Yang L et al. (2022), Evi5 is required for Xenopus limb and tail rege...

XB-ART-59684

Front Cell Dev Biol 2022 Jan 01;10:1027666. doi: 10.3389/fcell.2022.1027666.

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Evi5 is required for Xenopus limb and tail regeneration.

Yang L , Chen Y , Liu H , Liu Y , Yuan F , Li Q , Lin G .

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Amphibians such as salamanders and the African clawed frog Xenopus are great models for regeneration studies because they can fully regenerate their lost organs. While axolotl can regenerate damaged organs throughout its lifetime, Xenopus has a limited regeneration capacity after metamorphosis. The ecotropic viral integrative factor 5 (Evi5) is of great interest because its expression is highly upregulated in the limb blastema of axolotls, but remains unchanged in the fibroblastema of post-metamorphic frogs. Yet, its role in regeneration-competent contexts in Xenopus has not been fully analyzed. Here we show that Evi5 is upregulated in Xenopus tadpoles after limb and tail amputation, as in axolotls. Down-regulation of Evi5 with morpholino antisense oligos (Mo) impairs limb development and limb blastema formation in Xenopus tadpoles. Mechanistically, we show that Evi5 knockdown significantly reduces proliferation of limb blastema cells and causes apoptosis, blocking the formation of regeneration blastema. RNA-sequencing analysis reveals that in addition to reduced PDGFα and TGFβ signaling pathways that are required for regeneration, evi5 Mo downregulates lysine demethylases Kdm6b and Kdm7a. And knockdown of Kdm6b or Kdm7a causes defective limb regeneration. Evi5 knockdown also impedes tail regeneration in Xenopus tadpoles and axolotl larvae, suggesting a conserved function of Evi5 in appendage regeneration. Thus, our results demonstrate that Evi5 plays a critical role in appendage regeneration in amphibians.

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Species referenced: Xenopus laevis
Genes referenced: evi5 kdm6b kdm7a pcna
GO keywords: limb morphogenesis [+]
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???displayArticle.gses??? GSE218034: NCBI

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	FIGURE 1. Expression of evi5 during limb and tail regeneration in Xenopus and axolotl. (A) Detection of evi5 mRNA in NF stage 52 tadpole hindlimb by whole mount in situ hybridization (WISH). (B) Detection of evi5 mRNA in stage 52 tadpole hindlimb at 3,5,7 and 10 days post amputation (dpa). (C) Sagittal section of 3 and 7 dpa WISH specimens showing evi5 expression in wound epithelium and blastema region of regenerating Xenopus tadpole hindlimb. (D) RT-PCR analysis of evi5 in the NF stage 52 tadpole limb, froglet forelimb and tadpole tail. (E) WISH analysis of evi5 mRNA expression during Xenopus tadpole tail regeneration. (F) RT-PCR analysis of Evi5 in the regenerating axolotl limb and tail. Scale bars represent 500 μm in (A–B,E) and 3 dpa specimen in (C), 50 μm in 7 dpa specimen shown in (C). Data shown in (D,F) are mean with standard derivations, from 3 independent experiments, all with significant differences (p <0.05) analyzed with one-way ANOVA test.
	FIGURE 2. Effect of evi5 Mo on limb development of Xenopus. (A) Design of evi5 Mo against Xenopus evi5 (xl evi5) and axolotl Evi5 (am Evi5). Open reading frame indicated in red, targeted region of evi5 Mo shown in yellow. (B) Detection of Evi5 protein by Western blotting, in Xenopus and axolotl limb stump after injection and electroporation of evi5 Mo. Anti-Evi5 (ab 70790) was used for Evi5 protein. β-Actin used as loading control. (C–H) Knockdown of Evi5 expression during limb development. The Mo-injected limbs (C–E) showed significant developmental delay compared with the control group (F–H), and developmental abnormalities were observed at late developmental stages (white brackets indicate the length of the zeugopods). (I,J) Skeletal staining of hindlimbs at late developmental stages, with red indicating bone tissue and blue indicating cartilage tissue. Black brackets indicate zeugopods. Scale bars represent 500 μm in (C,D,F,G) and 1 mm in (E,H) and (I,J).
	FIGURE 3. Xenopus tadpole limb regeneration after Evi5 knockdown. (A–I) Stage 52–53 tadpole hindlimbs at 0, 3 and 7 dpa, after injection/electroporation of evi5 Mo (A–C), GFP DNA (D–F), or evi5 mRNA + evi5 Mo (G–I). Fluorescent images (insets) indicate site of injected materials. Bright field insets show enlarged views of the limb stump. Black * indicates the epithelium layer formed in evi5 Mo-injected tadpole limbs. White arrowheads indicate amputation levels. Scale bar: 0.5 mm. (J,K) HE staining of the evi5 Mo and GFP-injected 3 dpa limb regenerates, showing that blastema formation was defective in evi5 Mo-injected tadpole limb. Scale bar represents 0.2 mm. (L) Stacking graph of non-regenerative, partial regenerative and full regenerative tadpole limbs.
	FIGURE 4. Proliferation and apoptosis of Xenopus tadpole limb blastema after Evi5 knockdown. (A) Immunofluorescence staining of PCNA (red) in evi5 Mo-injected NF stage 52 tadpole hind limbs at 3 dpa. Nuclei were counterstained with DAPI, shown in blue. (B) Quantitative analysis of PCNA-positive cells in the limb stump mesenchyme, distal to the level marked by white arrowheads. indicates significant difference, p <0.01, n = 3, t-test. (C) Immunofluorescence staining of active Caspase3 (aCaspase3, shown in red) in evi5 Mo-injected NF stage 52 tadpole hind limbs at 3 dpa. (D) Quantitative analysis of aCaspase3-positive cells in the limb stump distal to the level marked by white arrowheads. indicates significant difference, p <0.01, n = 3, t-test. Scale bars represent 100 μm.
	FIGURE 5. Tail regeneration in Xenopus tadpoles after evi5 Mo injection. (A–B) Regenerating tadpole tail at 3 and 7 dpa in a control tadpole. (C–D) Regeneration of the Mo-injected tail was delayed compared to controls. * marks the tail blastema region lacking pigment cells. (E–F) Co-injection of evi5 Mo and evi5 mRNA partially rescued the inhibitory effect of evi5 Mo on tail regeneration. (A’,C’,E’) High magnification images of tadpole tails at 3 dpa. Fluorescence signal in inset (C’) indicates the delivery of Mo. Scale bars represent 500 μm in (A,C,E), 200 μm in (A’,C’,E’), and 1 mm in (B,D,F).
	FIGURE 6. RNA-seq analysis of evi5 Mo-injected Xenopus tadpole limbs at 3 dpa. (A) Volcano plot showing differential genes after Evi5 knockdown, with significant down-regulation markers in blue, significant up-regulation markers in red, and no significant difference markers in gray (B) GO enrichment analysis of the biological processes enriched by up- and down-regulated genes, histogram representation - Log10 (p value). (C) KEGG pathway enrichment analysis of pathways enriched by up- and down-regulated genes, histogram representation - Log10 (p value). (D) Gene set enrichment analysis (GSEA) of genes enrichment in histone demethylase activity and transforming growth factor beta receptor binding gene ontology. (E) Heatmap analysis of gene set for GSEA.
	FIGURE 7. kdm6b and kdm7a in Xenopus tadpole hindlimb development and regeneration. (A–F) Expression of kdm6b (A,C,E) and kdm7a (B,D,F) in NF stage 52 (A,B) and stage 54 (C,D) Xenopus tadpole hindlimb, and 3 dpa limb regenerates (amputated at stage 52) (E,F). (G–O) Representative images of tadpole hindlimbs at 3 dpa (G,J,M), 5 dpa (H,K,N), 7 dpa (I,L,O) after injection/electroporation of kdm6b Mo (G–I), kdm7a Mo (J–L) or control Mo (M–O). Images of tadpole limbs shown with dorsal up, anterior to the left. Scale bars represent 0.2 mm.
	Figure S1. Sequence comparison of Evi5. Comparison of human, mouse, axolotl and Xenopus Evi5 protein sequence.

	Figure S3. Injection and electroporation method. (A) Injection of Mo into the hind limb of NF stage 52 tadpoles using a pulled glass micropipette. (B) Tweezer-style electrodes placed on both sides of the limb for electroporation. (C) Fluorescence detection after electroporation.
	Figure S4. Skeletal preparation of Evi5 knockdown tadpoles. Shown are examples of skeletal staining of the hindlimbs of Xenopus froglets, 1 month after Evi5 knockdown at NF stage 52/53. While hindlimbs injected and electroporated with Control Mo regenerate 3-4 digits, hindlimbs with Evi5 knockdown regenerate fewer digits, forming 0 to 2 digits. Stage 52/53 tadpole hindlimbs were amputated through the knee level.

	Figure S6. Axolotl tail regeneration after Evi5 knockdown. (A-C) Axolotl tail regeneration was inhibited by evi5 Mo-injection. (D-F) Axolotl tail regeneration occurred normally in controls. White arrowheads indicate amputation level. Scale bar: 1mm.

	Figure S8. Overexpression of evi5 in the hind limbs of NF stage 54-55 tadpoles. (A-H) Injection of the evi5-2A plasmid into the hind limbs of tadpoles at NF stage 54-55 showed that evi5 overexpression promoted tissue growth but not enough to induce regeneration. (I) The area of the regenerated blastema was measured and analyzed for significance by t-test. Scale bar: 500 μm. * indicates p < 0.05 and ** indicates p < 0.01.
	Figure S9. Effect of evi5 Mo on wound healing in the Xenopus skin punch model. (A-H) Skin punches performed in the hindlimb of the Xenopus froglets. (I, J) HE staining of the control and Mo groups at day 10. (I’, J’) Enlargements of the squared areas in (I) and (J), respectively. (K) Measurement of the area of punch. (L) Counting of the number of pigment cells in wounds. Scale bars represent 500 μm in (A-H), 200 μm in (I) and (J), and 50 μm in (I’) and (J’). * indicates p < 0.05. One-way ANOVA, and multiple between-group t-test.Skin punches were performed with a 1 mm diameter biopsy punch (Acuderm Inc., USA).

References [+] :

Aztekin, Tissues and Cell Types of Appendage Regeneration: A Detailed Look at the Wound Epidermis and Its Specialized Forms. 2021, Pubmed