Fig. 1: Single-cell RNA-seq identifies a perichondrial cluster as a putative cell origin in the fetal cartilage.

a–e Single-cell RNA-seq analysis of Col2a1-cre-marked chondrocytes and perichondrial cells at E13.5. a: Col2a1-cre; R26R^tdTomato femur immunostained for SOX9. R round layer, F flat layer, PH prehypertrophic layer, PC perichondrium, Grey DIC. Scale bar: 200 µm. n = 3 mice. b, c FACS (b) and scRNA-seq (c) strategy for Col2a1^cre-tdTomato⁺ cells (red box). Shown are cells isolated from Col2a1-cre; R26R^tdTomato limbs (left) or R26R^tdTomato control limbs (right). d UMAP plot of major classes of Col2a1^cre-tdTomato⁺ cells (Cluster 0 – 9, 7,889 cells). Pooled from n = 5 mice. Dotted box: Feature plot of tdTomato. e Feature plots of representative genes enriched in each cluster. Cluster 0,1,2: Sox9⁺ (green dotted contour), Cluster 1: Col2a1⁺, Acan⁺, Fgfr3⁺, Cluster 1,5: Sp7⁺, Cluster 4,5: Prrx1⁺, Dlx5⁺, Cluster 5: Runx2⁺. Violet: high expression, yellow: low expression. f Fgfr3-GFP; Osx-mCherry femur immunostained for SOX9. Scale bar: 200 µm. n = 4 mice. g RNAScope analyses of Dlx5. Scale bar: 200 µm. n = 4 mice. h RNA velocity analysis. Dynamical model-based RNA velocity vectors superimposed on the UMAP plot. The origins of black arrows represent the inferred initial states, namely Cluster 1 (blue arrowhead) and Cluster 5 (red arrowhead). i Initial state and root cell inference. Top: colored by GPCCA-based, CellRank-computed initial state probability. Violet indicates low probability while yellow indicates high probability. Bottom: Cluster 5 is the inferred root cell population.