Quantification of transport in early embryonic pattern formation through analysis of lipid-droplet motion in Drosophila and BMP patterning in zebrafish
Abstract
Transport processes are crucial in biological systems, and there are multiple transport mechanisms. In this study, we investigated two biological transport processes as specific examples for (i) active transport and (ii) diffusion, which are lipid-droplet motion powered by microtubule motors during early Drosophila embryogenesis, and Bone Morphogenetic Protein gradient activity of the zebrafish blastula embryo. To study lipid-droplet motion, we used a newly developed technique termed "femtosecond-stimulated Raman loss microscopy" for long-term time-lapse imaging of the live embryo. Individual droplet motion was tracked and analyzed to measure each droplet's velocity and turning frequency at different apical-to-basal depths and stages of development. We then simulated droplet motion using a velocity-jump mathematical model incorporating the velocity and turning rate parameters. This model yielded droplet density distributions that agreed well with experimental observations without any model optimization or unknown parameter estimation, demonstrating the sufficiency of a velocity-jump process for droplet trafficking dynamics in blastoderm Drosophila embryos. To study BMP patterning, we fine-tuned an imaging and image analysis strategy to segment and extract the fluorescent intensity profiles of P-Smad immunostaining in fixed zebrafish embryos of different types at different time stages. We observed directly from quantitative intensity profiles the functional redundancy of Noggin and Follistatin to Chordin in determining BMP signaling gradient formation. We then constructed, optimized and screened a mathematical model system to investigate the combined function of key BMP regulators in generating the robust BMP gradient. Model output was consistent with experimental data and indicated a dorsally concentrated Noggin/Follistatin distribution, which worked as a BMP "sink" functioning redundantly in response to Chordin levels in the embryo. We then studied current normalization methods used to quantify BMP activity profiles. We found that normalization tended to lower the number of samples required to establish statistical significance between profiles in controls and experiments, but it might go too far and eliminate some natural bio- logical variability. Additionally, we examined the tradeoffs for normalizing versus not normalizing, and discussed their impacts on experimental design and the interpretation of resultant data.
Degree
Ph.D.
Advisors
Umulis, Purdue University.
Subject Area
Physiology|Developmental biology
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