Conditional Control of Protein Function in Aquatic Embryos

The work discussed in this dissertation covers a wide array of diverse projects with one unifying theme: engineering conditional control over biomolecular function in the developing Danio rerio and Xenopus laevis embryo. The zebrafish and African clawed frog are both well utilized aquatic vertebrate animal models. Both embryos have been used extensively to yield foundational knowledge about vertebrate development and disease. Development is a highly dynamic, and in the case of fish and frog embryos, a very rapid process that depends on orchestrated signaling events and genetic programs to work in harmony. Anything temporally or spatially out of tune can disrupt important developmental steps and cause disease. When studying the effects of a particular protein during development, most rely on overexpressing or depleting the protein of interest, and with the exception of conditional Cre/loxp transgenic lines, this disruption impacts all tissues and all stages of development. To truly understand a biological process as complex as gastrulation, tissue differentiation, and organ development, we need to understand the spatial and temporal context of the individual players (biomolecules) and their role. For many proteins, the initial excavation into their function during development has already been complete, but now there is a need for more fine and precise tools to unearth new insight. In this dissertation, I discuss the development of conditionally controlled proteins and miRNA to dissect their spatiotemporal context in the developing embryo. To make these new tools, I rely on genetic code expansion for the site-specific incorporation of unnatural amino acids, caged RNA oligonucleotides for the optical control of Cas9 and dCas9 function, circularized morpholino oligonucleotides for the conditional knockdown of miRNA, and a photocleavable fluorescent protein to induce targeted apoptosis in live embryos