Genetic, epigenetic, and post‐transcriptional basis of divergent tissue regenerative capacities among vertebrates

Abstract Regeneration is widespread across the animal kingdom but varies vastly across phylogeny and even ontogeny. Adult mammalian regeneration in most organs and appendages is limited, while vertebrates such as zebrafish and salamanders are able to regenerate various organs and body parts. Here, we focus on the regeneration of appendages, spinal cord, and heart—organs and body parts that are highly regenerative among fish and amphibian species but limited in adult mammals. We then describe potential genetic, epigenetic, and post‐transcriptional similarities among these different forms of regeneration across vertebrates and discuss several theories for diminished regenerative capacity throughout evolution.


| INTRODUCTION TO REGENERATION IN THE ANIMAL KINGDOM
Regenerative capacity varies widely in the animal kingdom. Invertebrate species such as planarians are champions of regeneration, capable of regenerating the entire organism. 1,2 Within vertebrate species, those most closely resembling their fossil ancestors-"phylogenetically primitive" organisms such as fish (with zebrafish being a prominent example) and urodele amphibians-are capable of regenerating the limb, fin, spinal cord, heart, and other organs. Meanwhile, adult mammalian species are largely limited to regenerating specific tissues such as the skin, liver, and skeletal muscle and not body parts such as the heart, spinal cord, and limbs. [3][4][5] Strikingly, neonatal mammals retain a lot of the regenerative capacity observed in fish and amphibian species. Whether this regenerative trait is a unique regeneration response, a modified injury response, or a combination of both remains unclear. The subsequent critical question is whether regeneration is an ancestral trait lost through evolution or a novel trait gained.
Despite evidence supporting both theories, the ancestral trait theory has been gaining popularity with increasing evidence of conserved genetic and molecular pathways governing regeneration across phylogeny. 6 This review will first briefly introduce regeneration in invertebrate species, then focus on examining the diverse regenerative capacities of appendage, spinal cord, and heart in key model organisms including zebrafish (Danio rerio), axolotl salamanders (Ambystoma mexicanum), frogs (Rana spp., Xenopus spp.), and mice (laboratory strains of Mus musculus unless otherwise identified). We hope to highlight some genetic and epigenetic commonalities of regeneration across vertebrate species and address a few theories seeking to explain the general decline in regenerative capacity accompanying evolutionary development (Figures 1 and 2).

| Regeneration in invertebrates
Unlike complex vertebrates, most invertebrates demonstrate a robust ability to regenerate. 7 For instance, the planarian flatworm (Schmidtea mediterranea) is prized for its astonishing regenerative potential. 1 Planaria maculata fully restore their bodies when left with a mere 1/279th fraction of the original body. 8 Throughout development, planaria rely on neoblast stem cells to regenerate tissues; upon injury, positional control genes, such as notum and wnt1, are activated to reestablish positional information and serve as guides for progenitor cell differentiation. 9 On the other hand, insects demonstrate limited regenerative capacity-possibly due to their complex, derived morphogenesis-and are an anomaly amongst invertebrates. 10 For example, Drosophila melanogaster imaginal discs, which develop into limbs, appendages, and other parts, are highly regenerative during early larval stages but lose the ability to regenerate in larval development as they differentiate exoskeletal cuticle. 11 Still, many invertebrates of both the protostome and deuterostome lineages, such as flatworms and echinoderms, retain high regenerative potential. 7

| Appendage regeneration in vertebrates
Contemporary vertebrate appendage regeneration models follow a similar order of events: epithelial closure of the wound site, establishment of a wound epithelium, dedifferentiation or activation of cells leading to epimorphic blastema formation, and proliferation and redifferentiation of progenitor cells to regenerate various appendage cell types in a lineage-restricted manner (or what appears to be lineage-restricted in species thoroughly investigated thus far). 12 The model is not allencompassing: morphologically simpler vertebrates can fully regenerate appendages, while more complex vertebrates generally possess extremely limited appendage regeneration.
Regeneration of the teleost fin was first documented by French naturalist Broussonet, then further explored by Thomas Hunt Morgan. [13][14][15][16] To date, the signaling pathways essential for zebrafish fin regeneration include many that are necessary for vertebrate embryonic development. Specifically, Wnt/β-catenin, RA, IGF, and FGF signaling pathways drive blastema formation and expression of fgf20a, essential for initiating fin regeneration and subsequent proliferative outgrowth. 14,17,18 Axolotl salamanders (Ambystoma mexicanum) are among the oldest studied regenerative models with the first evidence of whole adult tail and limb regeneration documented by Spallanzani. 19 Molecular pathways similar to those in zebrafish fin regeneration also drive salamander limb regeneration. For instance, FGF signaling is required for blastema formation, with fgf8 and fgf10 expression essential for successful completion. 20 In addition, Wnt/β-catenin signaling is necessary during early stages of limb regeneration but not after blastema formation. 21,22 Although complete limb regeneration is not observed in mammals, neonatal mice, adult mice, and humans can regenerate digit tips amputated through the distal region of the terminal phalanx. [23][24][25] Amputations transecting more proximal regions, however, result in regenerative failure and scarring instead. 26 Mammalian digit tip regeneration correlates with regions that express Msx-1 and Msx-1-mutant mice cannot regenerate the digit tip. 27,28 However, BMP-4 can rescue this phenotype, suggesting the importance of the BMP/Msx signaling pathway in digit tip regeneration. 28 Antlers in all Cervidae and female reindeer (Rangifer tarandus) are the only mammalian appendages that can fully regenerate, not only once, but many times throughout the host's lifetime. Scholars still debate the classification of this form of annual tissue replacement since seasonal hormonal changes, rather than injuries, prompt antler F I G U R E 1 Phylogenetic distribution of species demonstrating varied heart, appendage, and spinal cord regenerative capacities across development regeneration. Regardless, deer antlers are used as a model to understand complete mammalian appendage regeneration being a unique example of stem cell-based epimorphic regeneration that also depends on blastema-based appendage regeneration pathways. 29-32

| Spinal cord regeneration in vertebrates
Spinal cord injuries in adult mammals generate permanent glial scars and lead to motor and sensory impairment. 33,34 Studying organisms capable of spinal cord regeneration can provide insights into various injury responses permitting pro-regenerative environments. Here, we will discuss zebrafish and salamanders, species regenerative throughout their lifetimes, as well as frogs and mice, species only regenerative soon after birth.
Upon injury, the adult zebrafish spinal cord triggers proliferation and differentiation of resident neural progenitor cells called ependymoradial glial cells. 35 Spinal cord regeneration reuses major developmental pathways including Notch, Hh, Wnt, and FGF signaling. 36,37 FGF signaling in particular induces glial cell bridge formation, which provides a scaffold for regenerating axonal growth. Moreover, connective tissue growth factor a (ctgfa) is necessary and sufficient to initiate glial cell bridging and regeneration. 38,39 Spallanzani first described axolotl salamander spinal cord regeneration in 1768; subsequent studies of the field appeared in the mid-20th century. [40][41][42][43][44] After tail amputation, salamanders can regenerate their spinal cord through ependymoradial glial cell proliferation and differentiation, similar to zebrafish. [45][46][47] Radial glial cells in axolotl tail regeneration can switch lineages to generate surrounding muscle and cartilage tissue in addition to regenerating a functional spinal cord. 48 This phenomenon contrasts the emerging theme that transdifferentiation is not a major mechanism governing limb and fin regeneration.
Xenopus, more phylogenetically complex than fish and salamanders, are able to regenerate their spinal cords as larvae but not post-metamorphosis. 49 Unlike the axolotl, each main tissue group in the regenerated tail-notochord, spinal cord, and myofibersarises from its respective remaining tissue in a lineage-restricted fashion (again, given current limitations on genetic tools). 50,51 Regeneration-organizing cells are present and essential during tail development and regeneration. 52 These cells express multiple regeneration-supportive genes (Wnt5a, Fgf10, Fgf20, and Msx) and simultaneously upregulate ligand expression for common proregenerative signaling pathways. 52 Although similar cell types have yet to be discovered in other regeneration models, regenerationorganizing cells offer a fresh perspective to current known regenerative mechanisms.
Recent studies have revealed the capability of neonatal mice to heal, scar-free, after spinal cord injuries. This discovery dispels the long-standing theory that mammals are incapable of such regeneration. 53 Following injury, microglia transiently secrete fibronectin and binding proteins to form bridges across the severed spinal cord (like that in zebrafish) and express peptidase inhibitors to help resolve inflammation. 53 Future studies can help identify key genetic and molecular factors regulating pro-regenerative traits of neonatal, but not adult, microglia.

| Cardiac regeneration in vertebrates
Similar to spinal cord regeneration, adult mammalian hearts have limited regenerative potential compared to certain fish and amphibians.
The general consensus within available models across phylogeny is that cardiac regeneration depends on the dedifferentiation and proliferation of pre-existing cardiomyocytes. [54][55][56][57][58][59] Zebrafish-and other teleost fish including Giant Danio (Devario aequipinnatus) 60 and goldfish (Carassius auratus) 61 but not medaka (Oryzias latipes) 62 -retain robust regenerative capacity throughout life; adult zebrafish can regenerate their heart without scarring within 2 months post-injury after 20% ventricular amputation. 63 Although zebrafish reuse many developmental signaling pathways seen in other models of regeneration, Jak/Stat, BMP, and Notch pathways are regeneration-specific and not essential for cardiac development. 64 All three signaling pathways regulate cardiomyocyte proliferation after injury; bmp2b overexpression, in particular, is sufficient to enhance cardiomyocyte dedifferentiation and proliferation. 65 Mexican tetra (Astyanax mexicanus) are teleost fish whose surface populations can regenerate the heart. Groups of Mexican tetra diverged into caves and evolved independently 1.5 million years ago, gaining traits not seen in surface fish and losing traits such as their eyes, pigment, and, at least in the Pach on cavefish population, cardiac tissue regeneration. 66,67 Intra-species breeding and genetic analyses between these distinct populations provide genomic explanations for differences in cardiac regenerative capacity. Lrrc10, a highly conserved gene unique to the heart, is differentially expressed and positively regulates cardiac regeneration in surface fish. Pach on cavefish, which exhibit low endogenous levels of lrrc10, and mutant zebrafish with lrrc10 knocked out, both demonstrate impaired cardiac regeneration. 67 Initial studies on adult newt (Notophthalmus viridescens) cardiac regeneration report incomplete regenerative capacity 30 days postinjury. 68,69 Newer studies suggest that adult newts can fully regenerate their hearts 60 days post-injury, with damaged tissue completely restored structurally and functionally. 57,58 Molecular pathways governing adult newt cardiac regeneration are not well established due to difficulties in sequencing their complex genome. Transcriptional approaches, however, show strong gene expression changes associated with the extracellular matrix post-injury, including matrix metalloproteinases, collagen, keratin, and tenascin-C. [70][71][72] The extracellular matrix may be important in signaling to the regenerating myocardium. For example, tenascin-C can promote cell cycle re-entry of newt cardiomyocytes in vitro. 73 The adult mammalian heart is mainly thought to be a post-mitotic organ incapable of regeneration. In contrast, neonatal mice can fully regenerate their hearts without scarring within the first week after birth. 74 Significant molecular pathways that mediate cardiac regeneration in neonatal mice include Hippo, neuregulin, FGF, and thyroid hormone signaling, each altering cardiomyocyte cell cycle activity. 72,75,76 Positive cell cycle regulators can promote dedifferentiation and proliferation of post-mitotic cardiomyocytes, with the combination of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1 (CCNB), and cyclin D1 (CCND) inducing post-mitotic cell proliferation. 77 Deletion of Meis1-normally required for transcriptional activation of CDK inhibitors-is also sufficient to extend the perinatal cardiomyocyte proliferative window. 78

| Liver Regeneration in Vertebrates
Although more complex vertebrates exhibit limited regenerative potential overall, most, if not all, are capable of liver regeneration. 79 Upon partial hepatectomy, the liver undergoes both hyperplasia and hypertrophy to regenerate and restore homeostasis. 80,81 Once signaling pathways targeting hepatocyte growth factor are activated, hepatocytes enter the cell cycle and restore the liver to its normal size. 82 Rats can regenerate the liver even after 70% partial hepatectomy, while zebrafish are able to after 30% partial hepatectomy. 80

| SIMILARITIES IN REGENERATION ACROSS VERTEBRATES
Despite divergent tissue regeneration capacities observed across the animal kingdom, commonalities exist among appendage, heart, and spinal cord regeneration in fish, amphibians, and mammals. Here, we first highlight how regeneration is generally dependent on nerves, and proceed to discuss conserved genetic, epigenetic, and posttranscriptional machinery governing regenerative potential.

| Nerve dependence
Various organ regeneration in diverse species is nerve-dependent. 85 Marcus Singer's neurotrophic hypothesis of regeneration suggests that nerves influence regeneration by producing factors promoting progenitor cell proliferation and differentiation. 86 These factors are still not clearly characterized today but suggest an evolutionarily conserved role and function of nerves in regeneration across vertebrates.
Seminal work by Tweedy John Todd demonstrates salamander limb regeneration's nerve-dependent nature. 87 Limb and fin regeneration occur through blastema formation adjacent to the wound epithelium. If the nerve is severed at the base of the limb prior to or shortly after amputation, the wound epithelium still forms but the apical epithelial cap does not, and limb regeneration fails due to loss of blastema cell proliferation. 85,88 Proliferation is mediated by the apical epithelial cap dependence on nerves and its interaction with the blastema. In the newt, regenerating axons in the apical epithelial cap synthesize and secrete anterior gradient proteins, which then bind to its receptor Prod1 in the blastema to promote cell cycling. 86 Local expression of the anterior gradient protein after electroporation into a denervated newt limb blastema is sufficient to induce distal structure regeneration. Moreover, in vitro studies show mesenchymal blastema cells can re-enter S phase when exposed to recombinant anterior gradient protein. 89 There are around 100 differentially expressed genes between innervated and denervated Mexican axolotl limbs, half of which are annotated to cell cycle and mitosis-related ontologies. 90 Genes encoding cell cycle regulators such as ccna2 (a positive regulator of cardiac regeneration in mice and pigs 91,92 ) and those associated with cell proliferation and differentiation are enriched and only affected during the formation of the blastema following denervation. 90 F I G U R E 2 Table of essential genetic, epigenetic, and post-transcriptional regulators of regeneration categorized by heart, appendage, or spinal cord regeneration. Arrows indicate whether overexpression or inhibition promotes regeneration In zebrafish fin regeneration, denervation affects mesenchymal cell proliferation, apical epithelial cap formation and signaling, and blastema formation and outgrowth. Genes essential for blastema formation, including msxb, msxc, fgfr1, and fgf20a, are significantly altered in denervated fins, suggesting the role of nerves in FGF signal regulation. 88 In addition, lef1 and wnt5b expression levels are significantly altered in denervated fins, suggesting the nerves' importance in regulating Wnt signaling during regeneration as well. 88 Although less understood mechanistically, mouse digit tip regeneration is also dependent on innervation. Following amputation, Schwann cell-derived precursor cells from intact peripheral nerves secrete pro-proliferative factors that are necessary for digit tip regeneration. These mitogenic factors include oncostatin M and platelet derived growth factor AA, which are sufficient to rescue digit tip regeneration following denervation. 93 Despite the general consensus that appendage regeneration is dependent on nerves, antler regeneration is in fact nerve independent. Denervation of antlerogenic regions does not affect the pedicles' or first antlers' formation and growth, and denervation of pedicles is not essential for antler regeneration. [94][95][96] In both cases, however, denervation affects antler size and shape, meaning nerves may help determine antler growth specifically. 94,95 This may be due to the stem cell-based, rather than blastema-based, nature of antler regeneration. Overall, current literature suggests that nerves are crucial for blastema formation in appendage regeneration.
The heart is extensively innervated. Multiple studies show that nerve function is essential for complete cardiac regeneration in adult zebrafish and neonatal mice. 97 Recently, more regeneration-associated enhancers have been discovered. [101][102][103] Transcriptome analysis in the regenerating zebrafish fin and heart reveal leptin b (lepb) transcription is significantly upregulated during both forms of regeneration. A sequence upstream of lepb, described as lepb-linked regeneration enhancer (LEN), directs regeneration-specific gene expression without developmental activity.
This enhancer sequence is also separable into regulatory elements with distinct tissue preferences-heart or fin-which are activated by the respective injuries. 103 An evolutionary emergence or loss of regeneration enhancers through natural selection may explain the wide variance in regenerative capacity across phylogeny. Limited DNA sequence conservation of zebrafish LEN is found upstream of the leptin gene in mice. Despite this, zebrafish LEN is able to direct injury-dependent gene expression following cardiac injury and digit tip amputation. 103,104 This suggests that although the regeneration-specific enhancer was lost in mammals during evolution, regulatory mechanisms to activate zebrafish LEN still exist and can be reactivated in the context of an injury. There thus exists the possibility that LEN acts more like an injury-responsive enhancer in mammals.
An outstanding question is whether an enhancer element that is activated following an injury is an injury-responsive enhancer or a regeneration-specific enhancer. Moreover, is the regenerationspecific enhancer function important for tissue regeneration? A recent comprehensive study addresses these questions by comparing zebrafish and killifish fin and heart regeneration. 105 A set of evolutionarily conserved enhancer regulatory sequences is activated in both species and during both processes. Deletion of a conserved inhibin beta A (inhba) enhancer element in killifish delays fin regeneration and abrogates heart regeneration. While the predicted zebrafish inhba enhancer recapitulates killifish inhba enhancer activity and can functionally replace it for effective fin and heart regeneration, the predicted human inhba enhancer neither shows the same spatiotemporal activity nor rescues defective fin regeneration in killifish. 105 Thus, these findings present an example in which both of the following occur simultaneously: enhancer element conservation leading to regenerative competency in simpler vertebrates and enhancer sequence variation resulting in regenerative failure in more complex vertebrates.

| Epigenetic modifications
Regardless of the cellular mode of regeneration-self-renewing stem cells, cell dedifferentiation, or proliferation of pre-existing cellsextensive epigenetic reprogramming occurs to generate an appropriate regenerative response. 106 Gene expression is regulated through chromatin structure modifications, cis-regulatory DNA elements, and transcriptional machinery. Understanding evolutionarily conserved gene regulatory mechanisms may, in the future, allow researchers to reactivate dormant regeneration-associated genes in mammals.
Regenerative processes often reuse genes and pathways involved in development. To ensure that these genes are only activated in a particular spatiotemporal context, epigenetic modifications are required to alter gene accessibility. To illustrate, caudal fin amputation in zebrafish leads to the reactivation of developmental regulators such as Shh and Msx. 107,108 Histone demethylases can activate these developmental regulatory genes by removing H3K27me3 marks. 109 Similarly, Shh enhancer methylation status dictates limb regenerative capacity in the Xenopus; here, hypomethylation contributes positively to regeneration in tadpoles, but negatively in adults. 110 Epigenetic modifications also regulate spinal cord regeneration.
Histone deacetylases are necessary for both axolotl and Xenopus tail regeneration. 111,112 Histone deacetylase inhibition in the Xenopus tail leads to aberrant expression of notch1 and bmp2 and regenerative failure. 111 Likewise, multiple Notch and BMP pathway targets are also dysregulated when a histone deacetylase inhibitor is added to the axolotl tail regeneration model. 112 Igf2bp3, a regulator of IGF2 signaling, is highly expressed in the regenerative neonatal mouse heart and significantly downregulated in the nonregenerating adult heart. 113 This positive regulator of cardiomyocyte proliferation and cardiac regeneration is initially modified with H3K27ac activators but is then replaced with H3K27me3 silencers during postnatal development. 113 In zebrafish, H3K27me3 deposition is necessary for silencing structural genes in the heart to complete regeneration. 114 These findings suggest the critical role epigenetic modifications play in regenerative processes; modifications may both stimulate and stymie regeneration. This miRNA program is present but dormant in mammals. The inability to endogenously downregulate these miRNAs following an injury correlates with low mammalian cardiac regenerative capacity. Guided manipulation with anti-miRs can lead to significantly reduced fibrotic scarring and enhanced cardiomyocyte dedifferentiation and proliferation in mice. 128

| POTENTIAL EXPLANATIONS FOR DIFFERENCES IN REGENERATIVE CAPACITIES AMONG VERTEBRATES
There are several potential explanations for the widely varied regenerative capacities observed across phylogeny and among vertebrates.
Some of these theories include the evolutionary gain or loss of regeneration-specific genes and molecular machinery, oxygenation and increase in oxidative damage, and the development of a more advanced immune system. Here, we will focus on the last two theories.

| Acquisition of endothermy
Simpler vertebrates, such as fish and amphibians, are generally ectotherms, while more complex vertebrates, such as mammals and birds, are endotherms. Thyroid hormones are key regulators of energy metabolism and thermogenesis and also drive the evolutionary ectotherm-to-endotherm transition. 129,130 Regarding ontogenic development, a sharp postnatal increase in serum thyroid hormone levels coincides with the loss of cardiac regenerative capacity in mice. 74,131 Neonatal mice and other ectotherms-including zebrafish, axolotls and newts-have higher diploid cardiomyocyte content (indicative of higher cardiac regenerative potential) than their adult or endothermic counterparts. 131 This trend also inversely correlates with standard metabolic rate, serum thyroid hormone levels, and body temperature.
In other words, ectothermic animals demonstrate higher cardiac regenerative potential. 131 Genetic inhibition of functional thyroid hormone receptor alpha in the heart directly shows how thyroid hormones affect mammalian cardiac regenerative potential. In this model, postnatal and adult mice demonstrate enhanced cardiomyocyte proliferation, a higher percentage of diploid cardiomyocytes, and regeneration after injury. 131 In contrast to this, when exposed to exogenous thyroid hormones, zebrafish, which typically demonstrate robust regeneration, experience pronounced scarring, a 45% reduction in cardiomyocyte proliferation, and increased polyploidization (more reminiscent of the nonregenerative mammalian heart). These results recapitulate an evolutionarily conserved function of thyroid hormone in regulating cardiac regeneration and suggest that the decline in cardiac regenerative potential may have been a trade-off for endothermy acquisition.
Xenopus laevis (African clawed frog) serves as an excellent model for exploring the relationship between thyroid hormone and ectotherm regeneration. In this species, a transient surge in thyroid hormone drives the complete metamorphosis from a highly regenerative larva to a minimally regenerative adult. 132,133 Xenopus tadpoles can regenerate their hindlimbs and tail but lose this capability as metamorphosis proceeds. 134,135 In respect to spinal cord axonal regeneration, accelerating metamorphosis through pharmacological thyroid hormone treatment leads to axonal growth inhibition and paralysis. 136 Xenopus laevis tadpoles can also regenerate the heart but again, not after thyroid hormone-regulated metamorphosis. 137

| Development of an advanced immune response
Similar to changes in thyroid hormone levels across phylogeny and ontogeny, the development of a more complex and advanced immune system parallels the regenerative decline observed across vertebrate species and within an animal's development. 145,146 Xenopus have simpler immune systems during highly regenerative larval stages but develop a more advanced immune system akin to that of mammals after metamorphosis. 147 As adults, Xenopus are protected from viruses that kill axolotls (Ambystoma mexicanum). In turn, Xenopus lose regenerative capacity that even sexually mature but morphologically larval axolotls retain. 148,149 This may be in part due to the increase in proinflammatory immune cells and cytokines that accompany an adaptive immune response. Although unclear mechanistically, this different immune response is observed during the refractory period when Xenopus larval tails fail to regenerate. At this time, there is a chronic immune response accompanied by a delay in local inflammation resolution and T cell maturation. 150 Immunosuppressants can restore regenerative capacity during this refractory period, implying a correlation between a subdued or underdeveloped immune system and regeneration. More detailed studies are needed to show this relationship is causal.
That being said, an innate immune response is necessary for blastema formation in zebrafish fin and urodele limb regeneration. 151 Successful axolotl limb regeneration requires proper inflammation modulation and macrophage recruitment. 152 Ablating these macrophages reduces MMP9 and MMP3 activation and ultimately results in scarring. 153,154 Besides salamander limb regeneration, macrophages are also necessary for zebrafish fin 155 and spinal cord 156 regeneration as well as neonatal mouse digit tip 157 and cardiac 158 regeneration.
Within mammalian regeneration, neonatal mice lose their high regenerative capacity throughout development with concomitant immune system maturation. 146,159 Although pro-regenerative macrophages with properties similar to those present in zebrafish and urodeles are found in neonatal mice hearts, they are progressively replaced with bone marrow-derived macrophages which hinder regeneration. 160,161 As such, immunosuppression in neonatal mice inhibits cardiac regeneration, with macrophage-secreted IL-6 essential to initiate cardiomyocyte proliferation. 162 To further illustrate, IL-10 is another anti-inflammatory cytokine which regulates the neonatal inflammatory response. After dermal injury, IL-10 deletion in neonates results in scar tissue formation, and overexpression in postnatal mice allows for scarless wound healing. [163][164][165] Besides skin wounds, IL-10 can also improve cardiac function after myocardial infarction. [166][167][168] Although present in adult mice, IL-10 may not be upregulated enough following an injury, yielding a more pro-inflammatory response that accelerates wound healing via fibrosis instead.
In addition to innate immunity, jawed fish possess adaptive immunity, albeit less evolved and diversified than mammals. [169][170][171] Having a conserved adaptive immune system coupled with genetic tractability, zebrafish are an increasingly popular model to uncover ancestral pro-regenerative immune signals and pathways that may have been lost or modified in mammals. In established models of spinal cord, heart, and retina regeneration, zebrafish regulatory T (T reg ) cells modulate inflammation and promote precursor cell proliferation by secreting organ-specific regenerative factors. 172 Mature mouse T reg cells can also decrease inflammation and promote cell differentiation and proliferation but only in regeneration-competent tissues. [173][174][175] The critical role of zebrafish T reg cells in tissues normally regeneration-incompetent in mammals has alluded to possible evolutionary pressures that limited mammalian T reg cell pro-regenerative capacity and/or altered the microenvironments in nonregenerative tissues so that they no longer stimulate T reg cell pro-regenerative activity.
Experiments in T cell-deficient neonatal mice show that the latter hypothesis is more likely. T reg cells are necessary for neonatal cardiac regeneration. 176 Not only are T reg cells from neonates proregenerative, those from adult mice can also induce cardiomyocyte proliferation and regeneration in nonregenerative T cell-deficient neonatal mice. 176

| CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Are regenerative traits gained or lost throughout evolution? Are these traits positively, negatively, or neutrally selected by natural selection?
Lastly, what is the major driving force that results in divergent tissue regenerative capacities across phylogeny and ontogeny? Among many theories that attempt to address these questions, ones suggesting regeneration is an ancestral trait have garnered increasing support with recent scientific discoveries. 6 Seeing how regenerative traits are conserved throughout phylogeny, one can argue that regeneration is positively selected for if this trait correlates with increased survival. In some species, the ability to recover from physical injuries via regeneration can help individuals successfully reach maturity and pass down genes to next generations. Evidently, most simpler vertebrates retain the ability to regenerate various whole organs and body parts throughout their lifetime. At the same time, the only whole complex organ most adult mammals can regenerate is the liver. Yet, natural selection may favor traits that improve fitness to a greater extent in some species. Thus, it is possible that other traits may be selected over regenerative traits and that regeneration is lost neutrally throughout evolution.
Across evolution, regenerative potential declines with increasing phylogenetic complexity. However, while invertebrates are considered more primitive than vertebrates, insects-arguably more evolutionarily complex than flatworms but still invertebrates-do not demonstrate the same robust regeneration. There are thus parallel trends in regeneration even within invertebrate and vertebrate species, respectively.
Furthermore, this evolutionary trend also manifests as developmental changes in some vertebrates: losing regenerative traits and simultaneously gaining other evolutionary traits like endothermy and more advanced immune systems with maturation. The inverse correlation between heart regenerative potential and endothermic adequacy is an apt example. 131 In an evolutionary perspective, the ability to regulate body temperature likely permitted early mammals to occupy new ecological niches, allowing them to thrive in colder climates and also nocturnally. 182 Cardiac regeneration may not have been negatively selected for in this case, but rather neutrally lost as endothermy posed greater advantages in evolution. Still, why cardiac regeneration potential had to wane alongside the acquisition of endothermy across phylogeny, and why it continues to wane throughout mammalian ontogeny, requires further study.