Cell lineage consists of cell division timing, cell migration and cell fate, and is highly conserved during development of nematode species. An outstanding question is how differentiated cells are genetically and physically regulated in order to migrate to their precise destination among individuals. Here, we first generated a reference embryo using time-lapse 3 dimensional images of 222 wild-type C. elegans embryos at about 1.5-minute interval. This was achieved by automatic tracing and quantitative analysis of cellular phenotypes from 4-to 24-cell stage, including cell cycle duration, division orientation and migration trajectory. We next characterized cell division timing and cell kinematic state, which suggests that eight groups of cells can be clustered based on invariant and distinct division sequence. Cells may still be moving while others start to divide, indicating strong robustness against motional noise in developing embryo. We then devised a system-level phenotyping method for detecting mutant defect in global growth rate, cell cycle duration, division orientation and cell arrangement. A total of 758 genes were selected for perturbation by RNA interference followed by automatic phenotyping, which suggests a cryptic genetic architecture coordinating early morphogenesis spatially and temporally. The high-quality wild-type reference supports a conceptual close-packing model for cell arrangement during 4-to 8-cell stage, implying fundamental mechanical laws regulating the topological structure of early C. elegans embryo. Also, we observed a series of remarkable morphogenesis phenomena such as induced defect or recovery from defect in mutant embryo. To facilitate use of this quantification system, we built a software named STAR 1.0 for visualizing the wild-type reference and mutant phenotype. It also allows automatic phenotyping of new mutant embryo. Taken together, we not only provide a statistical wild-type reference with defined variability, but also shed light on both genetic and physical mechanisms coordinating early embryonic morphogenesis of C. elegans. The statistical reference permits a sensitive approach for mutant phenotype analysis, with which we phenotype a total of 1818 mutant embryos by depletion of 758 genes. Figure 1. Establishment of wild-type morphogenesis reference with spatio-temporal properties at single-cell level. A. A pipeline consisting of data acquisition, quality control, data processing and data integration. B. Time-lapse 3D in vivo imaging, embryo reconstruction and automatic cell-position tracing on a C. elegans embryoexpressing GFP in nucleus (green) and PH(PLC1d1) in membrane (red). The whole duration lasted from 4-to 350-cell stage ; the membrane marker here is only for illustration purpose, as most of the data in this work was not obtained from this strain ; 2-cell, 4-cell and 8-cell stages are presented (Supplementary Material 2). C. Cell-lineage tree up to 51-cell stage with tissue-differentiation information [1,21] and cell grouping based on invariant division ordering...

The early embryogenesis in the nematode Caenorhabditis elegans is well-known for its stereotypic precision of cell arrangements and their lineage relationship. Much research has been focused on how biochemical processes achieve the highly reproducible cell lineage tree. However, the origin of the robustness in the cell arrangements is poorly understood. Here, we set out to provide a mechanistic explanation of how combining mechanical forces with the order and orientation of cell division ensures a robust arrangement of cells. We used a simplified mechanical model to simulate the arrangement of cells in the face of different disturbances. As a result, we revealed three fail-safe principles for cell self-organization in early nematode embryogenesis: ordering, simultaneity, and the division orientation of cell division events. Our work provides insight into the developmental strategy and contributes to the understanding of how robust or variable the cell arrangement can be in developing embryos.