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Evol Dev
2020 Jul 01;224:297-311. doi: 10.1111/ede.12332.
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von Willebrand factor D and EGF domains is an evolutionarily conserved and required feature of blastemas capable of multitissue appendage regeneration.
Leigh ND, Sessa S, Dragalzew AC, Payzin-Dogru D, Sousa JF, Aggouras AN, Johnson K, Dunlap GS, Haas BJ, Levin M, Schneider I, Whited JL.
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Regenerative ability varies tremendously across species. A common feature of regeneration of appendages such as limbs, fins, antlers, and tails is the formation of a blastema-a transient structure that houses a pool of progenitor cells that can regenerate the missing tissue. We have identified the expression of von Willebrand factor D and EGF domains (vwde) as a common feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative species, including axolotl (Ambystoma mexicanum), lungfish (Lepidosiren paradoxa), and Polpyterus (Polypterus senegalus). Further, vwde expression is tightly linked to the ability to regenerate appendages in Xenopus laevis. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent growth factor in the blastema. These data identify a key role for vwde in regenerating blastemas and underscore the power of an evolutionarily informed approach for identifying conserved genetic components of regeneration.
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88881.198758/2018-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, 12171 Paul G. Allen Family Foundation, 1DP2HD087953-01 NIH Office of the Director, F32HD092120 Eunice Kennedy Shriver National Institute of Child Health and Human Development, R01HD095494-01A1 Eunice Kennedy Shriver National Institute of Child Health and Human Development, R03HD083434 Eunice Kennedy Shriver National Institute of Child Health and Human Development, 403248/2016-7 Conselho Nacional de Desenvolvimento Científico e Tecnológico, F32 HD092120 NICHD NIH HHS , R03 HD083434 NICHD NIH HHS , DP2 HD087953 NICHD NIH HHS , R01 HD095494 NICHD NIH HHS
Figure 1. von willebrand factor D and EGFâ€like domains (vwde) is a blastemaâ€enriched gene that is found across deuterostomes. (a) vwde (contig c1084387_g3_i1) expression in FPKM across tissues sampled from Bryant et al. (2017). Proximal and distalblastema samples are combined. (b–e) RNA in situ hybridization for vwde at (b) wound healing (3 days postamputation), (c) earlyâ€bud blastema, (d) mediumâ€bud blastema, and (e) palette stage regenerating limbs. Black arrows indicate vwde expression, scale bar is 100 µm. (f) OrthoFinder 2.0 phylogeny with corresponding protein domain structure for putative Vwde orthologs. Protein domain pictures were generated with drawProteins (Brennan, 2018). Species and Uniprot ID or transcriptome contig number are included. Polypterus vwde contained multiple splice isoforms and the closest match to axolotl Vwde is shown here. Axolotl Vwde is denoted with “*†and other species Vwde that are described in this manuscript are marked with “#.†Orthologs to the Vwde studied in this study are indicated with brackets, paralog is also denoted with brackets. FPKM, fragments per kilobase of exon mapped [Color figure can be viewed at wileyonlinelibrary.com]
Figure 3. vwde expression is tightly linked with the regenerationâ€competent environment. In situ hybridization chain reaction probing for vwde in (a–f) regenerationâ€competent Xenopus laevis tails (a,b) before amputation, (c,d) blastema 24 hr postamputation, and (e,f) the peripheral edge of the amputation plane 24 hr postamputation. (g–l) Regenerationâ€refractory tails (g,h) before amputation, (i,j) blastema 24 hr postamputation, and (k,l) the peripheral edge of the amputation plane 24 hr postamputation. White arrows indicate the location of vwde expression. Scale bars = 100 µm. DAPI, 4′,6â€diamidinoâ€2â€phenylindole [Color figure can be viewed at wileyonlinelibrary.com]
Figure 4. Vwde is essential for limb regeneration. (a) Representative images of blastemas 16 days postamputation (9 days postmorpholino administration) from standard control morpholino (“Control (SC)â€), vwdeâ€targeting morpholino 1 (vwde MO1), and vwdeâ€targeting morpholino 2 (vwde MO2). Dotted line indicates amputation plane, blastemas are all tissuedistal to amputation plane. Scale bars = 1 mm. (b) Quantification of blastema length 16 days postamputation. Median and quartiles noted with dotted lines, **p < .01, *p < .05 by nested T test. (c) Representative EdU stained sections of blastemas 10 dpa (3 days postelectroporation) of vwde morpholino 1 inverted (“Control (INV)â€) and vwdeâ€targeting morpholino 1. Scale bars = 100 µm. (d) Quantification of percent of blastema cells positive for EdU in control (INV) and knockdown (vwde MO1). Each dot represents a limb, 4–5 animals per group. Median and quartiles noted with dotted lines, **p < .01 by nested T test. (e) Representative skeletal preparations of limbs after full regeneration after knockdown of Vwde at 7 dpa. From left to right, normal forelimb, normal hindlimb, spike, and loss of distal elements. Scale bars = 5 mm. (f) Donut plots of regenerative outcomes, pooled as abnormal versus normal from experiment with Control (SC), vwde MO1, and vwde MO2. *P < .05 by Fisher's exact test comparing Control (SC) versus vwde MO1 and Control (SC) versus vwde MO2. (g) Donut plots of regenerative outcomes, pooled as abnormal versus normal from experiment with Control (INV) and vwde MO1. *p < .05 by Fisher's exact test comparing outcomes from Control (INV) compared to vwde MO1. Edu, 5â€ethynylâ€2′â€deoxyuridine [Color figure can be viewed at wileyonlinelibrary.com]
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