A single-cell and tissue-scale analysis suite resolves Mixl1's role in heart development.

Perturbation studies using gene knockouts have become a key tool for understanding the roles of regulatory genes in development. However, large-scale studies dissecting the molecular role of development master regulators in every cell type throughout the embryo are technically challenging and scarce. Here, we systematically characterize the knockout effects of the key developmental regulators T/Brachyury and Mixl1 in gastrulation and early organogenesis using single-cell profiling of chimeric mouse embryos. For the analysis of these experimental data, we present COSICC, an effective suite of statistical tools to characterize perturbation effects in complex developing cell populations. We gain insights into T's role in lateral plate mesoderm, limb development, and posterior intermediate mesoderm specification. Furthermore, we generate Mixl1 (-/-) embryonic chimeras and reveal the role of this key transcription factor in discrete mesoderm lineages, in particular concerning developmental dysregulation of the recently identified juxta-cardiac field.