Prediction and control of Definitive Endoderm morphologies
in an embryo-like in-vitro model

Mammalian embryogenesis is highly robust in terms of morphogenesis. For example, all embryos develop a gut tube with certain dimensions, neural tube, and specific number of somites with a reproducible bilateral arrangement. What leads to this robustness of morphologies is still poorly understood. The use of in-vivo study for a better understanding of early mammalian embryonic development has multiple limitations. In-vitro embryo-like models based on embryonic stem cells have been improved greatly in recent years, and can be used for studying and manipulating early developmental processes. One such advanced model is the Trunk-like Structure (TLS), which uses external cues of biochemical signals and extra-cellular matrix to establish embryo-like features, like somites, neural tube and gut tube. Unlike in a developing embryo which follows a robust developmental path, the embryo-like features of the TLS are highly variable, both in the frequencies of each structure, and in the spatial pattern they make. These variabilities raise the need for a more stable differentiation protocol, with robust differentiation patterns. Here we are interested in the divergence of Definitive Endoderm morphologies in the TLS model. We have defined different endoderm morphologies, and identified early organoid-specific predictors for each one. Using external manipulations, we have decreased the divergence of TLS endoderm development, achieving better control over its morphologies and behaviors. Understanding and controlling in-vitro variabilities can help explain the robustness of embryonic development, as well as help in standardization of organoids for drug or genetic screens.