Development and application of light-controlled gene silencing methods in zebrafish embryos

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Embryonic development is a remarkable program of cell proliferation, migration, and differentiation that transforms a single fertilized egg into a complex multicellular organism. This process depends on spatial and temporal control of gene function, and deciphering the molecular mechanisms that underlie pattern formation requires novel methods for perturbing gene expression with similar precision. Synthetic reagents can help meet this demand, and in this thesis I describe the development and application of caged morpholino (cMO) oligonucleotides for inactivating genes in zebrafish and other optically transparent organisms with spatiotemporal control simply by irradiating embryonic tissues with a focused light beam. In chapter 1 I provide an overview of the zebrafish model system of vertebrate development and survey the capabilities and limitations of various oligonucleotide-based technologies for perturbing RNA function and tracking RNA expression in zebrafish. I examine various light-gated oligonucleotide technologies that exploit the optical transparency of zebrafish embryos, including cMOs, for achieving spatiotemporal control of RNA function. In chapter 2 we describe the initial synthesis of a cMO targeting expression of the no tail a (ntla) transcription factor. By permitting spatiotemporal gene regulation in zebrafish embryos, the ntla cMO was used to make initial observations into the time-dependent role of this gene in notochord formation. In chapter 3 we report optimized methods for the design and synthesis of hairpin cMOs, 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, I 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 established the generality of cMO technologies to facilitate the application of these chemical probes in vivo for functional genomic studies. Finally, in chapter 4 we illustrate the utility of the cMO technology in isolating spatiotemporally-distinct functions of transcription factors -- genes that play diverse roles during embryonic development, with each controlling multiple cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level; however, probing in vivo gene function with comparable spatiotemporal precision has been a technological challenge. To address this need, I have integrated cMOs with similarly caged fluorophores, fluorescence-activated cell sorting (FACS), and microarray technologies. Using this approach, I have dynamically profiled the No tail-a (Ntla)-dependent transcriptome at different stages of zebrafish mesoderm development, discovering discrete sets of genes that are associated with either notochord cell fate commitment or subsequent changes in cell function. Our studies elucidated the roles of several Ntla-regulated genes in notochord development and demonstrated the activation of multiple transcriptomes within a cell lineage by a single transcription factor.


Type of resource text
Form electronic; electronic resource; remote
Extent 1 online resource.
Publication date 2011
Issuance monographic
Language English


Associated with Shestopalov, Ilya
Associated with Stanford University, Department of Chemistry
Primary advisor Chen, James Kenneth
Thesis advisor Chen, James Kenneth
Thesis advisor Kool, Eric T
Thesis advisor Wender, Paul A
Advisor Kool, Eric T
Advisor Wender, Paul A


Genre Theses

Bibliographic information

Statement of responsibility Ilya Anatoliy Shestopalov.
Note Submitted to the Department of Chemistry.
Thesis Thesis (Ph.D.)--Stanford University, 2011.
Location electronic resource

Access conditions

© 2011 by Ilya Shestopalov
This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).

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