Ivanova AS et al. (2018), Ras-dva small GTPases lost during evolution of ...

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Sci Rep 2018 Aug 29;81:13035. doi: 10.1038/s41598-018-30811-0.

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Ras-dva small GTPases lost during evolution of amniotes regulate regeneration in anamniotes.

Ivanova AS , Korotkova DD , Ermakova GV , Martynova NY , Zaraisky AG , Tereshina MB .

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In contrast to amniotes (reptiles, birds and mammals), anamniotes (fishes and amphibians) can effectively regenerate body appendages such as fins, limbs and tails. Why such a useful capability was progressively lost in amniotes remains unknown. As we have hypothesized recently, one of the reasons for this could be loss of some genes regulating the regeneration in evolution of amniotes. Here, we demonstrate the validity of this hypothesis by showing that genes of small GTPases Ras-dva1 and Ras-dva2, that had been lost in a stepwise manner during evolution of amniotes and disappeared completely in placental mammals, are important for regeneration in anamniotes. Both Ras-dva genes are quickly activated in regenerative wound epithelium and blastema forming in the amputated adult Danio rerio fins and Xenopus laevis tadpoles' tails and hindlimb buds. Down-regulation of any of two Ras-dva genes in fish and frog resulted in a retardation of regeneration accompanied by down-regulation of the regeneration marker genes. On the other hand, Ras-dva over-expression in tadpoles' tails restores regeneration capacity during the refractory period when regeneration is blocked due to natural reasons. Thus our data on Ras-dva genes, which were eliminated in amniotes but play role in anamniotes regeneration regulation, satisfy our hypothesis.

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Species referenced: Xenopus laevis
Genes referenced: ag1 agr2 diras1 diras2 fgf20 fgf8 igf2bp3 msx1 nras psmd6 rab1a ran ras-dva1 ras-dva2 rasl10b rho rhoa
GO keywords: animal organ regeneration
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	Figure 1 From: Ras-dva small GTPases lost during evolution of amniotes regulate regeneration in anamniotes. Genes of Ras-dva family of small GTPases were eliminated during vertebrates’ evolution. (A) Schematic version of the phylogenetic tree (full version see at Supplementary Fig. S1) of Ras-dva small GTPases and nearest groups of small GTPases, Ras, Rho, Rab etc. All vertebrate’s Ras-dva proteins form a separate bunch (bootstrap 94). The nearest homologs (41% of homology) are invertebrate small GTPases. (B) Scheme of Ras-dva genes elimination during vertebrates’ evolution. The presence of Ras-dva1 or Ras-dva2 genes in different classes is marked by “plus”, absence - by “minus”. The gray triangle demonstrates the tendency of impairment in regenerative ability during the evolution. Oblique cross indicates the loss of Ras-dva1 or Ras-dva2 gene. Notably, lack of Ras-dva genes correlates with the regenerative capacity decrease. Drawings were done by M.B.T.
	Figure 2 From: Ras-dva small GTPases lost during evolution of amniotes regulate regeneration in anamniotes. The qRT-PCR analysis of Ras-dva1, Ras-dva2 and regeneration markers expression in X. laevis tadpoles and D. rerio adult fishes after the body appendages amputation. (A,B) Schemes of experiments. Drawings were done by M.B.T. (C,D) The results of qRT-PCR analysis of xRas-dva1, xRas-dva2 and marker genes (Msx1b, Fgf20 or Fgf8) expression dynamics during X. laevis tadpole (stage 51) tail (C) or hindlimb bud (D) regeneration at 0, 1, 2, 5 dpa (days post amputation). The geometric mean of expression of two reference housekeeping genes: ornithine decarboxylase (ODC) and elongation factor 1alpa (EF-1alpha) was used for normalization of the target genes expression levels. (E) The expression of xRas-dva genes and regeneration marker genes in tadpole tails amputated in the refractory period (stage 46) at 1, 2, 5 dpa in comparison to 0 dpa. (F) qRT-PCR analysis of dRas-dva1, dRas-dva2 genes and Fgf20a, Igf2b markers expression pattern during the D. rerio fins regeneration on 1, 2 5 dpa in comparison to 0 dpa (the color of the fin on B correspond to the color of columns representing the gene expression in respective fin on F). All graphs represent means of quantification using total RNA derived from three independent samples. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit (a.u.) in each series. Data are represented as mean ± SD, t-test, p < 0,05 (asterisk).
	Figure 3 From: Ras-dva small GTPases lost during evolution of amniotes regulate regeneration in anamniotes. Analysis of Ras-dva1 and Ras-dva2 expression patterns during regeneration by whole-mount in situ hybridization. (A–F) Expression of xRas-dva1 and xRas-dva2 in X. laevis tadpole’s tails at 1 and 2 dpa in whole-mount tails (A,B,D,E) and on tail cryosections (C,F). At 1 dpa xRas-dva1 transcripts were revealed in wound epithelium, notochord and notochord tip cells (A). At 2 dpa expression is shown in wound epithelium and blastema cells (B,C). Expression of xRas-dva2 in X. laevis tadpole’s tails on 1 dpa is very poor in wound epithelium and notochord tip cells (D) and by 2 dpa it is strongly activated in blastema cells (E,F). (G,N) Tail 1 dpa and hindlimb 2 dpa stumps after whole-mount in situ hybridization with control (sense xRas-dva1 + sense xRas-dva2) probe respectively. (H–M) Expression of xRas-dva1 and xRas-dva2 in X. laevis tadpole’s hindlimb stumps at 1 and 2 dpa respectively. At 1 dpa expression of both genes is seen in wound epithelium and by 2 dpa – in blastema cells as well (see cryosections J,M). Lateral view, distal to the left, dorsal to the top. (O–T) The D. rerio caudal fins with amputated left part after hybridization in situ with dRas-dva1 (O,Q,S) or dRas-dva2 (P,R,T) probe at 1, 2 and 5 dpa. The inserts on Q and R show ISH signal in 2 dpa fin cryosections. Black arrowhead – wound epithelium. Lateral view, distal to the top, dorsal to the left. Dashed red line marks the level of amputation, black dotted lines on C and F mark the wound epithelium boundary, we – wound epithelium, bl – blastema, nct – notochord tip.
	Figure 4 From: Ras-dva small GTPases lost during evolution of amniotes regulate regeneration in anamniotes. Promoter activation analysis in transgenic tadpoles, expressing EGFP under the xRas-dva1 promoter control. (A,A’,A”) The transmitted light (A) and fluorescent (A’) images of whole transgene tadpole on stage 46 at 3 dpa, showing strong activity of xRas-dva1 promoter in the notochord, gut and the head. The enlarged fluorescent image of head region (A”) demonstrates the EGFP fluorescence in ba - branchial arches, ep- epiphysis, gb - gall-bladder, hyp – hypophysis, nc – notochord, op - olfactory pits, st – stomach. (B–E) The transmitted light and fluorescent images of proRas-dva1-EGFP tadpole’s st.42 tail tip just after amputation 0 dpa (B), at 1 dpa (C), at 2 dpa (D) and its sagittal section (E). (F–H) The transmitted light and fluorescent images of proRas-dva1-EGFP tadpole st.52 hindlimb bud at 1 dpa (F), at 2 dpa (G) and its sagittal cryosection (H). Red dashed line indicates the amputation level. The yellow arrowheads point on cells of wound epithelium. The white arrows point on blastema cells. The yellow dashed lines on H indicate wound epithelium borders.
	Figure 5 From: Ras-dva small GTPases lost during evolution of amniotes regulate regeneration in anamniotes. Down-regulation of xRas-dva1 and xRas-dva2 genes functioning results in abnormal regeneration of tadpole’s tails. (A–E) The transmitted light and fluorescent images of regenerated tails of 8 dpa tadpoles injected by solution of control MO (A), mismatched mis-xRas-dva MO (B), xRas-dva1 MO (C), xRas-dva2 MO (D) or xRas-dva1 MO + xRas-dva2 MO (E) mixed with fluorescent tracer FLD. The red dashed line indicates the amputation level. Scale bar 0,5 mm. (F) Quantification of abnormal regenerates percentage of tadpoles, injected by different MO solutions or by the “rescue” mixtures of xRas-dva1 MO + xRas-dva1 RNA, xRas-dva2 MO + xRas-dva2 RNA. Error bars indicate SD. Statistical significance was determined with paired sample t-test, the results are statistically significant, p < 0,001 (asterisk). (G) qRT-PCR analysis of expression levels changes of regeneration markers Fgf20, Msx1b and also Xenopus Ag1 homolog, xAg2, during the regeneration process (at 0 and 2 dpa) in amputated tails of tadpoles injected by control, xRas-dva1 or xRas-dva2 MO solution. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit (a.u.) in each series. Data are represented as mean ± SD, t-test, p < 0,05 (asterisk).
	Figure 6 From: Ras-dva small GTPases lost during evolution of amniotes regulate regeneration in anamniotes. Down-regulation of Ras-dva genes by vivo-morpholino oligonucleotides inhibits the D. rerio fin regeneration. (A–D) Transmitted light images of regenerating D. rerio caudal fins injected in the right half by control vivoMO (A), dRas-dva1 vivoMO (B), mis-dRas-dva1 vivoMO (C) or dRas-dva2 vivoMO (D) at 3 dpa. Red dashed line indicates amputation level. Distal is upside, dorsal is to the left. Scale bar 200 µm. (E) Regeneration efficiency is calculated as normalized area of regenerated part of the caudal fin at 3 dpa injected by vivoMO divided by the normalized area of regenerated part that was not injected. Resulting values were taken as a percentage of the value obtained for control vivoMO. The scheme demonstrates how normalized length value <L> was calculated. S – square mean, W-width mean, ni- non-injected part, inj-injected part. Error bars indicate SD. Statistical significance was determined with two-tailed t-test, the results are statistically significant, p < 0,001 (asterisk). (F) qRT-PCR analysis of the expression of early regeneration marker genes Igf2b and Fgf20a, as well as Agr genes, dAg1 and dAgr2, during the regeneration process (at 0 and 2 dpa) in the D. rerio caudal fins injected with control, dRas-dva1 or dRas-dva2 vivoMO. The scheme of experiment is the same as described in Fig. 2A. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit (a.u.) in each series. Dpa - days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk).
	Figure 7 From: Ras-dva small GTPases lost during evolution of amniotes regulate regeneration in anamniotes. Analysis of mitotic cells patterns in amputated tails of tadpoles with normal and inhibited xRas-dva1 or 2 functioning during 1–4 dpa period. (A–C) Transmitted light and fluorescent images of distal areas of 1–4 dpa tails of tadpoles, developed from embryo injected by control MO (A) or xRas-dva2 MO (B) or xRas-dva1 vivoMO (C). (A) Immunostaining with primary rabbit anti-pH3 and secondary anti-rabbit antibodies conjugated with red fluorescent protein CF568 demonstrate gradual increase of mitotic activity in the regenerating area of tadpoles injected by control MO starting from 2 dpa (see B’ for statistics). (B) Transmitted light and fluorescent images of tadpoles injected by xRas-dva2 MO show inhibition of mitotic activity during 2–4 dpa. Scale bar 0,1 mm. (B’) Quantification of pH3-labbeled mitotic cells number in regenerates of 1–4 dpa tadpoles, developed from embryos injected by control, xRas-dva1 or xRas-dva2 MO. Data of five independent experiments (5 tadpoles of each injection type were used in 1 experiment) were used for statistical analysis, statistical significance was determined by paired t test, p < 0,05 (asterisk). Error bars indicate SD. (C) Transmitted light and fluorescent images of tadpoles injected by xRas-dva1 vivoMO show strong inhibition of mitotic activity during 2–3 dpa. (C’) Quantification of mitotic cells number in 1–4 dpa regenerates of tadpoles injected at 0, 1,2 dpa by control, xRas-dva1 or xRas-dva2 vivoMO. Data of five independent experiments (5 tadpoles of each injection type were used in 1 experiment) were used for statistical analysis, statistical significance was determined by paired t test, p < 0,05 (asterisk) Error bars indicate SD.
	Rescue of the ability to regenerate tails during the refractory period in the X. laevis tadpoles. (A) Different rates of regeneration success in the refractory tadpoles with or without Ras-dva1/2 overexpression. The average percent mean values of regenerating tadpoles with different success rates marked by colors: gray - no regeneration, pink – partial regeneration, red – normal regeneration, in tadpole butches injected with fluorescein (FLD, as control) or overexpressing xRas-dva1 and xRas-dva2 mRNAs. n – number of injected tadpoles in three independent experiments. Differences of percent of normal regeneration between regenerates injected by FLD and mRNA are statistically significant, two-tailed t-test, p < 0,05. Percent differences of not regenerating tadpoles in FLD and mRNA injected tadpoles is statistically significant, two-tailed t-test, p < 0,05. (B) The measurements of the length of regenerated tail tips by 8 day after amputation of refractory tadpoles, developed from embryos injected by FLD (control) or xRas-dva1 or xRas-dva2 mRNA. n – number of injected tadpoles in three independent experiments. Two-tailed t-test, p < 0,001 (asterisk). The transmitted light (C) and fluorescent (D) images of regenerated tails of 8 dpa refractory tadpoles injected with fluorescein (FLD) or xRas-dva1 mRNA + FLD and xRas-dva2 mRNA + FLD. FLD refractory tadpoles show regeneration arrest which is common for refractory period. Refractory tadpoles with Ras-dva genes overexpression effectively regenerate their tails. (E–G) The transmitted light images of regenerating tail tips of control tadpoles (FLD) or xRas-dva1 mRNA and xRas-dva2 mRNA at 4 dpa in refractory period show regeneration arrest in control tip but normal regeneration of notochord (nt), spinal cord (sc), muscles (m), melanophores (mp) in the tadpole’s tails with xRas-dva1 or xRas-dva2 overexpression. The red dashed line indicates the amputation level. Scale bar 1 mm. (H) qRT-PCR analysis of the expression of early regeneration marker genes Fgf20 and Msx1b during the regeneration process (at 0 and 2 dpa) after amputation in refractory period in tadpoles tails injected with FLD or xRas-dva1 or xRas-dva2 mRNAs at early developmental stages. The scheme of samples harvesting is the same as described in Fig. 2A. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit (a.u.) in each series. Dpa - days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk).
	Figure S1. Phylogenetic tree of Ras-dva small GTPases and representatives of other separate groups of small GTPases. Ras-dva small GTPases were identified in all vertebrates classes except placental mammals. These GTPases form a group (bootstrap 94) separate from Ras, Rab, Ran and other small GTPases. The subgroup of Ras-dva2 is very close to Ras-dva1 subgroup. For phylogenetic tree details and sequences numbers see Supplementary Material and Methods.
	Figure S2. Outgrowth dynamics during regeneration of different Danio rerio fins. To verify of regeneration score we calculated average fins height (h) outgrown by 1, 2 and 5 day post amputation (h=S/l, S- area, l - length) and compare it with each other. Drawings were done by M.B.T. The mean of the fin’s h at 1dpa was used for normalization. All calculations were done with ImageJ program. For statistical analysis we carried out 5 independent experiments. Bars indicate SD. All outgrowth data at 2-5 were statistically significant, t-test, p<0,001.
	Figure S3. Analysis of xRas-dva genes activity in tails and hindlimbs during non-regenerating stages. (A and B) Expression pattern of xRas-dva1 and xRas-dva2 at 0, 1 and 2 days post amputation at refractory 46 stage revealed by in situ hybridization. At 0dpa we detected Ras-dva1 and Ras-dva2 expression in notochord. Strong activity of gene is seen in wound epithelium and notochord tip cells at 1 dpa, but by 2 dpa expression level decreases. Nc - notochord, nct - notochord tip cells, we - wound epithelium. Dashed red line – amputation level. Dorsal to the right, distal to the top (C, D, E) The in vivo imaging of proRas-dva1-EGFP transgenic tadpoles after amputation in refractory period. (C) At 0dpa we detected EGFP expression only in notochord (nc). (D and E) Strong xRas-dva1 promoter activation was determined in wound epithelium (yellow arrowheads) and notochord tip cells at 1 dpa, but by 2 dpa expression level decreased and presented only in thin wound epithelium layer and notochord proximal to the amputation line. Dorsal to the top, distal to the left. We – wound epithelium, nc – notochord, nct – notochord tip cells. (F) The qRT-PCR analysis of xRas-dva and Fgf8 expression dynamics in hindlimbs stumps after amputation at prometamorphic stages (57 stage and later). As can be seen only xRas-dva1 is slightly activated at 1dpa.
	Figure S4. Xenopus laevis morpholino oligonucleotides (MO) and vivo-morpholino oligonucleotides (vivoMO) efficiency and specificity tests. (A) Scheme of experiments on testing MO/vivoMO. mRNA encoding for Flag-tagged xRas-dva 1 or 2 was injected into each blastomere of 2-cell Xenopus laevis embryos (100 pg/blastomere), either alone or with control mis-xRas-dva1 vivoMO (mis-xRAs-dva2 vivoMO) or with specific xRas-dva1 MO/vivoMO (xRas-dva2 MO/vivoMO) (4nl of 0,25mM MO solution or 4 nl of 0,4 mM water vivoMO solution per blastomere). The injected embryos were collected at the late gastrula stage and analyzed for presence of 3Flag-xRas-dva proteins by Western blotting with anti-Flag antibody. Tubulin was used as loading control (see Methods). Drawings were done by M.B.T. (B) Results of western blotting with conjugated anti-flag alkaline phosphatase antibody (Sigma) and monoclonal anti-tubulin antibody demonstrate specific and effective inhibition of flag-xRas-dva1 synthesis by xRas-dva1 MO, but not by xRas-dva2 MO. The common results are obtained for xRas-dva2 MO demonstrating their effectiveness and specificity. (C) Results of western blotting with conjugated anti-flag alkaline phosphatase antibody (Sigma) and monoclonal anti-tubulin antibody demonstrate specific and effective inhibition of flag-xRas-dva1 synthesis by xRas-dva1 vivoMO, but not by xRas-dva2 vivoMO or mis-xRas-dva1 vivoMO. The common results are obtained for xRas-dva2 vivoMO.
	Figure S5. Down-regulation of xRas-dva1 and xRas-dva2 genes functioning in tail tip just after amputation results in malformations of regenerated tadpole’s tails. (A) Scheme of the experiment. The wild type tadpoles were incubated till stage 40-42. Then tadpoles’ tail’s distal parts were amputated and tails were injected by vivo-Morpholino oligonucleotides solution (vivoMO). VivoMO are able to penetrate cell membranes and can sequence-specifically inhibit translation of correspondent mRNA. Injections were performed once per day at 0, 1, 2 dpa. At 4dpa the tadpole’s regeneration success was analyzed basing on morphological parameters of restored tails. Drawings were done by M.B.T. (B) Quantification of abnormal regenerates percentage of tadpoles, injected by different vivo-MO solutions: control vivoMO, mis-xRas-dva vivoMO, xRas-dva1-vivoMO and xRas-dva2-vivoMO. Error bars indicate SD. Statistical significance of results from 5 independent experiments (1 experiment comprise 15-20 tadpoles for injection by each variant of vivoMo) was determined with paired sample t-test, the results are statistically significant, p < 0,001 (asterisk). (C-E) The transmitted light images of regenerated tails of 4dpa tadpoles injected by solution of mis-xRas-dva vivoMO (C), xRas-dva1-vivoMO (D) and xRas-dva2-vivoMO (E). The red dashed line indicates the amputation level. Scale bar 0,5 mm.
	Figure S6. Danio Ras-dva vivo-morpholino oligonucleotides efficiency and specificity test. TUNEL assay of apoptosis in the regenerating caudal fins. (A) Results of western blotting with conjugated anti-flag alkaline phosphatase antiboby (Sigma) and monoclonal anti-tubulin antibody demonstrate effective inhibition of flag-dRas-dva1 synthesis by dRas-dva1vivo MO, but not by mis-dRas-dva1 vivoMO. The same results are obtained for dRas-dva2 vivoMO. (B) dRas-dva1 vivoMO specifically inhibit dRas-dva1 protein synthesis, but not dRas-dva2 and vice versa. The scheme of the experiment was as in Supplementary Figure S4. (C-E) Transmitted light and fluorescent images of distal areas of 3dpa caudal fins injected by fluorescent tracer (FLD) (C) or dRas-dva1 vivoMO and dRas-dva2 vivoMO (D and E). Scale bar 250μm. The bright green dots on fluorescent images indicate the apoptotic nuclei. (F) The density value of the numbers of apoptotic TUNEL-labeled nuclei in the injected fins as calculated in the regenerate’s area marked by yellow dashed line. Data are represented as mean ±SD. N – number of fins used in the assay.
	Figure S7. Histological analysis of tadpoles’ tails at 1, 2 dpa upon xRas-dva1 or xRas-dva2 knock-down. (A-C) Hematoxylin staining of sagittal cryosections of 1dpa tails of tadpoles injected by control vivoMO (A) or by xRas-dva1 vMO (B) or xRas-dva2 vMO (C) tadpoles. Knock-down of Ras-dva genes results in problems in wound epithelium formation. (D-E) Hematoxylin staining of sagittal cryosections of 2dpa tails of control (D) or injected by xRas-dva1 MO (E) or xRas-dva2 MO (F) tadpoles. Down-regulation of Ras-dva genes result in reduction of spinal cord and notochord regrowth and lower density of blastemal cells in contrast to control regeneration. Dashed black line indicates the amputation level. Scale bar 0,1 mm. Bc – blastemal cells, na – neural ampule, nc – notochord, sc – spinal cord, we – wound epithelium. Cryosections width 18μm.
	Figure S8. Apoptosis profiles in amputated tail of tadpoles with normal and inhibited Ras-dva1 or 2 functioning at 1dpa. (A-C) Transmitted light and fluorescent images of distal areas of 1dpa tail of the tadpole, developed from embryo injected by control MO (A) or xRas-dva1 MO (B) or xRas-dva2 MO (C). The bright green dots on fluorescent images indicate the apoptotic nuclei after TUNEL assay. Scale bar 0,25 mm. Distal to the right, dorsal to the top. Red dashed line marks the amputation level. (D) The density value of the numbers of apoptotic TUNEL-labeled nuclei in the tadpoles tails injected by control or Ras-dva-specific MO was calculated using ImageJ software. Data are represented as mean ±SD. N – number of tails used in the assay in three independent experiments.
	Figure S9. Detection of injected ectopic xRas-dva1 and xRas-dva2 mRNAs in refractory tadpoles tails after amputation. (A and A’) Transmitted light and fluorescent tail images of tadpoles developed from embryos injected by EGFP-xRas-dva1 mRNA and amputated in refractory period (st. 46). Fluorescent signals in regenerating refractory tail in fins and tip demonstrate that injected mRNA is still functions. Scale bar 2mm. (B and C) In situ hybridization staining of control refractory tadpole’s tails at 2dpa for xRas-dva1 and xRas-dva2 expression show weak signal in the tail tip. (D and E) In situ hybridization staining of refractory tadpole’s tails developed from embryos, which were injected at 2-4 cell stage by synthetic xRas-dva1 or xRas-dva2 mRNA, demonstrate xRas-dva1 (D) or xRas-dva2 (E) ectopic mRNA presence in tail tip, fins and muscles (yellow arrowheads) at 2dpa.
	Figure S10. Synthetic mRNA viability test. Results of western blotting with conjugated anti-flag alkaline phosphatase antiboby (Sigma) demonstrate translation activity of synthetic mRNA xRas-dva1-3flag and xRas-dva2-3flag in samples of refractory tail’s tips at 2dpa of tadpoles, developed from embryos injected by correspondent mRNAs.
	Figure 1. Genes of Ras-dva family of small GTPases were eliminated during vertebrates’ evolution. (A) Schematic version of the phylogenetic tree (full version see at Supplementary Fig. S1) of Ras-dva small GTPases and nearest groups of small GTPases, Ras, Rho, Rab etc. All vertebrate’s Ras-dva proteins form a separate bunch (bootstrap 94). The nearest homologs (41% of homology) are invertebrate small GTPases. (B) Scheme of Ras-dva genes elimination during vertebrates’ evolution. The presence of Ras-dva1 or Ras-dva2 genes in different classes is marked by “plus”, absence - by “minus”. The gray triangle demonstrates the tendency of impairment in regenerative ability during the evolution. Oblique cross indicates the loss of Ras-dva1 or Ras-dva2 gene. Notably, lack of Ras-dva genes correlates with the regenerative capacity decrease. Drawings were done by M.B.T.
	Figure 2. The qRT-PCR analysis of Ras-dva1, Ras-dva2 and regeneration markers expression in X. laevis tadpoles and D. rerio adult fishes after the body appendages amputation. (A,B) Schemes of experiments. Drawings were done by M.B.T. (C,D) The results of qRT-PCR analysis of xRas-dva1, xRas-dva2 and marker genes (Msx1b, Fgf20 or Fgf8) expression dynamics during X. laevis tadpole (stage 51) tail (C) or hindlimb bud (D) regeneration at 0, 1, 2, 5 dpa (days post amputation). The geometric mean of expression of two reference housekeeping genes: ornithine decarboxylase (ODC) and elongation factor 1alpa (EF-1alpha) was used for normalization of the target genes expression levels. (E) The expression of xRas-dva genes and regeneration marker genes in tadpole tails amputated in the refractory period (stage 46) at 1, 2, 5 dpa in comparison to 0 dpa. (F) qRT-PCR analysis of dRas-dva1, dRas-dva2 genes and Fgf20a, Igf2b markers expression pattern during the D. rerio fins regeneration on 1, 2 5 dpa in comparison to 0 dpa (the color of the fin on B correspond to the color of columns representing the gene expression in respective fin on F). All graphs represent means of quantification using total RNA derived from three independent samples. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit (a.u.) in each series. Data are represented as mean ± SD, t-test, p < 0,05 (asterisk).
	Figure 3. Analysis of Ras-dva1 and Ras-dva2 expression patterns during regeneration by whole-mount in situ hybridization. (A–F) Expression of xRas-dva1 and xRas-dva2 in X. laevis tadpole’s tails at 1 and 2 dpa in whole-mount tails (A,B,D,E) and on tail cryosections (C,F). At 1 dpa xRas-dva1 transcripts were revealed in wound epithelium, notochord and notochord tip cells (A). At 2 dpa expression is shown in wound epithelium and blastema cells (B,C). Expression of xRas-dva2 in X. laevis tadpole’s tails on 1 dpa is very poor in wound epithelium and notochord tip cells (D) and by 2 dpa it is strongly activated in blastema cells (E,F). (G,N) Tail 1 dpa and hindlimb 2 dpa stumps after whole-mount in situ hybridization with control (sense xRas-dva1 + sense xRas-dva2) probe respectively. (H–M) Expression of xRas-dva1 and xRas-dva2 in X. laevis tadpole’s hindlimb stumps at 1 and 2 dpa respectively. At 1 dpa expression of both genes is seen in wound epithelium and by 2 dpa – in blastema cells as well (see cryosections J,M). Lateral view, distal to the left, dorsal to the top. (O–T) The D. rerio caudal fins with amputated left part after hybridization in situ with dRas-dva1 (O,Q,S) or dRas-dva2 (P,R,T) probe at 1, 2 and 5 dpa. The inserts on Q and R show ISH signal in 2 dpa fin cryosections. Black arrowhead – wound epithelium. Lateral view, distal to the top, dorsal to the left. Dashed red line marks the level of amputation, black dotted lines on C and F mark the wound epithelium boundary, we – wound epithelium, bl – blastema, nct – notochord tip.
	Figure 4. Promoter activation analysis in transgenic tadpoles, expressing EGFP under the xRas-dva1 promoter control. (A,A’,A”) The transmitted light (A) and fluorescent (A’) images of whole transgene tadpole on stage 46 at 3 dpa, showing strong activity of xRas-dva1 promoter in the notochord, gut and the head. The enlarged fluorescent image of head region (A”) demonstrates the EGFP fluorescence in ba - branchial arches, ep- epiphysis, gb - gall-bladder, hyp – hypophysis, nc – notochord, op - olfactory pits, st – stomach. (B–E) The transmitted light and fluorescent images of proRas-dva1-EGFP tadpole’s st.42 tail tip just after amputation 0 dpa (B), at 1 dpa (C), at 2 dpa (D) and its sagittal section (E). (F–H) The transmitted light and fluorescent images of proRas-dva1-EGFP tadpole st.52 hindlimb bud at 1 dpa (F), at 2 dpa (G) and its sagittal cryosection (H). Red dashed line indicates the amputation level. The yellow arrowheads point on cells of wound epithelium. The white arrows point on blastema cells. The yellow dashed lines on H indicate wound epithelium borders.
	Figure 5. Down-regulation of xRas-dva1 and xRas-dva2 genes functioning results in abnormal regeneration of tadpole’s tails. (A–E) The transmitted light and fluorescent images of regenerated tails of 8 dpa tadpoles injected by solution of control MO (A), mismatched mis-xRas-dva MO (B), xRas-dva1 MO (C), xRas-dva2 MO (D) or xRas-dva1 MO + xRas-dva2 MO (E) mixed with fluorescent tracer FLD. The red dashed line indicates the amputation level. Scale bar 0,5 mm. (F) Quantification of abnormal regenerates percentage of tadpoles, injected by different MO solutions or by the “rescue” mixtures of xRas-dva1 MO + xRas-dva1 RNA, xRas-dva2 MO + xRas-dva2 RNA. Error bars indicate SD. Statistical significance was determined with paired sample t-test, the results are statistically significant, p < 0,001 (asterisk). (G) qRT-PCR analysis of expression levels changes of regeneration markers Fgf20, Msx1b and also Xenopus Ag1 homolog, xAg2, during the regeneration process (at 0 and 2 dpa) in amputated tails of tadpoles injected by control, xRas-dva1 or xRas-dva2 MO solution. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit (a.u.) in each series. Data are represented as mean ± SD, t-test, p < 0,05 (asterisk).
	Figure 6. Down-regulation of Ras-dva genes by vivo-morpholino oligonucleotides inhibits the D. rerio fin regeneration. (A–D) Transmitted light images of regenerating D. rerio caudal fins injected in the right half by control vivoMO (A), dRas-dva1 vivoMO (B), mis-dRas-dva1 vivoMO (C) or dRas-dva2 vivoMO (D) at 3 dpa. Red dashed line indicates amputation level. Distal is upside, dorsal is to the left. Scale bar 200 µm. (E) Regeneration efficiency is calculated as normalized area of regenerated part of the caudal fin at 3 dpa injected by vivoMO divided by the normalized area of regenerated part that was not injected. Resulting values were taken as a percentage of the value obtained for control vivoMO. The scheme demonstrates how normalized length value <L> was calculated. S – square mean, W-width mean, ni- non-injected part, inj-injected part. Error bars indicate SD. Statistical significance was determined with two-tailed t-test, the results are statistically significant, p < 0,001 (asterisk). (F) qRT-PCR analysis of the expression of early regeneration marker genes Igf2b and Fgf20a, as well as Agr genes, dAg1 and dAgr2, during the regeneration process (at 0 and 2 dpa) in the D. rerio caudal fins injected with control, dRas-dva1 or dRas-dva2 vivoMO. The scheme of experiment is the same as described in Fig. 2A. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit (a.u.) in each series. Dpa - days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk).
	Figure 7. Analysis of mitotic cells patterns in amputated tails of tadpoles with normal and inhibited xRas-dva1 or 2 functioning during 1–4 dpa period. (A–C) Transmitted light and fluorescent images of distal areas of 1–4 dpa tails of tadpoles, developed from embryo injected by control MO (A) or xRas-dva2 MO (B) or xRas-dva1 vivoMO (C). (A) Immunostaining with primary rabbit anti-pH3 and secondary anti-rabbit antibodies conjugated with red fluorescent protein CF568 demonstrate gradual increase of mitotic activity in the regenerating area of tadpoles injected by control MO starting from 2 dpa (see B’ for statistics). (B) Transmitted light and fluorescent images of tadpoles injected by xRas-dva2 MO show inhibition of mitotic activity during 2–4 dpa. Scale bar 0,1 mm. (B’) Quantification of pH3-labbeled mitotic cells number in regenerates of 1–4 dpa tadpoles, developed from embryos injected by control, xRas-dva1 or xRas-dva2 MO. Data of five independent experiments (5 tadpoles of each injection type were used in 1 experiment) were used for statistical analysis, statistical significance was determined by paired t test, p < 0,05 (asterisk). Error bars indicate SD. (C) Transmitted light and fluorescent images of tadpoles injected by xRas-dva1 vivoMO show strong inhibition of mitotic activity during 2–3 dpa. (C’) Quantification of mitotic cells number in 1–4 dpa regenerates of tadpoles injected at 0, 1,2 dpa by control, xRas-dva1 or xRas-dva2 vivoMO. Data of five independent experiments (5 tadpoles of each injection type were used in 1 experiment) were used for statistical analysis, statistical significance was determined by paired t test, p < 0,05 (asterisk) Error bars indicate SD.
	Figure 8. Rescue of the ability to regenerate tails during the refractory period in the X. laevis tadpoles. (A) Different rates of regeneration success in the refractory tadpoles with or without Ras-dva1/2 overexpression. The average percent mean values of regenerating tadpoles with different success rates marked by colors: gray - no regeneration, pink – partial regeneration, red – normal regeneration, in tadpole butches injected with fluorescein (FLD, as control) or overexpressing xRas-dva1 and xRas-dva2 mRNAs. n – number of injected tadpoles in three independent experiments. Differences of percent of normal regeneration between regenerates injected by FLD and mRNA are statistically significant, two-tailed t-test, p < 0,05. Percent differences of not regenerating tadpoles in FLD and mRNA injected tadpoles is statistically significant, two-tailed t-test, p < 0,05. (B) The measurements of the length of regenerated tail tips by 8 day after amputation of refractory tadpoles, developed from embryos injected by FLD (control) or xRas-dva1 or xRas-dva2 mRNA. n – number of injected tadpoles in three independent experiments. Two-tailed t-test, p < 0,001 (asterisk). The transmitted light (C) and fluorescent (D) images of regenerated tails of 8 dpa refractory tadpoles injected with fluorescein (FLD) or xRas-dva1 mRNA + FLD and xRas-dva2 mRNA + FLD. FLD refractory tadpoles show regeneration arrest which is common for refractory period. Refractory tadpoles with Ras-dva genes overexpression effectively regenerate their tails. (E–G) The transmitted light images of regenerating tail tips of control tadpoles (FLD) or xRas-dva1 mRNA and xRas-dva2 mRNA at 4 dpa in refractory period show regeneration arrest in control tip but normal regeneration of notochord (nt), spinal cord (sc), muscles (m), melanophores (mp) in the tadpole’s tails with xRas-dva1 or xRas-dva2 overexpression. The red dashed line indicates the amputation level. Scale bar 1 mm. (H) qRT-PCR analysis of the expression of early regeneration marker genes Fgf20 and Msx1b during the regeneration process (at 0 and 2 dpa) after amputation in refractory period in tadpoles tails injected with FLD or xRas-dva1 or xRas-dva2 mRNAs at early developmental stages. The scheme of samples harvesting is the same as described in Fig. 2A. The value of normalized PCR signal in the 0 dpa sample, harvested immediately after amputation, was taken as an arbitrary unit (a.u.) in each series. Dpa - days post amputation. Error bars indicate SD, t-test, p < 0.05 (asterisk).

References [+] :

Bachy, Defining pallial and subpallial divisions in the developing Xenopus forebrain. 2002, Pubmed, Xenbase