Contact area–dependent cell communication and the morphological invariance of ascidian embryogenesis

INTRODUCTION Within each animal species, embryonic development is highly reproducible, ensuring the faithful production of a complex organism with precisely arranged and shaped organs. In most animal embryos, reproducibility is found at the tissue scale, the behaviors of individual cells being stochastic beyond the first cell divisions. Ascidians, a group of marine invertebrate chordates, show an extreme form of embryoni­­c reproducibility: Homologous cells can be found across individual embryos, and early emb­­ryonic cell lineages are considered invariant. Embryonic geometries are even conserved between species, which diverged 400 million years ago and have very dissimilar genomes. Because of their evolutionary conservation of early embryonic development and ability to buffer genetic divergence, ascidians constitute attractive model systems to study the mechanisms driving cellular reproducibility. RATIONALE To quantify embryonic reproducibility in the ascidian Phallusia mammillata, we first built a high-resolution atlas of embryonic cell lineages, cell shapes, and cell interactions. We imaged 10 live embryos every 2 min up to the end of the neurula stages using multiview light-sheet microscopy. To systematically measure the developmental variability of a range of temporal and spatial cellular features, we developed a robust and scalable adaptive segmentation and tracking of embryonic cells procedure (ASTEC) compatible with high-throughput multiview light-sheet imaging datasets. We related these features to cell fate specification, which in ascidians is mainly controlled by differential sister cell inductions. Inspired by previous work indicating that the area of contact to signaling cells controls ascidian neural induction, we integrated our geometric description with a signaling gene expression atlas. This integration allowed us to test, through computational and experimental approaches, the hypothesis that contact area–dependent cell communication imposes constraints on embryonic geometries. RESULTS We found that, up to the neurula stages, Phallusia embryos develop without cell growth, programmed cell death, or cell neighbor exchanges. Beyond cell position, cell cycle duration, and cell lineages, we observed a high reproducibility of cell arrangements: 75% of cells shared at least 80% of their neighbors in all 10 embryos studied. Furthermore, the areas of contact between homologous cells varied by less than 20% across embryos. Mechanistically, we uncovered a tight link between the control of cell arrangements and asymmetric cell divisions, which give rise to sister cells of distinct fates. We then combined computational and experimental approaches to reveal that areas of cell contact between signaling and responding cells have sufficient encoding potential to explain all known early embryonic inductions, without the need to invoke gradients of ligand concentration. Finally, using geometrical perturbations of embryonic development we demonstrated that precise areas of cell-cell contact were important for mesendodermal and neural fate specification. CONCLUSION Our work establishes the highly reproducible ascidian embryo as a framework to bridge cell behaviors, morphogenesis, and the underlying regulatory program. The ASTEC pipeline allows systematic automated whole-cell segmentation and tracking across whole embryos in high-throughput light-sheet datasets. Second, we establish the geometric control of embryonic inductions as an alternative to classical morphogen gradients and suggest that the range of cell signaling events sets the scale at which embryonic reproducibility is observed. Finally, our study suggests that the high level of reproducibility of ascidian embryonic geometries may paradoxically lift constraints on the evolution of ascidian genomes, thereby contributing to rapid molecular evolution.