Identifying cross-lineage dependencies of cell-type specific regulators in gastruloids

Correct gene expression levels in space and time are crucial for normal development. Advances in genomics enable the inference of gene regulatory programs that are active during development. However, this approach cannot capture the complex multicellular interactions that occur during embryogenesis. Compared to model organisms such as fruit flies and zebrafish, the growth of mammalian embryos in utero further complicates the analysis of cell-cell communication during development. However, in vitro models of mammalian development such as gastruloids can overcome this limitation. Using time-resolved single-cell chromatin accessibility analysis, we have delineated the regulatory landscape during gastruloid development and thereby identified the critical drivers of developmental transitions. We observed that gastruloids develop from pluripotent cells driven by the transcription factor (TF) dimer OCT4-SOX2 and differentiate along two main branches. A mesoderm branch characterized by the TF MSGN1 and a spinal cord branch characterized by CDX1, 2, 4 (CDX). Consistent with our lineage reconstruction, {Delta}CDX gastruloids fail to form spinal cord. Conversely, Msgn1 ablation inhibits the development of paraxial mesoderm, as expected. However, this also abolished spinal cord cells, which is surprising given that MSGN1 is not associated with differentiation along this branch. Therefore, formation of paraxial mesoderm is required for spinal cord development. To validate this, we generated chimeric gastruloids using {Delta}MSGN1 and wildtype cells, which formed both spinal cord and paraxial mesoderm. Strikingly, {Delta}MSGN1 cells specifically contributed to spinal cord, suggesting that cell-cell interactions between paraxial mesoderm and spinal cord are necessary for the formation of the latter. Our work has important implications for the study of cell-cell communication in development and how gene regulatory programs are functionally executed to form complex multicellular developmental structures.