THESIS
2014
iv leaves, v-xvi, 137 pages : illustrations (some color) ; 30 cm
Abstract
Ca
2+ signaling has been extensively studied in the mature heart. In addition to
excitation-contraction
coupling, Ca
2+ also plays a major role in regulating heart development
via excitation-transcription
coupling. Downstream targets of the latter include myocardium
transcription factors, such as Mef2, Gata4 and Nkx2.5. Ca
2+ has been reported to act via CaM
kinase and calcineurin, and subsequent Ca
2+ effectors, to modulate the transcription or activity
of these myocardium transcription factors, which in turn activate multiple downstream targets.
It is not clear, however, how Ca
2+ initiates and/or coordinates these various different cardiac
myogenic programs in situ within complex multicellular developing embryos.
My goal was to study Ca
2+ signaling in the developing zebrafish he...[
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Ca
2+ signaling has been extensively studied in the mature heart. In addition to
excitation-contraction
coupling, Ca
2+ also plays a major role in regulating heart development
via excitation-transcription
coupling. Downstream targets of the latter include myocardium
transcription factors, such as Mef2, Gata4 and Nkx2.5. Ca
2+ has been reported to act via CaM
kinase and calcineurin, and subsequent Ca
2+ effectors, to modulate the transcription or activity
of these myocardium transcription factors, which in turn activate multiple downstream targets.
It is not clear, however, how Ca
2+ initiates and/or coordinates these various different cardiac
myogenic programs in situ within complex multicellular developing embryos.
My goal was to study Ca
2+ signaling in the developing zebrafish heart in situ. I first
improved our ‘photon imaging microscope system’ acquisition program to allow more
accurate detection of the signals generated from bioluminescent Ca
2+ indicators. I also
attempted to visualize Ca
2+ signals in developing heart using the bioluminescent Ca
2+ indicator
aequorin. The possibility of using aequorin reconstitution to improve light output was
investigated. Results suggest that aequorin is quickly consumed over 24 hours, and is not efficiently reconstituted by coelenterazine incubation for long term Ca
2+ imaging. I therefore
sought to achieve Ca
2+ imaging using Nano-Lantern(Ca
2+) a novel bioluminescent Ca
2+
indicator. A line of transgenic zebrafish that express Nano-Lantern(Ca
2+) driven by the nkx2.5
promoter was generated to facilitate the study of Ca
2+ signaling in the developing heart.
Another transgenic line of fish expressing rat parvalbumin-α
(again driven by the nkx2.5
promoter) was also generated as an additional tool for studying Ca
2+ disruption-related phenotypes.
These two lines of transgenic fish will prove to be useful for studying Ca
2+ signaling
during the development of the zebrafish heart.
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