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Abstract

During the development of an organism, chemokine signaling provides long range guidance for migrating cells and tissues. Using an intricate system of promiscuous receptor-ligand interactions, the embryo coordinates the robust positioning of its future organs in space and time. Cells both sense and actively shape a highly dynamical and diverse signaling environment, thus making it challenging to systematically study cell signaling regulation and function in vivo.

To approach this problem, I combined novel methods from the fields of microscopy, cell biology and image analysis. Specifically, this includes multiple-view light sheet microscopy for imaging developmental processes simultaneously at the cell and organism scales and tandem fluorescent lifetime reporters as an emerging tool to visualize signaling receptor turnover in vivo. To quantitatively compare three dimensional embryo acquisitions across genetic conditions and multiplex fluorescent readouts, I used nonlinear image registration to perform spatial normalization. Further, the so obtained sample deformation maps were exploited to perform morphometric phenotype analysis. Together, these tools led to the development of a computational analysis framework enabling quantification of chemokine signaling activity and function during embryonic development.

This framework was then applied to search for novel interactions between the chemokine scavenger CXCR7 (or ACKR3) and the signaling receptor CXCR4 in the early zebrafish embryo. In recent years, the competition of these receptors for their shared ligands has been shown to be implicated in several developmental processes as well as tumor progression. In my analysis, loss of the scavenger was revealed to exert a strong activating effect on CXCR4 expressing tissues across the entire embryo. The remarkable up-regulation of signaling activity in the absence of the scavenger was however found to be aphenotypic in many target tissues. Therefore, I proceeded to investigate at which level the phenotypic impact of this genetic perturbation is compensated for and found receptor desensitization to likely be dispensable in this context.

In conclusion, this work provides an example of how spatial normalization and multiplexing of in toto light sheet images can be used to investigate the general logic and functional output of chemokine-scavenger interactions during embryonic development.