Sindelka R et al. (2024), Characterization of regeneration initiating cel...

XB-ART-60969

Genome Biol 2024 Oct 01;251:251. doi: 10.1186/s13059-024-03396-3.

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Characterization of regeneration initiating cells during Xenopus laevis tail regeneration.

Sindelka R , Naraine R , Abaffy P , Zucha D , Kraus D , Netusil J , Smetana K , Lacina L , Endaya BB , Neuzil J , Psenicka M , Kubista M .

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BACKGROUND: Embryos are regeneration and wound healing masters. They rapidly close wounds and scarlessly remodel and regenerate injured tissue. Regeneration has been extensively studied in many animal models using new tools such as single-cell analysis. However, until now, they have been based primarily on experiments assessing from 1 day post injury. RESULTS: In this paper, we reveal that critical steps initiating regeneration occur within hours after injury. We discovered the regeneration initiating cells (RICs) using single-cell and spatial transcriptomics of the regenerating Xenopus laevis tail. RICs are formed transiently from the basal epidermal cells, and their expression signature suggests they are important for modifying the surrounding extracellular matrix thus regulating development. The absence or deregulation of RICs leads to excessive extracellular matrix deposition and defective regeneration. CONCLUSION: RICs represent a newly discovered transient cell state involved in the initiation of the regeneration process.

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Species referenced: Xenopus laevis
Genes referenced: aldh1l2 ank3 ano1 apoc1 axin2 bcl3 bmp1 bmp4 bmp5 bmp7 capn8 cd274 chd1 ctnnb1 cxcl8a dkk1 dkk3 dlat dlx3 egr1 epcam esyt3 fgf7 fgf9 fibin fn1 fos fosl1 frzb gadd45g glud1 got1 got2 hpse inhba jun junb klf3 krt12 lamb3 lamc2 lep lrp1 lrp4 mak16 mmp1 mmp11 mmp13 mmp19 mmp8 mmp9 msx2 muc1 myh11 myh13 myh4 myh8 myl1 myl4 ncapg2 ndufs1 notch1 nuak1 palld pdpr plat plk2 pmepa1 pthlh ran rgcc runx1 sdhc sdhd shh smad1 smad4 smad9 sod1 sox11 sp8 tgfb1 tgfb2 timp1 timp3 tp73 vim wasf2 wnt10a wnt11 wnt5b
GO keywords: Notch signaling pathway [+]
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???displayArticle.morpholinos??? mmp9.1 MO1 pmepa1 MO1

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	Fig. 1 Study design of regeneration initiation using Xenopus laevis tail amputation model. Green positive sign represents the regeneration experiment. Red negative sign represents the refractory experiment
	Fig. 2 Temporal bulk RNA-Seq comparison of regenerative and refractory embryos. A Scheme of experiment. Green positive sign indicates regeneration. Red negative sign refractory experiment. B Regeneration is divided into three phases: early, intermediate, and late. Heatmaps showing the z-score for the mean normalized expression of selected genes together with enriched GO terms (red: not significantly enriched in refractory samples). C Bulk expression of five genes from the intermediate group (later identified as RICs markers) in regenerative (orange) and refractory (purple) samples. D Expression (z-score) of selected intermediate genes measured with RT-qPCR in other regenerative models
	Fig. 2 Temporal bulk RNA-Seq comparison of regenerative and refractory embryos. A Scheme of experiment. Green positive sign indicates regeneration. Red negative sign refractory experiment. B Regeneration is divided into three phases: early, intermediate, and late. Heatmaps showing the z-score for the mean normalized expression of selected genes together with enriched GO terms (red: not significantly enriched in refractory samples). C Bulk expression of five genes from the intermediate group (later identified as RICs markers) in regenerative (orange) and refractory (purple) samples. D Expression (z-score) of selected intermediate genes measured with RT-qPCR in other regenerative models
	Fig. 3 Single-cell analysis of regeneration at early phase. A Scheme of the scRNA-Seq experiment. B UMAP visualization of the integrated data sets, identifying the regeneration initiating cells (RICs), regeneration organizing cells (ROCs), small secretory cells (SSCs), and multiciliated cells (MCCs). Temporal changes in the epidermal cell populations (tp63 +) for time separated and integrated data. C Expression profiles of selected RIC markers within the different cell populations. Top ten enriched GO terms for the RIC markers. D In situ hybridization of selected RIC markers at 1 dpa. Representative bright field images from at least seven biological replicates (scale size 300 μm). Green positive sign indicates the regeneration experiment
	Fig. 3 Single-cell analysis of regeneration at early phase. A Scheme of the scRNA-Seq experiment. B UMAP visualization of the integrated data sets, identifying the regeneration initiating cells (RICs), regeneration organizing cells (ROCs), small secretory cells (SSCs), and multiciliated cells (MCCs). Temporal changes in the epidermal cell populations (tp63 +) for time separated and integrated data. C Expression profiles of selected RIC markers within the different cell populations. Top ten enriched GO terms for the RIC markers. D In situ hybridization of selected RIC markers at 1 dpa. Representative bright field images from at least seven biological replicates (scale size 300 μm). Green positive sign indicates the regeneration experiment
	Fig. 4 Trajectory, regulon and cell–cell communication pathway analysis associated with RICs. A CellRank analysis showing the UMAP projections of the clustered locations for the selected cell states. "Basal epidermal (tp63 +) 1" represents the initiating population. UMAP showing the cells that are identified as terminal states and the fate probability of each cell towards a given cell state. B Gene regulatory network of the RIC’s specific transcription factors that are differentially (fold change > 2x, Padj < 0.05) expressed. Shown are examples of the correlated associated regulons. Heatmap is based on z-score of the AUC (area under the curve) scores for each cell. Columns are ordered by cell type. C Cell–cell communication network comparison between 1 and 3 hpa showing the major sources and targets for the signals, all signal changes associated with the RICs, and the differential pathways involving RICs
	Fig. 5 Spatial transcriptomics of tail regeneration. A Scheme of the spatial transcriptomics experiment. B Clusters formed based on enriched expression in regenerative and refractory samples. Ten clusters in the Loupe software were manually annotated based on top cluster markers and comparison with scRNA-Seq data. C Volcano plot showing differential expression between regenerative bud and the remaining spots. Bar plot shows the top 10 enriched GO terms associated with the overexpressed genes. D Comparison of spatial expression of selected RIC markers in regenerative and refractory samples at 1 dpa. E Distribution of RICs in regenerative and refractory samples at 1 dpa based on deconvolution using scRNA-Seq data. F ROC and myeloid markers (based on scRNA-Seq) in regenerative and refractory samples at 3 dpa. Green positive and red negative signs indicate regenerative and refractory sample, respectively. Sections are shown from anterior (left) to posterior (right) of the tail bud region
	Fig. 5 Spatial transcriptomics of tail regeneration. A Scheme of the spatial transcriptomics experiment. B Clusters formed based on enriched expression in regenerative and refractory samples. Ten clusters in the Loupe software were manually annotated based on top cluster markers and comparison with scRNA-Seq data. C Volcano plot showing differential expression between regenerative bud and the remaining spots. Bar plot shows the top 10 enriched GO terms associated with the overexpressed genes. D Comparison of spatial expression of selected RIC markers in regenerative and refractory samples at 1 dpa. E Distribution of RICs in regenerative and refractory samples at 1 dpa based on deconvolution using scRNA-Seq data. F ROC and myeloid markers (based on scRNA-Seq) in regenerative and refractory samples at 3 dpa. Green positive and red negative signs indicate regenerative and refractory sample, respectively. Sections are shown from anterior (left) to posterior (right) of the tail bud region
	Fig. 6 Comparative analysis of descriptive datasets. Overlap of significantly expressed transcripts and their associated enriched GO terms in: A intermediate bulk RNA-Seq, RICs in single cell, and regenerative bud in spatial datasets; B specific regenerating genes identified by comparison of bud clusters from spatial transcriptomics data in regenerative and refractory samples; C RIC and ROC marker genes in spatial transcriptomics data, and D in single-cell data sets. Significance was assessed using a hypergeometric test (1-tailed) (ns not significant, * p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001). Green positive and red negative sign represents regenerative and refractory sample, respectively
	Fig. 6 Comparative analysis of descriptive datasets. Overlap of significantly expressed transcripts and their associated enriched GO terms in: A intermediate bulk RNA-Seq, RICs in single cell, and regenerative bud in spatial datasets; B specific regenerating genes identified by comparison of bud clusters from spatial transcriptomics data in regenerative and refractory samples; C RIC and ROC marker genes in spatial transcriptomics data, and D in single-cell data sets. Significance was assessed using a hypergeometric test (1-tailed) (ns not significant, * p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001). Green positive and red negative sign represents regenerative and refractory sample, respectively
	Fig. 7 Functional validation of regenerative RICs. A Scheme of functional experiments. B Removal of the regenerative bud at 6 hpa and its phenotype (11 dpa), scoring, fibrosis (fibronectin staining, 8 hpa), and defective ROC migration (RT-qPCR of ROC markers 1 dpa). C Loss of function of three RIC markers: pmepa1, mmp8, and mmp9 using Vivo MO. D Phenotypes and scoring with extensive fibrosis (fibronectin IHC) and defective ROC migration (RT-qPCR for ROC markers). E Extensive fibrosis and defective ROC migration in refractory samples. Brightfield and confocal images scale size is 300 μm. RT-qPCR was prepared from at least biological triplicates, each containing at least five dissected regenerating tails. Significance tested with Student’s t-test (ns not significant, * p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001). Fibronectin staining is shown using one representative sample from at least seven biological replicates. Green positive sign indicates regenerative while red negative sign indicates refractory sample
	Fig. 7 Functional validation of regenerative RICs. A Scheme of functional experiments. B Removal of the regenerative bud at 6 hpa and its phenotype (11 dpa), scoring, fibrosis (fibronectin staining, 8 hpa), and defective ROC migration (RT-qPCR of ROC markers 1 dpa). C Loss of function of three RIC markers: pmepa1, mmp8, and mmp9 using Vivo MO. D Phenotypes and scoring with extensive fibrosis (fibronectin IHC) and defective ROC migration (RT-qPCR for ROC markers). E Extensive fibrosis and defective ROC migration in refractory samples. Brightfield and confocal images scale size is 300 μm. RT-qPCR was prepared from at least biological triplicates, each containing at least five dissected regenerating tails. Significance tested with Student’s t-test (ns not significant, * p ≤ 0.05, p ≤ 0.01, * p ≤ 0.001). Fibronectin staining is shown using one representative sample from at least seven biological replicates. Green positive sign indicates regenerative while red negative sign indicates refractory sample