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Graphical Abstract.
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Fig. 1. Hif1α is required for regeneration of muscle and axons. A-B) Quantification of regeneration length normalized to DMSO clutchmates across dose curves for 2ME (A) and Ech (B). C) Dapi counterstained tails at 24 and 72hpa following treatment with DMSO, 5 ​μM 2ME, or 0.5 ​μM Ech. D) Quantification of regeneration length normalized to DMSO clutchmates. Statistical significance between groups was determined by ANOVA (p ​< ​2e-16) followed by Tukey's posttest. ∗ indicates p ​< ​0.001. E) Regeneration scores binned to complete (full tail regeneration), strong (incomplete fin regeneration), poor (very little regeneration), or none. The treatments have statistically significant distribution of phenotypes (chi-square test, p ​< ​2.2e-16). F) Immunohistochemical stains for 12/101 (muscle) and neurofilament (axons) at 72hpa after treatment with DMSO, 2ME, or Ech. G-H) in situ hybridization for fgf20 (G) or cdx4 (H) at 24hpa following DMSO, 2ME, or Ech treatment. Indicated numbers in F–H represent number of tails with prevented phenotype over total number assayed. Scale bars in C and F are 100 ​μm, scale bars in G-H are 75 ​μm. Arrowheads in C and F–H indicate amputation plane.
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Fig. 2. RNA-sequencing reveals shared gene regulatory roles for Hif1α and Wnt. A) Schematic depicting experimental setup for RNA-sequencing. Tails were amputated and treated with DMSO, 2ME, Ech, or IWR and collected at 0 or 24hpa for sequencing. Purple chevrons in tails represent somites, green line indicates spinal cord, and grey box marks tissues collected for sequencing. Amputation plane is marked with an orange dotted line. B) MDS plot depicting relationship between samples. C) Heatmap of gene expression for genes differentially expressed in all 3 treatment groups as log2FC relative to DMSO clustered by expression. ∗Lower bound of color scale represented values from −6 to −15. D) Venn diagram showing overlap in downregulated genes (FDR < 0.05 and log2FC ​< ​−0.2). E) Selected GO terms from PANTHER gene ontology enrichment of 1443 downregulated genes plotted by -log10FDR.
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Fig. 3. Posterior hox gene expression in regeneration requires Hif1α and Wnt. A) Pie chart of hox gene expression changes following 2ME, Ech, or IWR treatment. B) Schematic representation of hox gene organization in Xenopus. Color coding is as in (A). C) Schematic depicting hox gene expression in tailbud stage tadpoles. Color coding is as in (A). Boundaries were determined by lining up in situ expression patterns from Xenbase at the noted stages and using structural landmarks to determine the anterior and posterior domains. D) Heatmap displaying all detected hox genes across a regeneration timecourse. E) Heatmap displaying all detected hox genes clustered by expression. Genes significantly differentially expressed in all treatments are indicated by ∗. F) in situ hybridization for selected hox genes at 24hpa following treatment with DMSO, 2ME, Ech, or IWR. Arrowheads indicate amputation plane. Scale bars in E are 150 ​μm.
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Fig. 4. Hif1α directs WRE activity in regeneration. A) Heatmap displaying genes of the Wnt signaling pathway ordered by expression in IWR and sorted by position in pathway. Genes differentially expressed in all treatments are indicated by ∗. B) Timecourse of WRE-driven GFP transcript in pbin7:GFP tadpoles via in situ hybridization. C) Visualization of GFP transcripts at 24hpa following treatment with DMSO, 2ME, Ech, or IWR. D) Heatmap displaying posterior neural patterning Wnt target genes. Genes differentially expressed in all treatments are indicated by ∗. Indicated numbers in C represent number of tails with prevented phenotype over total number assayed. Scale bars in B–C are 150 ​μm. Arrowheads indicate amputation plane.
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Fig. 5. Hif1α regulates WRE expression and AP patterning at neurula stages. A) Dorsal and lateral views of in situ hybridization for snai2 in a stage 23 embryo injected unilaterally with hif1α morpholino. Miniruby fluorescent tracer was used to identify the injected side (dorsal view). snai2 expression in the migrating neural crest streams is reduced on the injected side, noted with white arrowheads. B) Control and 5 ​ng hif1α morpholino injected tails at 72hpa. C) Tracer and 10 ​ng vivo-hif1α morpholino injected tails at 72hpa. D) Length of regenerated tissue normalized to average control length in control, 5 ​ng, and 10 ​ng hif1α morpholino injected tails at 72hpa. E) Regeneration score for control, 5 ​ng, and 10 ​ng hif1α morpholino injected tails at 72hpa. F) Length of regenerated tissue normalized to average tracer-only length in tracer and 10 ​ng vivo-hif1α morpholino injected tails at 72hpa. G) Regeneration score for tracer-only and 10 ​ng vivo-hif1α morpholino injected tails at 72hpa. Legend as in (E). H) Dorsal views of in situ hybridization for pbin7:GFP, sox2, otx2, and en2 in stage 18–20 embryos. Uninjected controls (upper panels) and stage matched, unilaterally injected Hif1α morphants (lower panels) are shown. Morpholino injected side is marked with ∗ in each lower panel. Scale bars in A, H are 150 ​μm and in C,D are 100 ​μm.
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Supplemental Figure S1: Optimization of small molecule treatments for Xenopus tropicalis tail regeneration
A,C,E) Regeneration scores binned to complete (full tail regeneration), strong (incomplete fin regeneration), poor (very little regeneration), or none across different concentrations of 2ME (A), Ech (C), and IWR (E). B,F) Percentage of tadpoles alive at 72hpa following treatment with different concentrations of 2ME (B) and IWR (F). D) Images of tadpoles at 72hpa following treatment with DMSO or Ech. The highest concentration of Ech tested results in curling of the fin epidermis and unhealthy tadpoles.G) Quantification of regeneration length normalized to DMSO clutchmates across dose curves for IWR.
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Supplemental Figure S2: RNA-seq reveals regeneration induced changes in gene expression and Hif1α and Wnt dependent genes
A) MDS plot depicting relationship between samples including 0hpa. B) Selected GO terms from PANTHER gene ontology enrichment of all 3259 genes differentially expressed in all 3 treatments relative to DMSO, plotted by -log10FDR. C) Venn diagram showing overlap in injury induced genes (log2FC(0hpa/ DMSO24hpa) < -0.2 and FDR < 0.05) downregulated by treatments (FDR < 0.05 and log2FC < -0.2).
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Supplemental Figure S3: pbin7:GFP transcripts are uniquely detected in transgenic animals and axial expression declines over regenerative time
A) In situ hybridization for pbin7:GFP in WT and transgenic tadpoles. There is no detected transcript in the WT while the transgenic have patterns of expression as previously described. B) Expression in the whole tadpole from 0 to 72hpa. Axial staining declines broadly while staining in the regenerating tissue is prominently seen at 72hpa. Staining in the anterior tail tissue declines in the time between amputation (stage 41) and 72hpa (equivalent to stage 46). All transgenic tadpoles can be identified at NF stage 41 by staining in the brain.
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