Hierarchical TBX6-FOXC Regulatory Logic Governs Human Trunk Mesoderm Diversification

Summary Mesoderm forms during gastrulation and diversifies to generate many embryonic tissues, yet the regulatory logic governing early human mesodermal lineage decisions remains poorly defined. We use human iPSC-derived 3D trunk-like structures (hTLS) to model lineage specification during early post-gastrulation development, a stage experimentally inaccessible in humans. Developing hTLS generate neural and mesodermal lineages and recapitulate progressive mesodermal diversification into paraxial (somitic) and intermediate (renal) identities. Using genetic perturbations and temporally controlled transcription factor activation, we identify duration-dependent TBX6 activity as a critical determinant of mesodermal diversification. Downstream, the forkhead box transcription factors FOXC1 and FOXC2 stabilise somitic identity and are required for sclerotome differentiation. These findings reveal a duration-dependent hierarchical regulatory logic in early human mesoderm, in which competence for diversification is established before downstream lineage identities are stabilised, positioning hTLS as a powerful platform to experimentally dissect human developmental programs. Highlights Human trunk-like structures model mesodermal diversification during early post-gastrulation human trunk development TBX6 duration acts as a temporal gate controlling mesodermal fate potential FOXC1/2 function downstream of TBX6 to lock in somitic identity and restrict alternative fates DegTrace enables lineage tracing following transient transcription factor activation