Abstract
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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The manuscript was comprehensively revised and now includes several new experiments and data points, so that the manuscript now encompasses four main and four supplementary figures. Mainly some basic descriptions of the experimental model is now included as well as a previously missing experiment to explain suggested cell non-autonomous effects of Brachyury-deficiency on body axis elongation via feedback loops with Wnt3a.