UC San Diego

The freshwater cnidarian Hydra owes its long history in experimental biology to its unique potential to address questions regarding the formation of complex patterns in biological systems. Its regenerative abilities enable grafting and transplantation experiments in the adult animal, which allowed researchers to probe and quantify the signaling gradients defining its body axis even prior to the advent of modern biochemical techniques. Furthermore, its ability to regenerate a complete animal from cell aggregates offers an easily studied de novo axis specification event. Hydra's body plan is sufficiently simple that quantitative modeling is feasible. Several such models of pattern formation have been developed that can reproduce experimental observations of transplantations and regeneration. However, the quantitative experimental data needed to definitively test their core premises have remained largely out of reach, representing a major limitation on Hydra's broader relevance.

In this dissertation, I leverage recent advances in molecular biology combined with adaptations of classical experiments to provide some of this quantitative validation. Existing models of Hydra patterning make two core assumptions: that a shift in oscillation pattern is a close approximation for biochemical specification of the body axis, and that tissue pieces can be used in lieu of cell aggregates for model testing and validation. We show the oscillation pattern shift is the result of a functional mouth structure rather than a close marker for a biochemical axis specification event. Further, we demonstrate that morphogen gradients encode axis information in the body of Hydra at a scale of several hundred microns, indicating that small tissue fragments do not exhibit an axis specification event as previously assumed. These findings demonstrate the need to reevaluate existing models. In addition, I aimed to develop new tools to enable further research. I developed transgenic strains introducing a new fluorescent protein to the system, and created plasmid constructs for a fluorescent fusion protein reporter for in vivo quantitative imaging. I also gathered preliminary data in support of innexins as regulators of patterning. This dissertation thus presents several valuable experimental findings on the nature of axis specifications in Hydra, and lays foundations for future quantitative studies.