Engineered dual-gastruloid model for foregut-pharyngeal mesoderm co-development

During mammalian gastrulation, progenitors of craniofacial and cardiopharyngeal muscles arise from anterior mesoderm adjacent to foregut endoderm, and they are controlled by gene regulatory networks and signalling pathways distinct from those of trunk mesoderm. While in vitro models capture aspects of posterior and heart development, a spatially organised interface between foregut-like endoderm and cranial-mesoderm-like tissue has not been described. Here we introduce a dual-aggregate gastruloid model, formed by fusing two mouse embryonic stem cell aggregates patterned under distinct Nodal, FGF, and Wnt signalling. These structures reproducibly establish an anterior-posterior axis and position a Foxa2⁺/Sox17⁺/Cdh1⁺ foregut-like epithelium proximal to Pax1⁺/Pax9⁺ pharyngeal-like endoderm and Tbx1⁺ cranial-mesoderm-like cells, resembling the E8.5 pharyngeal region. Reciprocal chimeras and Foxa2 loss-of-function indicate that Foxa2⁺ endoderm is required for Tbx1 expression in adjacent anterior mesoderm. Pharmacological perturbation of Hedgehog disrupts Foxa2 and Tbx1 expression. The model provides a tractable platform for dissecting endoderm-mesoderm signalling and congenital craniofacial disease mechanisms.