Studying zebrafish development and regeneration with chemical probes
- Embryonic development and tissue regeneration are regulated by complex genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control gene function with spatial and temporal precision. Synthetic chemistry has been increasingly used to create reagents that can enable such genetic manipulations. In this thesis I describe the development and application of caged morpholino (cMO) oligonucleotides and small molecules to study zebrafish embryonic development and larvae tail regeneration. In Chapter 1, we survey how chemical tools and engineered proteins have been used to perturb developmental processes at the DNA, RNA, protein, and cellular levels. We discuss the design principles, experimental capabilities, and limitations of each method, as well as future challenges for the chemical and developmental biology communities. In Chapter 2, we describe optimized methods for the design and synthesis of hairpin cMO oligonucleotides incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies. In Chapter 3, I first describe our genome-wide microarray analysis and whole-mount in situ hybridization studies that led us to the identification of 18 genes, including hyaluronan synthase 3 (has3), that were expressed during zebrafish larvae tail regeneration. I also report the functional studies of hyaluronan (HA) synthesis using small molecule inhibitor 4-methylumbelliferone (4-MU), in which we showed that 4-MU inhibited tail regeneration by preventing injury-induced cell proliferation. In addition, 4-MU also reduced the expressions of dlx5a and junbb, markers of the wound epithelium and blastema-like mesenchymal cells. We also illustrated that simultaneous dosing of Gsk3[beta] inhibitor BIO and 4-MU could rescue the 4-MU-induced regeneration defects. Our results indicated that the crosstalk between hyaluronan synthesis and Gsk3[beta] signaling played an important role in regulating tail regeneration.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Stanford University, Department of Chemical and Systems Biology.
|Longaker, Michael T
|Longaker, Michael T
|Statement of responsibility
|Submitted to the Department of Chemical and Systems Biology.
|Ph.D. Stanford University 2012
- © 2012 by Xiaohu Ouyang
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Also listed in
Loading usage metrics...