Neural Innervation Invigorates Yolk Sac Biological Functions beyond Nutrient Reservoir during Zebrafish Embryo Development

The zebrafish yolk sac (YS) is traditionally viewed as a nutrient reservoir. By reconstructing the complete progression of embryonic neural development via live-cell imaging, we previously observed that axonal projections extending from brain cranial neurons come to cover the yolk sac surface, hinting at its uncharacterized functional roles beyond nutrient storage. Using transgenic lines and long-term live imaging, we characterized a dynamic neuro-vascular-metabolic interface on the YS surface. We observed that peripheral neural networks expand radially and mature through hierarchical integration, sharing the same structural and dynamical features as those in the brain and spinal cord. Unexpectedly, YS surface cells harbor transient calcium flashes, reflecting primitive intercellular communication in response to neural innervation. Furthermore, we characterized activity-dependent neuronal pruning and stress-induced lipid droplet crystallization as indicators of developmental refinement and homeostatic collapse, respectively. Finally, we identified directional blood flow occurring before the formation of endothelial tubes, indicating a pre-vascular transport mechanism. These findings demonstrate that neural innervation enables the YS to serve as a coordinated developmental hub, facilitating complex crosstalk between neural, vascular, and metabolic systems during early vertebrate embryogenesis. Significance StatementTraditional paradigms view the embryonic yolk sac simply as a nutrient reservoir. Using advanced live-cell imaging in zebrafish, we uncover that the yolk sac can be innervated by brain-expanded axonal networks during development, invigorating its biological functions, including neural signal, vascular flow, and metabolic activity. Rather than maintaining local neuronal bodies, these long-distance nerve extensions map out an early topological scaffold that may guide future internal organ innervation during yolk resorption. Furthermore, we capture pre-vascular blood transport and stress-induced metabolic shifts on the yolk sac surface. These findings redefine the yolk sac as a neural-controlled functional hub before organs form.