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

Stem cell-derived 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 utilizing 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 wildtype cells. We showcase this experimental approach in chimeric gastruloids of mESCs carrying homozygous deletions of the Tbx transcription factors 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 unrecognized phenotypes such as tissue sorting preference of Brachyury -deficient cells to endoderm, and cell non-autonomous effects of Brachyury -deficiency on Wnt3a -patterning along the embryonic axis, demonstrating some of the advantages of chimeric gastruloids as efficient tool for studies of mammalian gastrulation.