PROJECT SUMMARY From the moment of conception until death, metazoans face relentless assault on their tissues, their cells, and their genomes. The ability of intricately patterned biological tissues to dynamically withstand these challenges is essential for adult life but is tested first – and perhaps most critically – during embryonic development. Early failures of embryonic patterning can lead to rapid and premature lethality, long-lasting birth defects, and/or devastating developmental disorders. Over the years, developmental biologists have made great progress in understanding many of the links connecting environmental and genetic perturbations to their consequences in embryos, generally by applying reductionist approaches (e.g. single-gene mutant phenotypes, single-clone fate mapping). However, a significant proportion of human pregnancies still result in developmental defects or miscarriages of unknown cause. At present, we also often fail to understand why certain perturbations result in failed embryogenesis in some individuals, but not in others. One persisting challenge is that any model of developmental patterning must simultaneously take into account many elements of a complex system: multiple cell types, each exerting both intrinsic and non-autonomous behaviors, cell turnover dynamics, lineage relationships, and many hundreds of genetic factors. To address these challenges, we are establishing a new experimental paradigm for studies of developmental biology that leverages microfluidics, single-cell genomics, computational biology, and a powerful in vivo molecular genetic model of vertebrate embryogenesis, the laboratory zebrafish (Danio rerio). Under this paradigm, we seek a new form of biological reductionism: comprehensive, molecular decomposition of vertebrate embryonic tissues into their constituent cell states, at an experimental scale not previously accessible to developmental biologists (e.g. 104–106 single-cell measurements per experiment, combined with whole-genome or whole-transcriptome resolution). In this application, we present our motivation and our vision for the whole-embryo single-cell state landscape as a versatile experimental and conceptual platform for systems- level interrogation of vertebrate embryogenesis. Using this landscape approach, we will systematically quantify developmental robustness (i.e. “canalization”) of all embryonic tissues to identify reoccurring bottlenecks and vulnerabilities that drive human birth defects. In parallel, we will dissect mechanisms for cell fate control that direct the “flow” of cells down the embryonic landscape, particularly in contexts where individual cells differ in their genetic and/or developmental fitness. Our experimental vision will therefore revisit fundamental questions in developmental biology, formulated nearly 8 decades ago, but which have persisted until now as abstract principles rather than as testable hypotheses. We anticipate this research program will accelerate our collective efforts to better understand organizing principles of developmental ontogeny in vertebrates in health and disease.

PROJECT NARRATIVE The research proposed here will provide crucial insights into how individual cells of vertebrate animals, such as humans, differentiate into mature tissues. This work will promote better understanding of: (1) cellular principles underlying fate restriction in development, (2) the genetic basis for embryonic failures, birth defects, and developmental disorders, and (3) developmental mechanisms that impact how individual cells compete and interact in embryonic tissues. Overall, the proposed research will accelerate our understanding of error- correction processes that take place naturally in embryos, and will generate molecular predictions for how these processes might be one day harnessed or modulated in human patients.