Fig 1.
Sample information and sampling strategy.
A. Table summary of human embryonic limb bud and chicken hindlimb samples with detailed scRNA-seq information. B. Human embryonic cells from 5 weeks post-conception (WPC) limb buds and 8 WPC long bones were sampled. C. Chicken embryonic cells from different stages of hind limb development were sampled.
Fig 2.
Cluster analysis of cell types of human embryonic limb buds and embryonic chicken hindlimbs.
A. Integration analysis of human embryonic limb bud (5 WPC) and chicken hindlimb (HH25). B. Integration analysis of human embryonic long bone (8 WPC) and chicken hindlimb (HH25). C. Integration analysis of human embryonic limb bud (5 WPC) and chicken hindlimb (HH29). D. Integration analysis of human embryonic long bone (8 WPC) and chicken hindlimb (HH29). E. Integration analysis of human embryonic limb bud (5 WPC) and chicken hindlimb (HH31). F. Integration analysis of human embryonic long bone. The degree of overlap reflects transcriptional similarity after CCA-based integration and should not be interpreted as a precise temporal alignment between human and chicken developmental stages. All figures were generated by the authors using original data in R.
Fig 3.
Integration analysis of human embryonic limb buds (5 WPC and 8 WPC) and chicken hindlimb (HH29).
A. Integration analysis of human embryonic limb buds (5 WPC and 8 WPC) and chicken hindlimb (HH29). B. T-Distributed Stochastic Neighbor Embedding (t-SNE) plots showing different cell types from 16498 limb bud bone cells from humans and chickens. C. Pearson correlation analysis showing the relationship among the 18 subsets. Hierarchical clustering according to Pearson correlation distinguished skeletogenic (clusters 1-6, 8, 10, and 13) and non-skeletogenic subsets (clusters 7, 9, 11, 12, and 14-17). D. Dot plots showing the expression of curated feature genes in 18 subsets, colored by relative gene expression. Each dot represents a gene, and its size indicates the percentage of cells expressing that gene. Cluster numbers on the x-axis correspond to the annotated cell types shown in Fig 3B. E. Proportion of cells from human limb buds and chicken limb buds. All figures were generated by the authors using original data in R.
Fig 4.
Cell subset clustering and pseudotime analysis of human and chicken limb buds.
A. t-SNE visualization of 16 subsets in human embryonic limb buds (5 WPC) and long bones (8 WPC). B. Dot plots showing the expression of feature genes for each subset of human limb buds. C. Developmental trajectory of 16 subsets inferred by monocle3 and visualized on the uniform manifold approximation and projection (UMAP). D. t-SNE visualization of 13 subsets in chicken embryonic hindlimb (HH29). E. Dot plots showing the expression of feature genes for each subset of chicken hindlimb. F. Developmental trajectory of 13 subsets inferred by monocle3 and visualized on the UMAP projection. All figures were generated by the authors using original data in R.
Fig 5.
Gene Ontology (GO) functional enrichment analysis of mesenchymal stem cells and gene expression profiling of human bone marrow stromal cells (BMSCs).
A. GO analysis of mesenchymal progenitor cells (LBM2). B. GO analysis of LBM1 cells. C. GO analysis of LBM3 cells. D. GO analysis of BMSC cells. E. GO analysis of perichondrial mesenchymal stromal cells (PMSCs). F. GO analysis of osteo-chondrogenic progenitors (OCPs). G. t-SNE plot of PDGFRA gene expression levels. H. t-SNE plot of CXCL12 gene expression levels. I. t-SNE plot of CD200 gene expression levels. All figures were generated by the authors using original data in R.
Fig 6.
Analysis of differentially expressed genes (DEGs) in chicken BMSCs.
A. Interaction network between BMSCs and other cells. B. Protein-protein interaction network (PPI) constructed from DEGs in BMSCs. C. Top 10 hub genes with higher degree of connectivity in BMSCs. D. Significantly enriched GO terms and KEGG pathways were identified for the DEGs in BMSCs. E. Heatmap of DEGs in BMSCs.
Fig 7.
Immunofluorescence images showing the expression of COL5A2, COL1A2, PRRX1, and TGFβ2 in chicken hindlimb bud sections.
Expression of COL5A2, COL1A2, PRRX1, and TGFβ2 was detected in the periosteal region and the primary ossification centers. A. Merged and single-channel images of COL5A2 (red) and DAPI (blue) staining. B. Merged and single-channel images of COL1A2 (red) and DAPI (blue) staining. C. Merged and single-channel images of PRRX1 (red) and DAPI (blue) staining. D. Merged and single-channel images of TGFβ2 (red) and DAPI (blue) staining. All experiments were independently repeated at least three times. Scale bars in snapshot image, 200 μm; scale bars in magnified images, 50 μm.