Establishing a Genetic and Exogenous Toolbox for Studying Multiple Stages of Vertebrate Development in vivo

Author: Dempsey, William P.

Year: 2012

Degree: Dissertation (Ph.D.)

Advisor: Fraser, Scott E.

Committee Members: Phillips, Robert B.; Fraser, Scott E.; Gharib, Morteza; Yang, Changhuei

Option: Bioengineering

DOI: 10.7907/Z98S4MVS

Abstract

Understanding of cell behavior during vertebrate development and repair has been greatly facilitated by advances in biological imaging. Importantly, more powerful tools to generate contrast within the tissue make in vivo analyses of these processes in time and space more tractable. Here, I present my efforts to develop and refine an imaging toolbox to study in vivo cell shape, dynamics, structure, and behavior in the zebrafish vertebrate model system. Mosaic analysis and targeted photoconversion illuminate fine morphological details as cells migrate during gastrulation, revealing cell connections that span several cell diameters across the embryo. These intercellular bridges link cells between lineage boundaries and allow cells to share membrane components on a developmentally relevant time scale. The PhOTO zebrafish transgenic lines combine the strengths of sparse and global cell labeling to monitor cell dynamics and morphology at any stage in the lifetime of the zebrafish. I demonstrate targeted and instantaneous sparse cell tracking in the context of global cell behavior in the embryo, and I also isolate a subset of slowly dividing cells populating a regenerating adult tail fin. Combining fluorescence and endogenous second harmonic generation (SHG) imaging as a tool to study early muscle structure and organization within whole zebrafish muscle compartments uncovers the source of vernier-patterned signal in highly ordered myosin arrays. Instead of being physical distortions in muscle sarcomeres, these patterns may result from an optical artifact of SHG imaging, since comparable signal is not visible in both the SHG and fluorescence channels. To complement the aforementioned genetic labeling and endogenous contrast tools, barium titanate SHG nanoprobes — exogenous and nontoxic SHG-capable nanomaterial tags — are refined for cell labeling in the zebrafish. Silane functionalization acts as a platform for further surface modifications, including: multistep chemical additions, non-reactive surface coating modifications, and antibody linkages for cell targeting applications. The power of each of these tools lies in their compatibility with one another: combining the fluorescence and SHG contrast approaches described here may enable high-resolution imaging at a variety of developmental stages to appreciate the multifaceted cell behaviors governing vertebrate developmental programs more completely.

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