Dynamic cell transition and immune response landscapes of axolotl limb regeneration revealed by single-cell analysis

doi:10.1007/s13238-020-00763-1

. 2021 Jan;12(1):57-66.

doi: 10.1007/s13238-020-00763-1.

Dynamic cell transition and immune response landscapes of axolotl limb regeneration revealed by single-cell analysis

Hanbo Li^#^{1

2}, Xiaoyu Wei^#^{1

3

4}, Li Zhou^#^{1

2}, Weiqi Zhang^#^{5

6

7}, Chen Wang^{1

2}, Yang Guo², Denghui Li², Jianyang Chen², Tianbin Liu^{1

4

8}, Yingying Zhang², Shuai Ma^{6

7

9}, Congyan Wang², Fujian Tan², Jiangshan Xu^{1

3

4}, Yang Liu^{1

3

4}, Yue Yuan^{1

3

4}, Liang Chen¹⁰, Qiaoran Wang^{5

6}, Jing Qu^{6

7

11}, Yue Shen^{1

4

8}, Shanshan Liu^{1

2}, Guangyi Fan^{1

2}, Longqi Liu^{1

4}, Xin Liu^{1

2}, Yong Hou^{1

4}, Guang-Hui Liu^{12

13

14}, Ying Gu^{15

16

17}, Xun Xu^{18

19

20

21

22}

Affiliations

¹ BGI-Shenzhen, Shenzhen, 518083, China.
² BGI-Qingdao, Qingdao, 266555, China.
³ BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, 518083, China.
⁴ China National Gene Bank, BGI-Shenzhen, Shenzhen, 518120, China.
⁵ CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, China National Center for Bioinformation, Chinese Academy of Sciences, Beijing, 100101, China.
⁶ University of Chinese Academy of Sciences, Beijing, 100049, China.
⁷ Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China.
⁸ Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, Shenzhen, 518083, China.
⁹ National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
¹⁰ Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, 40072, China.
¹¹ State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
¹² University of Chinese Academy of Sciences, Beijing, 100049, China. ghliu@ioz.ac.cn.
¹³ Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China. ghliu@ioz.ac.cn.
¹⁴ State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China. ghliu@ioz.ac.cn.
¹⁵ BGI-Shenzhen, Shenzhen, 518083, China. guying@genomics.cn.
¹⁶ China National Gene Bank, BGI-Shenzhen, Shenzhen, 518120, China. guying@genomics.cn.
¹⁷ Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, Shenzhen, 518083, China. guying@genomics.cn.
¹⁸ BGI-Shenzhen, Shenzhen, 518083, China. xuxun@genomics.cn.
¹⁹ China National Gene Bank, BGI-Shenzhen, Shenzhen, 518120, China. xuxun@genomics.cn.
²⁰ Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China. xuxun@genomics.cn.
²¹ Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, Shenzhen, 518083, China. xuxun@genomics.cn.
²² Synthetic Biology Technology Innovation Center of Shandong Province, Qingdao, 266003, China. xuxun@genomics.cn.

^# Contributed equally.

PMID: 32748350
PMCID: PMC7815851
DOI: 10.1007/s13238-020-00763-1

Dynamic cell transition and immune response landscapes of axolotl limb regeneration revealed by single-cell analysis

Hanbo Li et al. Protein Cell. 2021 Jan.

. 2021 Jan;12(1):57-66.

doi: 10.1007/s13238-020-00763-1.

Authors

Affiliations

¹ BGI-Shenzhen, Shenzhen, 518083, China.
² BGI-Qingdao, Qingdao, 266555, China.
³ BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, 518083, China.
⁴ China National Gene Bank, BGI-Shenzhen, Shenzhen, 518120, China.
⁵ CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, China National Center for Bioinformation, Chinese Academy of Sciences, Beijing, 100101, China.
⁶ University of Chinese Academy of Sciences, Beijing, 100049, China.
⁷ Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China.
⁸ Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, Shenzhen, 518083, China.
⁹ National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
¹⁰ Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, 40072, China.
¹¹ State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
¹² University of Chinese Academy of Sciences, Beijing, 100049, China. ghliu@ioz.ac.cn.
¹³ Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China. ghliu@ioz.ac.cn.
¹⁴ State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China. ghliu@ioz.ac.cn.
¹⁵ BGI-Shenzhen, Shenzhen, 518083, China. guying@genomics.cn.
¹⁶ China National Gene Bank, BGI-Shenzhen, Shenzhen, 518120, China. guying@genomics.cn.
¹⁷ Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, Shenzhen, 518083, China. guying@genomics.cn.
¹⁸ BGI-Shenzhen, Shenzhen, 518083, China. xuxun@genomics.cn.
¹⁹ China National Gene Bank, BGI-Shenzhen, Shenzhen, 518120, China. xuxun@genomics.cn.
²⁰ Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China. xuxun@genomics.cn.
²¹ Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, Shenzhen, 518083, China. xuxun@genomics.cn.
²² Synthetic Biology Technology Innovation Center of Shandong Province, Qingdao, 266003, China. xuxun@genomics.cn.

^# Contributed equally.

PMID: 32748350
PMCID: PMC7815851
DOI: 10.1007/s13238-020-00763-1

No abstract available

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Figures

**Figure 1**
**Integrated transcriptional cell state Atlas of the axolotl limb regeneration revealed a putative EMT-MET process.** (A) Schematic of the scRNA-seq workflow used for the upper arm tissues collected from the homeostatic (uninjured control, 0 h), trauma (3 h), wound healing (1 d), early-bud blastema (3 d), mid-bud blastema (7 d), late-bud blastema (14 d), palette stage (22 d) and re-differentiated stage (33 d). (B) UMAP visualization of the scRNA-seq data from 41,376 single cells collected from 8 sampling stages. Each dot represents a single cell; the colors distinguish clusters. The cell-type annotation is determined by published cell-lineage specific markers. (C) UMAP distribution of cells from each sampling stage. Red dots represent the cells from the corresponding time point, and gray dots represent all the cells from 8 stages. (D) Cell number proportion of each cell type at the corresponding time point. The x-axis represents the time points. The y-axis represents the cell ratio. (E) Pseudotime analysis of epidermal and mesenchymal populations during the regeneration process. Each dot corresponds to a single cell. The gradient bar reflects the pseudotime. The cell-type components are indicated by different colors. (F) Pseudotime analysis reveals the distribution of *Prdx2*⁺ blastema, BE, IE and CT cells along the pseudotime trajectory. The cell-type components are indicated by different colors. (G) *Prdx2*⁺ blastema, BE, IE and CT cells are divided into 8 separate pseudotime states in the pseudotime analysis. Colored dots represent different states. (H) Cell type shift patterns according to real-time points. Red dots represent the cells from the corresponding time point, and gray dots represent all involved cells. (I) Expression of EMT and MET related genes on a single-cell trajectory plot. Colored dots represent single cells from individual sampling time points. Black curves reflect the fitted smooth spline curves

**Figure 2**
**The cellular and molecular dynamics of ECM and immune microenvironment during axolotl limb regeneration.** (A) UMAPs reflect the cellular distribution of fibroblasts during different stages of axolotl limb regeneration and mouse digit wound healing. Each dot represents a cell. Violin plots reflect the expression of the cell type identify markers. (B) Violin plots reflect the gene expression of ECM deposition and modeling related genes during axolotl limb regeneration and mouse digit wound healing. (C) Violin plots reflect the gene expression of fibrosis pathway related genes during axolotl limb regeneration and mouse digit wound healing. (D) Violin plots reflect the gene expression of cellular senescence related genes during axolotl limb regeneration and mouse digit wound healing. (E) Expression and distribution of marker genes and key genes of macrophages. Each dot represents a cell, and the color corresponds to the expression level (log₂(CPM + 1)). (F) Expression of macrophage marker genes in each macrophage cluster at different time points. Error bars indicate the standard deviation to the expression level (log₂(CPM + 1)). (G) Cell number distribution of the macrophage subtypes at each time point during axolotl limb regeneration (left) and mouse digit wound healing (right)

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References

1. Doyle SL, O'Neill LA. Toll-like receptors: from the discovery of NFkappaB to new insights into transcriptional regulations in innate immunity. Biochem Pharmacol. 2006;72:1102–1113. doi: 10.1016/j.bcp.2006.07.010. - DOI - PubMed
1. Erickson JR, Gearhart MD, Honson DD, Reid TA, Gardner MK, Moriarity BS, Echeverri K. A novel role for SALL4 during scar-free wound healing in axolotl. NPJ Regener Med. 2016;1:1–11. doi: 10.1038/npjregenmed.2016.16. - DOI - PMC - PubMed
1. Forte E, Chimenti I, Rosa P, Angelini F, Pagano F, Calogero A, Giacomello A, Messina E. EMT/MET at the crossroad of stemness, regeneration and oncogenesis: the Ying-Yang equilibrium recapitulated in cell spheroids. Cancers. 2017;9:98. doi: 10.3390/cancers9080098. - DOI - PMC - PubMed
1. Gerber T, Murawala P, Knapp D, Masselink W, Schuez M, Hermann S, Gac-Santel M, Nowoshilow S, Kageyama J, Khattak et al (2018). Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration. Science (New York, NY) 362 - PMC - PubMed
1. Godwin JW, Pinto AR, Rosenthal NA. Macrophages are required for adult salamander limb regeneration. Proc Natl Acad Sci USA. 2013;110:9415–9420. doi: 10.1073/pnas.1300290110. - DOI - PMC - PubMed

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[1] Doyle SL, O'Neill LA. Toll-like receptors: from the discovery of NFkappaB to new insights into transcriptional regulations in innate immunity. Biochem Pharmacol. 2006;72:1102–1113. doi: 10.1016/j.bcp.2006.07.010. - DOI - PubMed

[2] Doyle SL, O'Neill LA. Toll-like receptors: from the discovery of NFkappaB to new insights into transcriptional regulations in innate immunity. Biochem Pharmacol. 2006;72:1102–1113. doi: 10.1016/j.bcp.2006.07.010. - DOI - PubMed

[3] Erickson JR, Gearhart MD, Honson DD, Reid TA, Gardner MK, Moriarity BS, Echeverri K. A novel role for SALL4 during scar-free wound healing in axolotl. NPJ Regener Med. 2016;1:1–11. doi: 10.1038/npjregenmed.2016.16. - DOI - PMC - PubMed

[4] Erickson JR, Gearhart MD, Honson DD, Reid TA, Gardner MK, Moriarity BS, Echeverri K. A novel role for SALL4 during scar-free wound healing in axolotl. NPJ Regener Med. 2016;1:1–11. doi: 10.1038/npjregenmed.2016.16. - DOI - PMC - PubMed

[5] Forte E, Chimenti I, Rosa P, Angelini F, Pagano F, Calogero A, Giacomello A, Messina E. EMT/MET at the crossroad of stemness, regeneration and oncogenesis: the Ying-Yang equilibrium recapitulated in cell spheroids. Cancers. 2017;9:98. doi: 10.3390/cancers9080098. - DOI - PMC - PubMed

[6] Forte E, Chimenti I, Rosa P, Angelini F, Pagano F, Calogero A, Giacomello A, Messina E. EMT/MET at the crossroad of stemness, regeneration and oncogenesis: the Ying-Yang equilibrium recapitulated in cell spheroids. Cancers. 2017;9:98. doi: 10.3390/cancers9080098. - DOI - PMC - PubMed

[7] Gerber T, Murawala P, Knapp D, Masselink W, Schuez M, Hermann S, Gac-Santel M, Nowoshilow S, Kageyama J, Khattak et al (2018). Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration. Science (New York, NY) 362 - PMC - PubMed

[8] Gerber T, Murawala P, Knapp D, Masselink W, Schuez M, Hermann S, Gac-Santel M, Nowoshilow S, Kageyama J, Khattak et al (2018). Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration. Science (New York, NY) 362 - PMC - PubMed

[9] Godwin JW, Pinto AR, Rosenthal NA. Macrophages are required for adult salamander limb regeneration. Proc Natl Acad Sci USA. 2013;110:9415–9420. doi: 10.1073/pnas.1300290110. - DOI - PMC - PubMed

[10] Godwin JW, Pinto AR, Rosenthal NA. Macrophages are required for adult salamander limb regeneration. Proc Natl Acad Sci USA. 2013;110:9415–9420. doi: 10.1073/pnas.1300290110. - DOI - PMC - PubMed

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Dynamic cell transition and immune response landscapes of axolotl limb regeneration revealed by single-cell analysis

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Dynamic cell transition and immune response landscapes of axolotl limb regeneration revealed by single-cell analysis

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