We found that binding of the Slit

C-terminal domain to dystroglycan requires Ca2+, since addition of EDTA is sufficient to abolish this Slit-dystroglycan interaction (Figure 6E). Moreover, a version of the Slit2 C-terminal domain in which two basic residues adjacent to the Ca2+ binding site are mutated to alanine (K1177A, R1179A, referred to here as Slit2 C-term AVA) is incapable of binding to Fc-dystroglycan (Figure 6F). Thus, the Slit2 LG domain mediates its association with dystroglycan and, similar to other LG modules, the Slit2 LG domain requires a Ca2+ binding site surrounded by a basic patch for this interaction. Our findings that Slit can bind directly to dystroglycan in vitro raise the intriguing possibility that dystroglycan Roxadustat mouse present in the floor plate and basement membrane serves as a scaffold for the proper localization of Slit in vivo. Consistent with this idea, dystroglycan and slit are required for proper cardiac tube formation in Drosophila, and Slit protein appears to be mislocalized in dystroglycan mutant cardioblasts

( Medioni et al., 2008). We first verified that the expression patterns of Slit1 and Slit2 mRNA are indistinguishable in control and B3gnt1 mutants ( Figure S7A), demonstrating that dystroglycan is not required for floor plate development or expression of these axonal guidance cues. To test whether dystroglycan I-BET151 regulates Slit localization, an AP-section binding assay was employed to visualize the location of endogenous C-terminal Slit binding sites in vivo. Incubation of transverse spinal cord sections from E11 control embryos with the AP-Slit C-term ligand showed robust binding to the basement membrane surrounding the spinal cord and the floor plate ( Figure 7A), regions that are enriched for dystroglycan protein expression ( Figures 3C and 3D). Importantly, binding of AP-Slit C-term is absent in B3gnt1LacZ/M115T mutants, demonstrating that glycosylation of dystroglycan is essential for Slit C-terminal domain binding in vivo. Since Slit binds directly

to glycosylated dystroglycan via its C-terminal LG domain, Dichloromethane dehalogenase we hypothesized that dystroglycan present in both the floor plate and basement membrane are required for organization of endogenous Slit proteins within these locations. Therefore, we developed a method to assess the sites of Slit protein localization in tissue sections to ask whether loss of glycosylated dystroglycan in the B3gnt1 mutants alters the distribution of endogenous Slit protein in vivo. The lack of antibodies suitable for mammalian Slit immunolocalization necessitated the development of an alternate approach. Therefore, we modified the AP-ligand section binding assay by using an AP-Robo ectodomain fusion protein that is capable of binding to Slit protein on tissue sections ( Jaworski and Tessier-Lavigne, 2012).