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

Li, Hanbo; Wei, Xiaoyü; Zhou, Li; Zhang, Weiqi; Wang, Chen; Guo, Yang; Li, Denghui; Chen, Jianyang; Liu, Tianbin; Zhang, Yingying; Ma, Shuai; Wang, Congyan; Tan, Fujian; Xu, Jiangshan; Liu, Yang; Yuan, Yue; Chen, Liang; Wang, Qiaoran; Qu, Jing; Shen, Yue; Liu, Shanshan; Fan, Guangyi; Liu, Longqi; Liu, Xin; Hou, Yong; Liu, Guanghui; Gu, Ying; Xu, Xun

doi:10.1007/s13238-020-00763-1

Cited by 47 publications

(70 citation statements)

References 15 publications

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“…However, current results suggest that limb regeneration-competent salamanders lack a developmental AER ( Purushothaman et al, 2019 ). Moreover, recent findings (including single-cell transcriptomic data) have provided conflicting results on epidermal Fgf8 expression during axolotl limb regeneration ( Gerber et al, 2018 ; Han et al, 2001 ; Leigh et al, 2018 ; Li et al, 2020 ; Nacu et al, 2016 ; Qin et al, 2020 ; Rodgers et al, 2020 ; Vincent et al, 2020 ). Therefore, it is unclear whether cells within AEC tissue use a novel transcriptional programme for regeneration, or whether they re-deploy a transcriptional programme associated with developmental AER.…”

Section: Introductionmentioning

confidence: 99%

Secreted inhibitors drive the loss of regeneration competence in Xenopus limbs

et al. 2021

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Absence of a specialized wound epidermis is hypothesized to block limb regeneration in higher vertebrates. However, the factors preventing its formation in regeneration-incompetent animals are poorly understood. To characterize the endogenous molecular and cellular regulators of specialized wound epidermis formation in Xenopus laevis tadpoles, and the loss of their regeneration-competency during development, we used single-cell transcriptomics and ex vivo regenerating limb cultures. Transcriptomic analysis revealed that the specialized wound epidermis is not a novel cell state, but a re-deployment of the apical-ectodermal-ridge (AER) program underlying limb development. Enrichment of secreted inhibitory factors, including Noggin, a morphogen expressed in developing cartilage/bone progenitor cells, are identified as key inhibitors of AER cell formation in regeneration-incompetent tadpoles. These factors can be overridden by Fgf10, which operates upstream of Noggin and blocks chondrogenesis. These results indicate that manipulation of the extracellular environment and/or chondrogenesis may provide a strategy to restore regeneration potential in higher vertebrates.

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Section: Introductionmentioning

confidence: 99%

Secreted inhibitors drive the loss of regeneration competence in Xenopus limbs

et al. 2021

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“…We found that MS-275 interfered with some regeneration-associated genes, which may have directly led to limb regeneration failure ( Figures 3E,G ). At the molecular level, the expression levels of extracellular matrix (ECM) deposition-related genes, including spalt-like transcription factor genes ( sall1 and sall4 ) ( Stewart et al, 2013 ; Erickson et al, 2016 ; Li et al, 2021 ) and collagens ( col1a1 , col5a1 , and col11a1 ), and ECM remodeling-related genes, including matrix metallopeptidase 13 ( mmp 13 ) ( Rayagiri et al, 2018 ), were low at the beginning of axolotl regeneration but increased at the blastema formation stage ( Figures 3E,G ). Twist1 is an early marker of the limb blastema mesenchyme ( Kragl et al, 2013 ).…”

Section: Discussionmentioning

confidence: 99%

Timing Does Matter: Nerve-Mediated HDAC1 Paces the Temporal Expression of Morphogenic Genes During Axolotl Limb Regeneration

Wang

Hsu

et al. 2021

Front. Cell Dev. Biol.

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Sophisticated axolotl limb regeneration is a highly orchestrated process that requires highly regulated gene expression and epigenetic modification patterns at precise positions and timings. We previously demonstrated two waves of post-amputation expression of a nerve-mediated repressive epigenetic modulator, histone deacetylase 1 (HDAC1), at the wound healing (3 days post-amputation; 3 dpa) and blastema formation (8 dpa onward) stages in juvenile axolotls. Limb regeneration was profoundly inhibited by local injection of an HDAC inhibitor, MS-275, at the amputation sites. To explore the transcriptional response of post-amputation axolotl limb regeneration in a tissue-specific and time course-dependent manner after MS-275 treatment, we performed transcriptome sequencing of the epidermis and soft tissue (ST) at 0, 3, and 8 dpa with and without MS-275 treatment. Gene Ontology (GO) enrichment analysis of each coregulated gene cluster revealed a complex array of functional pathways in both the epidermis and ST. In particular, HDAC activities were required to inhibit the premature elevation of genes related to tissue development, differentiation, and morphogenesis. Further validation by Q-PCR in independent animals demonstrated that the expression of 5 out of 6 development- and regeneration-relevant genes that should only be elevated at the blastema stage was indeed prematurely upregulated at the wound healing stage when HDAC1 activity was inhibited. WNT pathway-associated genes were also prematurely activated under HDAC1 inhibition. Applying a WNT inhibitor to MS-275-treated amputated limbs partially rescued HDAC1 inhibition, resulting in blastema formation defects. We propose that post-amputation HDAC1 expression is at least partially responsible for pacing the expression timing of morphogenic genes to facilitate proper limb regeneration.

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“…The institutional review board approved all experiments in this study on the ethics committee of BGI (permit BGI-IRB 19059). Axolotl breeding, housing, and tissue isolation were performed as previously described (Li et al, 2020). Briefly, we anesthetized the axolotls with 0.2% Tricaine (ethyl 3-aminobenzoate methane sulfonate) before the amputation surgery.…”

Section: Sample Collectionmentioning

confidence: 99%

“…To investigate the correlation between chromatin dynamics and gene expression changes, we took advantage of singlecell RNA-seq data of these eight stages we published previously and calculated the average expression of each gene to construct a pseudobulk gene expression matrix (Li et al, 2020). Correlation analysis was done between the chromatin accessibility of promoters (TSS ± 2 kb) and closest genes' expression.…”

Section: Pseudobulk Rna-seq Analysismentioning

confidence: 99%

“…Since the first axolotl genome assembly, multiple studies have been carried out to investigate the transcriptomic patterns of axolotl limb regeneration at single-cell resolution (Gerber et al, 2018;Leigh et al, 2018;Qin et al, 2020). These studies used single-cell gene expression variations to reflect dynamic cell population changes and cell fate transitions, as well as unique immune responses during regeneration (Tsai et al, 2019;Li et al, 2020;Rodgers et al, 2020). Standing on the shoulder of these studies and looking forward, analysis of the epigenetic regulations, which are responsible for the dynamic gene expression changes, will help scientists to better understand the underlying mechanisms of the regenerative process.…”

Section: Introductionmentioning

confidence: 99%

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