Vacuole Formation Guides the Regenerative Path of the Zebrafish Notochord

Abstract

<p>The notochord is a defining feature of our phylum Chordata and has critical roles in human development that are highly conserved in vertebrates. The notochord functions as a hydrostatic scaffold to provide structural rigidity needed for anterior-posterior axis elongation and later for proper spine development. The notochord’s mechanical properties depend on its unique structure. In zebrafish, the notochord consists of a core of giant vacuolated cells surrounded by an epithelial -like sheath. Previous research from our lab has shown that during early development, the notochord vacuole rapidly accumulates fluid and expands within the inelastic notochord sheath. In this work we first investigated the molecular processes by which large vacuolated cells of the notochord maintain integrity while being subjected to a significant amount of stress. We determined that caveolae play a mechanoprotective role in the zebrafish notochord and are crucial in preserving notochord integrity. Upon loss of caveolae, the vacuolated cell collapses at discrete positions under the mechanical strain of locomotion then sheath cells invade the inner notochord and differentiate into vacuolated cells thereby restoring notochord function and allowing normal spine development. Findings from our caveolae work next allowed us to investigate the arrangement of vacuolated cells within the zebrafish notochord. During notochord morphogenesis, the vacuolated cells in wild-type zebrafish arrange themselves in a staircase pattern. However, in both caveolae and vacuole mutants, this pattern is disrupted. We investigated the basis of this pattern and found that it can be described by simple physical principles. We modeled the arrangement of vacuolated cells using a system composed of silicone tubing and sodium polyacrylate jelly beads demonstrating that what we observe in vivo can be described by the theory developed for the packing of spheres in cylinders. We determined that the organization of vacuolated cells within the zebrafish notochord is controlled by the density of fluid filled vacuoles and the diameter of the notochord tube. Lastly, based on our finding that sheath cells of the notochord can form de novo vacuoles, we wanted to identify key factors contributing to notochord vacuole biogenesis and integrity. We used a two-pronged transcriptomics and proteomics approach to identify proteins involved in de novo vacuole formation. We find that loss of a protein previously linked to lysosome related organelle function, Lyst, leads to fragmentation of notochord vacuoles and impaired axis elongation. Interestingly, upon injury of the notochord, sheath cells fail to form a fully inflated vacuole and continue to grow outside of notochord boundaries, forming a tumor-like mass. The tumor-like mass appears very similar to a rare tumor type called chordoma, which is characterized by overgrowth of intervertebral disc tissue. This work suggests that Lyst is important for notochord vacuole biogenesis in zebrafish and may play an important role in chordoma formation. Our work has elucidated novel mechanisms of cell surface integrity and has shown how proper vacuolated cell inflation leads to a structurally intact notochord. Additionally, we have demonstrated the remarkable regenerative capacity of the zebrafish notochord and identified potential regulators of both vacuole biogenesis and chordoma formation. </p>