Nanobodies as novel tools to study morphogen function "in vivo"

Abstract

Nanobodies are small, monomeric antibody mimetic proteins produced by members of the camelid family (camels and llamas), that can be engineered by fusion to proteins carrying a specific function. These “functionalized” protein binders emerge as novel tools for protein manipulation in vivo. During my PhD studies I have generated scaffold-bound nanobodies (SBNs) specific to EGFP in order to interfere with gradient formation of a EGFP-tagged version of the Decapentaplegic (EGFP::Dpp) morphogen. Morphogens are secreted signaling molecules forming concentration gradients and controlling organ patterning and growth during animal development. Drosophila Decapentaplegic (Dpp) is one of the best studied morphogens, but it remains unclear how its concentration gradient is established and how it and controls patterning and growth of the Drosophila wing imaginal disc. In this PhD Thesis I summarize the development and characterization of SBNs and their applications in studying the formation and function of the Decapentaplegic morphogen gradient in the Drosophila melanogaster wing imaginal disc. In the first part of this Thesis, I will discuss how SBNs allowed us to investigate the importance of the Dpp gradient on proliferation and growth control of the wing imaginal disc. Using morphotrap, a SBN that localizes to the outer cell surface, we could completely block gradient formation and study the effect of a loss of the Dpp gradient on patterning and growth. We find that induction of Dpp target genes, and hence patterning, directly depends on the spreading of Dpp. Furthermore, we show that the Dpp gradient is crucial for growth and size control of the medial wing disc region. Moreover, we find that the Dpp gradient is not necessary for proliferation and size control of the lateral region of the wing disc. This data challenges previously published growth models, in which growth control solely depends on the signaling dynamics of Dpp. In the second part of this Thesis I investigate the mechanism of Dpp gradient formation in the wing disc. The wing disc is a complex three-dimensional structure, consisting of two contiguous epithelial layers. How the long-range Dpp gradient is established in the wing disc remains controversial. I have created different SBNs that localize to specific subcellular regions along the apicobasal axis. These SBNs allow us to reduce or block the dispersal of specific gradient subfractions and assess their contribution to wing development. We find that EGFP::Dpp disperses along three main routes: within the epithelial plane of the wing disc, in the luminal cavity between the two epithelial layers and along the basal lamina. Preliminary results suggest that these subfractions encode for different functions of Dpp. While we find that the patterning function of Dpp is encoded by the basolateral subfractions, the growth function of Dpp seems to be influenced by all three subfraction. Further experiments will investigate how target cells perceive and integrate Dpp input from these different subfractions.