# The roles of TGF-β, Wnt, and MAPK signaling pathways in joint lineage specification in vitro and ex vivo

## TL;DR

Researchers created a model using stem cells to study how joint tissues develop, finding that specific signaling pathways control joint cell formation.

## Contribution

A novel dual reporter mouse embryonic stem cell line was developed to study joint lineage specification and signaling pathways.

## Key findings

- TGF-β signaling is necessary and sufficient for inducing joint markers Gdf5 and Prg4.
- Inhibiting Wnt or MAPK signaling promotes joint lineage formation in vitro and ex vivo.
- mESC-derived joint lineages are transcriptionally similar to primary limb bud cells.

## Abstract

The initiation of synovial joint development and subsequent differentiation of progenitor cells toward anatomically and functionally distinct joint tissues are not well understood, despite being highly relevant to joint health and disease. We generated a dual reporter mouse embryonic stem cell (mESC) line to quantify cells expressing growth differentiation factor five (Gdf5), an early marker of joint formation, and Prg4, a lubricating proteoglycan found in joint tissues. Transforming growth factor β (TGF-β) signaling was necessary and sufficient for the induction of Gdf5-RFP and Prg4-GFP. Inhibition of either Wnt or MAPK signaling significantly increased the induction of Gdf5-RFP, while activation of either pathway prohibited this induction. Single cell transcriptomics demonstrated the chondrogenic identity of Gdf5+ cells in in vitro cultures and in mouse embryonic limb buds. We validated the roles of these signaling pathways in joint-specific ex vivo limb bud cultures. Thus, this in vitro model enhances our understanding of joint development and offers new insights into potential therapeutic approaches for joint disorders.

•A dual reporter mESC line was created to study joint lineage cell differentiation•mESC-derived joint lineages are transcriptionally similar to primary limb bud cells•TGF-β signaling is required and sufficient to induce Gdf5 and Prg4 in mESCs•Inhibition of Wnt or MAPK pathways promotes joint lineages in vitro and ex vivo

A dual reporter mESC line was created to study joint lineage cell differentiation

mESC-derived joint lineages are transcriptionally similar to primary limb bud cells

TGF-β signaling is required and sufficient to induce Gdf5 and Prg4 in mESCs

Inhibition of Wnt or MAPK pathways promotes joint lineages in vitro and ex vivo

Craft and colleagues introduce two developmental models used to delineate signaling pathways for promoting joint cell identities, thereby illuminating a poorly understood area of cell fate determination. This work represents a significant step toward recapitulating lineage commitment steps and those occurring downstream in pluripotent stem cell cultures to generate clinically relevant joint cells and tissues for repair.