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Dev Growth Differ
2022 Aug 01;646:266-278. doi: 10.1111/dgd.12795.
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Cellular responses in the FGF10-mediated improvement of hindlimb regenerative capacity in Xenopus laevis revealed by single-cell transcriptomics.
Yanagi N
,
Kato S
,
Fukazawa T
,
Kubo T
.
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Xenopus laevis tadpoles possess regenerative capacity in their hindlimb buds at early developmental stages (stages ~52-54); they can regenerate complete hindlimbs with digits after limb bud amputation. However, they gradually lose their regenerative capacity as metamorphosis proceeds. Tadpoles in late developmental stages regenerate fewer digits (stage ~56), or only form cartilaginous spike without digits or joints (stage ~58 or later) after amputation. Previous studies have shown that administration of fibroblast growth factor 10 (FGF10) in late-stage (stage 56) tadpolehindlimb buds after amputation can improve their regenerative capacity, which means that the cells responding to FGF10 signaling play an important role in limb bud regeneration. In this study, we performed single-cell RNA sequencing (scRNA-seq) of hindlimb buds that were amputated and administered FGF10 by implanting FGF10-soaked beads at a late stage (stage 56), and explored cell clusters exhibiting a differential gene expression pattern compared with that in controls treated with phosphate-buffered saline. The scRNA-seq data showed expansion of fgf8-expressing cells in the cluster of the apical epidermal cap of FGF10-treated hindlimb buds, which was reported previously, indicating that the administration of FGF10 was successful. On analysis, in addition to the epidermal cluster, a subset of myeloid cells and a newly identified cluster of steap4-expressing cells showed remarkable differences in their gene expression profiles between the FGF10- or phosphate-buffered saline-treatment conditions, suggesting a possible role of these clusters in improving the regenerative capacity of hindlimbs via FGF10 administration.
Figure 1. Single-cell transcriptomics on the FGF10-treated and PBS-treated hindlimb blastemas. (a1-2) UMAP plot of cells detected in the scRNA-seq. (a1) cells from the FGF10-treated and PBS-treated samples were combined and divided into 35 clusters depending on the similarity of the gene expression profiles in each cell. Colors indicate cluster identity. Expression profiles of several marker genes in each cluster are shown in Figure S2. (a2) UMAP plot with colors indicating sample condition. (b1-2) The scRNA-seq data showed expansion of the AEC cluster and fgf8-expressing cells in the FGF10-treated sample. (b1) UMAP plot of the AEC and basal epidermal cell clusters of the PBS-treated (top) and FGF10-treated (bottom) samples. (b2) Normalized expression levels (transcripts per 104) of fgf8.L and fgf8.S in the epidermal clusters of each cell of the PBS-treated (top) and FGF10-treated (bottom) samples are plotted in natural-log transformed scale shown by color depth of blue.
Figure 2. Effect of FGF10 treatment on gene expression profiles of each cluster. (a) Mouse orthologs of all the downregulated DEGs (Fc <0.5) detected in each cluster were searched, and the identified mouse orthologs were subjected to enrichment analysis. The terms with the five lowest q-values in the enrichment analysis are shown. (b1–3) Comparison of the expression levels of upregulated DEGs (Fc >2) detected in the epidermal (b1), reparative myeloid (b2), and steap4+ population (b3) clusters between PBS-treated and FGF10-treated conditions. Average expression level is shown as z-score (quotient of the difference between the mean expression value of cells in the cluster and the mean expression value of all cells, and the standard deviation of expression of all cells) calculated with the mean expression value of cells in indicated clusters of each condition (PBS or FGF10) and of both conditions, indicated by dot color depth. Percentage of expressing cells in each cluster is indicated by dot size. Mouse orthologs of loc100049121.S in (b1) and loc100489423.L in (b2) are cystatin B (cstb) and toll-like receptor 5 (tlr5), respectively.
Figure 3. Characterization of the steap4+ population. Expression of several lineage marker genes across all clusters is shown. Average expression level is shown as z-score calculated using the mean expression value of the indicated cluster in both conditions (PBS and FGF10) and of all cells, indicated by dot color depth. Percentage of expressing cells in each cluster is indicated by dot size. This cluster hardly/weakly expressed epidermal (epcam.L/S), endothelial (cd34.L, pecam1.L), myofibroblast (acta2.L), and leukocyte (ptprc.L) markers, and expressed a fibroblastic gene (pdgfra.S), chemokines (cxcl8e.L, cxcl8.S, Xelaev18009501m.G [cxcl1], Xelaev18005828m.G [cxcl5], Xelaev18009504m.G [cxcl5]), and immune-related genes (myd88.S, il5ra.S, il13ra2.S, arg1.L, cebpb.L, Xelaev18017526m.G [irak3]). Mouse orthologs of each unnamed gene are shown in square brackets.
