Aztekin C , Hiscock TW , Butler R , De Jesús Andino F , Robert J , Gurdon JB , Jullien J .

Wellcome Trust , R24 AI059830 NIAID NIH HHS , 101050/Z/13/Z Wellcome Trust , 105031/D/14/Z Wellcome Trust , C6946/A14492 Cancer Research UK, 092096/Z/10/Z Wellcome Trust

             inflammatory response
             regeneration

	Fig 1. Depleting myeloid lineage impedes Xenopus tail regeneration. (A) Experimental design for assessing the effect of myeloid lineage depletion on regeneration. Regeneration-competent tadpoles were injected with either control (Encapsome), or clodronate containing lipids (Clodrosome) before tail amputation. Regeneration efficiency was assessed at 7 days post tail amputation (dpa). (B) Clodrosome injection decreases myeloid gene expression compared to Encapsome injection. Expression of myeloid lineage genes after Encapsome or Clodrosome injections were assessed by RT-qPCR analysis on amputated tails. All values were normalized to that of the Uninjected controls. n ≥ 3 biological replicates for each gene expression quantification, error bars represent ±standard error of the mean (s.e.m.). (C) Representative images of Tg(Slurp1l:EGFP) tadpoles post Encapsome or Clodrosome injection. Decrease in Slurp1l:EGFP myeloid lineage signal is particularly seen at dorsal and ventral vein regions. Scale bar= 250 µM. (D) Regeneration outcomes at 7 dpa in uninjected, Encapsome or Clodrosome injected regeneration-competent tadpoles. Samples were obtained from three biological replicates: Uninjected n = 30; Encapsome n= 34; Clodrosome n= 46.
	Fig 2. The myeloid lineage is required for tissue remodelling, reduction of apoptosis levels, and relocalization of regeneration-organizing-cells. (A) (Left) Representative images of remodelling events occurring at 1 dpa in Encapsome or Clodrosome injected regeneration-competent tadpoles. Red lines indicate the posterior somitic region. Post amputation histolysis and remodelling of tissue is impaired in Clodrosome injected tadpoles. Scale bar= 250 µm. (Right) Remodelling index quantification. All samples were obtained from at least three biological replicates: Encapsome n= 39; Clodrosome n= 37. Each dot represents an individual tadpole. Student`s t-test was used to assess statistical significance, the red line denotes the median, *p<0.001. (B) (Left) Representative images of apoptosis occurring at 1 dpa in Encapsome or Clodrosome injected regeneration-competent tadpoles. The red arrows indicate the location of the Lysosensor signal used to report apoptosis. Scale bar= 250 µm. (Right) Apoptotic area quantification. All samples were obtained from at least three biological replicates: Encapsome n= 49; Clodrosome n= 53. Student`s t-test was used to assess statistical significance, red line denotes median, *p<0.001.A.U.: arbitrary unit. (C) (Left) Representative images of ROCs relocalization at 1 dpa for Encapsome or Clodrosome injected regeneration-competent tadpoles. The 7pbin:EGFP transgenic line was used to assess ROCs relocalization. Red arrows show the location of the leading ROC cells. Scale bar= 250 µm. (Right) ROCs relocalization quantification. All samples were obtained from at least three biological replicates: Encapsome n= 19; Clodrosome n= 16. Student`s t-test was used to assess statistical significance; red line denotes median. *p<0.001.
	Fig 3. Sequential involvement of apoptosis, tissue remodelling and regeneration- organising- cells mobilisation following tail amputation. (A) Experimental setup for assessing the relationship between the cellular mechanisms controlled by amputation induced myeloid lineage activity. Cellular events downstream of myeloid lineage activity were assessed after drug treatments. NS3694 (apoptosis inhibitor) and 4-MU (hyaluronic acid synthesis inhibitor) were used to block apoptosis, and tissue remodelling, respectively. (B) ROCs relocalization levels at 1 dpa in DMSO control, or after 4-MU, or NS3694 treatment in regeneration-competent tadpoles. All samples were obtained from two biological replicates: DMSO n = 13; 4-MU n= 11; NS3694 n= 17. One-way ANOVA was used to assess statistical significance, red line denotes median, *p<0.001. (C) Apoptotic area at 1 dpa in DMSO or 4-MU treated regeneration-competent tadpoles. All samples were obtained from four biological replicates: DMSO n = 29; 4-MU n= 25. Student`s t-test was used to assess statistical significance, red line denotes median, *p<0.001. (D) Quantification of remodelling index at 1 dpa in, DMSO, 4-MU or NS3694 treatment in regeneration-competent tadpoles. All samples were obtained from at least 3 biological replicates: DMSO n = 32; 4-MU n: 43, NS3694 40 µM n= 26; NS3694 60 µM n= 18. One-way ANOVA was used to assess statistical significance, red line denotes median, *p<0.001. (E) Epistasis experiments suggest the myeloid lineage controls apoptosis levels which influences tissue/ECM remodelling through HA deposition and finally, ROCs relocalization.
	Fig 4. A post amputation shift from inflammatory to reparative myeloid activity characterises regeneration-competent tadpoles. (A) Heatmap showing known myeloid, reparative, and inflammatory gene expression levels in the Xenopus tail cell atlas myeloid cell clusters. Values for each gene were normalized to that of the highest expressing cluster. Data is reanalysed from Aztekin C. et al. (2019). (B) Relative abundance of inflammatory and reparative myeloid lineage cells post amputation in regeneration-competent and -incompetent tadpoles. Data is reanalysed from Aztekin C. et al. (2019). (C) Myeloid cell cluster 1 and 2 gene expressions were tested 1 day after treatment of regeneration-incompetent tadpoles with immune suppressing drugs, FK506 or Celastrol. n ≥ 3 biological replicates for each gene expression quantification, error bars represent mean ±s.e.m..
	Fig 5. Suppression of inflammation enables the emergence of a regeneration- permissive environment in regeneration-incompetent tadpoles. (A) Remodelling index at 1 dpa in FK506 or Celastrol-treated regeneration-incompetent tadpoles. All samples were obtained from 3 biological replicates: DMSO n = 22; FK506 n= 21; Celastrol n= 23. Red line denotes median, and black lines denote quartiles. Note that regeneration-competent 1 dpa DMSO data from Fig 3B were replotted for comparison. DMSO vs Celastrol: p<0.05 (t-Test), p<0.01 (Mann Whittney U test), DMSO vs FK506: p<0.01 (tTest), p<0.01 (Mann Whittney U test), (B) Apoptotic area levels at 1 dpa in FK506 or Celastrol treated regeneration-incompetent tadpoles. All samples were obtained from 3 biological replicates: DMSO n = 19; FK506 n= 20; Celastrol n= 20. Red line denotes median, and black lines denote quartiles. Note that regeneration-competent 1 dpa DMSO data from Fig 3A were replotted for comparison. DMSO vs Celastrol: p<0.05 (t-Test), p=0.065 (Mann Whittney U test), DMSO vs FK506: p<0.01 (t-Test), p<0.01 (Mann Whittney U test), (C) ROCs relocalization levels at 2 dpa in FK506 or Celastrol treated regeneration-incompetent tadpoles. All samples were obtained from two biological replicates: DMSO n = 10; FK506 n= 15; Celastrol n= 10. Red line denotes median, and black lines denote quartiles. (D) Immune suppressing drugs mediated rescue of regeneration requires apoptosis and tissue remodelling. Regeneration–incompetent tadpoles were treated with FK506 or Celastrol either alone or in combination with inhibitor of tissue remodelling (4-MU to block HA pathway) or inhibitor of apoptosis (NS3694). All samples were obtained from 4 biological replicates: FK506 + DMSO n = 33; FK506 + 4-MU n= 20; FK506 + NS3694 n= 13; Celastrol + DMSO n = 29; Celastrol + 4-MU n= 15; Celastrol + NS3694 n= 28. Red line denotes median, and black lines denote quartiles. (E) Model of myeloid lineage involvement in regeneration-competent and – incompetent tadpoles. The myeloid lineage activity controls hierarchical cellular mechanisms that create a regeneration-permissive environment required for regeneration.
	Fig S1. Clodrosome injection decreases myeloid lineage and inhibits regeneration. (A) Myeloid depletion efficiency following Encapsome or Clodrosome injections was assessed by quantifying the amount of Slurp1l expressing cell in the tail vein regions in regeneration competent tadpoles 2 days after the last liposome injection. Student`s t-test was used to assess statistical significance; *p<0.05. (B) Average regeneration index in Uninjected, Encapsome, and Clodrosome injected tadpoles. n = 3 biological replicates for each sample, error bars represent±s.e.m.. One-way ANOVA was used to assess statistical significance, *p<0.05. Note that this measurement was carried out on the same data as in Fig 1D.
	Fig S2. Genetic ablation of Slurp1l expressing cells decreases regeneration. (A) Experimental design for ablating Slurp1l expressing myeloid cells using NTR/MTZ method. F0 transgenic tadpoles that express NTR under the control of Slurp1l promoter were generated. MTZ was added one day before, immediately after, and two-day after amputation in regeneration-competent tadpoles. Regeneration efficiency was assessed at 7 dpa. (B) NTR/MTZ mediated ablation of Slurp1 expressing cells decreases myeloid cell lineage expression. Myeloid lineage gene expressions were assessed by RT-qPCR in Slurp1l:NTR expressing F0 tadpoles 1 day post MTZ treatment. Tail samples were collected at the time of amputation. All values were normalized to that of the controls without MTZ treatment. n ≥ 3 biological replicates for each gene expression quantification, error bars represent ±s.e.m.. ttest was used to assess statistical significance; *p<0.001.(C) Regeneration-outcomes at 7 dpa following Slurp1l mediated NTR/MTZ myeloid cell ablation. All samples were obtained from three independent biological batches: Empty without (wo) MTZ n = 31; Empty with (w) MTZ n= 34; Slurp1l:NTR wo MTZ n= 47; Slurp1l:NTR w MTZ n= 44.(D) Average regeneration index at 7 dpa following Slurp1l mediated NTR/MTZ myeloid cell ablation. n = 3 biological replicates for each sample, error bars represent mean ±s.e.m. Student`s t-test was used to assess statistical significance, *p<0.05. Note that this measurement was carried out on the same data as in Fig 2C. (E) Slurp1l promoter gets activated upon amputation in non-myeloid lineage cells. (Left) In an intact tail Tg(Slurp1l:EGFP) transgenic line labels cells that resemble macrophages. (Middle) Upon tail amputation at 1 dpa Tg(Slurp1l:EGFP) signal is also seen in non-myeloid lineages. (Right) Examples of cells with different morphologies that are labelled by Slurp1l promoter. All samples were stained against EGFP (green), and DAPI (blue). Number 5 is also stained for TP63. ROCs reside at the edges of TP63 positive epidermal cells, and Slurp1l signal can also be seen in these cells. Scale bar= 250 µm for intact tail and 1 dpa image, 50 µm for zoomed images
	Fig S3. CRISPR/Cas9 mediated Spib knock-out decreases myeloid lineage gene expression and impairs regeneration. (A) Experimental design for generating F0 tadpoles with a defective myeloid lineage via CRISPR/Cas9 mediated Spib knock-out and for testing regeneration in regeneration competent tadpoles. Regeneration efficiency was assessed at 7 dpa. (B) Myeloid lineage gene expression was assessed by RT-qPCR in F0 tadpoles injected with guide RNAs targeting either Tyrosinase (gTyr) as control, or Spib (gSpib). Spib-1 and Spib-2 denotes two different guide RNA mixes. Samples were collected at the time of tail amputation. All values were normalized to guide RNAs against Tyrosinase injected sample. Guide RNAs targeting Spib were able to decrease myeloid lineage gene expression. n ≥ 3 biological replicates for each gene expression quantification, error bars represents ±s.e.m.. (C) Guide RNA efficiencies were assessed via TIDE analysis. At least 10 tails of tadpoles injected with gRNAs targeting Tyrosinase or Spib mixes were used for TIDE. (D) Regeneration- outcomes at 7 dpa for guide RNA injected tadpoles. (E) Regeneration was assessed in individual tadpoles by comparing regenerated tail to body length ratio at the 7 dpa. All samples were obtained from at least three independent biological batches: gTyr n = 39; gSpib-1 n= 24; gSpib-2 n= 11. One-way ANOVA used to assess statistical significance, red line denotes median, *p<0.05, **p<0.001. Note that this measurement was carried out on the same data as in Fig S3D. (F) Myeloid lineage gene expressions were assessed by RT-qPCR in F0 tadpoles injected with guide RNAs targeting either Tyrosinase (as control), or Spib1. Spib-1 and Spib-2 denotes two different guideRNA mix. Samples were collected 7 day post tail amputation. All values were normalized to one sample of gTyr sample. GuideRNAs targeting Spib had no major difference in myeloid gene expression compared to gTyr control. n = 3 biological replicate for each gene expression quantification, and graphed, representing mean with ±s.e.m..
	Fig S4. Remodelling index captures morphological differences between the amputation plane of regeneration-competent and -incompetent tadpoles. (A) Schematics for amputation plane morphology immediately after amputation (left), and examples at 24 hours post amputation. Upon amputation, the somitic region is exposed to the outside medium and present a rectangular shape. Fin regions do not show any inward curvatures towards the amputation plane. Within 24 hours, somitic regions undergo histolysis and muscle tissue degeneration (Beck et al., 2003), while fin cells from the ventral and dorsal sides of the tail start covering the amputation plane. In regeneration-incompetent tadpoles, limited changes are observed (right, bottom). (B) Ratio of the length of the amputation plane surface to length of open somitic region used to estimate “remodelling index”. (C) Examples of images with corresponding regeneration index scores. (D) Representative images of regeneration-competent and –incompetent tadpoles, 0-hour post amputation and 24 hours post amputations. Competent tadpole present extensive remodelling of the somatic and fin tissues. Scale bar= 250 µm. (E) Quantification of remodelling index for regenerationcompetent and –incompetent 0 hpa, and 24 hpa samples. All samples were obtained from at least two biological replicates: Regeneration-competent 0 hpa n= 15; Regeneration competent 24 hpa n= 9; Regeneration-incompetent 0 hpa n= 15; Regeneration-incompetent 24 hpa n= 28. One-way ANOVA used to assess statistical significance, red line denotes median, *p<0.05.
	Fig S5. Lysosensor as an apoptosis reporter in amputated tail. (A) Representative images for multiple reagents tested to detect apoptosis levels at 1 dpa in regeneration-competent tadpoles. Caspase 3/7 sensor and Lysotracker, two commonly used apoptosis detection methods present multiple off target signal in the tadpole fin region. Meanwhile, Mitotracker, and Lysosensor label more specifically the regions previously reported to have amputation induced apoptosis (Tseng et al., 2007). Scale bar= 250 µm. (B) Apoptosis inhibitor (NS3694, 60 µM) treatment decreases Lysosensor signal at the amputation plane. (C) Apoptosis area quantitation in control and 20, 40 ,60 µM NS3694 treated regeneration-competent tadpoles at 1 dpa. NS3694 treatments diminished the Lysosensor signal at 1 dpa. DMSO n = 35; 20 µM NS3694 n= 8; 40 µM NS3694 n= 15; 60 µM NS3694 n= 8. One-way ANOVA used to assess statistical significance, red line denotes median, *p<0.05. (D) Apoptotic area at 1 dpa in regeneration-competent and –incompetent tadpoles. Observed apoptosis levels are in agreement with the previously published reports (Tseng et al., 2007). All samples were obtained from at least two biological replicates: Regeneration-competent n = 29; Regeneration-incompetent n = 28. Student`s t-test was used to assess statistical significance, red line denotes median, *p<0.001.
	Fig S6. CRISPR/Cas9 Spib knock-out impairs relocalization of regeneration-organizing cells, apoptosis levels, and tissue remodelling (A) Quantification of ROCs relocalization at 1 day post amputation in regeneration-competent gTyr control and gSpib injected tadpoles. All samples were obtained from four independent biological batches: gTyr n= 39; gSpib-1 n= 37; gSpib-2 n=20. One-way ANOVA used to assess statistical significance, red line denotes median. *p<0.05, **p<0.001. (B) Quantification of apoptosis levels at 1 day post amputation in regeneration-competent gTyr control and gSpib injected tadpoles. All samples were obtained at least from two independent biological batches except gSpib-2 was obtained from one batch: gTyr n= 21; gSpib-1 n= 14; gSpib-2 n=5. Oneway ANOVA used to assess statistical significance, red line denotes median. *p<0.05. (C) Quantification of remodelling index for the samples at 1 day post amputation in regeneration competent gTyr control and gSpib injected tadpoles. All samples were obtained from two biological replicates: gTyr n= 24; gSpib-1 n= 17; gSpib-2 n=13. One-way ANOVA used to assess statistical significance, red line denotes median, *p<0.05.
	Fig S7. Adult X. laevis frogs infected with the ranavirus FV3 or stimulated with heatshock killed bacteria upregulates inflammatory and reparative myeloid gene expression in peritoneal leukocytes. (A) Experimental design for assessing the gene expression of different myeloid clusters upon Frog virus 3 infection (Large double strand DNA iridovirus). (B) Myeloid lineage gene expressions were assessed 1, 3, 6 days post-FV3 infection in peritoneal leukocytes. Inflammatory and reparative myeloid marker genes were enriched upon infection. (C) Experimental design for assessing the gene expression of different myeloid clusters of elicited peritoneal macrophages upon heat-killed E. coli stimulation. MHC Class II+, MHC Class II+/MPEG+, or double negative populations were sorted by flow cytometry from peritoneal leukocytes and tested for myeloid gene expressions. (D) Myeloid lineage gene expressions were assessed 3 days post-elicitation of in peritoneal macrophages by heat-killed E. coli. (Left) Myeloid 1 and (Right) Myeloid 2 cluster genes are enriched upon infection.
	Fig S8. Immune suppressing drugs FK506 and Celastrol rescue the no-regeneration phenotype. (A) Regeneration-outcomes at 7 dpa in FK506, or Celastrol treated tadpoles in regeneration incompetent tadpoles. All samples were obtained from five biological replicates: DMSO n=40; Celastrol n=37; FK506 n=37. (B) Regeneration was assessed in individual tadpoles by comparing regenerated tail to body length ratio at 7 dpa. Note that this measurement was carried out on the same data as in Fig 8A. (C) ROCs mobilisation initiates at 1 dpa in immune suppressing drugs treated regeneration-incompetent tadpoles. Quantification of ROCs relocalization at 1 day post amputation. All samples were obtained from two biological replicates: DMSO n = 13; FK506 n= 10; Celastrol n= 12. Red bar denotes the mean. (D) Immune suppressing drugs mediated rescue of regeneration requires apoptosis and tissue remodelling. Regeneration-outcome at 7 dpa in regeneration-incompetent tadpoles treated with FK506 or Celastrol either alone or in combination with inhibitor of tissue remodelling (4-MU to block HA pathway) or inhibitor of apoptosis (NS3694). Note that this measurement was carried out on the same data as in Fig 5D.
	Fig. 1. Depleting the myeloid lineage impedes Xenopus tail regeneration. (A) Experimental design for assessing the effect of myeloid lineage depletion on regeneration. Regeneration-competent tadpoles were injected with either control (Encapsome), or clodronate-containing lipids (Clodrosome) before tail amputation. Regeneration efficiency was assessed at 7 days post-tail amputation (dpa). (B) Clodrosome injection decreases myeloid gene expression compared with Encapsome injection. Expression of myeloid lineage genes after Encapsome or Clodrosome injections was assessed by RT-qPCR analysis on amputated tails. All values were normalized to that of the uninjected controls. n≥3 biological replicates for each gene expression quantification, error bars represent s.e.m. (C) Representative images of Tg(slurp1l:EGFP) tadpoles post Encapsome or Clodrosome injection. A decrease in the slurp1l:EGFP myeloid lineage signal is particularly seen at dorsal and ventral vein regions. Scale bars: 250 µm. (D) Regeneration outcomes at 7 dpa in uninjected, Encapsome-injected or Clodrosome-injected regeneration-competent tadpoles. Samples were obtained from three biological replicates: uninjected n=30; Encapsome n=34; Clodrosome n=46.
	Fig. 2. The myeloid lineage is required for tissue remodelling, reduction of apoptosis levels, and relocalization of regeneration-organizing cells. (A) Left: Representative images of remodelling events occurring at 1 dpa in Encapsome- or Clodrosome-injected regeneration-competent tadpoles. Red lines indicate the posterior somitic region. Post-amputation histolysis and remodelling of tissue is impaired in Clodrosome-injected tadpoles. Scale bars: 250 µm. Right: Remodelling index quantification. All samples were obtained from at least three biological replicates: Encapsome n=39; Clodrosome n=37. Student's t-test was used to assess statistical significance; *P<0.001. (B) Left: Representative images of apoptosis occurring at 1 dpa in Encapsome- or Clodrosome-injected regeneration-competent tadpoles. The red arrowheads indicate the location of the LysoSensor signal used to report apoptosis. Scale bars: 250 µm. Right: Apoptotic area quantification. All samples were obtained from at least three biological replicates: Encapsome n=49; Clodrosome n=53. Student's t-test was used to assess statistical significance; *P<0.001. A.U., arbitrary unit. (C) Left: Representative images of ROC relocalization at 1 dpa for Encapsome- or Clodrosome-injected regeneration-competent tadpoles. The 7pbin:EGFP transgenic line was used to assess ROCs relocalization. Red arrowheads show the location of the leading ROC cells. Scale bars: 250 µm. Right: ROC relocalization quantification. All samples were obtained from at least three biological replicates: Encapsome n=19; Clodrosome n=16. Student's t-test was used to assess statistical significance; *P<0.001. In graphs, each dot represents individual tadpoles; red line denotes median.
	Fig. 3. Sequential involvement of apoptosis, tissue remodelling and regeneration-organizing cell mobilization following tail amputation. (A) Experimental setup for assessing the relationship between the cellular mechanisms controlled by amputation-induced myeloid lineage activity. Cellular events downstream of myeloid lineage activity were assessed after drug treatments. NS3694 (apoptosis inhibitor) and 4-MU (hyaluronic acid synthesis inhibitor) were used to block apoptosis, and tissue remodelling, respectively. (B) ROC relocalization levels at 1 dpa in DMSO control, or after 4-MU or NS3694 treatment in regeneration-competent tadpoles. All samples were obtained from two biological replicates: DMSO n=13; 4-MU n=11; NS3694 n=17. One-way ANOVA was used to assess statistical significance; *P<0.001. (C) Apoptotic area at 1 dpa in DMSO- or 4-MU-treated regeneration-competent tadpoles. All samples were obtained from four biological replicates: DMSO n=29; 4-MU n=25. Student's t-test was used to assess statistical significance; *P<0.001. n.s., not significant. A.U., arbitrary unit. (D) Quantification of remodelling index at 1 dpa in DMSO-, 4-MU- or NS3694-treated regeneration-competent tadpoles. All samples were obtained from at least three biological replicates: DMSO n=32; 4-MU n: 43, NS3694 40 µM n=26; NS3694 60 µM n=18. One-way ANOVA was used to assess statistical significance; *P<0.001. In B-D, each dot represents individual tadpoles; red line denotes median. (E) Epistasis experiments suggest that the myeloid lineage controls apoptosis levels, which influence tissue/ECM remodelling through HA deposition and, finally, ROCs relocalization.
	Fig. 4. A post-amputation shift from inflammatory to reparative myeloid activity characterizes regeneration-competent tadpoles. (A) Heat map showing known myeloid, reparative and inflammatory gene expression levels in the Xenopus tail cell atlas myeloid cell clusters. Values for each gene were normalized to that of the highest expressing cluster. Data were re-analysed from Aztekin et al. (2019). (B) Relative abundance of inflammatory and reparative myeloid lineage cells post-amputation in regeneration-competent and -incompetent tadpoles. Data were re-analysed from Aztekin et al. (2019). (C) Myeloid cell cluster 1 and 2 gene expressions were tested 1 day after treatment of regeneration-incompetent tadpoles with the immune-suppressing drugs FK506 or Celastrol. n>3 biological replicates for each gene expression quantification; error bars represent s.e.m.
	Fig. 5. Suppression of inflammation enables the emergence of a regeneration-permissive environment in regeneration-incompetent tadpoles. (A) Remodelling index at 1 dpa in FK506- or Celastrol-treated regeneration-incompetent tadpoles. All samples were obtained from three biological replicates: DMSO n=22; FK506 n=21; Celastrol n=23. Red line denotes median, and black lines denote quartiles. Note that regeneration-competent 1 dpa DMSO data from Fig. 3B were re-plotted for comparison. DMSO versus Celastrol: P<0.05 (t-test), P<0.01 (Mann–Whitney U-test); DMSO versus FK506: P<0.01 (t-test), P<0.01 (Mann–Whitney U-test). (B) Apoptotic area levels at 1 dpa in FK506- or Celastrol-treated regeneration-incompetent tadpoles. All samples were obtained from three biological replicates: DMSO n=19; FK506 n=20; Celastrol n=20. Red line denotes median, and black lines denote quartiles. Note that regeneration-competent 1 dpa DMSO data from Fig. 3A were re-plotted for comparison. DMSO versus Celastrol: P<0.05 (t-test), P=0.065 (Mann–Whitney U-test); DMSO versus FK506: P<0.01 (t-test), P<0.01 (Mann–Whitney U-test). A.U., arbitrary unit. (C) ROC relocalization levels at 2 dpa in FK506- or Celastrol-treated regeneration-incompetent tadpoles. All samples were obtained from two biological replicates: DMSO n=10; FK506 n=15; Celastrol n=10. Red line denotes median, and black lines denote quartiles. (D) Immune-suppressing drug-mediated rescue of regeneration requires apoptosis and tissue remodelling. Regeneration-incompetent tadpoles were treated with FK506 or Celastrol either alone or in combination with an inhibitor of tissue remodelling (4-MU to block the HA pathway) or an inhibitor of apoptosis (NS3694). All samples were obtained from four biological replicates: FK506+DMSO n=33; FK506+4-MU n=20; FK506+NS3694 n=13; Celastrol+DMSO n=29; Celastrol+4-MU n=15; Celastrol+NS3694 n=28. Red line denotes median, each dot represents individual tadpoles. (E) Model of myeloid lineage involvement in regeneration-competent and -incompetent tadpoles. The myeloid lineage activity controls hierarchical cellular mechanisms that create the regeneration-permissive environment required for regeneration.

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