How precisely patterned gradients of secreted guidance cues form in vivo is not well understood, although components of the extracellular matrix (ECM) are likely to play an essential role. In principle, components of the ECM can influence interactions between secreted cues and their receptors in several ways, including controlling cue

diffusion, concentrating cues in particular locales, Afatinib solubility dmso affecting ligand-receptor binding affinity, modulating ligand or receptor processing, or influencing ligand stability (Lee and Chien, 2004; Müller and Schier, 2011). In Drosophila, ECM components have well-established roles in generating gradients of secreted morphogens in vivo ( Yan and Lin, 2009). Furthermore, the localization of Slit is regulated by the proteoglycan syndecan in Drosophila and the ECM protein Collagen IV in zebrafish ( Johnson et al., 2004; Xiao et al., 2011). How specific ECM components affect

guidance cue distribution and function in vivo in the developing mammalian nervous system is largely unknown. Using a forward genetic KPT-330 molecular weight screen in mice, we have identified two genes, β-1,3-N-acetyl-glucosaminyltransferase-1 (B3gnt1) and Isoprenoid synthase domain containing (ISPD), as regulators of axon guidance. We show that B3gnt1 and ISPD are essential for glycosylation of the extracellular matrix protein dystroglycan in vivo and that B3gnt1 and ISPD mutants develop severe neuronal migration defects commonly associated with defective dystroglycan function. We find that dystroglycan is also required for spinal cord basement membrane integrity and that axon tracts growing in close proximity to the basement membrane are severely disorganized in B3gnt1, ISPD, and dystroglycan mutants. Remarkably, we find

that glycosylated dystroglycan also binds directly to the axon guidance cue Slit to organize its protein distribution in the floor plate and the basement membrane, thereby regulating Slit-mediated axon guidance. These findings reveal a fundamental role for dystroglycan in organizing axon guidance cue distribution and function within the ECM and identify novel mechanisms underlying human pathologies. We conducted an ENU-based, three-generation, forward genetic screen in order to identify mutations that affect 4-Aminobutyrate aminotransferase the organization of PNS and CNS axonal tracts (Merte et al., 2010; see Figure S1 available online). Utilizing a recessive breeding strategy, axonal tracts of E11.5–12.5 embryos were visualized using a whole-mount anti-neurofilament-based assay (Figure 1A). Screening of 235 G1 mouse lines led to the identification of 10 distinct lines harboring mutations resulting in axon guidance and axon branching defects (Figures 1B and 1E; data not shown). Lines 1157 and 9445 were initially identified based on similar defects in the development of longitudinal axonal tracts in the hindbrain.