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Figure 1. Downregulation of ag1and/or agr2 genes leads to regeneration blockage. (A,A’) Imaging of Xenopus laevis tadpoles developed from embryos injected with control morpholino oligonucleotides (control MO) and regenerating tail tip at two developmental timepoints corresponding to 2 and 4 days post amputation (dpa). Lateral view, dorsal to the top. Dashed red line indicates amputation level. Sc, spinal cord; nt, neural tube; m, muscles. Tail tip regeneration is dramatically reduced if ag1 and/or agr2 genes are downregulated by injection of embryos with ag1 vivo-MO (B,B’), agr2 vivo-MO (C,C’), or both (D,D’). (E) Quantification of normal regenerates percentage among controls and ag1/agr2 morphants. N—number of tails analyzed. Error bars indicate SD. Statistical significance was determined with t-test for independent samples; the results are statistically significant, p < 0.001 (asterisk). (F) qRT-PCR analysis of expression levels changes of regeneration markers wnt5a, msx1 and fgf20 during the regeneration process (at 0 and 2 dpa) in amputated tails of tadpoles injected with control, ag1 and/or agr2 vivo-MO. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit in each series. Dpa—days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk).
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Figure 2. Cell proliferation is inhibited in regenerating tails under ag1 and/or agr2 downregulation conditions. (A,A’) The transmitted light and fluorescent images of regenerating tails of tadpoles injected with solution of control vivo-MO after immunostaining with primary rabbit anti-pH3 and secondary anti-rabbit antibodies conjugated with red fluorescent protein CF568 demonstrate mitotic activity in the regenerating area at 2 and 4 dpa, respectively (see E for statistics). Transmitted light and immunostained fluorescent images of tadpoles injected with ag1 vivo-MO (B,B’), agr2 vivo-MO (C,C’), or a mixture of them (D,D’), show strong inhibition of mitotic activity at 2 and 4 dpa. Dashed yellow line indicates amputation level. (E) Quantification of number of mitotic cells per 1 mm2 of tail regenerating area. Data of five independent experiments (10 tadpoles of each injection type were used in 1 experiment) were used for statistical analysis; statistical significance was determined by t-test for independent samples, p < 0.05 (asterisk). Error bars indicate SD. (F) qRT-PCR analysis of expression levels changes of cell cycle markers cyclin d1, cdk4, and cdca9 during the regeneration process (at 0, 1, 2, and 4 dpa) in amputated tails of tadpoles injected with control, ag1 and/or agr2 vivo-MO. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit in each series. Dpa—days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk).
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Figure 3. Downregulation of ag1 and agr2 during regeneration is accompanied by cell cycle regulator cyclin d1 expression blockage but does not affect apoptosis activity in the regenerating tail. (A,A’). Results of in situ hybridization of non-injected control regenerating tails as well as tails injected with control vivo-MO (B,B’) demonstrate active expression of cyclin D1 at 2 and 3 dpa predominantly in blastema cells. Knock-down of ag1 (C,C’) as well as agr2 (D,D’) by specific vivo-MOs result in a high reduction of cyclin d1 expression at 2 and 3 dpa. Bl—blastema, we—wound epithelium. Dashed red line indicates amputation level. Lateral view, distal to the right. (E–G) TUNEL analysis of apoptotic cells pattern in 2 dpa regenerating tails injected with control vivo-MO (E), ag1 vivo-MO (F) or agr2 vivo-MO (G). (H) Statistical analysis of number of TUNEL-labeled nuclei per 1 mm2 of regenerating region, distal to the amputation level (yellow dashed line). N—number of tails analyzed.
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Figure 4. Regeneration blockage during tadpoles’ refractory period can be unlocked by ag1/agr2 over-expression or incubation in solution with purified Ag1/Agr2 proteins. (A) The qRT-PCR results show the difference in expression dynamics of ag1 and agr2 at 0–5 dpa in tadpole regenerates upon amputation at stage 40–42 or in the refractory period. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit in each series. Dpa—days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk). (B) Scheme of the experiment with ag1/agr2 over-expression and types of analysis of the regeneration process in the refractory period. (C) Statistical analysis of regeneration success of tadpoles, developed from embryos injected with a solution of FLD, either ag1 or agr2 mRNA (or both), and amputated in the refractory period. The picture shows the average values. (D) The transmitted light images of tails of corresponding tadpoles on 7 day post amputation in the refractory period demonstrate total regeneration of tails in tadpoles over-expressing either ag1 or agr2 mRNA (or both). (E) qRT-PCR analysis of expression levels changes of regeneration markers msx1b, wnt5a, and fgf20 and cell cycle markers cyclin d1, cdk4 and cdca9 during the regeneration process (at 0 and 2 dpa) in amputated tails of tadpoles injected with RDA, ag1 or agr2 mRNA solution. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit in each series. Dpa—days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk). (F) Scheme of the experiment with tadpoles amputated in refractory period and incubated in solution with BSA, Ag1 or Agr2 purified proteins (see Supplementary Material for the procedure of the recombinant proteins preparation and Supplementary Figure 3B for testing the integrity of the proteins in the medium with tadpoles). (G,G’) The transmitted light images of tadpoles tails on day 7 post amputation in the refractory period after incubation in BSA solution (G) or in solution with Ag1 or Agr2 proteins (G’) demonstrate total regeneration only in the latter variants. (H) Statistics of normally regenerated tails percentage among tadpoles amputated in refractory period and incubated with BSA or purified Ag1 or Agr2 proteins. N—total number of tadpoles used in three independent experiments.
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Figure S1. Vivo- and photo-morpholino oligonucleotides (MO) efficiency tests. (A) The MO (conventional and vivo-MO) and complementary photo-MO sequences and scheme of their sites on ag1 and agr2 mRNAs. (B)Results of Western blotting with rabbit anti-tRFP (Evrogen) and anti-rabbit alkaline phosphatase antibody (Sigma-Aldrich) and monoclonal anti-tubulin antibody (Sigma-Aldrich) demonstrate specific and effective inhibition of Ag1-tagRFP/Agr2-tagRFP synthesis by ag1/agr2 vivo-MO, but not by control vivo-MO. (C) Results of Western blotting demonstrate specific and effective inhibition of Ag1-tagRFP synthesis by ag1 vivo-MO, but not by agr2 vivo-MO and vice versa. (D) Results of Western blotting demonstrate specific and effective inhibition of Ag1-tagRFP or Agr2-tagRFP synthesis by ag1 (photo-MO+MO)or agr2 (photoMO+MO) only after illumination at 365 nm (violet lamp).
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Figure S2. Tail tips of tadpoles developed from embryos injected with ag1 and agr2 mRNAs contain high level of these mRNAs in the refractory period.
The qRT-PCR analysis of ag1 and agr2 expression in the tips of tails amputated in the refractory period demonstrates much higher level of ag1 and agr2 mRNAs in the tips of tadpoles developed from embryos injected with these synthetic mRNA comparing to the tips of control tadpoles. Statistical significance was determined by t-test for independent samples, p<0.05 (asterisk). Error bars indicate SD.
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Figure S3. Analysis of recombinant proteins. (A) The main stages of protein purification are shown in the example Agr2. (B) Recombinant Ag1 precipitated from 1ml of 0.1XMMR after incubation with tadpoles for 0, 24 and 72 hours, respectively (Martynova et al., 2021).
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Figure S4. The apoptotic cell density in the area proximal to amputation level does not change in response to ag1 and agr2 downregulation. (A) scheme of the experiment: tadpoles tail stumps at st.40-41 were injected by control vivo-MO, ag1-vivo MO or agr2-vivo-MO. After 2 days, the tadpoles' tails were stained for the presence of apoptotic cells using the TUNNEL analysis, and then in the area located proximal to the level of amputation (between the black and red lines, the distance between which was approximately equal to half the tail width), the density of apoptotic cells was determined per 1 mm2. (B) No statistically significant difference in the apoptotic cell density was revealed between control tails and those injected with ag1-vivo MO or agr2-vivo-MO. Error bars indicate SD.
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Figure S5. Overexpression of ag1/agr2 is sufficient to reactivate cell prolifertion and regeneration in the refractory period when they are naturally blocked. (A) The transmitted light and fluorescent images of tail tips of tadpoles injected with solution of RDA amputated in the refractory period after immunostaining with anti-pH3-fluorescein demonstrate very low mitotic activity in the tail tip area at 2dpa. In contrast, tadpoles injected with the solution of ag1 RNA or agr2 RNA after amputation in the refractory period demonstrate much higher proliferative activity in distal area of the tail. (B) Quantification of number of mitotic cells per 1 mm2 of tail regenerating area. Data of five independent experiments (10 tadpoles of each injection type were used in 1 experiment) were used for statistical analysis; statistical significance was determined using t-test for independent samples, p<0.05 (asterisk). Error bars indicate SD.
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FIGURE 1. Downregulation of ag1and/or agr2 genes leads to regeneration blockage. (A,A’) Imaging of Xenopus laevis tadpoles developed from embryos injected with control morpholino oligonucleotides (control MO) and regenerating tail tip at two developmental timepoints corresponding to 2 and 4 days post amputation (dpa). Lateral view, dorsal to the top. Dashed red line indicates amputation level. Sc, spinal cord; nt, neural tube; m, muscles. Tail tip regeneration is dramatically reduced if ag1 and/or agr2 genes are downregulated by injection of embryos with ag1 vivo-MO (B,B’), agr2 vivo-MO (C,C’), or both (D,D’). (E) Quantification of normal regenerates percentage among controls and ag1/agr2 morphants. N—number of tails analyzed. Error bars indicate SD. Statistical significance was determined with t-test for independent samples; the results are statistically significant, p < 0.001 (asterisk). (F) qRT-PCR analysis of expression levels changes of regeneration markers wnt5a, msx1 and fgf20 during the regeneration process (at 0 and 2 dpa) in amputated tails of tadpoles injected with control, ag1 and/or agr2 vivo-MO. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit in each series. Dpa—days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk).
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FIGURE 2. Cell proliferation is inhibited in regenerating tails under ag1 and/or agr2 downregulation conditions. (A,A’) The transmitted light and fluorescent images of regenerating tails of tadpoles injected with solution of control vivo-MO after immunostaining with primary rabbit anti-pH3 and secondary anti-rabbit antibodies conjugated with red fluorescent protein CF568 demonstrate mitotic activity in the regenerating area at 2 and 4 dpa, respectively (see E for statistics). Transmitted light and immunostained fluorescent images of tadpoles injected with ag1 vivo-MO (B,B’), agr2 vivo-MO (C,C’), or a mixture of them (D,D’), show strong inhibition of mitotic activity at 2 and 4 dpa. Dashed yellow line indicates amputation level. (E) Quantification of number of mitotic cells per 1 mm2 of tail regenerating area. Data of five independent experiments (10 tadpoles of each injection type were used in 1 experiment) were used for statistical analysis; statistical significance was determined by t-test for independent samples, p < 0.05 (asterisk). Error bars indicate SD. (F) qRT-PCR analysis of expression levels changes of cell cycle markers cyclin d1, cdk4, and cdca9 during the regeneration process (at 0, 1, 2, and 4 dpa) in amputated tails of tadpoles injected with control, ag1 and/or agr2 vivo-MO. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit in each series. Dpa—days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk).
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FIGURE 3. Downregulation of ag1 and agr2 during regeneration is accompanied by cell cycle regulator cyclin d1 expression blockage but does not affect apoptosis activity in the regenerating tail. (A,A’). Results of in situ hybridization of non-injected control regenerating tails as well as tails injected with control vivo-MO (B,B’) demonstrate active expression of cyclin D1 at 2 and 3 dpa predominantly in blastema cells. Knock-down of ag1
(C,C’) as well as agr2
(D,D’) by specific vivo-MOs result in a high reduction of cyclin d1 expression at 2 and 3 dpa. Bl—blastema, we—wound epithelium. Dashed red line indicates amputation level. Lateral view, distal to the right. (E–G) TUNEL analysis of apoptotic cells pattern in 2 dpa regenerating tails injected with control vivo-MO (E), ag1 vivo-MO (F) or agr2 vivo-MO (G). (H) Statistical analysis of number of TUNEL-labeled nuclei per 1 mm2 of regenerating region, distal to the amputation level (yellow dashed line). N—number of tails analyzed.
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FIGURE 4. Regeneration blockage during tadpoles’ refractory period can be unlocked by ag1/agr2 over-expression or incubation in solution with purified Ag1/Agr2 proteins. (A) The qRT-PCR results show the difference in expression dynamics of ag1 and agr2 at 0–5 dpa in tadpole regenerates upon amputation at stage 40–42 or in the refractory period. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit in each series. Dpa—days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk). (B) Scheme of the experiment with ag1/agr2 over-expression and types of analysis of the regeneration process in the refractory period. (C) Statistical analysis of regeneration success of tadpoles, developed from embryos injected with a solution of FLD, either ag1 or agr2 mRNA (or both), and amputated in the refractory period. The picture shows the average values. (D) The transmitted light images of tails of corresponding tadpoles on 7 day post amputation in the refractory period demonstrate total regeneration of tails in tadpoles over-expressing either ag1 or agr2 mRNA (or both). (E) qRT-PCR analysis of expression levels changes of regeneration markers msx1b, wnt5a, and fgf20 and cell cycle markers cyclin d1, cdk4 and cdca9 during the regeneration process (at 0 and 2 dpa) in amputated tails of tadpoles injected with RDA, ag1 or agr2 mRNA solution. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit in each series. Dpa—days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk). (F) Scheme of the experiment with tadpoles amputated in refractory period and incubated in solution with BSA, Ag1 or Agr2 purified proteins (see Supplementary Material for the procedure of the recombinant proteins preparation and Supplementary Figure 3B for testing the integrity of the proteins in the medium with tadpoles). (G,G’) The transmitted light images of tadpoles tails on day 7 post amputation in the refractory period after incubation in BSA solution (G) or in solution with Ag1 or Agr2 proteins (G’) demonstrate total regeneration only in the latter variants. (H) Statistics of normally regenerated tails percentage among tadpoles amputated in refractory period and incubated with BSA or purified Ag1 or Agr2 proteins. N—total number of tadpoles used in three independent experiments.
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