Introduction: The cardiopharyngeal mesoderm (CPM) gives rise to multiple cranial and cardiac muscle lineages. While bi-potent progenitors contributing to both compartments have been identified, the early specification steps of CPM remain poorly understood. Key questions persist: Do multipotent progenitors contribute to all CPM derivatives, including endothelial cells? When and how are lineage decisions made? These questions are especially relevant in the context of velocardiofacial syndrome, a rare congenital disorder affecting both heart and head structures, yet early CPM specification has not been thoroughly investigated.

Objectives: We aim to elucidate the mechanisms governing CPM lineage segregation under normal conditions and in disease models with craniofacial and cardiac defects.

Methods: We use a PolyloxExpress clonal analysis system that results in transcriptomic barcoding to study cell fate. Combined with 3D gastruloid culture, we simultaneously track cell lineage and transcriptomic identity during early development.

Results: Gastruloids derived from PolyloxExpress; MerCreMer mouse embryonic stem cells recapitulate CPM specification. We have successfully induced barcodes and tracked lineage emergence at three key time points, confirming robust barcode induction and lineage stability.

Conclusion: Our approach will shed light on the mechanisms of CPM specification and provide new insights into the developmental origins of complex congenital syndromes, ultimately improving diagnosis and patient care.