Williams MC et al. (2021), Nutrient availability contributes to a graded r...

XB-ART-57745

Dev Biol 2021 May 01;473:59-70. doi: 10.1016/j.ydbio.2021.01.005.

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Nutrient availability contributes to a graded refractory period for regeneration in Xenopus tropicalis.

Williams MC , Patel JH , Kakebeen AD , Wills AE .

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Xenopus tadpoles are a unique model for regeneration in that they exhibit two distinct phases of age-specific regenerative competence. In Xenopus laevis, young tadpoles fully regenerate following major injuries such as tail transection, then transiently lose regenerative competence during the "refractory period" from stages 45-47. Regenerative competence is then regained in older tadpoles before being permanently lost during metamorphosis. Here we show that a similar refractory period exists in X. tropicalis. Notably, tadpoles lose regenerative competence gradually in X. tropicalis, with full regenerative competence lost at stage 47. We find that the refractory period coincides closely with depletion of maternal yolk stores and the onset of independent feeding, and so we hypothesized that it might be caused in part by nutrient stress. In support of this hypothesis, we find that cell proliferation declines throughout the tail as the refractory period approaches. When we block nutrient mobilization by inhibiting mTOR signaling, we find that tadpole growth and regeneration are reduced, while yolk stores persist. Finally, we are able to restore regenerative competence and cell proliferation during the refractory period by abundantly feeding tadpoles. Our study argues that nutrient stress contributes to lack of regenerative competence and introduces the X. tropicalis refractory period as a valuable new model for interrogating how metabolic constraints inform regeneration.

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Species referenced: Xenopus tropicalis
Genes referenced: mtor vtga2 vtgb1
GO keywords: cell proliferation [+]

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	Fig. 1. A graded refractory period in Xenopus tropicalis shows reduced regenerative capacity at stages 43–46 and lost regenerative capacity at stage 47. A-E, U) Brightfield images of X. tropicalis across a developmental timecourse. Animals were reared at 22 °C. Nieuwkoop and Faber developmental stages as well as days post fertilization (dpf) are indicated. Scale bar is 1 mm. F-J, V) Brightfield images of the regenerating tail at 24 h post amputation (hpa). Amputations were performed at stage 41 (F), 43 (G), 45 (H), 46 (I), 47 (J), or 49 (V) and removed the distal third of the tail measured from the vent. Scale bar is 0.1 mm. K–O, W) Brightfield images of the regenerating tail at 72 hpa. Amputations were performed at stage 41 (K), 43 (L), 45 (M), 46 (N), 47 (O) or 49 (W). Scale bar is 0.1 mm. P-T, X) Brightfield images of the regenerating tail at 7 days post amputation (dpa). Amputations were performed at stage 41 (P), 43 (Q), 45 (R), 46 (S), 47 (T) or 49 (X). Scale bar is 0.1 mm. Y) Box-and-whisker plot showing the length of the tadpole across a developmental timecourse. Z, A′) Box-and-whisker plots showing the length of the regenerated tail at 72 hpa (Z) or 7 dpa (A′), normalized to vent-to-tail tip length of the tadpole at that timepoint. B′) Regeneration quality index showing regeneration quality over development at 7 dpa. Regeneration scores are shown as complete, robust, poor, or none. The stage indicated represents the stage at amputation. For (Y-A′), the midline represents the median of all tadpoles measured, box size represents the interquartile range, and the lines represent the range. Outliers are indicated as points. Stage numbers above a condition indicated statistical significance (p < 0.05) between the given group and those stages as determined by ANOVA (p = 1.59 × 10-13) followed by Tukey’s post-test. For (Z) n ranges 3–10; for (A′) n range is 7–29; for (B′) n ranges 14–15. White arrows indicate amputation site.
	Fig. 2. Proliferation in the posterior of the tail declines over development. A-E) Immunohistochemistry for pH3 in uninjured tails at stage 41 (A), 43 (B), 45 (C), 46 (D), 47 (E) counterstained with DAPI. Scale bar is 150 μm. F,G) Box-and-whisker plots showing total numbers of pH3 positive cells (F) or density of pH3 positive cells per 10,000 μm2 (G). For (F,G), the midline represents the median of all tadpoles assayed, box size represents the interquartile range, and the lines represent the range. Outliers are indicated as points. n ranges 7–9 per group. Stage numbers above a condition indicated statistical significance (p < 0.05) between the given group and those stages as determined by ANOVA (p < 2 × 10-16 for F) and G) followed by Tukey’s post-test. Area of quantification is indicated by dashed white outline.
	Fig. 3. Yolk is redistributed during development and regeneration. A) Western blot for vitellogenin and β-actin in uninjured tails at stage 41, 43, 45, 46, and 47. Immunohistochemistry for vitellogenin in uninjured tails at stage 41 (B), 43 (C), 45 (D), 46 (E), 47 (F) counterstained with DAPI. In G-O, amputation was performed at stage 41 (G,L), 43 (H,M), 45 (I,N), 46 (J,O), 47 (K,P) and tadpoles were collected at 24 hpa (G–K) or 72 hpa (L–P). White arrows indicate amputation site.
	Fig. 4. Rapamycin treatment inhibits growth, proliferation, and yolk distribution over development. A) Schematic of treatment and collection strategy. Tadpoles are treated with either DMSO (B,D,K,M), 10 μM Rapamycin (C,E,L,N) or 1 μM Torin1 before collection at indicted stages. B-E) Brightfield images of tadpoles at stage 43 (C,D) and stage 46 (E,F) following DMSO or Rapamycin treatment. Scale bar is 0.5 mm. F) Western blot for vitellogenin and β-actin at stages 43 and 46, of either DMSO or Rapamycin treated tadpoles. G) Box-and-whisker plot showing the length of tadpoles over development following DMSO or Rapamycin treatment. H) Box-and-whisker plot showing the length of tadpoles at stage 46 following either DMSO or Torin1 treatment. I) Box-and-whisker plot showing the density of pH3+ cells across groups. J-N) Immunohistochemistry for pH3 and vitellogenin in uninjured, untreated stage 41 (J) or treated (K–N) tadpoles at indicated stages with DAPI counterstain. Scale bar is 150 μm. For (G–I), the midline represents the median of all tadpoles assayed, box size represents the interquartile range, and the lines represent the range. For (G–I), statistical significance between DMSO and Rapamycin or Torin1 groups is determined with a two-sided t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). For (G) n ranges 7–13 per group; for (H) n ranges 18–19 per group: for (I) n ranges 5–10 per group.
	Fig. 5. Rapamycin treatment reduces regeneration and proliferation while disrupting regeneration induced yolk mobilization. A) Schematic of treatment and collection strategy. Tadpoles are amputated then treated with either DMSO (F,H), 10 μM Rapamycin (G,I), or 1 μM Torin1 before collection at indicted timepoints. B) Box-and-whisker plot showing the length of the regenerated tail normalized to vent-to-tail tip length of the tadpole at 72 hpa following DMSO or Rapamycin treatment. C) Box-and-whisker plot showing the length of the regenerated tail normalized to vent-to-tail tip length of the tadpole at 72 hpa following DMSO or Torin1 treatment. D) Regeneration quality index showing regeneration quality of DMSO vs Rapamycin treated tadpoles at 72 hpa. Regeneration scores are shown as complete, robust, poor, or none. E) Box-and-whisker plot showing total numbers of pH3 positive cells at 72 hpa. F–I) Immunohistochemistry for pH3 and vitellogenin at 24 hpa (F,G) or 72 hpa (H,I) after treatment with DMSO or 10 μM Rapamycin with DAPI counterstain. Scale bar is 150 μm. For (B,C, E), the midline represents the median of all tadpoles assayed, box size represents the interquartile range, and the lines represent the range. For (F–H), statistical significance between DMSO and Rapamycin groups is determined with a two-sided t-test (∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). For (B) n ranges 8–10; for (C) n ranges 18–19; for (D) n ranges 6–8; for (E) n ranges 9–10. White arrows indicate amputation site. Note autofluorescence of axial tissue in 5G was consistent across samples in this treatment group.
	Fig. 6. Proliferation and regeneration are promoted by feeding during the refractory period. A) Schematic of feeding and regeneration assay. Unfed group are provided no food for the duration of the experiment. Fed group are fed daily starting at stage 46 (24 h before amputation for regeneration assays). B) Box-and-whisker plot showing regeneration length normalized to vent-to-amputation plane length. C-J) Brightfield images of tadpoles under indicated conditions. For regeneration assays, tadpoles were collected uninjured at stage 47 (C, D), 72 hpa (E,F), 96 hpa (G, H) or 7 dpa (I,J). C-J) Brightfield images of tadpoles under indicated conditions. Scale bars are 1 mm or 0.15 mm. K) Regeneration quality index showing regeneration quality of unfed and fed tadpoles at 7 dpa. Regeneration scores are shown as complete, robust, poor, or none. L-S) Immunohistochemistry for pH3 of uninjured tails at 7 dpf (L,M), 8 dpf (N,O), and 9 dpf (P,Q) under indicated unfed or fed conditions with DAPI counterstain. R,S) Immunohistochemistry for pH3 of 7 dpa tails under unfed (R) or fed (S) conditions with DAPI counterstain. Scale bar is 150 μm. T) Box-and -whisker plot showing density of pH3 positive cells in uninjured tails following feeding protocol at 7, 8, and 9 dpf. U) Box-and -whisker plot showing number of pH3 positive cells 7 dpa. For (B, T- U), statistical significance between unfed and fed groups is determined with a two-sided t-test (∗p < 0.05, ∗∗p < 0.01, ns ‘not significant’). For (B–K), n ranges 11–12; for (L–T), n ranges 3–9; for (U), n is 3 per condition.
	Supplemental Figure S1: Xenopus tropicalis staging series. A-E) Brightfield, ventral view images of uninjured tadpoles at stages 41 (A), 43 (B), 45 (C), 46 (D), and 47 (E). Scalebar is 0.5 mm. Days post fertilization (dpf) for respective stages is indicated in top right corner of each image.F) Schematic showing length metrics for uninjured tadpoles. G) Schematic showing length metrics for regenerating tadpoles; normalized regeneration length = regeneration length / vent-to-tail tip length. H) Box-and-whisker plot showing tadpole length and vent-to-tail tip lengths of uninjured tadpoles over development. I) Box-and-whisker plot showing the vent-to-tail tip lengths and regeneration lengths of 7 dpa tadpoles amputated at indicated stages. For (H,I), the midline represents the median of all tadpoles measured, box size represents the interquartile range, and the lines represent the range. Outliers are indicated as points. For (H), n ranges 3 – 10; for (I) n ranges 7 - 29.
	Supplemental Figure S2: Regeneration is restored after the refractory period at stage 48. A) Schematic of standard rearing practices. Tadpoles are not fed until stage 47 and then reared until stage 48 or 49 before amputation. B-C) Brightfield images of stage 48 tadpoles before injury (B) and 72 hpa (C). Scale bars are 1 mm (B) or 0.25 mm (C). White arrows indicate amputation site.
	Supplemental Figure S3. Proliferation following injury declines during refractory period. A-J) Immunohistochemistry for pH3 in 24 hpa (A-E) and 72 hpa (F-J) tails at stage 41 (A,F), 43 (B,G), 45 (C,H), 46 (D,I), 47 (E,J) counterstained with DAPI. Scale bar is 150 µm. K,L) Box-and-whisker plots showing total numbers of pH3 positive cells at 24 hpa (K) or 72 hpa (L). For (K,L), the midline represents the median of all tadpoles assayed, box size represents the interquartile range, and the lines represent the range. Outliers are indicated as points. Stage numbers above a condition indicate statistical significance (p<0.05) between the given group and those stages as determined by ANOVA (p=4.39x10-16 for K and p=1,19x10-7 for L) followed by Tukey’s post-test. For (K) n > 9 per stage; for (L) n ranges 3 - 11 per stage. White arrows indicate amputation site. Area of quantification, the regenerated tissue, is indicated by dashed white outline.
	Supplemental Figure S4. Alternative timepoints and quantification methods for proliferation assays. A, B) Box-and-whisker plots of pH3 density at 24 hpa (A) or 72 hpa (B) of tadpoles amputated at indicated stages. C-F) Box-and-whisker plots showing either number of pH3+ cells (C, D) or pH3 density (E, F) of DMSO (blue) or Rapamycin (orange) treated regenerates at indicated stages (C) or regeneration time points (D-F). G) Box-and-whisker plot showing the normalized regeneration length of tadples at 24 hpa following either DMSO or Rapamycin treatment. (H, I) Box-and-whisker plot showing number of pH3+ cells (H) or pH3 density (I) at indicated timepoints in unfed (blue) or fed (orange) conditions. For all plots, the midline represents the median of all tadpoles assayed, box size represents the interquartile range, and the lines represent the range. For (A,B) Stage numbers above a condition indicated statistical significance (p<0.05) between the given group and those stages as determined by ANOVA (p=1.59x10-13) followed by Tukey’s post-test. For (C-I), statistical significance between DMSO and Rapamycin, or unfed and fed groups is determined with a two-sided t-test (p<0.05, p<0.01, **p<0.001).
	Supplemental Figure S5 Feeding before amputation does not accelerate development past the refractory period and feeding after amputation is not sufficient to rescue regeneration during the refractory period. A) Bright field images of 7 dpa tadpoles 24 hours after initiating feeding protocol; both unfed and fed tadpoles are stage 47. Scale bar is 0.5 mm. B) Schematic of various feeding conditions. Tadpoles were fed 48 hours prior to amputation, 24 hours prior to amputation, after amputation, or left unfed. Tadpoles were amputated at stage 47, at 7 dpf. C) Box-and -whisker plot showing regeneration length normalized to vent-to-amputation plane length comparing stage 41 (unfed) and refractory regenerates of respective feeding schemes. For (C) characters above respective conditions indicate significant difference (p < 0.05) between the given group and those conditions as determined by ANOVA (p=4.39x10-16 for K and p=1,19x10-7 for L) followed by Tukey’s post-test. For (C), stage 41 n is 23; for refractory groups, n ranges 3 - 12 per group. C – stage 41; U – Unfed; A – post-amputation; 24 – 24hr pre-amputation; 48 – 48hr pre-amputation.

References [+] :

Aztekin, Identification of a regeneration-organizing cell in the Xenopus tail. 2019, Pubmed, Xenbase