Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Mary . Mullins

Abstract

A fundamental question in developmental biology is how morphogens, such as Bone Morphogenetic Protein (BMP), form precise signaling gradients to impart positional and functional identity to the cells of the early embryo. The primary goal of this research is to employ recent advances in the quantitative measurement and visualization of the BMP signaling gradient to elucidate the mechanisms that shape the zebrafish BMP morphogen gradient in space and time. Specifically, I investigated the roles of Bmp1a and Tolloid, metalloproteases that promote BMP signaling by cleaving the critical BMP antagonist Chordin, and their competitive inhibitor Sizzled.

I combined rigorous mutant analyses with quantitative immunofluorescence to determine that the proteases Bmp1a and Tolloid spatially restrict Chordin in the early zebrafish gastrula. I discovered that maternally-deposited Bmp1a plays an unexpected and non-redundant role in establishing the BMP gradient, while Sizzled is surprisingly dispensable. Combining mathematical models and in vivo analyses with an immobile Chordin construct, I demonstrate that Chordin diffusion is dispensable for BMP gradient formation and DV patterning. These data exclude a counter-gradient of Chordin and instead favor a Chordin sink, established by Bmp1a and Tolloid, as the primary mechanism that drives BMP gradient formation.

I applied quantitative immunofluorescence to wild-type embryos and determined that the BMP signaling gradient steepens by the end of gastrulation. I discovered that Tolloid and Sizzled play separate roles in shaping the BMP gradient during gastrulation: they are first required at different stages and Tolloid steepens the BMP gradient while Sizzled limits the lateral extent of the highest BMP levels. These results suggest that gastrulation represents a new signaling environment that requires additional regulation by Tolloid and Sizzled. Taken together, I have defined distinct spatiotemporal roles for Bmp1a, Tolloid, and Sizzled in both establishing and then later shaping the BMP signaling gradient during zebrafish DV patterning.

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