28/29 chick embryos. Protein carpets click here were produced using silicon matrices with a channel system (distributed by Dr. Martin Bastmeyer’s laboratory) as described (Knöll et al., 2007). Carpets contained an alternating stripe pattern deposited in the following order: the first stripe contained a mixture of ephrin/Eph-Fc- (or Fc- only as controls) and Fc-specific Cy3 conjugated (4:1 weight ratio) while the second

stripe contained only Fc reagents without Fc-specific Cy3 conjugated. Dissection of e5 chick spinal motor column was modified from previously described methods (Gallarda et al., 2008). See Supplemental Experimental Procedures for detailed description of motor column dissection and conditions of motor neuron explant and dissociated culture. In situ mRNA detection, immunofluorescence and live-cell staining, and ephrin/Eph-Fc overlay of limb sections were performed as described (Kania and Jessell, 2003, Kao et al., 2009, Schaeren-Wiemers and Gerfin-Moser, 1993 and Zimmer et al.,

2003). Probe sequences are available upon request. For ephrin/Eph-Fc overlays, tissue sections were incubated in blocking solution (1% heat inactivated horse serum in 0.1% Triton-X/PBS [Sigma]) for 5 min and incubated overnight at 4°C with recombinant ephrin/Eph-Fc chimaera (10 μg/ml in PBS, R&D systems). Samples were then washed with PBS and fixed with 4% paraformaldehyde in PBS for 2 min. Following PBS washes, samples were incubated with preheated PBS at 65°C for 1 hr. Tissues were then cooled to room temperature (RT) in PBS and incubated

with IgG conjugated with alkaline phosphatase secondary find protocol MycoClean Mycoplasma Removal Kit antibody (1:1000, Promega) for 1 hr. After PBS washes, samples were incubated with B3 buffer (0.1 M Tris [pH 9.5], 0.1 M NaCl, 0.05 M MgCl2 [Fisher Scientific]) for 5 min followed by detection of bound antibody in B4 buffer (100 mg/ml NBT, 50 mg/ml BCIP [Roche] and 400 mM Levamisol [Sigma] in B3). See Supplemental Experimental Procedures for detailed description of immunostaining of spinal motor column explants and dissociated neurons and Table S1 for antibodies and Fc reagents. Images were acquired using a Leica DM6000 microscope or a Zeiss LSM confocal microscope with Volocity imaging software (Improvision). GFP-labeled axonal projections, protein and mRNA expressions, and motor neuron numbers of limb section images were quantified using Photoshop (Adobe) or ImageJ (NIH) as described (Kania and Jessell, 2003 and Kao et al., 2009). Proportions of GFP- or EphA4-labeled neurites of cultured motor neuron explants or single cells growing on each type of stripes were quantified by combining over-threshold pixel quantification over either first or second types of stripes in multiple images using Photoshop (Adobe). Data from the experimental replicate sets were evaluated using Microsoft Excel.