Additional file 1 of Unravelling the limb regeneration mechanisms of Polypedates maculatus, a sub-tropical frog, by transcriptomics
Creators: name: Mahapatra, Cuckoo
name: Naik, Pranati
name: Swain, Sumanta Kumar
name: Mohapatra, Pratyush Paradarsita
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: S Fig. 1. Bioinformatics work flow. S Fig. 2. Formation of 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. S Fig. 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. S Fig. 4. Pie-chart showing maximum hits of P. maculatus CDS to Nanorana parkeri. S Fig. 5. GO annotated sequences and WEGO plot of genes of intact froglet hindlimb of P. maculatus. S Fig. 6. GO annotated sequences and WEGO plot of genes of 3 dpa post amputated froglet hindlimb blastema of P. maculatus. S Fig. 7. GO annotated sequences and WEGO plot of genes of intact hindlimb of stage 56 tadpoles of P. maculatus. S Fig. 8. GO annotated sequences and WEGO plot of 3 dpa hindlimb blastema of stage 56 tadpoles of P. maculatus. 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. S Fig. 10. Pearson’s correlation analysis between DGE seq and real time PCR data showing positive correlation. S Fig. 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), TGFßigh3 (F), MDK (G), IL-8-like (H), EPYC (I), INMT (J), LEF1 (K) of 1,3 and 5 dpa tadpole blastemas relative to homeostatic tadpole limb. Asterisks indicate statistical significance (*: p&lt;0.05; **: p&lt; 0.01; ***: p&lt;0.001; ****: p&lt;0.0001). Data are the mean of three biological replicates, each one with three technical replicates. S Fig. 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), TGFßigh3 (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&lt;0.05; **: p&lt; 0.01; ***: p&lt;0.001; ****: p&lt;0.0001). Data are the mean of three biological replicates, each one with three technical replicates. S Fig. 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 arrowheads). Black arrows show the plane of amputation. Bars=25μm. we- wound epithelium, st- limb stump. S Fig. 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. S Fig. 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 hybridisation of GAG+ and NAE+ cells with the genes). Bars= 125μm.