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The vascularization, innervation and myogenesis of early regenerated tail in Gekko japonicus

Abstract

Many species of lizards are capable of tail regeneration. There has been increased interest in the study of lizard tail regeneration in recent years as it is an amenable regeneration model for amniotes. In this study, Gekko japonicus was used as a model to investigate the initiation of vascularization, innervation and myogenesis during tail regeneration. We found that angiogenesis and axon regeneration occurred almost simultaneously within 4 days post amputation. The results showed that the endothelial cells of the original vasculature proliferated and extended into the blastema as capillary vessels, which inter-connected to form a capillary network. The nerve fibers innervated the regenerated tissue from the original spinal cord and dorsal root ganglia, and the fiber bundles increased during 14 days. Regenerating muscle tissues emerged 2 weeks after amputation. PAX3 and PAX7 expression were detected during myogenesis, with PAX7 showing a continuous increase in expression from day 3 until the day 14, whereas PAX3 reached a peak level on day 10 day post amputation, and then declined quickly to level as normal control on day 14. PCNA and PAX3 double-positive satellite cells were observed in the original rostral tissues, indicating the involvement of satellite cell proliferation during tail regeneration. Taken together, these data suggest that tail regeneration in Gekko japonicus involved rapid angiogenesis from the beginning to the day 10 and followed by capillary remodeling. The innervation of regenerated tail was significant on day 4 and increased gradually during regeneration, while the regenerated muscle tissues was obvious on day 14 after amputation.

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Acknowledgements

This work was supported by National Natural Science Foundation of China (Grant No. 31970412, 31472004 and 31071874); Natural science foundation of Jiangsu province (Grant No. BK20191446); the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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Correspondence to Jian Yao or Yan Liu.

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Supplementary Information

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10735_2021_10032_MOESM1_ESM.tif

Supplementary figure S1. Morphology of regenerating tail from day 4 to day 14. Hematoxylin and eosin-stained longitudinal sections from regenerates on the 4th (A) 7th (B), 10th (C) and 14th day (D) post amputation. A. On 4th day post amputation, the rostral epaxial (em) and hypaxial (hm) musculature was the predominant tissue, under the healing wound at the amputation plane (dashed line). A clot (cl) is evident at the tip of the regenerate covering a thin layer of wound epithelium. B. By 7th day post amputation the wound epithelium (we) has thickened and completely covered the amputation plane (dashed line). C-D The blastema grew significantly on 10th and 14th day, and blood vessels (bv) were clearly visible in the blastema (bl). The amputation plane is indicated in all figures by the dashed lines. Scale bar represents 200 μm. Supplementary file1 (TIF 22017 kb)

10735_2021_10032_MOESM2_ESM.tif

Supplementary figure S2. Endothelial cell proliferation during tail regeneration. Co-immunolabelling for CD34 (green) and BrdU (white), with Hoechst nuclear staining (blue) revealed proliferating endothelial cells or their progeny were present in both the rostral original tail stump and the distal blastema region on days 10 and 14 (A, C). The enlarged views of boxed area are shown in B, D respectively. The arrows indicate BrdU+/CD34+ proliferating endothelial cells. The dashed lines indicate amputation planes. Scale bars represent 200 μm in A and C, represent 50 μm in B and D. Supplementary file2 (TIF 7026 kb)

10735_2021_10032_MOESM3_ESM.tif

Supplementary figure S3. No evidence for myogenesis in the regenerated tail from days 4 to 12 post amputation. A-C In longitudinal sections stained with myosin heavy chain (MHC) antibody, fragmented muscle fibers were present in the distal end of the tail stump rostral to the amputation plane on the 4th day. B and C show magnified views of the boxed areas in A. The green fluorescence in the clot at the tip of regenerate is artefact (*) D-F. On the 7th day post amputation, blood vessels had penetrated the tissues under the wound epithelium but there was no reactivity for muscle fibers other than in the original epaxial (em) and hypaxial (hm) muscles. E and F show magnified views of the boxed areas in D. G-L. On days 10 and 12 post amputation, blood vessels had clearly penetrated the growing blastema (bl). However, there was no evidence for regenerating muscle cells in the blastema using the MHC antibody. H and I show enlarged views of the boxed areas in G. K and L show enlarged views of the boxed areas in J. The dashed lines indicate amputation planes in each regenerate. Scale bars represent 200 μm in A, D, G and J, and 100 μm in B, C, E, F, H, I, K and L. Supplementary file3 (TIF 14167 kb)

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Liu, Z., Huang, S., Xu, M. et al. The vascularization, innervation and myogenesis of early regenerated tail in Gekko japonicus. J Mol Histol 52, 1189–1204 (2021). https://doi.org/10.1007/s10735-021-10032-y

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Keywords

  • Tail regeneration
  • Gekko japonicus
  • Angiogenesis
  • Innervation
  • Myogenesis
  • PAX3