, 2008, Koch et al., 2011, Larrivée et al., 2007 and Lu et al., 2004), and Unc5B knockdown leads to increased vascular branching in the mouse retina (Koch et al., 2011). Netrin-1 and Robo-4 have been shown to interact with Unc5B in the vasculature BMS-387032 clinical trial (Koch et al., 2011 and Lu et al., 2004); however, neither Netrin1−/− nor Robo4−/− mice display the hypervascularization effects observed in Unc5B−/− retinas, indicating that other factors may play a role. Here we demonstrate that Flrt3−/− mice present with a vascularization phenotype that strongly resembles that reported for Unc5B−/−. Using our tip cell
collapse experiments, we show that soluble FLRT3 controls the extension of endothelial tip cell filopodia through its specific Unc5B-binding site, providing functional evidence for a direct interaction of FLRT3 and Unc5. These results suggest that FLRT3 is a major player in controlling vascularization via Unc5B, and may therefore explain the puzzling lack of effects in retinal vascularization after removing other Unc5B ligands. Our stripe assays showed that surface-tethered FLRT3 also repels endothelial cells through interaction with Unc5B. Further work is required to understand whether FLRT3 acts in its soluble or cell-bound form in vivo.
Further questions Venetoclax remain; how do FLRTs signal adhesion/attraction in response to homotypic interaction with other FLRTs? Are the downstream pathways activated by the FLRT intracellular domain similar to classical CAMs? Are small GTPases Endonuclease such as Rnd proteins (Chen et al., 2009 and Karaulanov et al., 2009) and cytoskeletal proteins involved? FLRTs
have also been reported to bind other proteins, for example, latrophilin (O’Sullivan et al., 2012). It will be important to understand the molecular determinants of these interactions and how they influence FLRT functions. The crosstalk of FLRT3-Unc5B interactions to other key vascular players, such as VEGF/VEGFR2, also remains to be investigated. In summary, we integrated information generated by a broad range of biological methods to understand the functions of FLRT and Unc5 receptors in cortical and vascular development. Our results reveal how FLRTs act as bimodal guidance molecules directing essential developmental processes through structurally distinct, combinatorial mechanisms. As FLRT and Unc5 are expressed in a wide range of tissues (Engelkamp, 2002 and Haines et al., 2006), the conserved functional mechanisms we report are likely to control cell adhesion and repulsion in tissues beyond those described here. We cloned constructs of mouse Flrt2 (UniProt Q8BLU) and Flrt3 (UniProt Q8BGT1), human Unc5A (UniProt Q6ZN44), mouse or human Unc5B (UniProt Q8K1S3 and Q8IZJ1), and rat Unc5D (UniProt F1LW30) into the Age1-Kpn1 or EcoR1-Kpn1 cloning site of vectors from the pHLSec family ( Aricescu et al., 2006), depending on whether the construct includes a native secretion signal sequence.