Shayan Shamipour

Multicellular self-organization depends on interactions across multiple length scales, yet mapping protein states at high spatial resolution in whole-mount embryos remains challenging. Here, we introduce high-throughput 3D in toto iterative immunofluorescence imaging (3D-4i) and a dedicated computer vision pipeline to quantify morphological and molecular features from subcellular to whole-embryo scales across hundreds of samples. Applying this pipeline to early zebrafish embryos undergoing mid-blastula transition, we determine the cell cycle phase for each cell across the embryo, and uncover the spatiotemporal dynamics by which global meta-synchronous mitotic waves transition to cell cycle desynchronization. Using statistical analysis, we find that the cell cycle phase is the major source of variability in transcription within a division cycle, and combining this with the analysis of key transcription factors and chromatin modifier state, included in our multiplexed dataset, enables accurate prediction of transcriptional output during zygotic genome activation in individual cells. Together, these findings establish 3D-4i as a powerful approach for quantifying multimodal, multiscale biological processes underlying multicellular self-organization.

Self-organization in multicellular systems emerges from reciprocal interactions across spatiotemporal scales. Understanding how subcellular organization, tissue remodeling and developmental outcome are coordinated, thus requires simultaneous profiling of biological processes spanning orders of magnitudes in space and time. Yet, a unified experimental and computational framework for capturing these multiscale properties across in vivo and stem cell-derived systems has been lacking. Here, we introduce Shield-4i, a high-throughput, versatile, and accessible method for automated in toto iterative immunofluorescence imaging of whole-mount structures at subcellular resolution. Through polyepoxide-mediated inter- and intramolecular crosslinking, Shield-4i preserves sample integrity during repeated SDS-based elution cycles. We benchmark this method in gastrulating zebrafish and post-implantation mouse embryos and demonstrate its applicability to stem cell-derived 3D gastruloids, achieving up to 30-plex measurements of proteins and their post-translational modifications across hundreds of samples. To enable scalable analysis, we developed a dedicated 3D workflow supporting OME-Zarr-based and FAIR-compliant data storage, standardized processing, and multiscale feature extraction. Applying this framework to investigate gastruloid self-organization, we quantify how cellular physicochemical state and signaling properties encode cell position along embryonic axes and connect molecular patterning and fate decisions to morphological symmetry breaking at the multicellular scale. Together, Shield-4i provides a high-content in toto spatial proteomics platform for dissecting multiscale information flow and self-organization in multicellular systems.

During embryonic development, mechanical forces are essential for cellular rearrangements driving tissue morphogenesis. Here, we show that in the early zebrafish embryo, friction forces are generated at the interface between anterior axial mesoderm (prechordal plate, ppl) progenitors migrating towards the animal pole and neurectoderm progenitors moving in the opposite direction towards the vegetal pole of the embryo. These friction forces lead to global rearrangement of cells within the neurectoderm and determine the position of the neural anlage. Using a combination of experiments and simulations, we show that this process depends on hydrodynamic coupling between neurectoderm and ppl as a result of E-cadherin-mediated adhesion between those tissues. Our data thus establish the emergence of friction forces at the interface between moving tissues as a critical force-generating process shaping the embryo.