Branching morphogenesis of sea cucumber ossicles in multi-cellular syncytial confinement

Biomineralization is in living systems and has remained elusive due to limitations in the simultaneous understanding of both physical and biomolecular processes. In holothurians, biomineralization occurs in the form of discrete ~100 um length scale structures called ossicles that diversify in shapes not only across species but even within an individual animal. Through live and fixed microscopy techniques, we establish that these structures grow from individual crystalline seeds inside a multi-cell syncytial complex with the biomineralized phase completely covered with a membrane-coated cytoplasmic sheath. We demonstrate that the initial seed transforms into a multi-holed structure through 4 key steps - instability in the seed, tip extension, tip splitting, and merging of two tips. Through large-scale statistics on microCT data, we probe the robustness of the processes described. Throughout the growth, we demonstrate that cytoplasmic and membranous activity restricted to the surface of the biomineralized phase rather than motility of participating cell bodies regulate the material transport and directional growth of the structure. By observing distinct developmental niches, we demonstrate differential symmetry breaking and seed cell-cluster dispersion as the structure grows, which acts as an additional layer of control. The system thus serves as a unique playground merging non-equilibrium solidification growth in melts/solutions and classical branching morphogenesis in living systems.