These findings establish an essential role for glycosylated dystroglycan in regulating axon guidance at the ventral midline of the spinal cord in vivo. The commissural axon guidance phenotypes observed in the B3gnt1, ISPD and dystroglycan mutants raised the possibility that dystroglycan binds to axon guidance cues within the floor plate to regulate their function. Previous studies have identified a number of ligands that bind directly to dystroglycan in a glycosylation-dependent manner, including laminin, agrin, perlecan, neurexin, and pikachurin ( Gee et al., 1994; Sato et al., 2008; Sugita et al., 2001; Talts et al., 1999). A common feature of these ligands

is the presence of a laminin G (LG) domain that mediates their association with carbohydrate moieties present on glycosylated dystroglycan. Intriguingly, of the axon guidance cues known to be expressed Akt inhibitor selleck chemicals in the floor plate, Slits contain an LG domain within their C-terminal regions. Thus, the overlapping expression patterns of dystroglycan and Slits in the floor plate,

the similarities in axon guidance phenotypes observed in the B3gnt1, ISPD, dystroglycan, and Slit/Robo mutants, and the presence of an LG domain in the Slit C terminus, led us to hypothesize that glycosylated dystroglycan binds directly to Slits to regulate their function. We first asked whether dystroglycan can bind directly to the C-terminal region of Slit, which harbors the LG domain, using an in vitro COS7 cell-binding assay. We generated constructs in which alkaline phosphatase (AP) is fused to either the Robo-binding leucine rich repeat domain 2 of Slit2 (AP-LRR) or the C-terminal region containing the LG domain of Slit2 (AP-Cterm). As expected, COS7 cells

MTMR9 transfected with a construct encoding full-length Robo-1 specifically bind to AP-LRR, but not to AP-Cterm or AP alone (Figure 6A, data not shown). Importantly, COS7 cells expressing full-length dystroglycan showed robust binding to AP-Cterm but not to AP-LRR or AP alone. These findings demonstrate that dystroglycan is capable of binding to the Slit C-terminal domain. To further test direct binding between dystroglycan and Slit, we generated an Fc-dystroglycan protein secreted from COS7 cells and determined whether it is capable of direct association with the different domains of Slit. We find that while Fc-dystroglycan fails to bind to AP-LRR, it does bind to AP-Cterm (Figure 6B). We next asked whether the Slit C-terminal fragment is able to bind to endogenous dystroglycan. Dystroglycan enriched membrane fractions isolated by WGA precipitation from mouse brain lysates were incubated with either AP-LRR or AP-Cterm. Indeed, the Slit C-terminal fragment, but not the Slit LRR, is able to associate with endogenous dystroglycan (Figure 6C). Previous studies indicate that the binding of laminin LG domains to dystroglycan requires a basic patch surrounding a Ca2+ binding site (Harrison et al., 2007).