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Fig. 1
Early wound closure and accumulation of progenitor cells are hallmarks of successful limb regeneration in tadpoles of P. maculatus. A Comparative morphology of post amputated limb blastemas of tadpole and froglet (Not in scale); Black lines indicate plane of amputation; B: 1 dpa tadpole limb; C 1 dpa froglet limb; D 3 dpa tadpole limb; E 3 dpa froglet limb; F 5 dpa tadpole limb; G 5 dpa froglet limb. we- wound epithelium, b-blastema, le- larval epithelium, ae- adult epithelium, st-limb stump. Bars B-G = 100 μm, Dashed lines in B-G showing plane of amputation
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Fig. 2
Differential gene expression analyses. A Heat map and B Volcano plot of normalized transcript levels between intact froglet and tadpole hindlimbs. C Heat map and D Volcano plot of normalized transcript levels between intact froglet hindlimb and 3 dpa froglet hindlimb blastema; E Heat map and F Volcano plot of normalized transcript levels between intact tadpole hindlimb and 3 dpa tadpole hindlimb blastema; G Heat map and H Volcano plot of normalized transcript levels between 3 dpa froglet and tadpole hindlimb blastemas
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Fig. 3
Differential expression patterns of transcripts by qPCR at different days post amputation tadpole blastemas relative to froglet blastemas. log2 values of normalized expression of TBFSG (A), KRT10 (B), NNMT-like (C), MMP-18like (D), FRLP (E), TGFβigh3 (F), MDK (G), IL-8 like (H) of 1,3 and 5 dpa tadpole blastemas relative to same day froglet blastemas. Asterisks indicate statistical significance (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001) Data are the mean of three biological replicates, each one with three technical replicates
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Fig. 4
Glycosaminoglycan positive (GAG+) cells in tadpole and froglet hindlimb blastemas. A 1 dpa tadpole blastema; B 3 dpa tadpole blastema; C 5 dpa tadpole blastema showing formation of extracellular matrix (m) in the blastema; D 1 dpa froglet blastema showing absence of wound epithelium and blastema; E 3 dpa froglet blastema showing an adult type epidermis underlined by basement membrane enclosing the wound area.; F 5 dpa froglet blastema; Bars in A-C = 50 μm, D-F = 25 μm we- wound epithelium; b- blastema, st- limb stump, bm- basement membrane, m- extracellular matrix. Black arrows showing GAG+ cells
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Fig. 5
Longitudinal sections through tadpole limb with 1 dpa blastema showing dermal GAG+ cells to be NAE+. A Pentachrome stained section showing GAG+ cells (Black arrowheads), B NAE stained serial section of (A) showing myeloid cells (NAE+) (Red arrowheads). Black arrows show the plane of amputation. Bars = 125 μm. b- blastema, st-stump
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Fig. 6
Expression of epiphycan (EPYC) during tadpole limb regeneration. A log2 values of normalized expression of EPYC in the 1,3 and 5 dpa tadpole blastemas relative to the same day froglet blastemas. B-E RNA in situ hybridization of EPYC in the tadpole hindlimb and blastemas. Bars in B = 250 μm; C-E, =50 μm. Black arrows indicate positive hybridization and red arrows indicate no hybridization
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Fig. 7
Expression of indole ethylamine n methyl transferase (INMT) during tadpole limb regeneration. A log2 values of normalized expression of INMT in the 1,3 and 5 dpa tadpole blastemas relative to the same day froglet blastemas. B-E RNA in situ hybridization of EPYC in the tadpole hindlimb and blastemas. Bars in B = 250 μm; C-E = 50 μm. Black arrows indicate positive hybridization and red arrows indicate no hybridization
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Fig. 8
Expression of lymphoid enhancer binding factor 1(LEF1) during tadpole limb regeneration. A log2 values of normalized expression of LEF1 in the 1,3 and 5 dpa tadpole blastemas relative to the same day froglet blastemas. B-E RNA in situ hybridization of LEF1in the tadpole hindlimb and blastemas. Bars in C = 250 μm; D-F = 50 μm. Black arrows indicate positive hybridization and red arrows indicate no hybridization
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Fig. 9
Expression of Sal-like 4 (SALL4) during tadpole limb regeneration. A log2 values of normalized expression of SALL4 in the 1,3 and 5 dpa tadpole blastemas relative to the homeostatic tadpole limb. B-E RNA in situ hybridization of SALL4 in the tadpole 1,3 and 5 dpa blastemas. Bars in B = 250 μm; C-E = 50 μm. Black arrows indicate mRNA hybridisation. Red arrows indicate no hybridisation
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SFig.1. Bioinformatics work flow
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SFig.2. Hypomorphic limbs (Red arrows) or no regeneration (White arrow) in P. maculatus on limb amputation at NF stage 57-59 tadpoles.
A: Reduced shank and incomplete differentiation of toes in the right hindlimb.The left hindlimb is unamputated. B: Reduced shank and incomplete number of toes in the left hindlimb.
C: Bent left hindlimb and no regeneration in the right hindlimb.
D: Reduced shank and incomplete differentiation of toes in the right hindlimb. Figures not to scale
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SFig.3. Regenerating limb blastemas of tadpole of P. maculatus at different intervals post amputation.
A: 6 hours post amputation, B: 1 day post amputation, C- 3 days post amputation, D- 5 days post amputation, E- 7 days post amputation, G- 10 days post amputation. we- wound epithelium; b- blastema; st- limb stump; d- developing digit
Blue arrowheads mark the plane of amputation Bar= A-E=100μm,F=250μm
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SFig.4. Pie-chart showing maximum hits of P. maculatus CDS to Nanorana parkeri.
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SFig.5. GO annotated sequences and WEGO plot of genes of intact froglet hindlimb of P. maculatus.
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SFig.6. GO annotated sequences and WEGO plot of genes of 3dpa post amputated froglet hindlimb blastema of P. maculatus.
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SFig.7. GO annotated sequences and WEGO plot of genes of intact hindlimb of stage 56 tadpoles of P. maculatus.
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SFig.8. GO annotated sequences and WEGO plot of 3dpa hindlimb blastema of stage 56 tadpoles of P. maculatus.
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S Fig.9: Summary of the differentially expressed genes of various combinations. Combination 1: Froglet limb vs Tadpole limb.
Combination 2: Froglet limb vs 3 dpa froglet limb blastema Combination 3: Tadpole limb vs 3 dpa tadpole limb blastema
Combination 4: 3 dpa froglet limb blastema vs 3 dpa tadpole limb blastema
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S Fig.10: Pearson’s correleation analysis between DGE seq and real time PCR data showing positive correlation.
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SFig.11. Differential expression patterns of transcripts by qPCR at different days post amputation tadpole blastemas relative to homeostatic tadpole limb. log2 values of normalized expression of TBFSG (A), KRT10 (B), NNMT-like (C), MMP-18-like (D), FRLP (E), TGFβigh3 (F), MDK (G),IL-8-like (H), EPYC (I), INMT (J), LEF1 (K) of 1,3 and 5dpa tadpole blastemas relative to homeostatic tadpole limb. Asterisks indicate statistical significance (*:p<0.05; **: p< 0.01; ***: p<0.001; ****: p<0.0001)
Data are the mean of three biological replicates, each one with three technical replicates.
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SFig.12. Differential expression patterns of transcripts by qPCR at different days post amputation froglet blastemas relative to homeostatic froglet limb. log2 values of normalized expression of TBFSG (A), KRT10 (B), NNMT-like (C), MMP-18-like (D), FRLP (E), TGFβigh3 (F),
MDK (G),IL-8-like (H), EPYC (I), INMT (J), LEF1 (K) of 1, 3 and 5 dpa froglet blastemas relative to homeostatic froglet limb. Asterisks indicate statistical significance (*:p<0.05; **: p< 0.01; ***: p<0.001; ****: p<0.0001)
Data are the mean of three biological replicates, each one with three technical replicates.
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SFig.13. Longitudinal sections through froglet limb with 3 dpa blastema showing GAG+ cells to be NAE+. A: Pentachrome stained section showing GAG+ cells (Black arrowheads), B: NAE stained section showing myeloid cells (Red arrow heads). Black arrows show the plane of amputation. Bars=25μm.
we- wound epithelium, st- limb stump.
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SFig.14. Myeloid and GAG+ cells in tadpole and froglet limb blastemas of P. maculatus. . A-C: NAE stained blastemas at different time points post tadpole limb amputation,
D-F: NAE stained blastemas at different time points post froglet limb amputation.
G: Number of NAE+ cells per mm2 of the tadpole blastema at different time points post limb amputation,H:Number of NAE+ cells per mm2 of the froglet blastema post limb amputation. (Black arrows indicate myeloid cells)
Bars in A-C= 50 μm; D-E=100 μm.
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SFig.15. Myeloid cells in the unamputated regions of tadpole limb are INMT+, EPYC+,
SALL4+ and LEF1+. A: Pentachrome stained tadpole limb with 5 dpa blastema (Black arrow heads showing GAG+ cells). b: NAE stained serial section of “A” (Blue arrows showing myeloid cells), C-D: in situ hybridisation of serial sections of “A” (Black arrows showing hybridisation of GAG+and NAE+ cells with the genes), E-F: in situ hybridisation of 5 dpa blastema of another limb (Black arrows showing hybridi- sation of GAG+and NAE+ cells with the genes). Bars= 125μm
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