Chimeric 3D gastruloids - a versatile tool for studies of mammalian peri-gastrulation development.

Stem cell-derived three-dimensional (3D) gastruloids show a remarkable capacity of self-organisation and recapitulate many aspects of gastrulation stage mammalian development. Gastruloids can be rapidly generated and offer several experimental advantages, such as scalability, observability and accessibility for manipulation. Here, we present approaches to further expand the experimental potency of murine 3D gastruloids by using functional genetics in mouse embryonic stem cells (mESCs) to generate chimeric gastruloids. In chimeric gastruloids, fluorescently labelled cells of different genotypes harbouring inducible gene expression or loss-of-function alleles are combined with wild-type cells. We showcase this experimental approach in chimeric gastruloids of mESCs carrying homozygous deletions of the Tbx transcription factor brachyury or inducible expression of Eomes. Resulting chimeric gastruloids recapitulate reported Eomes and brachyury functions, such as instructing cardiac fate and promoting posterior axial extension, respectively. Additionally, chimeric gastruloids revealed previously unrecognised phenotypes, such as the tissue sorting preference of brachyury deficient cells to endoderm and the cell non-autonomous effects of brachyury deficiency on Wnt3a patterning along the embryonic axis, demonstrating some of the advantages of chimeric gastruloids as an efficient tool for studies of mammalian gastrulation.

Eomes restricts Brachyury functions at the onset of mouse gastrulation.

Mammalian specification of mesoderm and definitive endoderm (DE) is instructed by the two related Tbx transcription factors (TFs) Eomesodermin (Eomes) and Brachyury sharing partially redundant functions. Gross differences in mutant embryonic phenotypes suggest specific functions of each TF. To date, the molecular details of separated lineage-specific gene regulation by Eomes and Brachyury remain poorly understood. Here, we combine mouse embryonic and stem-cell-based analyses to delineate the non-overlapping, lineage-specific transcriptional activities. On a genome-wide scale, binding of both TFs overlaps at promoters of target genes but shows specificity for distal enhancer regions that is conferred by differences in Tbx DNA-binding motifs. The unique binding to enhancer sites instructs the specification of anterior mesoderm (AM) and DE by Eomes and caudal mesoderm by Brachyury. Remarkably, EOMES antagonizes BRACHYURY gene regulatory functions in coexpressing cells during early gastrulation to ensure the proper sequence of early AM and DE lineage specification followed by posterior mesoderm derivatives.