Date of Completion

2015

Thesis Type

College of Arts and Science Honors

Department

Biochemistry

First Advisor

Paula Deming

Second Advisor

Alicia Ebert

Keywords

fgf8, fgfr1, zebrafish, embryonic development, growth factor signaling, retinal ganglion cell

Abstract

In the embryonic zebrafish, the fibroblast growth factor 8a (FGF8) signaling network is essential for proper development and maintenance of retinal ganglion cells (RGCs) as well as the hyaloid vasculature, the vessels that supply the eye with nutrients during development. Disruption of FGF8 signaling via knock down of FGF8 or pharmacologic inhibition of FGF receptors (FGFRs) results in extensive abnormalities throughout the developing eye. Our preliminary data indicated that in developing zebrafish, mRNA expression of fgf8a is present exclusively in the RGCs, while the fibroblast growth factor receptor 1 (fgfr1b) is expressed exclusively in the area of the hyaloid vasculature. These results led us to hypothesize that FGF8 signals from the RGCs to the vasculature of the developing eye, and that this signaling network is essential for proper eye development. In order to test this hypothesis, we demonstrated the ability to detect downstream phosphorylation events in response to acute FGF8 stimulation in cells that expressed FGFR1 using Western blot and immunofluorescence (IF). Next, we established a zebrafish eye explant culture system to treat the cells of the developing zebrafish eye in vitro. Using transgenic zebrafish lines expressing green fluorescent protein (GFP) tags in either the differentiating RGCs or the vascular cells of the eye, we attempted to identify the specific cells capable of responding to FGF8. Our data indicate that recombinant FGF8 is capable of activating detectable intracellular signaling cascades, such as ERK phosphorylation, in cultured endothelial cells. Furthermore, FGF8 is capable of inducing signaling in some of the cells from the developing zebrafish eye, but not in the RGCs. These findings support our proposed model in which FGF8 signals from the RGCs to the hyaloid vasculature, resulting in the activation of signaling pathways that are necessary for proper development of the hyaloid vasculature and RGCs.

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