DNA (B) sequences from brv+, brv1, brv2, and brv3 alleles are presented. The 14 bp long deletion in the brv1 cDNA (boxed) is a consequence of a G to A mutation in the splice acceptor (black box) and the use of a cryptic alternative splice acceptor (red box). A G to A mutation transforms a Trp codon (TGG) to a Stop codon (TAG) (C, boxed) in brv2. A T to A transversion changes the conserved, non-polar, Leu residue (CTG) into the polar Gln amino acid (CAG) (D, boxed) in brv3. (E) Partial alignment of several GlcAT-P orthologs shows that the brv3 mutation affects an evolutionarily conserved hydrophobic residue (boxed). (TIF) Figure S2 (A) Protein preparations from brv+ and Df(3L)GlcAT-P brains were blotted with anti-HNK-1 (HNK-1) and anti-GlcAT-S (GlcAT-S) antibodies. (B–C) Misexpression of UAS-GlcAT-P in L3 larva wing imaginal discs with Dppblink-GAL4. (B) Ectopic expression of GlcAT-P is detected by a-GlcAT-P immunohisto- chemistry in the typical pattern of dpp expression domain. The white box indicates the region shown in (C). (C) Co-labeling (arrows) of a-GlcAT-P (red) with Sqh::EYFP-Golgi (green) reveals that GlcAT-P protein is localized to the Golgi apparatus. Bars in B and C are 100 mm and 20 mm respectively. (TIF) Figure S3 GlcAT-P-GAL4 driven expression of a reporter gene (nuclear b-Gal) recapitulates aspects of endogenous GlcAT-P expression during embryogenesis. (A, B) Note that reporter gene expression mimics the described GlcAT-P in situ hybridization expression pattern (see BDGP;http://www.fruitfly.org/cgi-bin/ ex/bquery.pl?qtype = report&find = CG6207&searchfield = CG). LacZ expression was detected in the amnioserosa (A; arrows) of stage 14 embryos, and in salivary glands (B; arrowheads) and the gut (arrows) at stage 17. Bar: 100 mm. (TIF) Figure S4 The general structure the VNC is unaffected in brv mutant larvae. Labeling of L3 larva brains from wild-type (brv+) and brv1 with anti-22C10 (A, B), anti-BP102 (C, D), anti-Fas2 (E, F), anti-Elav (G, H) and anti-Repo (I, J) shows apparently normal VNC structure in brv mutants. Bar: 50 mm. (TIF) Figure S5 Mushroom body neurons and VUM motoneurons are apparently normal in brv mutants. (A, B) OK107-GAL4.UAS- References 1. Geraldo S, Gordon-Weeks PR (2009) Cytoskeletal dynamics in growth-cone steering. J Cell Sci 122: 3595–3604. 2. Dickson BJ (2002) Molecular mechanisms of axon guidance. Science 298: 1959–1964. 3. Dent EW, Gertler FB (2003) Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40: 209–227. 4. Weiss P (1941) Nerve patterns: the mechanics of nerve growth. In: Growth, Third Growth Symposium pp. pp 163–203. 5. Smith DH (2009) Stretch growth of integrated axon tracts: extremes and exploitations. Prog Neurobiol 89: 231–239. 6. Dagg AI, Foster JB (1982) The giraffe.Its biology, behavior and ecology. 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(C, D) 807-GAL4.UAS-mCD8-GFP showed that VUM neurons were present in brv1 mutants (arrowheads). Two consecutive abdominal neuromeres (dashed lines) are shown. A–D represent maximum projections of Z-stacks. Bar: 20 mm. (TIF) Figure S6 Peripheral glia spacing and distribution is not affected in brv mutant larvae. (A) Labeling of L3 larva brains from brv+ and brv1 mutants with anti-Repo antibody shows a similar spacing between glia in the peripheral nerves. (B) Nrv2-GAL4.GFP expression in L3 larva brains from wild-type (brv+) and brv1 mutants. (C) Merged frames of anti-Repo (red) and Nrv2- GAL4.GFP (green). (D) Histograms depicting the average number of glial cells in comparable stretches of the most posterior peripheral nerves of brv+ and brv1 (n = 30 for each) larvae. Bar: 50 mm. (TIF) Figure S7 Rescue of brv mutants by expression of GlcAT-P using UAS/GAL4 system. Quantification of the length of the L3 VNCs (A) and peripheral nerves (B) in different rescue experiment settings is provided (*: p,0.0001). (TIF) Acknowledgments We thank R. White, J. Casanova, F. Matsuzaki and S. Oka for antibodies and S. Luschnig for flies. The Elav, Repo, Nrt, Antp and AbdB monoclonal antibodies developed by G.M. Rubin, C. Goodman, D. Brower and S. Celniker, respectively, were obtained from the Develop- mental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. The Bloomington Drosophila Stock Center resources contributed to this work and FlyBase provided important information used in this work. We are grateful to C. Lehner, X.H. Yang, B. Reversade and B. Knowles for critical comments on the manuscript. Author Contributions Conceived and designed the experiments: RP JB GU. Performed the experiments: RP JB. Analyzed the data: RP JB GU. 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