# Stage- and cluster-specific regulation of chondrogenic gene programs by Sox9 in mouse embryonic limb buds

## TL;DR

This study explores how the Sox9 gene regulates cartilage development in mouse embryos, showing that its activity changes with developmental stages and cell types.

## Contribution

The research provides a stage-resolved regulatory map of Sox9 activity during chondrogenesis using integrated single-cell and chromatin profiling.

## Key findings

- Sox9 chromatin occupancy undergoes stage- and cluster-specific reconfiguration.
- RNA-velocity reveals independent maturation trajectories from progenitor clusters.
- Sox9 regulates distinct transcriptional networks across cartilage developmental stages.

## Abstract

Cartilage formation in the limb is initiated and sustained by Sox9, though how its regulatory outputs evolve across developmental time and cell states remains unclear. Here, we integrate single-cell RNA sequencing of mouse forelimb buds across five stages (E9.5–E13.5) with CUT&RUN profiling of Ty1-tagged Sox9 at E11.5 and E13.5. We identify four Sox9-high populations—three chondroprogenitor clusters and mature chondrocytes—with distinct dynamics, and RNA-velocity infers independent trajectories from each progenitor cluster to maturation. Sox9 chromatin occupancy shows a conserved motif signature but undergoes stage- and cluster-dependent reconfiguration, aligning with shifts in extracellular matrix, cell-cycle, and patterning programs. Integrative analysis links these binding differences with coordinated transcriptional changes, suggesting that Sox9 operates through context-associated regulatory modes rather than a single uniform program. Our study provides a stage-resolved regulatory map of Sox9-associated regulation during chondrogenesis and a resource for limb mesenchyme.

•Three chondroprogenitor clusters and mature chondrocytes show stage-dependent dynamics•CUT&RUN of Ty1-tagged Sox9 reveals stage- and cluster-specific chromatin occupancy•Integration of CUT&RUN and scRNAseq identifies Sox9 targets in chondrocyte maturation•Sox9 regulates transcriptional networks across cartilage developmental stages

Three chondroprogenitor clusters and mature chondrocytes show stage-dependent dynamics

CUT&RUN of Ty1-tagged Sox9 reveals stage- and cluster-specific chromatin occupancy

Integration of CUT&RUN and scRNAseq identifies Sox9 targets in chondrocyte maturation

Sox9 regulates transcriptional networks across cartilage developmental stages

Molecular biology; Developmental biology; Omics