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. Author manuscript; available in PMC: 2019 Jan 15.
Published in final edited form as: Dev Biol. 2017 Oct 31;433(2):461–472. doi: 10.1016/j.ydbio.2017.07.010

Systemic cell cycle activation is induced following complex tissue injury in axolotl

Kimberly Johnson 1, Joel Bateman 1, Tia DiTommaso 1, Alan Y Wong 1, Jessica L Whited 1
PMCID: PMC5750138  NIHMSID: NIHMS895608  PMID: 29111100

Abstract

Activation of progenitor cells is crucial to promote tissue repair following injury in adult animals. In the context of successful limb regeneration following amputation, progenitor cells residing within the stump must re-enter the cell cycle to promote regrowth of the missing limb. We demonstrate that in axolotls, amputation is sufficient to induce cell-cycle activation in both the amputated limb and the intact, uninjured contralateral limb. Activated cells were found throughout all major tissue populations of the intact contralateral limb, with internal cellular populations (bone and soft tissue) the most affected. Further, activated cells were additionally found within the heart, liver, and spinal cord, suggesting that amputation induces a common global activation signal throughout the body. Among two other injury models, limb crush and skin excisional wound, only limb crush injuries were capable of inducing cellular responses in contralateral uninjured limbs but did not achieve activation levels seen following limb loss. We found this systemic activation response to injury is independent of formation of a wound epidermis over the amputation plane, suggesting that injury-induced signals alone can promote cellular activation. In mammals, mTOR signaling has been shown to promote activation of quiescent cells following injury, and we confirmed a subset of activated contralateral cells is positive for mTOR signaling within axolotl limbs. These findings suggest that conservation of an early systemic response to injury exists between mammals and axolotls, and propose that a distinguishing feature in species capable of full regeneration is converting this initial activation into sustained and productive growth at the site of regeneration.

Keywords: Limb regeneration, axolotl, systemic, cell cycle, mTOR

INTRODUCTION

Differences in natural regenerative responses across the animal kingdom are profound, and underlying causes for these differences are poorly understood. Across species, an injury-inducing event prompts a systemic, global response across a myriad of tissues (reviewed in [1–3]). In mammals, an injury stimulus initially promotes systemic activation of immune cells and increased levels of cytokines and growth factors (reviewed in [4–6]), followed by proliferation and remodeling to close the wound (reviewed in [7]). Mammalian wounding responses can result in formation of scars, nonfunctional fibrotic tissue physically distinct from surrounding structures (reviewed in [8, 9]). In contrast, invertebrates such as hydra and planarians, and lower vertebrates, such as salamanders and fish, are capable of perfectly regenerating complex tissues following amputation (reviewed in [10–12]). The wound response in planarians, for example, results in the systemic activation of resident tissue stem cells that proliferate locally at the injury surface to replace lost tissues with functional, perfectly integrated counterparts [13, 14]. Whether the same pathways are active across species and how injury-induced signals are differentially incorporated between regeneration-competent and incompetent species is less clear.

As opposed to wounding models such as epidermal injury, amputation of an appendage results in the total loss of a complex structure composed of multiple tissue types. In highly regenerative tetrapods, such as axolotl salamanders, the entire limb can be regenerated regardless of amputation location (reviewed in [15]). This process is facilitated by two key structures: the wound epidermis and the blastema. The wound epidermis is a specialized skin that forms atop the cut stump soon after amputation via the migration of existing epidermal cells. The blastema is a bud-like structure that develops at the tip of the stump, beneath the wound epidermis, that houses the activated progenitor cells that will give rise to the new cells of the regenerate limb. These progenitor cells are activated either by stimulating cell-cycle re-entry of resident adult stem cells, cellular dedifferentiation, or a combination of both mechanisms [16, 17].

A long-standing goal in the field has been to identify molecular mechanisms of progenitor cell activation following amputation. A common assumption is that the wound epidermis is predominantly responsible for secreting the factors that are required to promote progenitor cell activation in the internal stump tissues. This idea largely stems from the observation that when wound epidermis formation is experimentally blocked, limbs fail to grow blastemas, and they do not regenerate [18–20]. Past studies using X-irradiation and lead shielding have demonstrated that the only cells required to divide during the process of limb regeneration are those left on the stump, but close to the amputation plane [21]. Together, these experiments have led to a model in which progenitor cell activation occurs locally, where the limb was lost, leading to cell proliferation and tissue growth specifically where the injury occurred.

Mammals such as mice and humans are very restricted in their ability to regenerate portions of their limbs following amputation. The distal digit tip can be regenerated in mice and in young children, but more proximal amputations past the first joint fail to regenerate [22, 23]. The extent to which mammals might employ similar post-amputation progenitor cell activation programs as other regeneration-competent species is largely unexplored. The possibility that some components of the early regeneration program are intact in mammals, but that later essential features are compromised, exists. More information is required about the very early responses to injury in highly regenerative species, such as axolotls, in order to compare these events to known post-injury or unidentified post-amputation responses in mammals. These comparisons will be critical for further understanding where in the trajectory of successful regeneration the mammalian response goes awry.

We sought to examine early cell activation events that occur globally post-amputation in axolotls, and we found that these cellular activations are not restricted to the site of amputation. Cell-cycle re-entry was observed on intact limbs contralateral to limbs that were amputated on the same animal. Activated cells were not restricted to any particular tissue lineage, and activated cells were additionally identified in several organs throughout the body. We also discovered that early cell cycle re-entry distant to the site of amputation does not rely upon the activity of the wound epidermis formed atop the regenerating limb. These results point to an early, systemic cellular activation in response to the amputation injury that is independent of local structures later required for successful regeneration of the amputated limb. Intriguingly, a fraction of distant, activated cells have engaged the mTOR signaling pathway, recently shown to be required to promote distant quiescent stem cell activation in mice following injury [24]. We propose that global cell cycle activation post-injury is an evolutionary conserved response amongst species, and we hypothesize that axolotls are capable of converting this early systemic response into productive, localized growth at the amputation plane to reproduce a functional limb.

RESULTS

Cells distant to the site of amputation are cued to re-enter the cell cycle

To identify cells that re-enter the cell cycle in response to limb amputation, we performed an EdU time course experiment to label cells actively synthesizing DNA throughout the regenerative process (Figure 1). The baseline level of EdU-positive nuclei in limbs in which the axolotl has not undergone an amputation anywhere at all (intact, uninjured) is <0.5% (Figure 1A,M; intact). Total cell nuclei were visualized by DAPI stain. We compared the fraction of EdU+ cells in unamputated limbs contralateral to amputated limbs from the same animal at various time points up to 28 days post-amputation (dpa), until digit reformation has begun. At each time point, animals received a single pulse of EdU at 18 hours before tissue harvest (Figure 1A’). In all cases, both the right forelimb and the right hindlimb were amputated, while the left limbs were both left unmanipulated (Figure 1A’). Left limbs therefore allowed for contralateral, distant effects to be examined.

Figure 1. Cell cycle re-entry occurs within limbs contralateral to regenerating limbs.

Figure 1

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(A) Baseline EdU staining was performed on intact, unamputated limbs. Tissue sections were counterstained with DAPI. (A’) Experimental design for assaying cell cycle re-entry in limbs contralateral to regenerating limbs. (B–G) Cell-cycle re-entry in the internal (non-epidermal) cells of the amputated and regenerating limb measured just proximal to the wound epidermis. (B–F) Representative tissue sections stained with EdU and DAPI. (G) Quantification. (H–M) Cell-cycle re-entry in the internal cells of the contralateral limbs at various time points post-amputation of the limb on the other side. (H–L) EdU and DAPI. (M) Quantification. (N–P) Anti-phospho-Histone-H3 stained tissue sections counterstained with DAPI. (N) Representative tissue section from an intact, unamputated limb. (O) Representative tissue section from a limb contralateral to a regenerating limb at 14 days post-amputation. (P) Quantification of the fraction of DAPI+ cell nuclei which are also positive for phosphorylated Histone H3 in limbs contralateral to a regenerating limb at various time points post-amputation. * denotes p<0.05; ** denotes p<0.01, *** denotes p<0.001, **** denotes p<0.0001, n.s., not significant. Scale bar in A is 100 microns and applies to all.

In the amputated limbs, local cellular activation responses were observed consistent with expectations. Cell cycle re-entry skewed toward epidermis in early time points (3 dpa and 5 dpa) (Figure 1B, G), while later time points (7dpa through 14 dpa) (Figure 1C, D) exhibited more proliferation within the underlying internal tissues of the stump. By 21 and 28 dpa, both epidermis and underlying tissues displayed similar percentages of proliferating cells (Figure E, F). In all cases, we quantified total EdU+ nuclei within a millimeter of the amputation plane and under the epithelium in amputated limbs (Figure 1G) to reflect the population of activated cells that would contribute to the regenerating limb.

Meanwhile, we also harvested and processed the contralateral limbs and performed the same histological analysis. We observed an upregulation of EdU+ cells in contralateral limbs at 3 dpa, and while this effect was largely restricted to the skin, significantly more nuclei in the internal tissues of the contralateral limb were EdU+ compared to intact controls (Figure 1H, p<0.05). This trend increased in contralateral limbs between 5 through 9 dpa, with cell cycle activation within contralateral limbs significantly higher than in limbs of intact, uninjured animals (Figures 1I, M, p<0.001 for all time points). By 14 dpa, the fraction of cells in S-phase began to decline (Figure 1J), and it continued to do so through the end of our sampling at 28 dpa (Figure 1K–L summarized in M). Single limb amputations were also sufficient to induce this effect (data not shown), suggesting the induction of cell cycle entry following amputation on contralateral, uninjured tissues is systemic throughout the axolotl.

To determine if cells that have been provoked to re-enter the cell cycle and synthesize DNA progress further in the cell cycle and undergo mitosis, we stained adjacent tissue sections from our above analysis with anti-phospho-histone H3 (pH3). We found that in intact limbs from animals with no prior amputations anywhere, pH3+ cells are exceedingly rare (Figure 1N). However, in limbs contralateral to regenerating limbs, a detectable fraction of cells are pH3+ as early as 3 dpa, and by 15 dpa, this effect is highly significant (Figure 1O, quantified in P). To rule out that there exists a compensatory cell death mechanism counteracting proliferation in contralateral limbs, we additionally stained for anti-activated caspase-3 and TUNEL to label for both early and late stages of cell death, respectively. With either technique, we did not detect significant cell death within control intact limbs or within limbs contralateral to regenerating limbs (Supplemental Figure 1). Thus, we conclude that an amputation leads to cellular proliferation in the stump of the amputated limb, as expected; however, it also leads to an upregulation in cellular proliferation in the uninjured, contralateral limbs, and that these activated cells undergo at least one round of mitosis in response to the distant amputation injury.

A subset of distantly-activated cells are muscle satellite cells

We sought to determine the types of cells in contralateral limbs that became activated to enter the cell cycle in response to amputation of another limb (Figure 2). We therefore examined the repertoire of responding cell types within limbs contralateral to amputations using EdU and DAPI (Figure 2A–C’). Using cell and tissue morphology and location, we separately quantified activated cells in contralateral epidermis (Figure 2D), skeletal elements (bone, cartilage; Figure 2E), and other internal tissues (dermis, muscle, nerve, perichondrium, joint, tendon; Figure 2F).

Figure 2. A subset of activated cells persists in all major tissue types in contralateral limbs.

Figure 2

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(A–C’) Tissue sections processed for EdU and counterstained with DAPI from limbs contralateral to a regenerating limb at several time points during regeneration. (A’–C’) Magnified view of insets depicted in (A–C). (A–A’) Representative tissue section from a limb contralateral to a regenerating limb at 3 days post-amputation. (B–B’) Representative tissue section from a limb contralateral to a regenerating limb at 9 days post-amputation. (C–C’) Representative tissue section from a limb contralateral to a regenerating limb at 14 days post-amputation. Arrowheads in C’ refer to glands. (D) Quantification of epidermis. (E) Quantification of skeletal elements (bone and cartilage). (F) Quantification of other internal tissues (including muscle, nerve, vasculature, joint, perichondrial, dermis). (G–J) Representative tissue sections processed for EdU and counterstained with Pax7 from limbs contralateral to regenerating limbs at several time points during regeneration. Arrowheads denote double positive nuclei. * denotes p<0.05; ** denotes p<0.01, *** denotes p<0.001. Scale bar is 100 microns and applies to all.

This analysis revealed that different tissues have distinct temporal patterns for cell cycle activation within contralateral limbs in response to amputation. We identified that cells within the contralateral epidermis were significantly activated between two distinct temporal phases: an early stage that correlated with wound epidermis formation and early blastema development (3–7 dpa) and a later stage that correlated with differentiation of the regenerated limb (28 dpa) (Figure 2D). Within the early stage, the fold change of activated cells within epidermis in limbs contralateral to amputated limbs ranged from 1.9- to 2.7-fold over intact limbs, which increased to nearly 3-fold by 28 dpa.

Skeletal elements (bone and cartilage) differed from other internal tissues by displaying a slightly delayed activation pattern within limbs contralateral to regenerating limbs. After amputation, significant activation of skeletal cells was initially identified at 5 dpa, and remained significantly activated between 5 and 28 dpa (Figure 2E). A remarkable fold change was observed in contralateral skeletal elements as compared to intact controls, particularly at 9 dpa (83-fold) and 28 dpa (207-fold) that correlated with peak blastema formation and differentiation of the regenerated limb, respectively, on the amputated side.

Intriguingly, cells comprising soft internal tissues were activated throughout the entire time course (Figure 2F), with a fold change ranging from 4.1- to 64-fold. Activated cells were found throughout a variety of tissue subtypes, including glands of the dermis (Figure 2C’), joint, perichondrium (Figure 2B’), and muscle. To determine if this activation was reflective of resident adult stem cell populations, we further analyzed the status of muscle satellite cells as their presence in adult salamander tissues has been well characterized [17, 25]. We stained tissue sections with anti-Pax7, a well-characterized marker for muscle satellite cells [17, 25] from animals with a contralateral amputation who had been administered EdU. In intact limbs from animals without any amputations, we did not observe any EdU+ cells that were also Pax7+ (Figure 2G). In contrast, in intact limbs from animals with a contralateral regenerating limb, we observed a significant fraction of the EdU+ cells to also be Pax7+ (Figure 2H–J). This fraction culminated at the 9 dpa sample time, with >35% of the EdU+ cells also being Pax7+ (p<0.001). By 14 dpa, the fraction of EdU+ cells in intact, contralateral limbs had waned, but it was still significantly higher than in uninjured axolotls (p<0.01). These data demonstrate that a sizeable fraction of cells induced to re-enter the cell cycle in intact limbs contralateral to regenerating limbs are muscle satellite cells.

We next interrogated other organs to determine if a limb amputation leads to distant cellular activation elsewhere, beyond simply other limbs. We harvested the heart, liver, and spinal cord from axolotls that were either intact (uninjured) or had received a unilateral amputation and had developed a blastema (Figure 3). Within all three organs, we were able to detect a significant increase of EdU incorporation in response to limb amputation (heart, p=0.0057; liver, p=0.0033; spinal cord, p=0.048). Taken together, these data indicate that multiple tissues have the capacity to respond to cues downstream of the amputation by activating resident cells to re-enter the cell cycle.

Figure 3. Cell cycle re-entry is promoted in organs of animals with regenerating limbs.

Figure 3

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(A–F) Representative tissue sections from internal organs harvested from intact, uninjured controls (A,C,E) or from axolotls with a regenerating limb at 14 dpa (B,D,F) stained with EdU and counterstained with DAPI. (A,B) Representative tissue sections from the heart. (C,D) Representative tissue sections from the liver. (E,F) Representative tissue sections from the spinal cord. (G) Quantification. * denotes p<0.05; ** denotes p<0.01, *** denotes p<0.001. Scale bar is 500 microns and applies to all.

Full contralateral response is intrinsic to amputation but not other types of injuries

We next asked if the systemic activation we observed in intact limbs contralateral to amputated limbs reflects a global injury response or whether it is predicated on amputation per se. We performed two types of more limited limb injuries that do not produce a blastema and a new limb: a limb crush injury and a full-thickness excisional skin wound (Figure 4A). As a control for baseline local responses, we assayed cellular activation within intact, uninjured limbs (Figure 4B) and within the wounded limbs themselves (Figure 4C–D). These results are quantified in Figure 4E–F with respect to total EdU+ population and with respect to tissue types.

Figure 4. Nonregenerative injuries vary in activation response in contralateral limbs.

Figure 4

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(A) Schematic of experiment. (B–F) EdU and DAPI stain on representative tissue sections from intact control limbs (B) and wounded limbs (C,D). (E) Quantification of (B–D). (F) Quantification of tissues. (G–J) EdU and DAPI stain on representative tissue sections from limbs contralateral to crushed (G) or limbs with excisional skin wounds (H) at 7 days post injury. (I) Quantification of (B,G,H). (J) Quantification of activated cells in distinct tissues of the limb. * denotes p<0.05; ** denotes p<0.01, *** denotes p<0.001; n.s. = not significant. Scale bar is 100 microns and applies to all.

For both crush injury and skin injury, we assayed contralateral responses via EdU incorporation at 7 days post-injury (dpi), a time when all tissues were significantly activated in limbs contralateral to amputation, and compared to intact, uninjured control limbs. When quantified en masse, there was no apparent activation in limbs contralateral to these two types of injuries as compared to intact, uninjured control limbs (Figure 4G–H, summarized in I). However, quantification of distinct cellular populations (epidermis, skeletal elements, and internal tissues) revealed significant differences with respect to these tissue types (Figure 4J).

Surprisingly, within the contralateral limbs of both injury models, at 7 dpi, cellular activation was significantly decreased in the epidermis as compared to control limbs (p=0.00017 for crush injury and 5.2×10−6 for skin wound) (Figure 4F). In contrast to our amputation results, we did not observe activation within skeletal elements in limbs contralateral to either crush injuries or skin wounds (Figure 4G–H, quantified in 4J).

Intriguingly, internal tissues responded differently between the two injury model systems based on vicinity. Crush wounds induced a systemic response within internal soft tissues as both the injured limbs and uninjured, contralateral limbs exhibited significant increases in cellular activation at 7 dpi (p=6.3×10−5 locally, Figure 4F, black bar; in the injured limb; p=3.5×10−5 in contralateral limbs, Figure 4J, black bar). In contrast, excisional skin wounding only activated cells within the internal soft tissues within the injured limb itself (p=0.00035 locally, Figure 4F, grey bar; in the injured limb; p=0.094 in contralateral limbs, Figure 4J, grey bar). Notably, neither of these injuries recapitulated the either the full repertoire or the extent of cellular activation following amputation at this time point.

Distant cell activation does not require wound epidermis

As amputation alone, and not crush or skin wounding, induced the activation of all three tissue types in the contralateral limbs, we sought to determine if the engagement of distantly-responsive cells requires amputation-specific structures. The wound epidermis that covers a cut stump has been shown to be molecularly distinct even from healing epidermis of skin wounds [16, 26]. Thus, is possible that amputation-specific wound epidermis may produce signals that can become systemic and activate or sustain proliferation of contralateral cells. We therefore asked if preventing amputation-specific wound epidermis from forming would interfere with the contralateral response. We employed a previously-described procedure in which freshly-amputated limb stumps are covered with a full-thickness epidermis flap and sutured in place (Figure 5A) [18], and we subsequently administered EdU and harvested as in our previous experiments. To verify successful suturing, we performed H&E stains on bona fide regenerating limbs at 14 dpa (controls) versus sutured limbs at 14 dpa (Figure 5B, C) and confirmed absence of blastema formation.

Figure 5. Cell cycle re-entry in contralateral limbs is independent of wound epidermis on the regenerating limb.

Figure 5

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(A) Schematic of experiment. (B–F) Response on the amputated limb in the unmanipulated, regenerating context versus the sutured context. (B–C) Hematoxylin and eosin stain on tissue sections from regenerating (B) and sutured (C) limbs at 14 days post-amputation. (D–E) EdU and DAPI stain on tissue sections from regenerating (D) and sutured (E) limbs at 14 days post-amputation. (F) Percentage of DAPI+ cell nuclei that are also EdU+ in regenerating limbs versus sutured limbs at 14 days post-amputation. (G–I) Representative tissue sections of intact control limbs versus limbs contralateral to regenerating or sutured limbs at various time points post-amputation. (J) Quantification of (G–I). * denotes p<0.05; ** denotes p<0.01; n.s. = not significant. Scale bar in (B) is 500 microns and applies to (B–C). Scale bar in (D) is 100 microns and applies to (D–E, G–I).

As expected, we observed a significant diminishment in the fraction of EdU+ cells in amputated limbs with full-thickness epidermal suturing versus bona fide regenerating controls harvested at the same time point (14 dpa, Figure 5D, E, quantified in Figure 5F, p<0.01). The difference in proliferative index was about 6-fold. This data is consistent with previous literature demonstrating the wound epidermis is required to sustain cells in the cell cycle during regeneration locally at the amputation plane.

Within intact contralateral limbs, we found no difference in the activation of internal tissues when the amputated contralateral limb is undergoing bona fide regeneration versus when it is blocked from regenerating by a full-thickness epidermis suture (Figure 5G–I, quantified in J). This data demonstrates that the systemic, cell-activating effect in internal tissues following limb loss elsewhere on the body is independent of the formation of a regeneration-competent wound epidermis at the site of injury.

Distantly-responding cells are engaged in mTOR signaling

Lastly, we sought to uncover potential signaling pathways that may be mediating cell cycle activation in response to amputation. Recently, a study using a mouse muscle-injury model uncovered a systemic response to distant injury in which quiescent resident stem cells are activated to enter a “GAlert” phase that was mediated by mTOR signaling [24]. Active mTOR signaling has additionally been shown to be required during tissue regeneration by regulating stem cell activation and blastema outgrowth in planarian and zebrafish regeneration models [27–30]. We therefore hypothesized that axolotls might be employing the same mechanism to promote cell activation following amputation, and assayed for mTOR activity in regenerating limbs and their corresponding contralateral intact limbs using an antibody that detects the phosphorylation of the S6 subunit of the ribosome (pS6, [30]) downstream of the mTOR complex (Figure 6). Within intact, uninjured limbs, the fraction of activated cells that colabeled with pS6 staining was 40% (Figure 6A–C’, quantified in V, white bar), suggesting that activated mTOR signaling is present at homeostasis. We found that activated mTOR signaling through pS6 staining was enriched within stump tissues at 3 dpa (Figure 6D–F’), with expression remaining high within cells closest to the wound epidermis as the blastema developed through 14 dpa (Figure 6J–L’, P–R’, higher magnification in F’, L’, R’). We additionally observed that between 92–99% of the activated cells present at the amputation plane and within the blastema were positive for activated mTOR signaling throughout blastema development (Figure 6V, grey bars). This persistence of activated mTOR signaling during blastema development is consistent with previous data in zebrafish [30], suggesting mTOR signaling may play an important role during appendage regeneration even in tetrapods.

Figure 6. mTOR activity is differentially regulated following amputation in injured versus intact tissues.

Figure 6

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(A–T’) Tissue sections processed with DAPI, EdU, and stained with anti-pS6 in intact, uninjured control limbs (A–C’) versus those contralateral to a regenerating limb at various time points post-amputation. (D–I’) Representative tissue section from a regenerating stump at 3 dpa (D–F’) and its contralateral intact limb (G–I’). (J–O’) Representative tissue section from a regenerating limb at 7 dpa (J–L’) and its contralateral intact limb (M–O’). (P–U’) Representative tissue section from a regenerate limb at 9 dpa at early blastema stage (P–R’) and its contralateral intact limb (S–U’). (V) Quantification of total EdU+ population expressing pS6. (W) Quantification of total DAPI+ population coexpressing EdU and pS6. * denotes p<0.05; ** denotes p<0.01, *** denotes p<0.001, **** denoted p<0.0001. Scale bar is 100 microns and applies to all.

Within the contralateral limbs of regenerating axolotls, we found that a fraction of activated cells colabeled with active mTOR staining, ranging from 28–46% of the activated population throughout the time course (Figure 6G–I, M–O, S–U, higher magnification in I’,O’,U’, quantified in V, black bars). When compared to intact, uninjured limbs, there was no change in the total number of EdU+ cells that colabeled with pS6 in limbs contralateral to a 3 dpa regenerate (p=0.199) (Figure 6W, black bars). However, we detected this population of double labeled cells significantly increased over time in limbs contralateral to regenerating limbs at 7 dpa through 14 dpa as compared to intact, uninjured controls (p<0.04 for all time points; Figure 6W, black bars). Although the double positive population had increased about 6-fold during this window, the total population of activated cells co-expressing pS6 represented no more than half of the total population of activated cells within contralateral limbs at any given time point. Taken together, this data suggests that although activated mTOR signaling is present in the contralateral intact limb following amputation, notably higher mTOR activity persists in amputated limbs that will proceed through regeneration.

DISCUSSION

Identifying the full repertoire of responsive cells following amputation, and how amputation injuries uniquely lead to the growth of a new limb, is important for understanding how regeneration happens. Here, we have unexpectedly uncovered an apparent systemic cellular activation response that occurs distant to the site of injury. Importantly, the extent of the activation response increases with the level of tissue injury inflicted, with amputation inducing the highest levels of cell cycle activation globally and among the largest diversity of tissues. Our data supports a model of limb regeneration for which the process of activation can be decoupled from the epimorphic regeneration program, as distant cellular activation was achieved in the absence of wound epidermis and blastema formation. While cells distant from the amputation site are cued to re-enter the cell cycle, only those residing locally within the stump of the amputated limb ultimately participate in the regrowth of the new limb. This model shares similarity to planarian regeneration as neoblasts throughout the body are initially activated, but eventually this activation becomes refined to the site of injury and blastema growth [14]. This model is also aligned with an earlier study in newt whereby blocking wound epidermis formation by suturing did not prevent initial cell cycle activation following amputation, but it did inhibit further proliferation and it blocked blastema formation later [18]. Thus, the process of limb regeneration may involve a sort of relay system whereby following this initial amputation-linked activation, the task of supporting continued cell proliferation is handed off to the wound epidermis and thus becomes a more localized event at that time.

Intriguingly, a limb amputation does not cue only distant cells in other limbs to activate. It is also capable of cuing distant cells in non-limb appendages to re-enter the cell cycle, such as those in the heart, liver, and spinal cord. This data implies that a common molecular signaling pathway may regulate early activation events across regenerative areas of the body, and it suggests testable hypotheses about how this signal might travel. As extensive research in the wound healing field has demonstrated a wide berth of signaling cascades are induced upon injury (reviewed in [31]), two obvious mechanisms to explore are the possible influences of circulating factors and nerves on systemic cell cycle activation in salamanders. Future work will be required to uncover the identity of systemic, amputation-induced signal(s) and how these signals are integrated to activate distant cells. Additionally, future studies uncovering the full repertoire of responsive cell types that are capable of responding to these signals will provide valuable insight into functionally distinguishing responsive cell types from non-responsive cell types during regeneration.

Due to the conserved responses in systemic activation within internal tissues of axolotl and mammalian limbs following injury, we sought to characterize the role of conserved signaling pathways that may be mediating this response. In response to muscle injury within mouse limbs, resident muscle satellite cells are cued to exit a quiescent G0 state and enter a primed Galert state; mTOR signaling is both necessary and sufficient to execute activation in a sizeable fraction of muscle satellite cells [24]. In zebrafish, growth of the blastema necessary for fin regeneration is dependent on mTOR signaling [30]. Furthermore, in planarians, mTOR signaling is essential for regeneration [27–29]. Though we observed an induction of mTOR signaling in axolotl in both regenerating limbs and their contralateral limbs, if and how mTOR orchestrates limb regeneration remains unknown. Importantly, we observed an increase in activated cells engaged in mTOR signaling post-injury in contralateral limbs in similar fashion as mice; although the ratio of double labeled cells did not reach the same threshold observed in mammalian injury [24], it is encouraging that conserved signaling pathways were observed between two species of differing regenerative potential. Thus, we speculate that mammals are indeed engaged in the same early response as axolotls, but that they cannot convert this early systemic response into the sustained, localized proliferative phase because they either lack the formation of functional regenerative structures or require further signaling programs to refine cellular responses at the site of amputation. One possibility is that the transition from G0 to Galert that occurs contralateral to injuries in mice represents a vestigial response that only in modern, fully-regenerative species is still converted to sustained, local tissue outgrowth at the injury site.

Future investigations into the roles of other known mammalian injury mediators will also further our understanding of progenitor activation mechanisms between species. With clues from mammals (reviewed in [32]), important candidates to consider include: hepatocyte growth factor [33–38], TNF-α [33, 39–41] IL-1β [40], IL-6 [40–42], HMGB1 [43, 44], and IGF [45–47], among others. Unbiased approaches toward identifying systemic cellular activation cues in highly-regenerative species should also be pursued.

The data yielded by these experiments also highlight the importance of using the right controls for probing the regenerative process. Though it is popular to perform amputations on one side of the animal and use the contralateral, unamputated limb as an “intact” control to conserve animals used in experiments, we have systematically shown that this is not an appropriate control. As compared to a truly intact animal, limbs from axolotls with contralateral amputations have significantly upregulated cell-cycle activation, proliferation, and intracellular signaling. Further, other injury models, such as crushing or excisional skin wounds, also invoke their own dynamic patterns of cellular activation within tissues contralateral to the manipulated limb. Though differing in severity, these data not only identify novel systemic reactions to injuries in axolotl, but should also serve as a cautionary tale for choosing contralateral limbs as inert controls.

A logical next step will be to determine if this systemic response is required for limb regeneration. One hypothesis suggested from the data is that perhaps the amputation injury signals the systemic activation of responsive cells, and these same cells continue on to create the blastema in the amputated limb. Another important question relates to how the cellular activation signal is quenched in the contralateral limbs. Notably, there is no evidence that contralateral limbs increase in overall size or in mass of particular tissues following an amputation elsewhere in the animal. We have shown that contralateral activated cells undergo at least one mitotic episode, as they become phospho-Histone-H3 positive. Further, we have not yet observed an increase in cell death within the limbs of animals with contralateral amputations, suggesting that the ultimate fate of these cells is yet to be determined. Following identification of molecular markers that label the population of cells capable of responding to systemic amputation signals, lineage tracing can be utilized to further probe the cell fates and requirements of this population to homeostasis and regeneration.

METHODS

Animal experimentation

All animal experiments were performed in accordance with Harvard Medical School’s Institutional Animal Care and Use Committee and Brigham & Women’s Hospital’s Institutional Animal Care and Use Committee regulations and in accordance with Animal Experimentation Protocol #04160. Axolotl limbs were amputated at the mid-stylopod and the bone was trimmed back from the amputation site. Crush injury was performed by grasping the limb at mid-stylopod with forceps and forcibly pinching for 30 seconds before release, as described previously [48, 49]. Skin wounds were performed by removing a 2mm by 2mm flap of full thickness skin from the limb at the mid-stylopod level. For all survival surgeries, animals were anesthetized in 0.1% tricaine and allowed to recover overnight in 0.5% sulfamerazine.

Limb suturing

Suturing was performed as described in [18] with few modifications. Axolotls were anesthetized and limbs were amputated at the mid zeugopod. Full thickness skin was recessed down from the underlying tissue. A secondary amputation was performed to remove the soft underlying tissue to the mid-stylopod. The skin was pulled up, the two internal halves of dermis were placed in contact with each other, and the entire flap of skin was pulled over the amputated stump. Skin was sutured down over stump tissues using Vicryl suture 8-0 synthetic filament (Ethicon, catalog number J405G).

EdU administration

Stock solutions of 5-ethynyl-2’-deoxyuridine (EdU) was dissolved in dimethyl sulfoxide were prepared by manufacturer instruction (Thermo Fisher). Axolotls were anesthetized and 400µM EdU in PBS at a volume of 20µL/g was delivered via intraperitoneal injection 18 hours prior to harvesting tissue. Crush and skin wounded animals were treated with EdU at 6 days post injury (dpi), and limbs were harvested at 7 dpi.

Specimen harvesting and processing

Intact and regenerating limbs were amputated proximally and immediately fixed in 4% paraformaldehyde for 1–2 hours at room temp. For internal organs, the heart and portions of the liver (adjacent to the gall bladder) and the spinal cord (base of the tail) was placed immediately into 4% paraformaldehyde. All tissues were rinsed in 1× PBS prior to cryopreservation in sucrose solutions in PBS, and frozen into molds using TissueTek Optimal Cutting Temperature (OCT) compound. Sections were collected at 16µm using a Leica cryostat and stored at −80°C until processed for immunohistochemistry.

Immunohistochemistry

EdU staining was visualized with the Click-iT EdU Alexa Fluor 594 Imaging Kit per manufacturers instructions (Thermo Fisher). Labeling with additional antibodies was performed after EdU labeling. Sections were blocked with 2% BSA in PBS for 30 minutes prior to incubation in primary antibodies overnight at 4°C. Primary antibodies used were mouse anti-Pax7-s (IgG1, 1:5; DSHB), rabbit anti-activated-caspase-3 (1:400; BD Pharmingen), rabbit anti-phospho-histone H3 (Ser10, 1:400; EMD Millipore), and rabbit anti-phospho-S6 ribosomal protein (Ser235/236, 1:200; Cell Signaling). Sections labeled for anti-pS6 were initially post-fixed with 4% paraformaldehyde for 20 minutes, rinsed in PBS, incubated in PBS with 0.5% Triton-X for 20 minutes, rinsed in PBS, and boiled in 0.1M sodium citrate prior to incubation in blocking solution. Slides were incubated in appropriate secondary antibodies conjugated to Alexa Fluor 488 for 1 hour, and subsequently counter stained with DAPI (Roche) for 5 minutes before cover slipping.

Imaging

All imaging was conducted on a Nikon Eclipse Ni microscope with a DS-Ri2 camera. Blastema sections were imaged at peak height with bone in the stump visualized in the section to demarcate the middle of the specimen. Intact and contralateral intact limbs were imaged in sections with the humerus present to act as a landmark for consistency. Because no internal landmarks are visible in the heart, liver, and spinal cord samples, three images were taken of each organ sample at distances of 160–200µm apart and data was subsequently averaged together to provide an average reading of that tissue type.

Quantification

All cell counts were conducted manually using ImageJ software. For EdU+ nuclei counts, averages reported reflect the total number of EdU+ nuclei out of the total DAPI+ population present within that section with the exception of images acquired with antipS6 antibody. For anti-pS6 labeling, pS6+ cells were identified by high levels of cytoplasmic pS6 staining adjacent to the nucleus to distinguish positive cells from background noise [24]. The averages for pS6 reported reflect the total number of pS6+, EdU+ nuclei out of the total EdU+ population present within that section. In blastemas, counts reflect the total number of cells present at the amputation plane that extend into the blastema, and exclude all cell counts from the wound epidermis. In samples lacking blastema development, cell counts were performed within 1mm of the basal layer of the epidermis and excluded cells present in stump tissues. In intact and contralateral limbs, cell counts are reflective of the total number of EdU+ nuclei within all tissue types. To reflect the different tissue types with EdU+ nuclei, cell nuclei were binned into either epidermis (excluding the dermis), “skeletal elements” (bone, cartilage), and “soft tissues” (perichondrial cells, muscle, joint, tendon, ligaments, dermis, vasculature, nerves) based on nuclear morphology and location as previously described [50]. For cell counts within organs, averages reported reflect the total number of EdU+ nuclei out of the total DAPI+ nuclei over an average of three sections per sample. Statistics were performed using Student’s T-test assuming equal variance and alpha value of 0.05.

Supplementary Material

supplement. Supplementary Figure 1. Activated cells in contralateral limbs do not undergo cell death.

(A,B) Tissue sections processed with DAPI, and stained with anti-activated caspase-3. (A) Representative tissue section in an intact, uninjured control limb. (B) Represnetative tissue section from a limb contralateral to a regenerating limb at 14 dpa. Arrowheads in merged images indicate caspase3-positive cells. (C–F) Representative tissue sections from intact (C), a regenerating limb at 3 dpa (D), and limbs contralateral to amputated limbs at 3 dpa (E) and 14 dpa (F). Scale bar in (B) is 500 microns and applies to (A,B). Scale bar in (C) is 500 microns and applies to (C–F).

HIGHLIGHTS.

  • Limb amputation induces cell cycle entry in uninjured tissues systemically.

  • This activation proceeds throughout the time course of regeneration.

  • Distant cell cycle activation is independent of the wound epidermis.

  • Activated uninjured cells in axolotl share a conserved signaling pathway with mice.

Acknowledgments

We thank William Ye, Rui Qun Miao, and Adam Gramy for animal care. We acknowledge the Ambystoma Genetic Stock Center for providing a subset of the animals (Lexington, KY, NIH grant P40-OD019794). This work was supported by the NIH Common Fund (1DP2HD087953, J.L.W.), NIAMS (1R03AR068126, J.L.W.), Brigham & Women’s Hospital, and the PRISE (J.B., A.Y.W.) and HSCI Internship Programs (J.B.) at Harvard College. We thank members of the Whited, Levin, and Tabin labs, and the HSCI community for helpful comments.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplement. Supplementary Figure 1. Activated cells in contralateral limbs do not undergo cell death.

(A,B) Tissue sections processed with DAPI, and stained with anti-activated caspase-3. (A) Representative tissue section in an intact, uninjured control limb. (B) Represnetative tissue section from a limb contralateral to a regenerating limb at 14 dpa. Arrowheads in merged images indicate caspase3-positive cells. (C–F) Representative tissue sections from intact (C), a regenerating limb at 3 dpa (D), and limbs contralateral to amputated limbs at 3 dpa (E) and 14 dpa (F). Scale bar in (B) is 500 microns and applies to (A,B). Scale bar in (C) is 500 microns and applies to (C–F).

NIHMS895608-supplement.tif (6.4MB, tif)

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