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...

Morphogenesis is a precise and robust dynamic process during metazoan embryogenesis, consisting of both cell proliferation and cell migration. Despite the fact that much is known about specific regulations at molecular level, how cell proliferation and migration together drive the morphogenesis at cellular and organismic levels is not well understood. Using Caenorhabditis elegans as the model animal, we present a phase field model to compute early embryonic morphogenesis within a confined eggshell. With physical information about cell division obtained from three-dimensional time-lapse cellular imaging experiments, the model can precisely reproduce the early morphogenesis process as seen in vivo, including time evolution of location and morphology of each cell. Furthermore, the model can be used to reveal key cell-cell attractions critical to the development of C. elegans embryo. Our work demonstrates how genetic programming and physical forces collaborate to drive morphogenesis and provides a predictive model to decipher the underlying mechanism.

Metazoan development demands not only precise cell fate differentiation but also accurate timing of cell division to ensure proper development. How cell divisions are temporally coordinated during development is poorly understood. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study this coordination due to its invariant development and widespread division asynchronies. One of the most pronounced asynchronies is a significant delay of cell division in two endoderm progenitor cells, Ea and Ep, hereafter referred to as E2, relative to its cousins that mainly develop into mesoderm organs and tissues. To unravel the genetic control over the endodermspecific E2 division timing, a total of 822 essential and conserved genes were knocked down using RNAi followed by quantification of cell cycle lengths using in toto imaging of C. elegans embryogenesis and automated lineage. Intriguingly, knockdown of numerous genes encoding the components of general transcription pathway or its regulatory factors leads to a significant reduction in the E2 cell cycle length but an increase in cell cycle length of the remaining cells, indicating a differential requirement of transcription for division timing between the two. Analysis of lineage-specific RNA-seq data demonstrates an earlier onset of transcription in endoderm than in other germ layers, the timing of which coincides with the birth of E2, supporting the notion that the endoderm-specific delay in E2 division timing demands robust zygotic transcription. The reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. The results suggest that a tissue-specific transcriptional activation is required to coordinate fate differentiation, division timing, and cell migration to ensure proper development.Proper development of metazoans depends not only on precise differentiation of cell fate but also on tight control over division timing or division pace between cells, which we refer to as temporal coordination. A normal fate specification without correct division timing may lead to catastrophes, for example, cancerous development (1). Therefore, metazoan development demonstrates stereotyped division timing (2, 3). Despite intensive studies on the regulation of cell fate differentiation, genetic control over temporal coordination of cell division during metazoan development is poorly understood. Timing of cell division is particularly critical during early developmental stages such as embryogenesis when cells undergo rapid division and migration, which is concomitant with cell fate differentiation.Because of technical challenges in quantifying cell division timing especially when an embryo undergoes rapid cell division, most of the studies on cell division timing focus on the earliest stage of development (4). For example, the first embryonic division in Caenorhabditis elegans produces two daughters, namely AB and P1, with differential developmental potential. The two cells also divide asynchronously with the ...

Nematode species are well-known for their invariant cell lineage pattern, including reproducible division timing, volume segregation, fate specification and migration trajectory for each and every cell during embryonic development. Here, we study the fundamental principle optimizing cell lineage pattern with Caenorhabditis elegans. Combining previous knowledge about the fate specification induced by asymmetric division and the anti-correlation between cell cycle length and cell volume, we propose a model to simulate lineage by altering cell volume segregation ratio in each division, and quantify the derived lineage's performance in proliferation rapidity, fate diversity and space robustness (PFS Model). The stereotypic pattern in early C. elegans embryo is one of the most optimal solutions taking minimum time to achieve the cell number before gastrulation. Our methods lay a foundation for deciphering principles of development and guiding designs of bio-system.

hk § These authors contributed equally to this work.Cell lineage consists of cell division timing, cell migration and cell fate, which are highly reproducible during the development of some nematode species, including C. elegans. Due to the lack of high spatiotemporal resolution of imaging technique and 1 reliable shape-reconstruction algorithm, cell morphology have not been systematically characterized in depth over development for any metazoan. This significantly inhibits the study of space-related problems in developmental biology, including cell segregation, cell-cell contact and cell shape change over development. Here we develop an automated pipeline, CShaper, to help address these issues. By quantifying morphological parameters of densely packed cells in developing C. elegans emrbyo through segmentation of fluorescene-labelled membrance, we generate a time-lapse framework of cellular shape and migration for C. elegans embryos from 4-to 350cell stage, including a full migration trajectory, morphological dynamics of 226 cells and 877 reproducible cell-cell contacts. In combination with automated cell tracing, cell-fate associated cell shape change becomes within reach. Our work provides a quantitative resource for C. elegans early development, which is expected to facilitate the research such as signaling transduction and cell biology of division. Introduction 1 Embryogenesis in metazoans is a spatio-temporal biological process formed by a series 2 of multicellular structure evolution including proliferation and morphogenesis. As "eu-3 tely" C. elegans has invariant and reproducible cell lineage consisting of division tim-4 ing, migration trajectory and fate specification for each cell 1 , it has been wildly used as 5 2 an animal model for developmental biology research 2 . Thanks to advanced imaging e-6 quipment with single-cell resolution as well as automatic cell-tracing softwares 3-5 , a few 7 researchers have made great effort to quantitatively reconstruct its developmental atlas 8 in several dimensions of developmental properties, including cell division timing 6 , gene 9 expression and morphogenesis 7, 8 , cell-cell contact mapping and signaling 9, 10 . Despite al-10 l this, little is known about cell morphology experimentally and systematically, due to 11 lack of high-resolution cell membrane signaling marker and reliable imaging-based algo-12 rithm for cell segmentation, in particular for late stage which has hundreds of cells 11, 12 . 13 Cell morphology (e.g. cell shape, cell volume, cell-cell contact) is also a set of critical 14 developmental properties and information for metazoan embryogenesis, which is tight-15 ly correlated to cell-cycle control 13-15 , spindle formation 16 , cell-fate symmetry break-16 ing and differentiation 17, 18 , intercellular signal transmission 10, 12, 19, 20 , cytomechanics and 17 morphogenesis 21-24 , etc. 18 Recent studies also emphasized the necessity of 3-dimensional cellular segmenta-19 tion aside from the nucleus 25, 26 . With large quantities of volumetri...