (1) stem cells and their niches in aged animals retain their intrinsic capacities to respond to injuries and promote regeneration; and (2) systemic factors circulating in the bloodstream can modulate tissue regeneration, either through inhibitory or stimulatory mechanisms.

These parabiosis experiments prompted a search for a candidate systemic factor with properties that impact tissue regeneration. A proteomic screen using plasma from young-isochronic (same age), old-isochronic and heterochronic (different age) parabionts identified the inhibitory factor CCL11 (also known as eotaxin) as being sufficient to suppress neurogenesis and impair learning and memory in young mice (Villeda et al., 2011). A subsequent study reported that the pro-regenerative factor oxytocin, a circulating hormone synthesized in the hypothalamus and released into the bloodstream by the neurohypophysis, decreases in plasma levels with age, and is sufficient to promote muscle regeneration in old mice and required to maintain muscle regenerative ability in young mice (Elabd et al., 2014). Although the exact mechanisms by which circulating CCL11 and oxytocin might exert their effect on regeneration have not been thoroughly investigated, they are of extreme interest to regenerative medicine. Not only have these findings provided therapeutic targets, but they have also prompted groups to consider pro-regenerative physiological environments as part of potential regenerative therapies.

Exercise is a prime example of how an external intervention modulates tissue regeneration through systemic signals. Physical exercise regulates stem cell activities in many organs to the extent that it can impact various age-related diseases in model systems. For instance, voluntary wheel running by mice enhances neurogenesis in the hippocampus and improves learning in aged cohorts (van Praag et al., 2005). Similar exercise routines can increase the activity of quiescent satellite cells in aged mice and improve skeletal muscle regeneration (Brett et al., 2020). Transferring plasma or serum from exercised mice to age-matched sedentary mice can also boost tissue regenerative potential (Brett et al., 2020; De Miguel et al., 2021; Horowitz et al., 2020). These studies provide direct evidence that exercise stimulates the production of blood-borne factors that can enhance the function of stem cells in old animals. The liver-derived circulating factor glycosylphosphatidylinositol-specific phospholipase D1 (Gpld1) and the complement cascade inhibitor clusterin are two examples of such circulating factors (De Miguel et al., 2021; Horowitz et al., 2020). Exercise is known to elicit mechanical signals to act directly on a broad range of organs (reviewed by Chen et al., 2022). However, which organ(s) secretes pro-regenerative circulating signals in exercised animals requires further exploration. Skeletal muscle has recently been identified as a center for endocrine signals that promotes crosstalk between organs under the stimulus of exercise (Hoffmann and Weigert, 2017; Pedersen, 2019; Severinsen and Pedersen, 2020). This tissue could be a promising future target of investigation for identifying new systemic regulators of regeneration during exercise.

Recent studies have revealed a potential relationship between the percentage of mononucleated diploid cardiomyocytes and the ability of heart muscle cells to divide after an injury and regenerate heart muscle (González-Rosa et al., 2018; Patterson et al., 2017). These studies found that animals with a higher complement of polyploid heart muscle cells, which is the predominant state in mammals, display weakened regenerative responses with major scarring upon injury. Forcing polyploidy in the normally diploid zebrafish using genetic tools weakens the regenerative response (González-Rosa et al., 2018). Interestingly, a phylogenetic analysis of 41 species from zebrafish to humans found that mononucleated diploid cardiomyocyte abundance declines concurrently with the evolutionary transition from ectotherms to endotherms, with the activity of thyroid hormone signaling implicated as an underlying factor (Hirose et al., 2019). Pharmacologically inhibiting thyroid hormone synthesis or function, or genetically expressing dominant-negative thyroid hormone receptors in cardiomyocytes, measurably increased cardiomyocyte cycling and boosted cardiomyocyte diploidy in perinatal mice and also enhanced cardiac repair after ischemia-reperfusion injuries in adult mice (Hirose et al., 2019). Conversely, treating zebrafish with triiodothyronine (T3) had inhibitory effects on cardiac regeneration (Hirose et al., 2019). These findings present an evolutionary explanation for the loss of cardiac regenerative capacity in mammals; that is, tissue regeneration responses become constrained by hormone-dependent acquisition of physiological and cellular traits.

A similar idea from a different perspective was presented by Grobstein in the 1940s. He reported that an amputated gonopodium, the copulatory organ of male platyfish, fails to regenerate, in contrast to the full regenerative ability of the anal fins of female platyfish and the immature anal fin of male platyfish (Grobstein, 1947). In male platyfish, androgen regulates the morphological changes of the anal fin into a gonopodium (Grobstein, 1940, 1942). Administration of androgen to female platyfish prior to amputation significantly reduces anal fin regeneration. This inhibitory effect of androgen is prolonged in masculinized female anal fins even when androgen application is discontinued before amputation. In adult male platyfish, castration does not deliver complete regenerative ability to the gonopodium, and those rays that do regenerate have immature features without gonopodium morphology (Grobstein, 1947). This finding suggests that androgen induces morphogenesis of the reproductive organ in platyfish, and it can permanently reduce the capacity of this organ to regenerate even without continuous presence. A similar dimorphism was described in adult zebrafish; female fish can regenerate near-perfect replicates of amputated pectoral fins but many male fish show regenerative failures in certain pectoral fin rays (Nachtrab et al., 2011). This disrupted regenerative capacity in male pectoral fins was later attributed to androgen-dependent acquisition of epidermal tubercles, the male-specific ornaments for spawning that adorn anteromedial rays of the pectoral fin (Kang et al., 2013). These ornaments produce the Wnt pathway inhibitor Dkk1b, ostensibly impeding Wnt-dependent proliferation and patterning events during fin regeneration (Kang et al., 2013). Notably, androgenic signals have been linked to tissue regeneration as a positive or negative regulator in various species and contexts (Aldahl et al., 2019; Ashcroft and Mills, 2002; Bielecki et al., 2016; Frenkel et al., 2010; Goss, 1968; Kierdorf and Kierdorf, 2011; MacKrell et al., 2015). These studies collectively suggest the intriguing idea that some animals may have gained advantages in reproduction during evolution at the cost of regenerative capacity.

The phylogenic divergence of capacities for tissue regeneration in animal taxa has been of major interest to regeneration biologists for centuries. However, mechanisms underlying species differences are still in many ways mysterious. Intrinsic factors, such as metabolic signatures, ploidy, or acquisition of specialized regulatory sequences for regeneration, have been implicated in the evolution of regeneration potential (Harris et al., 2016; Kang et al., 2016; Lopaschuk et al., 1992; Patterson et al., 2017; Puente et al., 2014). However, the idea that distal tissue responses to organ injury might be evolutionary targets for regenerative capacity is understudied and would be challenging to explore directly. Determining similarities and differences in distant organ responses to injury across phylogeny, and how remote injury responses change during development, might hint at conserved organ interactions that are preferential to highly regenerative species or stages.

We are far from fully understanding the distant changes elicited during regeneration in an animal. Methods for transcriptomics, epigenomics, proteomics and metabolomics allow unbiased mapping of cellular and molecular responses to the stimulus at various levels. The recent emergence of single-cell technologies further provides opportunities to identify heterogeneous responses within a single organ and responses of rare cell populations in distant tissues (Benham-Pyle et al., 2021). With the latest generation of molecular and genetic technologies, the field of regeneration is ripe to monitor dynamic changes in remote organs in real time during the course of regeneration and to manipulate distant responses in a targeted manner to study the impact of distant organs on tissue regeneration.

Even with a well-defined readout in a distant organ, it is still challenging to map signaling events regulating inter-organ communication networks during tissue regeneration, given that distances are large and signals travel quickly. Systematic genetic screens for discovering inter-organ communication factors cannot be easily performed in most animal models, owing to the number of animals needed and the lack of screening platforms to distinguish the local role from the distant role of a candidate gene. Proteomic or metabolomic analysis of the plasma of regenerating animals also loses information about the origin of signals. Multi-step signal relays and propagation might occur at the injury site before ultimately distributing functional long-range injury signals, which complicates signal mapping. Recently, multiple groups developed proximity labeling methods to capture proteins that traverse classic endoplasmic reticulum/Golgi secretory routes (Kim et al., 2021; Wei et al., 2021). This endoplasmic reticulum-based proximity labeling method can facilitate the discovery of tissue-specific secretomes and identify candidate signals in the circulatory system during regeneration (Liu et al., 2021; Wei et al., 2023). Fluorescent tagging of endogenous signaling molecules can further facilitate the visualization of the production, transmission and reception of long-range signals during regeneration.

Organoid and organ-on-a-chip platforms allow researchers to model tissue regeneration in vitro (Bhatia and Ingber, 2014; Huh et al., 2010; Wang et al., 2013; Wu et al., 2020). What is exciting about these systems is the possibility of connecting several organ models on a chip through microfluidic systems and simulating the organ interactions and physiological environments of a regenerating organ in vitro (Rajan et al., 2020; Wu et al., 2020). Recent breakthroughs with this multi-organ platform will facilitate mechanistic studies of factors such as fluid shear stress, mechanical stress, and gradients of signaling molecules for their roles in inter-organ communication networks and control of tissue regeneration. Platforms such as these, in particular using human organoid culture systems, will provide opportunities to test the translational potential of long-range pro-regenerative factors in human tissue repair. Thus, there is much to learn about and potentially gain from the crowdsourcing of tissue regeneration.

We thank Leslie Slota-Burtt for comments on the manuscript.