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Nagel, Daniel Matthias (2015): Analysis of adhesive and repulsive functions of FLRT proteins in central nervous system development. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)



Fibronectin-leucine-rich-repeat-transmembrane proteins (FLRTs) are a family of three single pass transmembrane proteins with extracellular leucine rich repeats and a short intracellular domain of largely unknown function. They are broadly expressed in the developing and adult nervous system as well as in other tissues. FLRTs have been implicated in a variety of different developmental processes mainly via two functions: as homophilic cell adhesion molecules, and as repulsive ligands for Unc5-positive cells. Furthermore, they can regulate cell adhesion via control of surface expression of C Cadherin and are involved in FGF signaling. Previously we found that all FLRTs are localized to synapses in the mouse brain and thus investigated a potential involvement of FLRTs in synapse formation. However, using different in vitro and in vivo approaches ranging from HEK293 cell-neuron coculture assays to ultrastructural analysis of synapse density in FLRT3 knock-out mouse brains, I did not find any evidence for an involvement of FLRTs in synapse development. This is in contrast to published results but can be explained by differences in the experimental approaches and timing of the experiments. In collaboration with structural biologists we solved the crystal structure of the FLRT and Unc5 extracellular domains and a complex of both, to gain insight into the structural basis of the adhesive and repulsive functions of FLRTs. We found that homophilic FLRT FLRT and heterophilic FLRT-Unc5 interactions both occur via the FLRT LRR domain but at distinct structural surfaces. Thus, the interactions can be uncoupled. Based on the structural results we developed FLRT and Unc5 glycosylation mutants that specifically inhibit FLRT-FLRT or FLRT-Unc5 interaction and validated them in vitro. I then used these mutants in in utero electroporation experiments to prove that the repulsive effect of Unc5D overexpression in radially migrating neurons that was discovered previously is indeed, at least partially, mediated by FLRT2. Furthermore I found that overexpression of FLRTs inhibits radial migration of cortical pyramidal neurons and this effect is dependent on FLRT-FLRT homophilic interaction and the FLRT intracellular domain but independent of FLRT-Unc5 binding. In summary, the work presented here provides new insights into adhesive and repulsive functions of the FLRT family of proteins in the regulation of cell migration during cortical development.