© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev169128. doi:10.1242/dev.169128

STEM CELLS AND REGENERATION RESEARCH ARTICLE Blastemal progenitors modulate immune signaling during early limb regeneration Stephanie L. Tsai1,2, Clara Baselga-Garriga1,2 and Douglas A. Melton1,*

ABSTRACT 2009). Researchers have shown that, although the blastema itself Blastema formation, a hallmark of limb regeneration, requires is transcriptionally similar to the limb bud during development, proliferation and migration of progenitors to the amputation plane. regenerating limbs exhibit unique transcriptional profiles during Although blastema formation has been well described, the early stages of regeneration before the blastema has formed (Knapp transcriptional programs that drive blastemal progenitors remain et al., 2013). Elucidating these signals, as well as their specific unknown. We transcriptionally profiled dividing and non-dividing cells activities, is crucial for understanding why and how salamanders in regenerating stump tissues, as well as the wound epidermis, during respond to amputations with blastema formation. Several important early axolotl limb regeneration. Our analysis revealed unique signaling molecules and pathways have already been identified transcriptional signatures of early dividing cells and, unexpectedly, during early stages of appendage regeneration. For example, axolotl repression of several core developmental signaling pathways MARCKS-like protein (axMLP), a molecule that is secreted from in early regenerating stump tissues. We further identify an the wound epidermis, is required for the induction of tail immunomodulatory role for blastemal progenitors through regeneration and is capable of inducing cell proliferation in intact interleukin 8 (IL-8), a highly expressed cytokine in subpopulations limbs (Sugiura et al., 2016). Moreover, several core developmental of early blastemal progenitors. Ectopic il-8 expression in non- signaling pathways, including FGF and Wnt, are required for regenerating limbs induced myeloid cell recruitment, while IL-8 appendage regeneration in axolotls and other species (McCusker knockdown resulted in defective myeloid cell retention during late et al., 2015; Haas and Whited, 2017; Stocum, 2017). wound healing, delaying regeneration. Furthermore, the il-8 receptor Several studies have investigated bulk transcriptional changes cxcr-1/2 was expressed in myeloid cells, and inhibition of CXCR-1/2 during different stages of axolotl limb regeneration and successfully signaling during early stages of limb regeneration prevented identified genes that may play important roles in blastema formation regeneration. Altogether, our findings suggest that blastemal and maintenance (Monaghan et al., 2009; Campbell et al., 2011; progenitors are active early mediators of immune support, and Knapp et al., 2013; Stewart et al., 2013; Wu et al., 2013; Voss et al., identify CXCR-1/2 signaling as an important immunomodulatory 2015; Bryant et al., 2017; Gerber et al., 2018). More recently, single pathway during the initiation of regeneration. cell analysis specifically in the connective tissue lineage has elucidated the molecular transitional states during dedifferentiation KEY WORDS: Blastemal progenitor, Limb regeneration, of mature connective tissue to a progenitor state during limb Transcriptional analysis, il-8, cxcr-1/2, Axolotl, Myeloid cells regeneration (Gerber et al., 2018). In the present study, we sought to investigate the distinct INTRODUCTION transcriptional programs active within blastemal progenitors Salamanders, including newts and axolotls, possess the ability to irrespective of lineage, as well as the surrounding tissues, to better fully regenerate their limbs throughout their lifespan. This understand the genetic programs and signaling interactions that regenerative capacity requires the formation of a transient cellular govern the initiation of blastema formation. We reasoned that structure distal to the amputation plane, known as the blastema, blastemal progenitors would be among early proliferating cells in which comprises progenitors derived from multiple different tissues stump tissues following amputation. Therefore, to enrich for (Kragl et al., 2009; Sandoval-Guzman et al., 2014; Currie et al., blastemal progenitors, we transcriptionally profiled dividing cells 2016). Although salamanders can perform this process regularly, from regenerating stump tissues during early stages of axolotl limb mammals are incapable of forming a blastema that can give rise to regeneration. This approach allowed us to examine the an entire limb. Understanding the mechanisms underlying the transcriptional signature of early dividing cells, inclusive of initiation of blastema formation may provide important insights into blastemal progenitors, and to identify potential novel modulators unlocking human regenerative potential. of early blastemal cell induction and maintenance. We additionally Blastema formation requires coordinated proliferation and profiled non-dividing cells in regenerating stump tissues and the migration of progenitors derived from muscle, bone, dermal wound epidermis at similar stages for comparison. Pathway analysis fibroblasts, connective tissue and other tissues (Kragl et al., of the discrete transcriptional programs of all three subpopulations revealed differential patterns of signaling pathway activation/ inhibition and unexpectedly uncovered the strong suppression of 1Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity several core developmental signaling pathways throughout Avenue, Cambridge, MA 02138, USA. 2Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA. regenerating stump tissues. Finally, we demonstrate that one dividing cell-enriched candidate, interleukin-8, is a blastemal *Author for correspondence ([email protected]) progenitor-derived regulator of myeloid cell dynamics during the D.A.M., 0000-0002-1623-5504 transition from wound healing into blastema formation, and that signaling through its cognate receptor, CXCR-1/2, is necessary for

Received 18 June 2018; Accepted 23 November 2018 limb regeneration. DEVELOPMENT

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RESULTS referred to as the 0 dpa timepoint, which was collected to serve as a Transcriptional profiling of dividing cells during the initiation non-regenerating control. of limb regeneration Principal component analysis (PCA) of the transcriptional Cell cycle re-entry is an integral event for the initiation of limb profiles of all samples revealed four clusters representing regeneration prior to blastema formation. We therefore reasoned that tissue type (stump-derived or epidermis) and regeneration status we would be able to exploit differences in the cell cycle, such as (Fig. S2A). Differential expression analysis of transcripts between DNA content, as a means of enriching for blastemal progenitors the 4N (proliferating) and 2N (non-dividing) fractions at all three during early stages of regeneration. We transcriptionally profiled timepoints showed the expected enrichment of cell cycle gene three total cellular fractions at 0, 4 and 5 days post-amputation expression in the proliferating fraction (Fig. S2B). Moreover, known (dpa): stump-derived dividing (4N) and non-dividing (2N) cells, as blastemal cell markers, including prrx-1 (Satoh et al., 2011; Gerber well as the whole wound epidermis (Fig. 1A). We developed and et al., 2018) and kazd1 (Bryant et al., 2017), were enriched in the optimized a protocol to perform DAPI staining in conjunction with stump-dividing cells. Genes known to be upregulated early upon FACS to separate 4N and 2N cells in the regenerating stump during amputation but more likely to be modulating the extracellular matrix early regeneration (Fig. 1A, Fig. S1A-C). Intact limb tissue is degradation rather than marking progenitor cells, such as mmp9 and

Fig. 1. Growth factor signaling pathways are inhibited in regenerating stump tissues. (A) Schematic of transcriptomic profiling experiment. Limbs were amputated and 2-3 mm of tissue proximal to the amputation site was collected at 0, 4 or 5 days post-amputation (dpa). Three fractions were isolated from each sample for sequencing: stump-derived dividing cells, non-dividing cells and the whole-wound epidermis. DAPI cell cycle analysis and FACS was performed to isolate dividing and non-dividing cells in the stump tissue. (B,C) Venn diagrams of the distribution of differentially expressed transcripts at 4 or 5 dpa are shown in B and C, respectively. (D) Ingenuity Pathway Analysis of differentially expressed transcripts in stump-derived dividing, non-dividing or wound epidermal cells. Positive Z-scores depict predicted activation, whereas negative Z-scores depict predicted inhibition of the respective pathway. Color coding of labels for each fraction of tissue were as follows: stump-derived 2N, blue; stump-derived 4N, green; wound epidermis, purple. DEVELOPMENT

2 STEM CELLS AND REGENERATION Development (2019) 146, dev169128. doi:10.1242/dev.169128 mmp3 (Vinarsky et al., 2005; Monaghan et al., 2012; Stewart et al., enriched in dividing cells and, of these, only 298 transcripts (265 2013), were enriched in non-dividing stump tissues (Fig. S2C). To genes) were annotated. A heatmap representing the top 75 annotated investigate the specificity of our strategy to enrich for blastemal and most highly expressed enriched transcripts, with little to no progenitors rather than other dividing cell types, such as immune expression in the wound epidermis, is shown in Fig. 2A. A complete cell infiltrates, we examined the predicted activation and inhibition list of all annotated enriched transcripts identified can be found in of immune signaling pathways by applying Ingenuity Pathway Table S3. The small fraction of annotated enriched transcripts Analysis (IPA) software to the differentially expressed transcripts in suggests that many key modulators of early regenerative events each cell population relative to the non-regenerating limb. Most might be novel genes or at least may not have identifiable innate immune signaling pathways were inhibited within dividing orthologous genes in other species present in existing datasets. cells (Fig. S3, Table S1), while activated in the other fractions, Among the most highly expressed and enriched transcripts in suggesting that we did not preferentially enrich for dividing immune dividing cells were regenerative ECM components, including cells. In all, these observations validated the enrichment of dividing tenascin (tena), collagens (co5a2, co5a1, co1a2, coba1 and blastemal progenitors using this protocol and allowed us to examine coca1), emilin1 (emil1) and fibrillin 2 ( fbn2), suggesting that unique gene expression patterns of these cells. dividing cells play an early role in building the blastemal niche. Overall, a total of 21,077 transcripts (19,417 genes at 4 dpa) and Most notably, the transcriptional signatures of early dividing cells 16,510 transcripts (15,205 genes at 5 dpa) were differentially suggest that they may be directly regulated by TGF-β signaling. expressed across all three fractions during early regeneration Pathway analysis revealed that TGF-β signaling was specifically (Fig. 1B,C). Of these, only a strikingly small percentage were activated in early dividing cells (Fig. 1D). Further examination of commonly up- or downregulated across all tissues relative to non- TGF-β signaling components revealed upregulation of both up- and regenerating tissue at 0 dpa (1.51% at 4 dpa and 0.58% at 5 dpa), downstream regulators (tgf-β1, tgf-β1r, smad2, ltbp1 and inhba)as strongly suggesting that all three subpopulations initiate well as direct targets of TGF-β signaling associated with epithelial- distinct transcriptional programs following amputation. Lists of to-mesenchymal transition (EMT), such as snail1 and twist1 differentially expressed transcripts for all analyses have been (Fig. 2B). These data indicate that an autoregulatory TGF-β deposited in GEO under accession number GSE111213. The most signaling network is established both intra- and extracellularly in highly expressed and commonly upregulated transcripts dividing cells, including blastemal progenitors, during early stages corresponded to enzymes involved in modulating extracellular of regeneration. matrix (ECM) degradation (mmp18, mmp2, timp1, tena and adam8) To validate our differential gene expression findings and to learn and to transcription factors involved in limb development and more about where these transcripts are expressed, we performed regeneration, including sall4 (Neff et al., 2005; Akiyama et al., time course RNA in situ hybridization on two candidates with high 2015; Erickson et al., 2016) and runx-1 (Umansky et al., 2015; enrichment in dividing cells: transmembrane protein 119 (tm119) Deltcheva and Nimmo, 2017), indicating that they may play a role in and E3 ubiquitin-protein ligase (lin41) (Fig. 2C-H). In situ orchestrating regeneration across all tissues. hybridization confirmed that these transcripts were specific to early dividing cells and likely expressed in blastemal progenitors Growth factor signaling pathways are largely repressed during early stages of regeneration. Tm119 has been shown to play a within regenerating stump tissues role in bone development and osteoblast proliferation (Kanamoto We examined signaling interactions between regenerating et al., 2009; Mizuhashi et al., 2012, 2015), whereas lin41 has a subpopulations through IPA analysis of differentially expressed highly conserved role in stem cell maintenance as well as cellular transcripts in each fraction at both timepoints. IPA software uses reprogramming (Slack et al., 2000; Worringer et al., 2014). Both algorithms that account for the expression levels of signaling candidates showed little to no expression in uninjured limbs components, activators and inhibitors of well-known signaling (Fig. 2C,F). Co-expression of tm119 and lin41 with a dividing cell pathways to predict whether a particular pathway is activated or marker, top2a, via double in situ hybridization at 7 dpa validated inhibited. Surprisingly, pathway analysis revealed that several that these transcripts were expressed within dividing cells growth factor (e.g. FGF, Notch) (Fig. 1D) and intracellular (e.g. (Fig. 2D,G). Moreover, in situ hybridization of tm119 and lin41 at calcium and mTOR) signaling pathways implicated in regeneration 21 dpa revealed strong expression of tm119 across the blastema, and were strongly inhibited in regenerating stump tissues during these also lower, but pan-blastemal, expression of lin41 (Fig. 2E,H), early timepoints (Table S2). Notable exceptions include a handful suggesting that they were indeed expressed within blastemal of pathways: Hippo, canonical Wnt and TGF-β signaling. Among progenitors at earlier stages. the repressed pathways were neurovascular signaling pathways (CNTF, NGF, neuregulin, VEGF signaling) that were activated in interleukin-8 (il-8) is expressed in early blastemal the wound epidermis and suppressed in stump tissues. These data progenitors further suggest that the wound epidermis orchestrates early Interestingly, we noticed enriched expression of several neurovascular regeneration and that early repression of these inflammatory cytokines within early mitotically active cells, pathways in regenerating stump tissues may be required. In all, suggesting that blastemal progenitors may play a role in immune these results show that exact spatiotemporal modulation of growth regulation. Among these was il-8, a secreted cytokine that has well factor signaling pathways in specific tissues occurs during early known roles in inflammation, angiogenesis and proliferation (Russo stages of regeneration. et al., 2014), but no current known role in limb regeneration. We therefore chose to focus on il-8 for further study. The transcriptional landscape of early dividing cells Time course in situ hybridization for il-8 during limb indicates roles in shaping the blastemal niche regeneration revealed a strong, transient upregulation during early We next used our dataset to identify transcripts enriched in dividing stages of regeneration (Fig. 3). il-8 was undetectable in non- cells at both regenerating timepoints (see Materials and Methods for regenerating limbs (Fig. 3A,A′) and highly induced upon filtering criteria). A total of 1217 transcripts (1181 genes) were amputation (Fig. 3B-E′). At 1 dpa, il-8 was expressed in cells DEVELOPMENT

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Fig. 2. Identification of highly enriched transcripts in dividing cells during early regeneration. (A) Heatmap of the expression levels (log2TPM) of the top 75 transcripts enriched in regenerating dividing cells in wound epidermis and non-dividing cells in the stump tissue. Green arrows indicate transcripts for the blastemal markers kazd1 and prrx-1; red arrows indicate tm119 and lin41. (B) Heatmap of expression levels of select TGF-β signaling pathway regulators, targets and downstream effectors. Color coding of labels in A and B for each fraction of tissue as follows: stump-derived 2N, blue; stump-derived 4N, green; epidermis or wound epidermis, purple. (C-E) In situ hybridization of tm119 at 0, 7 and 21 dpa. Double in situ hybridization of tm119 with top2a is depicted in D. Arrowheads in D indicate co-positive tm119+top2a+ cells. (F-H) In situ hybridization of lin41 at 0, 7 and 21 dpa. Double in situ hybridization of tm119 and top2a is depicted in G. Arrowheads indicate co-positive lin41+top2a+ cells. Scale bars: 20 µm. Images were taken at 40× magnification. Insets indicate where in the overall section the higher magnification image was taken. WE, wound epidermis. Dashed lines in E and H indicate the wound epidermis boundary. lining the bone (Fig. 3B,B′) and in the dermis. il-8 expression prrx-1 and il-8 expression. At 3 dpa, 99.0% of prrx-1+ cells peaked at 3 dpa in mesenchymal cells within regenerating stump expressed il-8 and 96.6% of il-8+ cells expressed prrx-1. At 7 dpa, tissues and began to decrease by 7 dpa (Fig. 3C-D′). By early only 67.9% of prrx-1+ cells expressed il-8, but 97.5% of il-8+ cells blastemal stages at 14 dpa, il-8 was weakly and sparsely expressed expressed prrx-1, consistent with a decrease in il-8 expression over throughout the blastema and basal layers of the apical epithelial cap time (Fig. 3H). In contrast, at 3 dpa, only 3.8% of il-8+ cells were (AEC) (Fig. 3E,E′). csf1r+ and 1.7% of csf1r+ cells were il-8+, whereas at 7 dpa, 4.4% of Previous studies have shown that il-8 is highly expressed in il-8+ cells were csf1r+ and 1.5% of csf1r+ cells were il-8+ (Fig. 3I). invading monocytes in wound healing and tumorigenic contexts (Fu il-8 was also expressed in a subset of kazd1+ blastemal progenitors at et al., 2015; Williams et al., 2016). In contrast, double in situ 7 dpa (Fig. 3J). Altogether, these data provide evidence that il-8 is hybridization of il-8 with two blastemal cell markers prrx-1 and strongly expressed within a subset of blastemal progenitors primarily kazd1, as well as csf1-r, a monocyte marker in mammals and during early stages of limb regeneration. amphibians (Grayfer et al., 2014; Stanley and Chitu, 2014), confirmed that il-8 was indeed expressed within blastemal il-8 is sufficient to induce myeloid cell recruitment and progenitors and not monocytes. At both 3 and 7 dpa, il-8 was co- proliferation in bone/perichondrium and epidermis in expressed in a subpopulation of prrx-1+ cells (Fig. 3F), and exhibited non-regenerating limbs little to no expression in csf1-r+ monocytes (Fig. 3G). Quantification Because il-8 was strongly expressed in blastemal progenitors, we + + of prrx-1 and il-8 populations revealed high concordance between examined whether il-8 expression was sufficient to induce cell DEVELOPMENT

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Fig. 3. il-8 is strongly expressed in blastemal progenitors during early stages of limb regeneration. (A-E′) Time course double in situ hybridization of il-8 at 0, 1, 3, 7 and 14 dpa. il-8 is not expressed in non-regenerating limbs (0 dpa), begins expression at 1 dpa, peaks at 3 dpa and remains strongly expressed, but in fewer cells, at 7 dpa. By early blastemal stages, 14 dpa, il-8 expression has largely diminished, with weak expression in sparse blastema cells and in the basal layers of the wound epidermis. The amputation plane is indicated by a solid line in A-E. Higher-magnification images (40×) of the boxed areas are shown in A′-E′. The dotted lines in B′ and E′ mark the wound epidermis boundary. (F) Double in situ hybridization of il-8 with the blastemal cell marker prrx-1 at both 3 and 7 dpa shows colocalization between il-8 and prrx-1. Examples of co-positive cells are indicated with arrowheads. (G) Double in situ hybridization of il-8 with the monocyte marker csf-1r at both 3 and 7 dpa shows little to no colocalization between il-8 and csf-1r. Representative single-positive csf-1r+ cells are indicated with arrows, whereas single-positive il-8+ cells are indicated by arrowheads. (H,I) Quantification of double in situ experiments shown in F,G. (H) Percentage breakdown of total counted il-8- and/or prrx-1-expressing cells (3 dpa, n=298 cells in total, 7 dpa, n=412 cells in total). (I) Percentage breakdown of total counted il-8- and/or csf-1r-expressing cells (3 dpa, n=500 cells in total, 7 dpa, n=630 cells in total). (J) Double in situ hybridization of il-8 (blue) and kazd1 (red), a highly expressed blastemal cell marker reveals co-expression of il-8 in a subset of kazd1+ blastemal cells (arrowheads). Image was taken at 40× magnification. Insets in F,G,J indicate where in the overall section the higher magnification image was taken. Scale bars: 200 µm in A-E; 50 µm in A′-E′; 50 µm in F,G,J. behaviors characteristic of the initiation of blastema formation, such performing α-Naphthol Acetate (NSE) staining and Naphthol as immune cell recruitment or cellular proliferation. We designed a AS-D Chloroacetate (NCAE) staining, respectively. A 2.3-fold myc-tagged il-8 overexpression vector (pCMV-IL8myc-T2A- increase in NSE+ monocytes (117.53 vs 51.94 cells/mm2, tdTomato) and control vector (pCMV-T2A-tdTomato), and P=0.0041) and a 2.0-fold increase in NCAE+ granulocytes validated secretion of myc-tagged IL-8 protein in 293T cells (121.54 versus 61.97 cells/mm2, P=0.0353) was observed in (Fig. S4A-E). We injected and electroporated either the control limbs expressing il-8 relative to controls (Fig. 4B-D′), indicating tdTomato vector or the il-8 overexpression vector into intact limbs of that IL-8 is sufficient to recruit myeloid cells. non-regenerating animals, briefly pulsed the animals with EdU at We also assessed whether IL-8 could induce cellular proliferation 3 days post-injection, and collected samples 24 h later (Fig. 4A). by comparing the percentage of total EdU+ cells between tdTomato As IL-8 is a well-known inflammatory cytokine (Zeilhofer and il-8-expressing limbs. We observed a modest, yet significant, and Schorr, 2000), we first examined whether ectopic expression increase in the total number of EdU+ cells in limbs overexpressing of il-8 induced monocyte and granulocyte recruitment by il-8 compared with control tdTomato limbs (13.92% versus 11.27%, DEVELOPMENT

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Fig. 4. il-8 induces recruitment of monocytes and granulocytes in intact limbs. (A) Experimental schematic of il-8 overexpression experiment. The left limbs of axolotls were injected and electroporated with a T2A-tdTomato control construct, whereas the right limbs were injected and electroporated with an il8myc-T2A-tdTomato construct. Animals were pulsed with EdU 24 h prior to tissue collection at 4 days post-injection (dpi). (B) Quantification of monocytes and granulocytes per mm2 revealed a statistically significant increase in both monocytes (**P=0.0041, n=9) and granulocytes (*P=0.0353, n=9) in il-8-overexpression limbs. Statistical analyses were performed using a two-tailed paired t-test. Data are mean±s.d. with individual data points shown. (C-D′) Representative 10× montage images of NSE/NCAE stained limbs in control or il-8-overexpression limbs are shown in C,C′ and D,D′, respectively. (C′,D′) Higher magnification (20×) images of the boxed areas in C,D, respectively. NSE+ monocytes are stained in black and NCAE+ granulocytes are stained in purple. Arrowheads indicate representative monocytes and arrows indicate representative granulocytes. Scale bars: 200 µm in C,D; 20 µm in C′,D′. Dotted lines indicate bone.

P=0.0015) (Fig. 5A,C). Quantification of dividing bone/ expression of il-8 during early stages of limb regeneration suggested perichondrial, epidermal, endothelial and satellite cells revealed that il-8 morphant limbs were likely exhibiting defects at earlier that this total increase in dividing cells was primarily due to an timepoints in regeneration. increase in the percentage of dividing bone/perichondrial cells (17.78% versus 8.71%, P=0.004), although there is a modest IL-8 knockdown leads to defective retention of myeloid cells increase in the percentage of dividing epidermal cells (24.60% during the wound healing transition to blastema formation versus 19.70%, P=0.0146) (Fig. 5B-E). No increase in proliferation As ectopic il-8 expression was sufficient to recruit immune cells, we was observed in CD34+ endothelial cells or pax7+ muscle satellite hypothesized that il-8 morphant limbs may exhibit deficiencies in cells. These data suggest that ectopic il-8 expression is sufficient to myeloid cell recruitment. Surprisingly, we observed no significant promote proliferation of bone/perichondrial and epidermal cells in a deficiency in recruitment of either NCAE+ granulocytes or NSE+ non-regenerative context, i.e. without amputation. monocytes to the amputation site at either 1 dpa or 5 dpa (Fig. 7A,D). However, the mean density of granulocytes was lower overall than Knockdown of IL-8 results in delayed blastemal outgrowth that of controls at both of these timepoints, suggesting that il-8 may and regeneration play a minor role in recruiting granulocytes during wound healing. We next examined whether loss of function of il-8 may impair limb More interestingly, there was a significant decrease in both regeneration. To this end, we performed a dual pulse morpholino granulocytes (374.16 versus 483.24 cells/mm2, P<0.01) and experiment (Fig. 6A) using translation-blocking il-8 targeted or monocytes (198.20 versus 290.92 cells/mm2, P<0.05) during later control morpholinos, injected into either the right or left forelimb, at stages of wound healing at 7 dpa (Fig. 7). We did not observe a 2 days pre-amputation and 3 dpa. This treatment produced a difference in overall cell proliferation or cell death at this timepoint. strong and transient knockdown of endogenous IL-8 protein levels Therefore, the data suggest that il-8 likely plays a role in retaining (Fig. S4F). il-8 morpholino-treated limbs displayed delayed both granulocytes and monocytes during the transition from wound blastemal outgrowth (Fig. 6B). Quantification at 21 dpa revealed a healing to blastema formation. 37% decrease in blastemal length (572.89 vs 915.40 µm, P<0.0001) To determine whether IL-8 was directly regulating myeloid cell and a 32% reduction in blastemal area (491.29 vs 723.90 µm, behaviors, we examined the expression pattern of its cognate receptors, P=0.0005) (Fig. 6C-F). In addition, control limbs reached digit cxcr-1 and cxcr-2, during early stages of regeneration. Although other stages of differentiation before il-8 morphant limbs (Fig. 6B), organisms, including mammals and zebrafish, have both receptors, indicating that transient knockdown of IL-8 delays limb analysis of publicly available transcriptomes for the axolotl (Bryant regeneration. At this time point, there was no defect in cell et al., 2017; Nowoshilow et al., 2018) revealed only one receptor with proliferation in either blastemal cells or the wound epidermis homology to both cxcr-1 and cxcr-2 in different species. We therefore (Fig. S5) and no visible phenotypes with the blastema aside from the refer to the detectable cognate receptor as cxcr-1/2. Double in situ difference in size. These observations in conjunction with the strong hybridization of cxcr-1/2 with csf1r or mpo, a neutrophil marker DEVELOPMENT

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chemokines, during wound healing, and further suggests that retention of myeloid cells during the transition to blastema formation is crucial.

CXCR-1/2 signaling is necessary for limb regeneration Finally, we asked whether inhibition of CXCR-1/2 signaling would also impair limb regeneration. We perturbed the pathway by treating regenerating axolotls with a small molecule inhibitor of CXCR-1/2: SB-225002 (White et al., 1998) (Fig. 9). Inhibitor treatment beginning from the time of amputation (0 dpa) completely inhibited limb regeneration, while DMSO-treated limbs regenerated normally (Fig. 9B-E). Owing to the well-documented role of CXCR-1/2 signaling in immediate wound-associated inflammatory responses (Ha et al., 2017), we hypothesized that failure to regenerate in this context was mainly due to prevention of immediate inflammatory responses stimulated by limb amputation. Therefore, we treated regenerating animals with SB-225002, beginning at later stages of wound healing (3 dpa) and found that this also prevented regeneration (Fig. 9F-I), suggesting that prolonged CXCR-1/2 signaling is essential. Yet, treatment beginning after blastema formation (15 dpa) led to normal limb regeneration (Fig. 9J,K). In all, these data suggest that CXCR-1/2 signaling is necessary during early stages of regeneration, but dispensable for later stages. Since il-8 morphants displayed defective retention of myeloid cells during late wound healing, we examined whether inhibition of CXCR-1/2 signaling affected myeloid cell behavior similarly during early stages of regeneration. Unexpectedly, monocytes and granulocytes in SB-225002-treated limbs (from 0 dpa) displayed unhealthy morphologies at 7 dpa. The majority of NSE+ and NCAE+ cells at the distal amputation site had formed apoptotic bodies, suggestive of myeloid cell death (Fig. 10A). Quantification of healthy NCAE+ and NSE+ myeloid cells revealed statistically significant decreases in both populations in SB-225002-treated limbs at 7 dpa compared with DMSO controls (Fig. 10B,C, NCAE+ granulocytes: 128.9 versus 362.3 cells/mm2, P<0.0001, NSE+ monocytes: 94.71 versus 240.6 cells/mm2, P=0.0022). In concurrence with these observations, TUNEL staining of inhibitor-treated limbs revealed higher levels of cell death near Fig. 5. il-8 is sufficient to induce proliferation of bone/perichondrial cells + the amputation plane at 7 dpa (6.03% versus 1.47%, P=0.0154) and epidermis. (A,B) Quantification of total EdU cells and cell type-specific (Fig. 10D), suggesting that myeloid cells were likely undergoing EdU+ cells in control versus il-8 overexpression limbs revealed a mild statistically significant increase in the total percentage of EdU+ cells apoptosis. Interestingly, increased cell death was not observed in (**P=0.0015, n=8), a strong significant increase in the percentage of dividing il-8 morphant limbs, suggesting that other cytokines in addition to bone/perichondrial cells (***P=0.0004, n=8) and a mild increase in the il-8 may synergistically modulate myeloid cell behaviors. percentage of dividing epidermal cells (*P=0.0146, n=8). No significant Altogether, these data newly identify CXCR-1/2 signaling as an increase was detected in CD34+ endothelial cells or pax7+ satellite cells. immunomodulatory pathway that is, in part, regulated by IL-8, and All statistical analyses were carried out using a two-tailed paired t-test. Data is crucial for successful blastema formation. are mean±s.d. Each point on the graphs in A and B represents a biological replicate. Each biological replicate is one limb from a different animal. The boxes in A and B represent the first to third quartile of the distribution. ns, non- DISCUSSION significant. (C) Representative 10× montage images from control or il-8- Elucidating the molecular mechanisms underlying the initiation of overexpression limbs. Scale bars: 200 µm. (D,E) High-magnification images of regeneration is key to understanding the difference in responses to tissue boxed areas in C. Representative 20× images of the bone and surrounding loss between regenerative and non-regenerative organisms. Here, perichondrium of control (D) or il-8 (E) overexpression limbs. Arrowheads we transcriptionally profiled distinct regenerating subpopulations indicate EdU+ perichondrial cells. Scale bars: 20 µm. The yellow dotted lines in C-E indicate bone within the tissue. during early pre-blastemal stages of limb regeneration to differentiate gene expression changes that occur in early dividing cells, inclusive of blastemal progenitors, from those of surrounding (Walters et al., 2010), at 3 dpa revealed co-expression of cxcr-1/2 with tissues. Using this dataset, we have gained insights into the patterns subpopulations of csf1r+ and mpo+ cells (Fig. 8), suggesting that IL-8 of suppression and activation of signaling pathways present within likely directly acts on subpopulations of monocytes and granulocytes, subsets of early regenerating limb tissues. Notably, our examination including neutrophils during wound healing. Altogether, these of the expression profiles of early dividing cells revealed an data provide one of the first examples of blastemal progenitors as immunomodulatory role for blastemal progenitors during early immunomodulators, specifically as the source of canonical stages of regeneration. DEVELOPMENT

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Fig. 6. IL-8 knockdown results in delayed blastemal outgrowth and regeneration. (A) Schematic of the dual-pulse morpholino experiment. The forelimbs of an axolotl were injected with either control morpholino (left limb) or translation-blocking il-8 morpholino (right limb) at 2 days pre-amputation and 3 days post-amputation. Limbs were then collected at 21 dpa. (B) Representative bright-field images of the delay in blastemal outgrowth and regeneration. Dotted and solid lines indicate the blastema and the amputation plane, respectively. Arrows indicate the digits. Scale bars: 1 mm. (C,D) Picromallory stained sections of control and il-8 MO-injected limbs. Scale bars: 500 µm. The dotted lines indicate the amputation plane. (E) Quantification of blastema length in control versus il-8 MO-injected limbs. il-8 MO-injected limbs exhibited a statistically significant decrease in blastema length (****P<0.0001, n=19). (F) Quantification of blastema area in control versus il-8 MO-injected limbs. il-8 MO-injected limbs exhibit a significant decrease in blastemal area (***P<0.0005, n=19). Paired two-tailed t-tests were employed for statistical analyses. Data are mean±s.d. Each individual point represents a biological replicate. Each replicate is a limb from a different animal. The boxes in F represent the first to third quartile of the distribution.

The transcriptional signatures of early dividing cells and Surprisingly, we observed the strong repression of many growth blastemal progenitors suggest that the formation of the early factor signaling pathways in early regenerating stump tissues. FGF, blastemal niche may be regulated by canonical Wnt, Hippo and Notch, IGF-1, PDGF and non-canonical Wnt signaling pathways TGF-β signaling. Dividing cells showed enriched expression of (PCP and Wnt/Ca+) all appear to be inhibited, yet many of these regenerative ECM components, many of which are important for signaling pathways are necessary and/or sufficient for appendage regeneration (Calve et al., 2010; Godwin et al., 2014), suggesting regeneration in amphibians as well as zebrafish (Poss et al., 2000; that they are drivers of the transition to a regenerative ECM. In Yokoyama et al., 2000, 2001; Stoick-Cooper et al., 2007; Chablais addition, Hippo, Wnt and TGF-β signaling pathways were activated and Jazwinska, 2010; Satoh et al., 2011; Grotek et al., 2013; within dividing cells. As downstream effectors of all three pathways Makanae et al., 2014; Rodrigo Albors et al., 2015; Currie et al., interact in development and tumorigenesis (McNeill and Woodgett, 2016; Nacu et al., 2016; Shibata et al., 2016). Furthermore, 2010; Attisano and Wrana, 2013), it is likely that synergy between signaling pathways involved in neuronal (e.g. neuregulin) and these pathways is essential for early blastemal cell establishment vascular (e.g. VEGF) regeneration (Yu et al., 2014; Farkas et al., and maintenance. Interestingly, TGF-β signaling, which is 2016; Farkas and Monaghan, 2017; Ritenour and Dickie, 2017) necessary for axolotl limb regeneration (Levesque et al., 2007; were active in the wound epidermis, yet suppressed in regenerating Denis et al., 2016), was specifically active in dividing cells. Our data stump tissues, signifying that early neurovascular regeneration is further suggest that TGF-β signaling is sustained through autocrine coordinated by the wound epidermis and that repression of these feedback in early dividing cells, which exhibit exclusive pathways within regenerating stump tissues may be important. A upregulation of tgf-β1, tgf-β1r and smad-2, as well as regulators potential explanation for this apparent dichotomy lies in the timing and direct targets, including ltbp1, twist1 and snail-1. snail-1 directs of activation. Premature activation of signaling pathways such as epithelial-to-mesenchymal transition (EMT) behaviors (Fuxe et al., Notch (Grotek et al., 2013) or non-canonical Wnt signaling (Stoick- 2010), and activates expression of both regenerative ECM Cooper et al., 2007) inhibits blastemal growth during zebrafish fin components and twist family members, which are expressed in regeneration. Therefore, our findings suggest that repression of limb blastemal cells (Kragl et al., 2013; Bryant et al., 2017). these pathways in early stages of regeneration may be necessary and Moreover, dividing cells highly expressed ECM components such that precise timely release of inhibition ensures successful as emilin-1 and fibrillin-2, which modulate TGF-β signaling regeneration. It is interesting to note that the analysis did not through interactions with ltbp1 (Neptune et al., 2003; Randell and reveal strong predictions for activation or repression of other Daneshtalab, 2017), indicating that both an intra- and extracellular pathways essential for limb regeneration, such as sonic hedgehog

TGF-β signaling network is established. (shh) signaling (Singh et al., 2015). These other pathways may be DEVELOPMENT

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Fig. 7. IL-8 knockdown results in defective retention of myeloid cells during the transition from wound healing into blastema formation. (A) Quantification of NCAE+ granulocytes in control or il-8 MO limbs at 1, 5 or 7 dpa revealed a statistically significant decrease at 7 dpa (**P<0.01, n=9). Data are mean±s.d. Each point represents a biological replicate from a different animal in A and D and the boxes represent the first to third quartile of the distribution. (B-C′) Representative 10× montage images of control and il-8 MO limbs at 7 dpa are shown in B and C, respectively. Boxed areas in B,C are shown at 40× magnification in B′ and C′. (D) Quantification of NSE+ monocytes in control or il-8 MO limbs at 1, 5 or 7 dpa revealed a statistically significant decrease at 7 dpa (*P<0.05, n=9). Data are mean±s.d. (E-F′) Representative 10× montage images of control and il-8 MO limbs at 7 dpa are shown in E,E′ and F,F′, respectively. Boxed areas in E,F are shown at 40× magnification in E′ and F′. Paired two-tailed t-tests were employed for statistical analyses. Scale bars: 200 µm in B,C,E,F; 50 µm in B′,C′,E′,F′. Asterisks in B,C,E,F indicate the bone. Dotted lines in B′,C′,E′,F′ indicate the boundary of the bone. downstream and act at later stages of regeneration (or are regulated (Arango Duque and Descoteaux, 2014), we found that the post-transcriptionally). Nevertheless, these findings could provide primary source of IL-8 in early stages of limb regeneration is a targetable insights for improving regenerative outcomes. subpopulation of blastemal progenitors. Furthermore, the high Most notably, we provide one of the first examples that blastemal concordance of expression between il-8 and prrx-1, recently progenitors play an early paracrine immunomodulatory role in demonstrated as a connective tissue blastemal cell marker (Gerber appendage regeneration. Others have shown that IL-8 recruits et al., 2018), suggests that il-8 is primarily expressed in early neutrophils and macrophages in zebrafish organ and appendage regenerating connective tissue. Meanwhile, its receptor cxcr1/2 is regeneration (de Oliveira et al., 2013; Xu et al., 2018); however, the expressed in myeloid cells. Our functional data further shows source of IL-8 was not examined. Contrary to other injury and the importance of signaling between blastemal progenitors and tumorigenic contexts, where IL-8 is secreted from macrophages the immune system. il-8 knockdown delayed regeneration and

Fig. 8. cxcr-1/2 is expressed in subsets of monocytes and granulocytes. Double in situ hybridization of cxcr-1/2 and either csf1r, a monocyte marker (A), or mpo, a neutrophil marker (B). Both co-positive cxcr-1/2+ csf1r+ cells and cxcr-1/2+ mpo+ cells were apparent at 3 dpa (orange arrows). However, not all csf1r+ and mpo+ cells were co-positive, suggesting cxcr-1/2 is expressed in a subset of monocytes and granulocytes. Insets show where in the section the 63× magnification image was taken. Scale bars: 20 µm. DEVELOPMENT

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Fig. 9. Early CXCR-1/2 signaling is necessary for limb regeneration. (A) Experimental schematic of different SB-225002 drug treatments beginning at 0, 3 or 15 dpa. (B-E) SB-225002 treatment beginning from 0 dpa inhibits limb regeneration. Bright-field images of regenerating limbs treated with DMSO or SB-225002 at 17 dpa are shown in B and C, respectively. Alcian Blue stained DMSO- or SB-225002-treated limbs at 40 dpa are shown in D and E. (F-I) SB-225002 treatment beginning at 3 dpa prevents limb regeneration. Bright-field images of DMSO- or SB- 225002-treated limbs at 17 or 40 dpa are shown in F-I. (J,K) Limbs regenerate normally if SB-225002 treatment begins after blastema formation (15 dpa). Bright-field images of DMSO- or SB-225002-treated limbs at 30 dpa are shown in J and K, respectively. Arrows in B-K indicate the amputation plane. Scale bars: 1 mm.

CXCR-1/2 inhibition prevented limb regeneration. We also necessary for successful appendage regeneration in the axolotl demonstrate that il-8 can recruit both myeloid cell-types in non- and other model systems, playing roles that include directing regenerating limbs. Thus, whereas leukocytes have been shown to blastemal outgrowth, clearing senescent cells and modulating regulate blastemal cells in other models (Nguyen-Chi et al., 2017), blastemal cell proliferation (Li et al., 2012; Godwin et al., 2013; our findings newly suggest that blastemal progenitors direct Petrie et al., 2014; Yun et al., 2015; Nguyen-Chi et al., 2017; Simkin immune support and that this bi-directional signaling is important et al., 2017). Others have demonstrated the importance of pro- for early stages of regeneration. regenerative (M2) rather than pro-inflammatory (M1) macrophage Failure to retain myeloid cells during the transition from wound subtypes in regeneration (Pei et al., 2016; Simkin et al., 2017). healing to blastema formation may account for the regenerative Therefore, it is possible that IL-8 may retain M2 macrophages delay in il-8 morphants. Monocyte-derived macrophages are during the initiation of blastema formation. The extent of the role of

Fig. 10. CXCR-1/2 inhibition impacts myeloid cell survival during early limb regeneration. (A) Images of NCAE+, NSE+ or TUNEL+ cells in DMSO- or SB-225002-treated limbs at 7 dpa with treatment beginning from 0 dpa (DMSO, n=4, SB-225002, n=5). Insets of fully stained sections show where the higher magnification image was taken. Scale bars: 50 µm. Black arrows indicate healthy NCAE+ or NSE+ cells, whereas black arrowheads indicate unhealthy NCAE+ or NSE+ cells exhibiting apoptotic bodies. White arrowheads indicate TUNEL+ nuclei. (B) Quantification of NCAE+ granulocytes with healthy morphology revealed a statistically significant decrease in SB- 225002-treated limbs (362.3 versus 128.9 cells, ****P<0.0001). (C) Quantification of NSE+ granulocytes with healthy morphology revealed a statistically significant decrease in SB-225002-treated limbs (240.6 versus 94.71 cells, **P=0.0022). (D) Quantification of TUNEL+ nuclei revealed higher levels of cell death in SB-225002- treated limbs (6.033% versus 1.46%, *P=0.0172). Unpaired two- tailed t-tests were employed for statistical analyses. Data are mean±s.d. Each point represents a biological replicate and the boxes represent the first to third quartile of the distribution. DEVELOPMENT

10 STEM CELLS AND REGENERATION Development (2019) 146, dev169128. doi:10.1242/dev.169128 granulocytes in regeneration, however, seems to be more context or 5 dpa. We chose to transcriptionally profile regenerating limbs at 4 and dependent (Nakayama et al., 2011; Li et al., 2012; Kurimoto et al., 5 dpa because preliminary experiments showed a distinct increase in cellular 2013; Paris et al., 2016; Lindborg et al., 2017). Granulocytes, proliferation in the regenerating stump beginning at 3 dpa (data not shown including neutrophils, clear debris during wound healing (Wang, here) and we wanted to capture the transcriptional signatures of dividing 2018). Therefore, low levels of granulocytes in il-8 morphants may cells during the duration of cell-cycle re-entry. In order to obtain enough material for the sorting and sequencing protocol, we chose to perform have slowed wound healing, delaying regeneration. Furthermore, in the experiments at 4 and 5 dpa. Twelve limbs were pooled for each the zebrafish, IL-8 can act as a potent neutrophil chemoattractant (de biological replicate per timepoint and the experiment was performed in Oliveira et al., 2013, 2016) or a chemokinetic molecule, aiding in biological triplicate. neutrophil reverse migration from the sites of injury through Cxcr2 To prep the tissue for FACS, the intact epidermis or wound epithelium signaling (Powell et al., 2017). In contrast, our findings suggest that was micro-dissected off and placed into 0.25% Trypsin-EDTA for 15 min IL-8 may retain granulocytes, including neutrophils, during late with agitation at room temperature to dissociate epithelial cells from the wound healing rather than facilitating their exit strategy as in the dermal layer. The remaining stump tissues were micro-dissected further into zebrafish, highlighting potential species-specific differences in small pieces with dissecting scissors and chemically dissociated in a immune responses during regeneration. As IL-8 morphant limbs solution composed of 5 mg/ml collagenase (Sigma-Aldrich), 7.3 mg/ml eventually regenerate, it is clear that other pathways compensate to dispase II (Roche Diagnostics) and 1.36 mg/ml D-glucose in 80% PBS (Kumar and Brockes, 2007) for 15 min with agitation at room temperature. ensure wound healing resolution. In order to obtain a representation of dermal cells in the stump tissue Last, we show that CXCR-1/2 signaling is necessary during early, fraction, we also chemically dissociated micro-dissected intact epidermis or but not late, stages of limb regeneration. CXCR-1/2 inhibitor-treated wound epithelia along with the stump tissues. For regenerating samples, the limbs beginning during early and late stages of wound healing failed wound epidermis region, which is visibly transparent, was carefully to form a blastema. The high level of myeloid cell death, which was removed to isolate adjacent full-thickness skin with both epidermal and not observed in il-8 morphants, during early regeneration was likely dermal layers, and then dissociated with the stump tissue fraction. the main cause of failure to regenerate, suggesting that CXCR-1/2 Dissociated cells were then transferred to a new tube (non-dissociated signaling may serve as an important survival pathway for monocytes chunks of tissue were left behind) and the dissociation was serum and granulocytes in early limb regeneration. Additionally, the inactivated. The following protocol for staining, FACS and RNA isolation difference in severity of phenotypes between il-8 morphant and was optimized and adapted from Hrvatin et al. (2014). The cell suspension was pelleted, re-suspended, passed through a 70 µM cell strainer, washed CXCR-1/2 inhibitor-treated limbs is likely attributed to the fact that twice with 80% PBS and fixed in 4% paraformaldehyde/0.1% saponin with CXCR-1/2 binds to other interleukins, including IL-1 and IL-6 a 1:50 dilution of RNasin plus RNase Inhibitor (Promega) for 30 min at 4°C. (Baggiolini et al., 1997). Therefore, it is possible that other cytokines Fixed cells were then washed twice with a 1% BSA/ 0.1% saponin (1:40 are acting in concert with IL-8 to control CXCR-1/2-specific myeloid dilution RNasin plus RNase Inhibitor, Promega) in PBS wash buffer and cell behaviors during early limb regeneration. Altogether, these data DAPI staining (10 µg/ml) of cells was performed for 30 min at 4°C in a highly suggest that CXCR-1/2 signaling may be key in bridging 0.1% saponin solution (1:20 RNasin plus RNase Inhibitor, Promega). The communication between early blastemal cells and the immune system stump fraction of DAPI-stained cells were immediately sorted into 2N and during the initiation of regeneration. 4N fractions using FACS, whereas the corresponding epithelial fraction was In conclusion, our approach reveals differential patterns of subjected to the same staining treatment, but not sorted. Cells were sorted activation/suppression of core developmental signaling pathways into RNAlater solution (Invitrogen) and RNA was isolated with the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Invitrogen). The within separate cellular subsets during early regeneration and a role quality of the RNA samples was assessed using the Agilent RNA 6000 Pico for early dividing cells, inclusive of blastemal progenitors, in kit on the Bioanalyzer 2100 (Agilent Technologies). shaping the regenerative niche. We demonstrate that blastemal progenitors play an early immunomodulatory role through IL-8 and cDNA library preparation and sequencing that signaling through its cognate receptor, CXCR-1/2, is necessary cDNA was synthesized from RNA samples using the NuGEN Ovation for limb regeneration. These findings highlight the importance of RNA-seq System V2 protocol (Integrated Sciences) according to the further characterizing the complexity of bi-directional crosstalk manufacturer’s instructions using 10 ng of RNA as starting material. The between the immune system and blastemal cells on a broader scale. quality and concentration of cDNA preps was then assessed using the Agilent DNA 1000 kit on the Bioanalyzer 2100 (Agilent Technologies). RT-PCR for cell cycle markers ccnb3, ccna2 and cdk1b was then performed MATERIALS AND METHODS on the cDNA generated from 4N and 2N cells to check for proper Animal procedures enrichment of dividing cells. Primer sequences are as follows: ccnb3-For, Axolotl (Ambystoma mexicanum) husbandry and surgeries were performed 5′-CACAAGAATCCAGTGCCACA-3′; ccnb3-Rev,5′-CCTCCTTT- in accordance with the Association for Assessment and Accreditation of GCAACAGTGTCC-3′; ccna2-For, 5′-GAACGTACAGCCTGGCAAG- Laboratory Animal Care (AAALAC) and Institutional Animal Care and Use 3′; ccna2-Rev, 5′-CTGACGGCTGCTCCTTTG-3′; cdk1b-For, 5′-GCCA- Committee (IACUC) guidelines at Harvard University. Sub-adult white and AACAACGAAATCTGGC-3′; cdk1b-Rev, 5′-AGGGTGGTTCAATGC- albino axolotls (15-18 cm) provided by the Ambystoma Genetic Stock CTCTT-3′. Center (AGSC, University of Kentucky) were used for the initial RNA- To prepare cDNA sequencing libraries, 200 ng of cDNA was first sheared sequencing experiment. Juvenile white axolotls (5-8 cm) were used for all to a peak size of 200 bp using the Covaris S220 according to the morpholino and overexpression experiments. For CXCR-1/2 inhibitor manufacturer’s protocol. Good quality and correct size distribution of experiments, animals (3-5 cm) were immersed in either 500 nM SB-225002 sheared DNA fragments was assessed with the Agilent DNA High (Tocris Bioscience) or DMSO (Sigma) beginning at either 0, 3 or 15 dpa and Sensitivity kit on the Bioanalyzer 2100 (Agilent Technologies). solutions were changed daily. Sequencing libraries were then generated using the Wafergen PrepX Complete ILMN DNA Library kit (Takara Bio) protocol on the Apollo 324 FACS and RNA isolation NGS Library Prep System (Takara Bio). The quality of the DNA sequencing Briefly, animals were anesthetized in 0.1% Tricaine (Sigma-Aldrich) and all libraries was performed with the Agilent DNA High Sensitivity kit on the four limbs were amputated at the mid-radius/ulna level. Bone was trimmed Bioanalyzer 2100 (Agilent Technologies). Concentration of the DNA back to facilitate wound closure and regeneration. Approximately 2-3 mm of libraries was doubly confirmed using the Qubit dsDNA HS Assay kit tissue directly proximal to the amputation plane was collected at either 0, 4 (ThermoFisher Scientific) and Kapa Illumina Library Quantification kit DEVELOPMENT

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(Kapa Biosystems). Libraries were multiplexed and sequenced on either the as above) and tdTomato using a polyclonal goat anti-tdTomato antibody Illumina Hiseq 2500 system (125 bp reads) or Nextseq 500 (150 bp reads) at (LS-C348313, LSBio, 1:1000). the Harvard Bauer Core Sequencing Facility. Ectopic expression of il-8 in intact limbs Sequencing analysis Intact limbs of axolotls were injected and electroporated with myc tagged Reads were trimmed using Trimmomatic (Bolger et al., 2014) to a minimum il-8 overexpression construct (1 µg/µl in PBS). At 3 days post-injection, length of 100 bp and poor quality reads were removed from the sequencing animals were injected intraperitoneally with EdU (Life Technologies) at a analysis. Alignment was performed on the trimmed reads using Kallisto (Bray concentration of 8 mg/kg 4 hours prior to tissue collection. Tissue was et al., 2016) and the previously published well-annotated axolotl prepped and embedded in OCT in a similar manner to previously described transcriptome (Bryant et al., 2017). Raw read data and the processed data above. The blocks were serially sectioned at 16 µm. EdU staining was matrix containing TMM-normalized TPM values for each sample have been performed using the Click-it EdU Alexa Fluor 488 Imaging Kit deposited in GEO under accession number GSE111213. Differential (ThermoFisher Scientific). For immunostaining, mouse anti-chick Pax7 expression analysis of genes and transcripts relative to the non-regenerating (DSHB, 1:200) and rabbit anti-human CD34 (ab81289, Abcam, 1:200) timepoint (0 dpa) for each fraction was performed using DESeq2 (Love et al., antibodies were used. 2014) with an adjusted P-value cutoff of 0.05. Core and comparison analysis The imaging analyses were all conducted blinded. For the quantification of differentially expressed transcript lists for each cellular population was of il-8 overexpression limbs, three or four sections were imaged per limb and performed using Ingenuity Pathway Analysis software (Qiagen). Uniprot IDs quantified. Total numbers of EdU+, CD34+, pax7+, CD34+ EdU+ and pax7+ of transcript blast hits (extracted using the associated Trinotate file by Bryant EdU+ cells were counted, and percentages were calculated using DAPI+ et al., 2017) were converted to human IDs for IPA analysis. We focused on nuclei totals for EdU percentages, pax7+ nuclei totals for dividing satellite strongly activated or inhibited growth factor signaling pathways, i.e. absolute cells and CD34+ cell totals for dividing endothelium. Dividing bone/ value of the Z-score>1 and signal detected across at least three differentially perichondrial or epidermal cells were counted based on EdU+ cells of the expressed analyses out of a total of six. cell type out of DAPI+ total of the cell type (counted by morphology). A To identify transcripts enriched within dividing cells at both timepoints, two-tailed paired t-test was used for statistical analysis. we first compared dividing and non-dividing cells averaged at both regenerating timepoints (4 and 5 dpa) and identified 6834 differentially NSE/NCAE staining and analysis of myeloid cells expressed transcripts (3510 of which were upregulated within dividing Staining of monocytes and granulocytes was performed using the α- cells). Transcripts that were normally differentially expressed in non- Naphthyl Acetate (Non-specific Esterase) (NSE) kit or Naphthol AS-D regenerating dividing and non-dividing cells (at 0 dpa), such as cell cycle- Chloroacetate (Specific Esterase) (NCAE) Kit (Sigma-Aldrich), associated transcripts, were filtered out. Of these, 628 total transcripts (583 respectively, according to the manufacturer’s instructions. The only genes) were regeneration specific, annotated and had at least a twofold modification to the protocol was a 10 min fixation step in citrate-acetone- change between dividing and non-dividing cells; only 298 transcripts in this methanol fixative. For the overexpression experiment, the area of each cross- list were upregulated in dividing cells. section was measured using ImageJ analysis software. For characterization of the il-8 morphant limbs, tissue was collected and prepared at 1, 5 and In situ hybridization and quantification 7 dpa. The area of the section 500 μm from the amputation plane was Tissue was collected at 0, 7 and 21 dpa and fixed in 4% paraformaldehyde measured and the quantification was performed blinded. Every effort was overnight at 4°C, washed in PBS, brought up a sucrose gradient to 30% made to ensure quantification around the same area in all limbs using the sucrose, and embedded in OCT. The blocks were serially sectioned and humerus and ulna as landmarks. A two-tailed paired t-test was used for 16 µm sections were collected. Custom RNAscope probes for the axolotl all statistical analyses and all of the quantification was performed blinded orthologs of top2a, cxcr-1, tm119, lin41, il-8, kazd1, mpo and prrx-1 were (averaging two or three sections/limb). For characterization of DMSO- or SB- generated (Advanced Cell Diagnostics) in either the C1 or C2 channels. 225002-treated limbs, limbs from animals treated from time of amputation Double chromogenic section in situ hybridization was performed on frozen were collected at 7 dpa, stained and analyzed as described above. cryosections using the RNAscope 2.5 HD Duplex Detection Kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions. IL-8 morpholino knockdown The total numbers of prrx-1+only, il-8+ only, csf1r+ only or double- Axolotls were anesthetized in 0.1% tricaine and intact forelimbs were positive cells were quantified by counting cells that exhibited at least 10 injected and electroporated with either control (left limb) or il-8-targeted puncta for either probe. These are categorized as strongly-expressing cells, morpholino (right limb). Approximately 3-4 µl of morpholinos were according to RNAscope standards (score of 4+). These numbers were used injected at a final concentration of 5 µM in PBS. At 2 days post-injection, to quantify the percentages of co-positive and single-positive cells. both forelimbs of axolotls were amputated at the mid-radius/ulna level and control or il-8 morpholinos were injected again at 3 days post-amputation Generation and validation of the myc-tagged il-8 overexpression into regenerating stump tissue. Both translation-blocking and five-point construct mutation control morpholino antisense oligonucleotides were designed and To generate an il-8 overexpression construct, the open reading frame for il-8 generated by GeneTools against the il-8 ORF with the following sequences: was amplified out of cDNA from regenerating limbs at 7 dpa with primers control, 5′-CCGATCTTGATGCTCACCTCCTG-3′; il-8,5′-CCGAT- that added on a NheI site, and a kozak sequence directly upstream of the GTTCATGGTGACCTGCTG-3′. To validate morpholino knockdown, ATG start codon and a HindIII site directly downstream of the coding il-8-targeted and control morpholino injected limbs from the same animal sequence. This PCR product was cloned into a pCMV-TVA-T2A-tdTomato were collected and protein was extracted using Trizol (ThermoFisher backbone (a gift from J. Whited, Harvard University, MA, USA) in place of Scientific). Western blotting was performed on protein extracts using the TVA. An empty pCMV-T2A-tdTomato vector was used as a control. anti-GAPDH antibody described above, as well as a cross-reacting polyclonal To validate the expression of the vector, 293T cells were transfected with mouse anti-chick IL-8 antibody (MBS2018201, MyBioSource, 1:400). 50 µg of either the il-8 overexpression or tdTomato control construct using For analysis at the blastemal stages, animals were injected Lipofectamine 2000. The media and cells were separately collected at 48 h intraperitoneally with EdU (Life Technologies) at a concentration of post-transfection (hpt). Media was concentrated with a 15 ml Amicon filter 8 mg/kg 4 hours prior to tissue collection. Tissue was collected at 21 dpa (3 K) (MilliporeSigma). Protein was extracted from cells using Trizol and prepared as described above. Picro-mallory staining was performed on (ThermoFisher Scientific). Western blotting was performed on both il-8 sections for histological analysis to analyze blastema length and area. Cell overexpression and control transfected cell lysates and media using a mouse proliferation was assessed using the Click-it EdU Alexa Fluor 488 Imaging monoclonal anti-GAPDH (MAB374, MilliporeSigma, 1:2000) and Kit (ThermoFisher Scientific). All imaging analysis was carried out in polyclonal rabbit anti-myc tag antibody (ab9106, Abcam, 1:2000). Cells ImageJ and conducted blinded. A two-tailed paired t-test was used for were additionally immunostained at 72 hpt for the myc tag (same antibody statistical analysis. DEVELOPMENT

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TUNEL staining de Oliveira, S., Reyes-Aldasoro, C. C., Candel, S., Renshaw, S. A., Mulero, V. TUNEL staining was performed using the In Situ Cell Death Detection Kit and Calado, A. (2013). Cxcl8 (IL-8) mediates neutrophil recruitment and behavior (Roche) as described previously (Zhu et al., 2012). The percentage of in the zebrafish inflammatory response. J. Immunol. 190, 4349-4359. + de Oliveira, S., Rosowski, E. E. and Huttenlocher, A. (2016). Neutrophil migration TUNEL nuclei (out of DAPI total) was quantified from two sections per in infection and wound repair: going forward in reverse. Nat. Rev. Immunol. 16, limb and averaged. 378-391. Deltcheva, E. and Nimmo, R. (2017). RUNX transcription factors at the interface of Alcian Blue staining stem cells and cancer. Biochem. J. 474, 1755-1768. Alcian Blue staining of DMSO or SB-225002-treated 40 dpa limbs was Denis, J.-F., Sader, F., Gatien, S., Villiard, E., Philip, A. and Roy, S. (2016). Activation of Smad2 but not Smad3 is required to mediate TGF-beta signaling performed as described previously (Whited et al., 2013). during axolotl limb regeneration. Development 143, 3481-3490. Erickson, J. R., Gearhart, M. D., Honson, D. D., Reid, T. A., Gardner, M. K., Acknowledgements Moriarity, B. S. and Echeverri, K. (2016). A novel role for SALL4 during scar-free We thank J. Whited for providing the overexpression vector (pCMV-TVA-T2A- wound healing in axolotl. NPJ Regen. Med. 1, 16016. tdTomato), and for providing scientific guidance and helpful comments on the Farkas, J. E. and Monaghan, J. R. (2017). A brief history of the study of nerve manuscript. We acknowledge I. Adatto, L. Krug and the Harvard Office of Animal dependent regeneration. Neurogenesis 4, e1302216. Resources (OAR) for their dedicated animal care in the axolotl facilities, as well as Farkas, J. E., Freitas, P. D., Bryant, D. M., Whited, J. L. and Monaghan, J. R. S. Ionescu and J. Lavecchio for performing all of the FACS in the SCRB department (2016). Neuregulin-1 signaling is essential for nerve-dependent axolotl limb flow cytometry core. In addition, we thank the Ambystoma Genetic Stock Center regeneration. Development 143, 2724-2731. (AGSC), which provided many of the animals for the experiments conducted here. Fu, X.-T., Dai, Z., Song, K., Zhang, Z.-J., Zhou, Z.-J., Zhou, S.-L., Zhao, Y.-M., Last, we greatly thank J. Davis, J. Rivera-Feliciano, E. Rosado-Olivieri, N. Sharon, Xiao, Y.-S., Sun, Q.-M., Ding, Z.-B. et al. (2015). Macrophage-secreted IL-8 C. Kayatekin, all of the members of the Melton lab and also R. Amamoto for helpful induces epithelial-mesenchymal transition in hepatocellular carcinoma cells by scientific discussion and review of the manuscript. activating the JAK2/STAT3/Snail pathway. Int. J. Oncol. 46, 587-596. Fuxe, J., Vincent, T. and Garcia de Herreros, A. (2010). Transcriptional crosstalk Competing interests between TGF-beta and stem cell pathways in tumor cell invasion: role of EMT The authors declare no competing or financial interests. promoting Smad complexes. Cell Cycle 9, 2363-2374. Gerber, T., Murawala, P., Knapp, D., Masselink, W., Schuez, M., Hermann, S., Gac-Santel, M., Nowoshilow, S., Kageyama, J., Khattak, S. et al. (2018). Author contributions Single-cell analysis uncovers convergence of cell identities during axolotl limb Conceptualization: S.L.T., D.A.M.; Methodology: S.L.T.; Validation: S.L.T.; Formal regeneration. Science 362, eaaq0681. analysis: S.L.T., C.B.-G.; Investigation: S.L.T.; Resources: D.A.M.; Data curation: Godwin, J. W., Pinto, A. R. and Rosenthal, N. A. (2013). Macrophages are S.L.T.; Writing - original draft: S.L.T.; Writing - review & editing: S.L.T., C.B.-G., required for adult salamander limb regeneration. Proc. Natl. Acad. Sci. USA 110, D.A.M.; Supervision: D.A.M.; Funding acquisition: D.A.M. 9415-9420. Godwin, J., Kuraitis, D. and Rosenthal, N. (2014). Extracellular matrix Funding considerations for scar-free repair and regeneration: insights from regenerative This research was performed using resources and/or funding from the Harvard Stem diversity among vertebrates. Int. J. Biochem. Cell Biol. 56, 47-55. Cell Institute and the Howard Hughes Medical Institute (HHMI). D.A.M. is an Grayfer, L., Edholm, E.-S. and Robert, J. (2014). Mechanisms of amphibian investigator of the HHMI. Deposited in PMC for release after 12 months. macrophage development: characterization of the Xenopus laevis colony- stimulating factor-1 receptor. Int. J. Dev. Biol. 58, 757-766. Data availability Grotek, B., Wehner, D. and Weidinger, G. (2013). Notch signaling coordinates The raw read data, the processed data matrix containing TMM-normalized TPM cellular proliferation with differentiation during zebrafish fin regeneration. values for each sample, and lists of differentially expressed transcripts for heatmaps Development 140, 1412-1423. and analyses in Figs 1 and 2 have been deposited in GEO under accession Ha, H., Debnath, B. and Neamati, N. (2017). Role of the CXCL8-CXCR1/2 axis in number GSE111213. cancer and inflammatory diseases. Theranostics 7, 1543-1588. Haas, B. J. and Whited, J. L. (2017). Advances in decoding axolotl limb Supplementary information regeneration. Trends Genet. 33, 553-565. Hrvatin, S., Deng, F., O’Donnell, C. W., Gifford, D. K. and Melton, D. A. (2014). Supplementary information available online at MARIS: method for analyzing RNA following intracellular sorting. PLoS ONE 9, http://dev.biologists.org/lookup/doi/10.1242/dev.169128.supplemental e89459. Kanamoto, T., Mizuhashi, K., Terada, K., Minami, T., Yoshikawa, H. and References Furukawa, T. (2009). Isolation and characterization of a novel plasma membrane Akiyama, R., Kawakami, H., Wong, J., Oishi, I., Nishinakamura, R. and Kawakami, protein, osteoblast induction factor (obif), associated with osteoblast Y. (2015). Sall4-Gli3 system in early limb progenitors is essential for the differentiation. BMC Dev. Biol. 9, 70. development of limb skeletal elements. Proc. Natl. Acad. Sci. USA 112, 5075-5080. Knapp, D., Schulz, H., Rascon, C. A., Volkmer, M., Scholz, J., Nacu, E., Le, M., Arango Duque, G. and Descoteaux, A. (2014). Macrophage cytokines: Novozhilov, S., Tazaki, A., Protze, S. et al. (2013). Comparative transcriptional involvement in immunity and infectious diseases. Front. Immunol. 5, 491. profiling of the axolotl limb identifies a tripartite regeneration-specific gene Attisano, L. and Wrana, J. L. (2013). Signal integration in TGF-beta, WNT, and program. PLoS ONE 8, e61352. Hippo pathways. F1000Prime Rep 5, 17. Kragl, M., Knapp, D., Nacu, E., Khattak, S., Maden, M., Epperlein, H. H. and Baggiolini, M., Dewald, B. and Moser, B. (1997). Human chemokines: an update. Tanaka, E. M. (2009). Cells keep a memory of their tissue origin during axolotl Annu. Rev. Immunol. 15, 675-705. limb regeneration. Nature 460, 60-65. Bolger, A. M., Lohse, M. and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Kragl, M., Roensch, K., Nusslein, I., Tazaki, A., Taniguchi, Y., Tarui, H., Hayashi, T., Agata, K. and Tanaka, E. M. (2013). Muscle and connective tissue progenitor Illumina sequence data. Bioinformatics 30, 2114-2120. populations show distinct Twist1 and Twist3 expression profiles during axolotl limb Bray, N. L., Pimentel, H., Melsted, P. and Pachter, L. (2016). Near-optimal regeneration. Dev. Biol. 373, 196-204. probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525-527. Kumar, A. and Brockes, J. P. (2007). Preparation of cultured myofibers from larval Bryant, D. M., Johnson, K., DiTommaso, T., Tickle, T., Couger, M. B., Payzin- salamander limbs for cellular plasticity studies. Nat. Protoc. 2, 939-947. Dogru, D., Lee, T. J., Leigh, N. D., Kuo, T.-H., Davis, F. G. et al. (2017). A tissue- Kurimoto, T., Yin, Y., Habboub, G., Gilbert, H.-Y., Li, Y., Nakao, S., Hafezi- mapped axolotl de novo transcriptome enables identification of limb regeneration Moghadam, A. and Benowitz, L. I. (2013). Neutrophils express oncomodulin and factors. Cell Rep 18, 762-776. promote optic nerve regeneration. J. Neurosci. 33, 14816-14824. Calve, S., Odelberg, S. J. and Simon, H.-G. (2010). A transitional extracellular Levesque, M., Gatien, S., Finnson, K., Desmeules, S., Villiard, E., Pilote, M., matrix instructs cell behavior during muscle regeneration. Dev. Biol. 344, 259-271. Philip, A. and Roy, S. (2007). Transforming growth factor: beta signaling is Campbell, L. J., Suárez-Castillo, E. C., Ortiz-Zuazaga, H., Knapp, D., Tanaka, essential for limb regeneration in axolotls. PLoS ONE 2, e1227. E. M. and Crews, C. M. (2011). Gene expression profile of the regeneration Li, L., Yan, B., Shi, Y.-Q., Zhang, W.-Q. and Wen, Z.-L. (2012). Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin epithelium during axolotl limb regeneration. Dev. Dyn. 240, 1826-1840. regeneration. J. Biol. Chem. 287, 25353-25360. Chablais, F. and Jazwinska, A. (2010). IGF signaling between blastema and Lindborg, J. A., Mack, M. and Zigmond, R. E. (2017). Neutrophils are critical for wound epidermis is required for fin regeneration. Development 137, 871-879. myelin removal in a peripheral nerve injury model of wallerian degeneration. Currie, J. D., Kawaguchi, A., Traspas, R. M., Schuez, M., Chara, O. and Tanaka, J. Neurosci. 37, 10258-10277. E. M. (2016). Live imaging of axolotl digit regeneration reveals spatiotemporal Love, M. I., Huber, W. and Anders, S. (2014). Moderated estimation of fold change

choreography of diverse connective tissue progenitor pools. Dev. Cell 39, 411-423. and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550. DEVELOPMENT

13 STEM CELLS AND REGENERATION Development (2019) 146, dev169128. doi:10.1242/dev.169128

Makanae, A., Mitogawa, K. and Satoh, A. (2014). Co-operative Bmp- and Fgf- Shibata, E., Yokota, Y., Horita, N., Kudo, A., Abe, G., Kawakami, K. and signaling inputs convert skin wound healing to limb formation in urodele Kawakami, A. (2016). Fgf signalling controls diverse aspects of fin regeneration. amphibians. Dev. Biol. 396, 57-66. Development 143, 2920-2929. McCusker, C., Bryant, S. V. and Gardiner, D. M. (2015). The axolotl limb blastema: Simkin, J., Sammarco, M. C., Marrero, L., Dawson, L. A., Yan, M., Tucker, C., cellular and molecular mechanisms driving blastema formation and limb Cammack, A. and Muneoka, K. (2017). Macrophages are required to coordinate regeneration in tetrapods. Regeneration 2, 54-71. mouse digit tip regeneration. Development 144, 3907-3916. McNeill, H. and Woodgett, J. R. (2010). When pathways collide: collaboration and Singh, B. N., Koyano-Nakagawa, N., Donaldson, A., Weaver, C. V., Garry, M. G. connivance among signalling proteins in development. Nat. Rev. Mol. Cell Biol. and Garry, D. J. (2015). Hedgehog signaling during appendage development and 11, 404-413. regeneration. Genes 6, 417-435. Mizuhashi, K., Kanamoto, T., Ito, M., Moriishi, T., Muranishi, Y., Omori, Y., Slack, F. J., Basson, M., Liu, Z., Ambros, V., Horvitz, H. R. and Ruvkun, G. Terada, K., Komori, T. and Furukawa, T. (2012). OBIF, an osteoblast induction (2000). The lin-41 RBCC gene acts in the C. elegans heterochronic pathway factor, plays an essential role in bone formation in association with between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol. Cell 5, osteoblastogenesis. Dev. Growth Differ. 54, 474-480. 659-669. Mizuhashi, K., Chaya, T., Kanamoto, T., Omori, Y. and Furukawa, T. (2015). Obif, Stanley, E. R. and Chitu, V. (2014). CSF-1 receptor signaling in myeloid cells. Cold a transmembrane protein, is required for bone mineralization and Spring Harb. Perspect Biol. 6, a021857. spermatogenesis in mice. PLoS ONE 10, e0133704. Stewart, R., Rascón, C. A., Tian, S., Nie, J., Barry, C., Chu, L.-F., Ardalani, H., Monaghan, J. R., Epp, L. G., Putta, S., Page, R. B., Walker, J. A., Beachy, C. K., Wagner, R. J., Probasco, M. D., Bolin, J. M. et al. (2013). Comparative RNA-seq Zhu, W., Pao, G. M., Verma, I. M., Hunter, T. et al. (2009). Microarray and cDNA analysis in the unsequenced axolotl: the oncogene burst highlights early gene sequence analysis of transcription during nerve-dependent limb regeneration. expression in the blastema. PLoS Comput. Biol. 9, e1002936. BMC Biol. 7,1. Stocum, D. L. (2017). Mechanisms of urodele limb regeneration. Regeneration 4, Monaghan, J. R., Athippozhy, A., Seifert, A. W., Putta, S., Stromberg, A. J., 159-200. Maden, M., Gardiner, D. M. and Voss, S. R. (2012). Gene expression patterns Stoick-Cooper, C. L., Weidinger, G., Riehle, K. J., Hubbert, C., Major, M. B., specific to the regenerating limb of the Mexican axolotl. Biol. Open 1, 937-948. Fausto, N. and Moon, R. T. (2007). Distinct Wnt signaling pathways have Nacu, E., Gromberg, E., Oliveira, C. R., Drechsel, D. and Tanaka, E. M. (2016). opposing roles in appendage regeneration. Development 134, 479-489. FGF8 and SHH substitute for anterior-posterior tissue interactions to induce limb Sugiura, T., Wang, H., Barsacchi, R., Simon, A. and Tanaka, E. M. (2016). regeneration. Nature 533, 407-410. MARCKS-like protein is an initiating molecule in axolotl appendage regeneration. Nakayama, E., Shiratsuchi, Y., Kobayashi, Y. and Nagata, K. (2011). The Nature 531, 237-240. importance of infiltrating neutrophils in SDF-1 production leading to regeneration Umansky, K. B., Gruenbaum-Cohen, Y., Tsoory, M., Feldmesser, E., of the thymus after whole-body X-irradiation. Cell. Immunol. 268, 24-28. Goldenberg, D., Brenner, O. and Groner, Y. (2015). Runx1 transcription Neff, A. W., King, M. W., Harty, M. W., Nguyen, T., Calley, J., Smith, R. C. and factor is required for myoblasts proliferation during muscle regeneration. PLoS Mescher, A. L. (2005). Expression of Xenopus XlSALL4 during limb development Genet. 11, e1005457. and regeneration. Dev. Dyn. 233, 356-367. Vinarsky, V., Atkinson, D. L., Stevenson, T. J., Keating, M. T. and Odelberg, S. J. Neptune, E. R., Frischmeyer, P. A., Arking, D. E., Myers, L., Bunton, T. E., (2005). Normal newt limb regeneration requires matrix metalloproteinase function. Gayraud, B., Ramirez, F., Sakai, L. Y. and Dietz, H. C. (2003). Dysregulation of Dev. Biol. 279, 86-98. TGF-beta activation contributes to pathogenesis in Marfan syndrome. Nat. Genet. Voss, S. R., Palumbo, A., Nagarajan, R., Gardiner, D. M., Muneoka, K., 33, 407-411. Stromberg, A. J. and Athippozhy, A. T. (2015). Gene expression during the first Nguyen-Chi, M., Laplace-Builhé, B., Travnickova, J., Luz-Crawford, P., Tejedor, 28 days of axolotl limb regeneration I: Experimental design and global analysis of G., Lutfalla, G., Kissa, K., Jorgensen, C. and Djouad, F. (2017). TNF signaling gene expression. Regeneration 2, 120-136. and macrophages govern fin regeneration in zebrafish larvae. Cell Death Dis. 8, Walters, K. B., Green, J. M., Surfus, J. C., Yoo, S. K. and Huttenlocher, A. (2010). e2979. Live imaging of neutrophil motility in a zebrafish model of WHIM syndrome. Blood Nowoshilow, S., Schloissnig, S., Fei, J.-F., Dahl, A., Pang, A. W. C., Pippel, M., 116, 2803-2811. Winkler, S., Hastie, A. R., Young, G., Roscito, J. G. et al. (2018). The axolotl Wang, J. (2018). Neutrophils in tissue injury and repair. Cell Tissue Res. 371, genome and the evolution of key tissue formation regulators. Nature 554, 50-55. 531-539. Paris, A. J., Liu, Y., Mei, J., Dai, N., Guo, L., Spruce, L. A., Hudock, K. M., White, J. R., Lee, J. M., Young, P. R., Hertzberg, R. P., Jurewicz, A. J., Chaikin, Brenner, J. S., Zacharias, W. J., Mei, H. D. et al. (2016). Neutrophils promote M. A., Widdowson, K., Foley, J. J., Martin, L. D., Griswold, D. E. et al. (1998). alveolar epithelial regeneration by enhancing type II pneumocyte proliferation in a Identification of a potent, selective non-peptide CXCR2 antagonist that inhibits model of acid-induced acute lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 311, L1062-L1075. interleukin-8-induced neutrophil migration. J. Biol. Chem. 273, 10095-10098. Pei, W., Tanaka, K., Huang, S. C., Xu, L., Liu, B., Sinclair, J., Idol, J., Varshney, Whited, J. L., Tsai, S. L., Beier, K. T., White, J. N., Piekarski, N., Hanken, J., G. K., Huang, H., Lin, S. et al. (2016). Extracellular HSP60 triggers tissue Cepko, C. L. and Tabin, C. J. (2013). Pseudotyped retroviruses for infecting regeneration and wound healing by regulating inflammation and cell proliferation. axolotl in vivo and in vitro. Development 140, 1137-1146. NPJ Regen. Med. 1, 16013. Williams, C. B., Yeh, E. S. and Soloff, A. C. (2016). Tumor-associated Petrie, T. A., Strand, N. S., Yang, C. T., Rabinowitz, J. S. and Moon, R. T. (2014). macrophages: unwitting accomplices in breast cancer malignancy. NPJ Breast Macrophages modulate adult zebrafish tail fin regeneration. Development 141, Cancer 2, 15025. 2581-2591. Worringer, K. A., Rand, T. A., Hayashi, Y., Sami, S., Takahashi, K., Tanabe, K., Poss, K. D., Shen, J., Nechiporuk, A., McMahon, G., Thisse, B., Thisse, C. and Narita, M., Srivastava, D. and Yamanaka, S. (2014). The let-7/LIN-41 pathway Keating, M. T. (2000). Roles for Fgf signaling during zebrafish fin regeneration. regulates reprogramming to human induced pluripotent stem cells by controlling Dev. Biol. 222, 347-358. expression of prodifferentiation genes. Cell Stem Cell 14, 40-52. Powell, D., Tauzin, S., Hind, L. E., Deng, Q., Beebe, D. J. and Huttenlocher, A. Wu, C.-H., Tsai, M.-H., Ho, C.-C., Chen, C.-Y. and Lee, H.-S. (2013). De novo (2017). Chemokine signaling and the regulation of bidirectional leukocyte transcriptome sequencing of axolotl blastema for identification of differentially migration in interstitial tissues. Cell Rep 19, 1572-1585. expressed genes during limb regeneration. BMC Genomics 14, 434. Randell, A. and Daneshtalab, N. (2017). Elastin microfibril interface-located protein Xu, S., Webb, S. E., Lau, T. C. K. and Cheng, S. H. (2018). Matrix 1, transforming growth factor beta, and implications on cardiovascular metalloproteinases (MMPs) mediate leukocyte recruitment during the complications. J. Am. Soc. Hypertens 11, 437-448. inflammatory phase of zebrafish heart regeneration. Sci. Rep. 8, 7199. Ritenour, A. M. and Dickie, R. (2017). Inhibition of vascular endothelial growth Yokoyama, H., Yonei-Tamura, S., Endo, T., Izpisúa Belmonte, J. C., Tamura, K. factor receptor decreases regenerative angiogenesis in axolotls. Anat. Rec. 300, and Ide, H. (2000). Mesenchyme with fgf-10 expression is responsible for 2273-2280. regenerative capacity in Xenopus limb buds. Dev. Biol. 219, 18-29. Rodrigo Albors, A., Tazaki, A., Rost, F., Nowoshilow, S., Chara, O. and Tanaka, Yokoyama, H., Ide, H. and Tamura, K. (2001). FGF-10 stimulates limb regeneration E. M. (2015). Planar cell polarity-mediated induction of neural stem cell expansion ability in Xenopus laevis. Dev. Biol. 233, 72-79. during axolotl spinal cord regeneration. Elife 4, e10230. Yu, L., Yan, M., Simkin, J., Ketcham, P. D., Leininger, E., Han, M. and Muneoka, Russo, R. C., Garcia, C. C., Teixeira, M. M. and Amaral, F. A. (2014). The CXCL8/ K. (2014). Angiogenesis is inhibitory for mammalian digit regeneration. IL-8 chemokine family and its receptors in inflammatory diseases. Expert Rev. Regeneration 1, 33-46. Clin. Immunol. 10, 593-619. Yun, M. H., Davaapil, H. and Brockes, J. P. (2015). Recurrent turnover of Sandoval-Guzman, T., Wang, H., Khattak, S., Schuez, M., Roensch, K., Nacu, E., senescent cells during regeneration of a complex structure. Elife 4, e05505. Tazaki, A., Joven, A., Tanaka, E. M. and Simon, A. (2014). Fundamental Zeilhofer, H. U. and Schorr, W. (2000). Role of interleukin-8 in neutrophil signaling. differences in dedifferentiation and stem cell recruitment during skeletal muscle Curr. Opin Hematol. 7, 178-182. regeneration in two salamander species. Cell Stem Cell 14, 174-187. Zhu, W., Pao, G. M., Satoh, A., Cummings, G., Monaghan, J. R., Harkins, T. T., Satoh, A., Makanae, A., Hirata, A. and Satou, Y. (2011). Blastema induction in Bryant, S. V., Randal Voss, S., Gardiner, D. M. and Hunter, T. (2012). Activation aneurogenic state and Prrx-1 regulation by MMPs and FGFs in Ambystoma of germline-specific genes is required for limb regeneration in the Mexican axolotl.

mexicanum limb regeneration. Dev. Biol. 355, 263-274. Dev. Biol. 370, 42-51. DEVELOPMENT

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