Neuregulin-1 signaling is essential for nerve-dependent axolotl limb regeneration
Johanna E. Farkas1, Polina D. Freitas1, Donald M. Bryant2, Jessica L. Whited2, and James
R. Monaghan1.
1Department of Biology, Northeastern University, Boston, MA, USA
2Regenerative Medicine Center and Department of Orthopedic Surgery, Brigham & Women’s Hospital, Harvard Medical School, Cambridge, MA, USA
Corresponding author: James R. Monaghan, [email protected]
Summary Statement
Denervation of the amputated axolotl limb prevents regeneration. We have found that the neuronally-secreted protein Neuregulin-1 is necessary for axolotl limb regeneration and capable of inducing regeneration in denervated limbs.
© 2016. Published by The Company of Biologists Ltd.
Abstract
The Mexican axolotl (Ambystoma mexicanum) is capable of fully regenerating amputated limbs, but denervation of the limb inhibits the formation of the post-injury proliferative mass called the blastema. The molecular basis behind this phenomenon remains poorly- understood, but previous studies have suggested that nerves support regeneration via the secretion of essential growth-promoting factors. An essential nerve-derived factor must be found in the blastema, capable of rescuing regeneration in denervated limbs, and its inhibition must prevent regeneration. Here we show that the neuronally-secreted protein Neuregulin-1 (NRG1) fulfills all these criteria in the axolotl. Immunohistochemistry and in situ hybridization of NRG1 and its active receptor ErbB2 found that they were expressed in regenerating blastemas but lost upon denervation. NRG1 was localized to the wound epithelium prior to blastema formation and was later strongly expressed in proliferating blastemal cells. Supplementation via implantation of NRG1-soaked beads rescued regeneration to digits in denervated limbs, while pharmacological inhibition of NRG1 signaling reduced cell proliferation, blocked blastema formation and induced aberrant collagen deposition in fully-innervated limbs. Taken together, our results show that nerve- dependent NRG1/ErbB2 signaling promotes blastemal proliferation in the regenerating limb and may play an essential role in blastema formation, thus providing insight into the longstanding question of why nerves are required for axolotl limb regeneration.
Introduction
Regeneration of the axolotl (Ambystoma mexicanum) limb is inhibited by denervation of the limb, but the molecular mechanisms underlying this nerve dependence remain largely unknown. Denervation of the amputated axolotl limb does not inhibit wound healing but blocks the formation of the post-injury proliferative mass called the blastema (Todd, 1823). Nerve dependence is a phenomenon observed during wound healing and regeneration across a wide range of phylogeny (Kumar and Brockes, 2012) and may be due to the secretion of essential growth-promoting factors from peripheral nerves at the wound site (Singer, 1952; Stocum, 2011). Though evidence has been gathered in support of numerous candidate factors including transferrin (Kiffmeyer et al., 1991; Mescher et al., 1997), fibroblast growth factors (Satoh et al., 2011) and anterior gradient protein (Kumar et al., 2007), no neuronal factor thus far has proven capable of rescuing regeneration in the denervated axolotl limb. Here we examined Neuregulin-1 (NRG1), a neuronally-secreted mitogen that promotes proliferation through ErbB2 signaling (Falls, 2003b) and has been found in the newt peripheral nervous system (Brockes and Kintner, 1986) and implicated in newt limb regeneration (Wang et al., 2000). We have shown that NRG1 is found in the axolotl peripheral nervous system and blastema, is capable of rescuing regeneration to the point of digit formation in denervated limbs, and that its inhibition inhibits blastema formation, suggesting that it is a vital upstream proliferative signal during the regenerative process.
NRG1 and ErbB2 are expressed in regenerating limbs
Blastema-specific expression of NRG1 isoforms and receptors (Figures 1A-1E) was observed via in situ hybridization (ISH) in blastemas collected at 16 days post amputation (DPA). Strong expression of the active EGF-like domain, which is found in all isoforms of NRG1, was observed in the mesenchyme as well as the basal wound epithelium of the blastema. Expression of the Ig-like domain of NRG1, which is common to types I and II NRG1, was strong in the distal mesenchyme and basal wound epithelium and was comparatively absent in cells located proximal to the site of amputation. The CRD domain of type III NRG1 was also observed in the blastema, though this expression was found in fewer cells compared to that of the Ig-like domain. Type I and type II NRG1 are capable of signaling in a paracrine manner while type III NRG1 is limited to juxtacrine signaling (Falls, 2003b), indicating that paracrine NRG1 isoforms in particular are strongly expressed in the blastema during limb regeneration. ISH of ErbB2 revealed that it was expressed in the mesenchymal cells of the distal blastema as well as the basal layer of the wound epithelium, though it was virtually absent from cells which were located proximal to the site of amputation. The ErbB2 co-receptor ErbB3 was similarly expressed in the distal blastema. Taken together, these ISH results suggest that expression of NRG1 and its receptors is blastema-specific during axolotl limb regeneration. RT-PCR of blastemal (21 DPA) and uninjured tissues found that all isoforms of NRG1 examined were present in the regenerating and uninjured limbs (Figure 1F). NRG1 types I&II were upregulated in injured vs. uninjured limbs while type III NRG1 was more highly expressed in uninjured limbs. The presence of NRG1 in intact limbs is consistent with its known roles in Schwann cell and neuromuscular junction maintenance (Falls, 2003a; Sandrock et al., 1997).
Immunohistochemical staining of 16 DPA blastemas further confirmed the nerve- dependent presence of NRG1 and its active receptor in the regenerating limb. At 16 DPA, NRG1-positive cells were found both in the wound epithelium and in 56.18% of mesenchymal blastemal cells (Figures 1G, 1H). By contrast, the percentage of mesenchymal NRG1-positive cells was significantly reduced to 29.87% in denervated limbs (Figures 1G, 1I). These findings indicate that NRG1 protein is found in the blastema and reduced upon denervation, suggesting that nerves support a positive feedback loop which sustains NRG1/ErbB2 expression. NRG1 antibody specificity was tested via Western blot analysis, which showed a band at the expected size of 47kDa and demonstrated stronger band intensity in blastemal tissue as compared to denervated tissue at 16 DPA (Figure 1T).
Immunohistochemical staining for the receptor ErbB2 was consistent with these findings, as ErbB2 was strongly expressed in both the mesenchyme and wound epithelium of blastemas at 16 DPA (Figure 1J) but reduced upon denervation (Figures 1G, 1K). Overall, these results show that RNA and protein of both NRG1 and ErbB2 are highly expressed in the blastema during axolotl limb regeneration.
Dorsal root ganglia, which are capable of rescuing regeneration if grafted into a denervated limb (Goldhamer et al., 1992; Kamrin and Singer, 1959; Tomlinson and Tassava, 1987), showed extensive NRG1 and ErbB2 staining (Figures 1M, 1O). NRG1 (Figure 1L) and ErbB2 (Figure 1N) were further observed in cross-sectioned peripheral nerves. NRG1 staining in the basal wound epithelium was observed before blastema formation and as early as 6 DPA (Figure 1P). Costaining with BrdU showed that at 6 DPA, NRG1 was also present in a subpopulation of proliferating mesenchymal cells located just underneath the wound epithelium. Though the mechanism behind blastema formation remains poorly-understood, previous studies have shown that signals from the basal wound epithelium may work in conjunction with nerves to induce the accumulation of dedifferentiated mesenchymal cells at the site of amputation (Goss, 1956a; Goss, 1956b; Loyd and Tassava, 1980; Tassava and Garling, 1979). The pre-blastemal presence of NRG1 in the wound epithelium as well as the proliferating mesenchyme thus indicates that it may play an important role in blastema induction.
NRG1/BrdU costaining was further observed in 16 DPA blastemas (Figure 1Q). BrdU-positive mesenchymal cells costained with NRG1 in 65.89% of cells in control limbs and 46.55% of cells in denervated limbs (Figure 1S), indicating a pro-proliferative function that is consistent with its known roles in cell proliferation and survival (Canoll et al., 1996; Flores et al., 2000; Garratt et al., 2000). Furthermore, cells which costained with NRG1 and BrdU were observed along peripheral nerves near the site of amputation, (Figure 1R) suggesting that Schwann cells may also be secreting NRG1. Taken together, these immunohistochemical results show that NRG1 and its active receptor are localized in peripheral nerves and the proliferating blastema and thus may promote nerve-dependent blastemal formation and proliferation.
NRG1 supplementation rescues regeneration in denervated limbs
To determine if NRG1 supplementation is sufficient to rescue regeneration in denervated limbs, NRG1β-1 peptide-soaked beads were implanted underneath the wound epithelium of limbs at 7 DPA (Figure 2A). Supplementation with NRG1 induced blastema formation in six of seven denervated limbs (Figures 2B-D), which regenerated significantly more tissue than PBS-treated denervated limbs but not innervated limbs across a span of two weeks (Figure 2E). Blastema formation was not the result of nerve survival or regeneration, as demonstrated by the fact that immunohistochemical staining for nerves was deficient in denervated and NRG1-treated limbs at 21 DPA (Figures 2F-H).
Because axolotl limbs cannot be reliably denervated for longer than approximately 20 days, in a separate experiment we denervated blastemas at 19 DPA and supplemented them with NRG1-soaked beads every four days in order to determine whether this treatment was sufficient to rescue limb regeneration all the way to digit formation. By 36 DPA, four of five NRG1-treated limbs had regenerated to the point of digit formation, while three of three controls and zero of three denervated limbs developed digits (Figures 2I-L, Figure S1). NRG1-supplemented limbs regenerated significantly more tissue than PBS-treated limbs, although they did not regenerate to the same degree as the fully-innervated controls (Figure 2M), suggesting that greater NRG1 supplementation or the inclusion of additional factors may be necessary to achieve total rescue. Alcian blue staining demonstrated the presence of chondrogenesis in the new digits in control and NRG1-treated limbs (Figures 2N, 2O), while beta-tubulin III immunohistochemistry further confirmed the lack of nerves in both denervated conditions (Figures 2P-2R).
NRG1 supplementation thus appears capable of bypassing the threshold of nerves required for blastema induction and limb regeneration, a finding with considerable
implications towards explaining the longstanding question of nerve-dependent regeneration. While it has been found that application of Gdf5 and Fgfs can induce limb formation in an accessory limb model of axolotl regeneration (Satoh et al., 2011), this is the first example to our knowledge of a single protein rescuing regeneration in the denervated axolotl limb. These results suggest that NRG1 acts as an essential link between nerves and the blastema, as it promotes blastemal growth and proliferation throughout the entire process of limb regeneration, from early blastemal growth to later digit formation.
Inhibition of NRG1/ErbB2 signaling blocks regeneration
NRG1 signaling was inhibited with the specific (Nagasawa et al., 2006; Ufkin et al., 2014) ErbB2 inhibitor Mubritinib. Submersion in 500nM Mubritinib did not impair wound healing but completely inhibited blastema formation in fully-innervated limbs, rendering them outwardly identical to denervated limbs (Figures 3A-C). Limbs treated with Mubritinib regenerated significantly less tissue than control but not denervated limbs (Figure 3M), lacked cellular accumulation at the wound site, and morphologically resembled denervated limbs (Figures 3G, H). BrdU cell counts of 12 DPA blastemas found that cellular proliferation was significantly decreased and in fact virtually abolished in drug-treated limbs (Figures 3I-J, N) despite the presence of healthy nerves, suggesting that the observed lack of proliferation was the direct result of ErbB2 inhibition rather than any inadvertent loss of innervation.
To further examine the similarities between denervated and Mubritinib-treated limbs, animals were bathed in 500nMol Mubritinib starting at 16 DPA, well after blastema formation. Previous studies have found that denervation after blastema formation substantially reduces cell cycling and proliferation (Goldhamer and Tassava, 1987; Maden, 1978; Tassava et al., 1974) and results in the formation of a miniature limb (Powell, 1969; Schotté and Butler, 1944; Singer and Craven, 1948), suggesting that nerves are required for blastemal proliferation but not limb differentiation and morphogenesis. We found that Mubritinib inhibited blastemal growth but not limb patterning over a span of 12 days, inducing the formation of miniature limbs that were phenotypically similar to those formed as a result of late denervation (Figures 3D-F). Limbs treated with Mubritinib regenerated significantly less tissue than control but not denervated limbs (Figure 3O), underlining the similarity between denervated and ErbB2-inhibited limbs. Our inhibition experiments thus indicate that ErbB2 signaling is necessary for promoting blastema formation and maintaining blastemal proliferation during the early tissue growth and late tissue patterning phases of regeneration. Long-term (23 DPA) exposure to 10µMol Mubritinib induced contraction of the wound epidermis similar to that observed in mice after injury (Dunn et al., 2013), contrary to the minimal wound contraction observed in control limbs (Figures 3K-L). Axolotl tissue regeneration is a typically scar-free process that occurs with minimal collagen deposition (Levesque et al., 2010; Seifert et al., 2012), but extensive and aberrant collagen deposition was observed in the mesenchyme of Mubritinib-treated limbs. The phenotype observed here after long-term ErbB2 inhibition indicates a disruption of these scar-preventing programs and resembles the phenotype observed in amputated limbs after total macrophage ablation
(Godwin et al., 2013).
As ErbB2 can also heterodimerize with EGFR, we pharmacologically inhibited EGFR in order to ensure that the effects of Mubritinib were due to NRG1 and not EGF signaling inhibition. Animals bathed in the specific (Goishi et al., 2003; Han et al., 1996) EGFR inhibitor AG1478 for six days post-amputation exhibited a markedly different phenotype from Mubritinib-treated animals, as EGFR inhibition resulted in improper wound healing and eventual tissue regression (Figures 4D-G). Strikingly, these animals also developed excessive numbers of iridophores after just ten days of treatment (Figure 4H). Furthermore, EGFR inhibition significantly reduced epidermal but not mesenchymal proliferation at 5-6 DPA, while ErbB2 inhibition significantly reduced mesenchymal but not epidermal proliferation (Figures 4A-C, I, J). These results suggest that inhibition via Mubritinib primarily blocks NRG1/ErbB2 signaling rather than EGF/ErbB2 signaling, which instead appears to play a critical role in wound healing and epidermal proliferation after amputation. Overall,
our data show that NRG1/ErbB2 signaling is essential for limb regeneration and may play a vital role in preventing scar formation during this process as well.
Conclusion
We have shown that a single nerve-derived protein, Neuregulin-1, is capable of supporting blastemal growth and tissue regeneration up to the point of digit formation in the denervated axolotl limb. We propose that nerve-dependent NRG1/ErbB2 signaling is crucial for blastemal proliferation and may also be an essential component of blastema formation and scar-prevention programs. Although NRG1 is the first protein to our knowledge that is capable of rescuing regeneration to digits in the axolotl limb, these findings do not rule out the possibility of other factors playing a crucial role in this process. Newt anterior gradient protein has been shown to rescue regeneration in denervated newt limbs (Kumar et al., 2007), and despite some prominent species differences between axolotls and newts- which demonstrate a different recovery response to denervation (Liversage and McLaughlin, 1983) as well as a phylogenetically unique method of regenerating muscular tissues (Sandoval- Guzman et al., 2014; Tanaka et al., 2016)- further exploration of the relationship between these two signaling pathways is necessary in order to fully characterize the underlying cause of nerve dependency in the axolotl limb. Given the conserved role of NRG1/ErbB2 signaling in the peripheral nerves as well as the burgeoning evidence of its necessity in other animal models of cardiac (Bersell et al., 2009; D’Uva et al., 2015; Gemberling et al., 2015) and peripheral nerve (Fricker et al., 2011; Ronchi et al., 2013; Ronchi et al., 2015) regeneration, elucidating the function and mechanism of this signaling pathway in the axolotl may have far- reaching impacts on the field of regenerative medicine.
Materials and Methods Surgical procedures
Leucistic axolotls (Ambystoma mexicanum) were bred and raised at Northeastern University
according to (Farkas and Monaghan, 2015). Animals were anesthetized in 0.01% benzocaine and amputation was performed just proximal to the elbow joint. Recombinant human NRG1β-1 peptide (0.5mg/mL in PBS; PeproTech) was incubated overnight with Affi- gel 50-100 mesh agarose beads (Bio-Rad) according to (Niswander, 2008). An incision was made 1-2mm above the site of amputation, then two beads were probed with forceps through the incision until they rested underneath the wound epithelium. Animals were denervated one hour later. Two more beads were implanted, limbs were re-denervated at 14 DPA, and blastemas were imaged three times a week. Area regenerated was assessed blind to the experimental condition via the tracing of blastemas in ImageJ. For the digit rescue experiment, three beads were implanted into the base of blastemas at 19 DPA, and denervations were performed one hour post-implantation. Three more beads were added every four days, limbs were re-denervated at 27 DPA and collected at 36 DPA. All experiments were conducted with the approval of and in accordance with the Northeastern University Institutional Animal Care and Use Committee.
Drug treatment
Mubritinib (TSZ Scientific) stock solution (10mM in DMSO) was diluted in salamander housing solution to 500nM for juveniles (3.5-6cm SVL) and 10µM for adults (20-25cm SVL), which are more capable of tolerating the drug. Juvenile animals were bathed in Mubritinib starting at either 0 or 16 DPA, while adult animals were treated from 6-23 DPA before tissues were collected and prepared for immunohistochemistry. AG1478 (Tocris) stock solution (10mMol in DMSO) was diluted to 10µMol and animals were bathed in either 500nM Mubritinib, 10µM AG1478, or 10µM DMSO for six days prior to tissue collection. BrdU (20mg/ml, Sigma) was injected I.P. at 1mg BrdU/ 1g animal. Limbs were collected at 24 hours post-injection.
Immunohistochemistry and histology
Tissues were fixed in 10% neutral buffered formalin at 4ºC overnight, washed 2 x in PBS, incubated in 10% EDTA at 4ºC for 48hr, processed for paraffin embedding, and sectioned at 10µm. Sections were deparaffinized and hydrated, pressure-cooked in 10% citrate buffer for
20 minutes (Cuisinart Electric Pressure Cooker CPC-600), washed 5 minutes in PBS, blocked for 30 minutes in 1.5% normal goat serum, incubated at 4°C overnight in primary antibody, washed, and incubated for 30 minutes at room temperature in secondary Alexafluor (Life Technologies) antibody, then mounted and coverslipped with Slowfade Diamond Antifade Mountant with DAPI (Life Technologies). Slides stained for ErbB2 were soaked for 30 minutes in 0.05% saponin (Sigma) then washed 3 x 10 minutes in PBS prior to the blocking step. Primary antibodies are listed in supplementary materials. Picrosirius (Polysciences, Inc.) and Masson’s trichrome (Thermo Scientific) stains were performed per the manufacturers’ protocol. Alcian blue staining was performed according to (Lee and Gardiner, 2012).
RT-PCR analysis and in situ hybridization
Total RNA was extracted from uninjured and 21 DPA limbs (QIAGEN RNEasy kit), converted to cDNA template (Life Technologies Maxima H Minus First Strand cDNA Synthesis kit), and PCR amplified (2X PCR Master Mix; Thermo Scientific) with 10ng cDNA template and 0.5uM of isoform-specific primers. PCR products were cloned into pGEM-T (Promega), sequence verified (Genewiz), and vectors used to generate digoxigenin-labeled probes. Limbs were collected at 16 DPA and ISH performed on 35µm thick cryosections according to (Monaghan et al., 2012). See supplementary materials for primer sequences.
Western blot
NRG1 primary antibody was diluted to 1:10000; secondary was goat anti-rabbit HRP at 1:5000 (Jackson ImmunoResearch). Mouse anti-alpha tubulin (1:5000, Sigma) was used as the loading control and was detected with goat-anti-mouse HRP (1:5000, Jackson ImmunoResearch). All antibodies were incubated in 5% BSA in TBST.
Competing interests
The authors declare no competing or financial interests.
Author contributions
J.E.F. and J.R.M. designed the research. J.E.F., P.D.F., D.M.B., and J.L.W. performed experiments and provided materials. J.E.F. and J.R.M. analyzed the data. J.E.F. wrote the paper with contributions from J.R.M., D.M.B. and J.L.W.
Funding
This research received funding from Northeastern University and D.M.B. was supported by an HHMI Gilliam Fellowship.
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Figures
Figure 1: NRG1 and ErbB2 are expressed in the PNS and the regenerating blastema. (A-E) ISH showing that NRG1 isoforms and receptors are expressed in the blastema at 14 DPA. Insets show sense controls. (F) rt-PCR analysis showing upregulation of type I and II NRG1 isoforms in regenerating vs. uninjured limbs. (G-K) NRG1 and ErbB2 are expressed in the mesenchyme of regenerating blastemas but lost upon denervation (n = 4 biological replicates each). Green and orange fluorescence is due to autofluorescent cellular debris.
(L-O) NRG1 and ErbB2 are expressed in dorsal root ganglia and peripheral nerves. (P) NRG1 is expressed in the wound epithelium and mesenchyme of 6 DPA limbs along with proliferating BrdU-positive cells. (Q) Extensive NRG1 expression and colocalization with BrdU in a 16 DPA blastema. (R) NRG1 and BrdU colocalization along peripheral nerves in a regenerating limb at 16 DPA. (S) Denervation significantly decreases the percentage of BrdU/NRG1 colocalizaiton in 16 DPA limbs (n = 4 biological replicates). (T) Western blot of NRG1 at 16 DPA showing a band at the expected size of 47kDa and greater band intensity in blastemal tissue relative to denervated tissue. Data is represented as mean +/- s.e.m., statistical analysis performed via Student’s t-test, ***p < 0.001, **p < 0.01, *p < 0.05. Scale bars at 100µm. Figure 2: Supplementation with NRG1 rescues regeneration in denervated limbs. (A) Timeline of early NRG1 supplementation experiment. (B-D) Supplementation with NRG1 rescues regeneration in denervated limbs at 20 DPA. (E) From 6-20 DPA, NRG1- supplemented (n = 7) limbs regenerated significantly more tissue than denervated (p < 0.05, n = 7), but not control limbs (p > 0.05, n = 3). (F-H) NRG1-supplemented limbs regenerated in the absence of hyperinnervation. (I) Timeline of late NRG1 supplementation experiment. (J-L) Implantation of NRG1-soaked beads into denervated limbs rescues regeneration to the point of digit formation at 36 DPA. (M) From 19-36 DPA, NRG1-supplemented (n = 5) limbs regenerated significantly more tissue than denervated (p < 0.05, n = 3) and significantly less tissue than innervated (p < 0.01, n = 3) limbs. (N-O) Alcian blue staining showing digit formation in control and NRG1-treated limbs. (P-R) NRG1 induced growth and digit formation in fully-denervated limbs. Data is represented as mean +/- s.e.m, statistical analysis performed via one-way ANOVA with Tukey’s post-hoc. Scale bars at 100µm. Figure 3: Inhibition of ErbB2 blocks regeneration, inhibits proliferation, and induces aberrant collagen deposition. (A-C) Inhibition of ErbB2 with 500nMol Mubritinib blocks blastema formation at 13 DPA. (D-F) Mubritinib application after 16 DPA blocks limb proliferation but not patterning and appears phenotypically similar to day 16 denervataion. (G-H) Picrosirius staining showing that 23 days of submersion in 10µMol Mubritinib results in contraction of the epidermis (e) and aberrant collagen deposition (c) in the mesenchyme, contrary to the minimal fibrotic deposition seen in control blastemas (b). Arrowheads indicate the contracted wound margin. (I-J) Masson’s trichrome staining of control and Mubritinib-treated limbs at 12 DPA show a lack of blastemal accumulation in the drug-treated limbs. (K-L, N) Treatment with Mubritinib does not reduce innervation but significantly decreases the proliferative index of amputated limbs (n = 5). (M) At 14 DPA Mubritinib- treated limbs (n = 5) had regenerated significantly less area than control (n = 8) but not denervated (n = 8) limbs. (O) Limbs that were either denervated (n = 8) or treated with Mubritinib (n = 8) at 16 DPA regenerated significantly less tissue than control limbs (p < 0.001, n = 7). Arrows indicate the planes of amputation. Data is represented as mean +/- s.e.m., statistical analysis performed via Student’s t-test, **p < 0.01. Scale bars at 100µm. Figure 4: EGFR inhibition inhibits wound closure and is phenotypically distinct from ErbB2 inhibition. (A-C) Proliferating cells are localized to the mesenchyme in AG1478- treated limbs and the epidermis in Mubritinib-treated limbs at 6 DPA. Arrowheads indicate autofluorescent cellular debris and red blood cells, dotted lines indicate the boundary between the wound epidermis and mesenchyme. (D-G) AG1478 treated limb showing aberrant wound closure over time compared to control limb at 3DPA and 8 DPA. (H) Limb treated with AG1478 for ten days demonstrating aberrant development of iridophores (i). (I) Control limb treated with DMSO for ten days showing lack of iridophores. (J-K) Percentage of proliferating epidermal and mesenchymal cells in control, Mubritinib-treated, and AG1478- treated limbs at 6 DPA (n = 5 biological replicates each). Data is represented as mean +/- s.e.m., statistical analysis performed via one-way ANOVA with Tukey’s post-hoc **p < 0.01. Scale bars at 100µm.TAK 165