Defense Date

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Integrative Life Sciences

First Advisor

Robert Tombes

Abstract

Despite external bilateral symmetry, the internal organs of vertebrates are asymmetrically arranged. The development of Left-Right (LR) asymmetry is dependent on the activity of a ciliated organ known as the Kupffer’s vesicle (KV) or the embryonic node, in zebrafish and mouse, respectively. Cilia beating within the KV are essential to generate a counterclockwise flow of fluid, which leads to left-sided Ca2+ elevation. Ca2+ is a crucial morphogenic second messenger during vertebrate development. Ca2+ elevation activates targets, such as CaMK-II, which is also known as the multifunctional Ca2+/calmodulin-dependent protein kinase. Studies have linked CaMK-II to secretion (mediated through synapsin I) and to bipolar spindle formation in mammalian cells, among other cellular functions. In mammals, CaMK-II is encoded by four genes: α, β, δ, and γ. In zebrafish, like other teleost fish, gene duplication has yielded eight genes (α, αKAP, β1, β2, δ1, δ2, γ1, and γ2), which are transcriptionally active during the first 72 hours post fertilization (hpf). This dissertation has demonstrated that suppression of at least three of these CaMK-II genes disrupts proper LR-axis formation. In particular, knockdown of γ1 and αKAP CaMK-II dramatically affects southpaw (spaw) expression in the Lateral Plate Mesoderm (LPM). Interestingly, activated CaMK-II (P-CaMK-II) appears transiently in cells on the left side of the KV following Ca2+ elevation, and prior to spaw expression in the LPM. These findings indicate a role for CaMK-II in the processing or secretion of Spaw to the left LPM. In addition, cilia length and number are diminished in all three morphants, suggesting a role for CaMK-II in the process of ciliogenesis. CaMK-II is a binding partner of Kif2C, a Kinesin 13 member. Kinesin 13 members control flagellar length in Chlamydomonas, the blue-green algae and protists. The roles of Kif2C in zebrafish have never been explored, but could potentially explain cilia shortening seen in CaMK-II morphants. Zebrafish has served as an excellent model organism for the determination of the relative contributions of calcium signaling CaMK-II and Kif2C to the cellular and molecular mechanisms of LR-axis formation.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

12-11-2012

Available for download on Sunday, December 11, 2022

Included in

Life Sciences Commons

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