DSCAM functions as a receptor in commissural pathfinding

Guofa Liua,1, Weiquan Lib,1, Lei Wanga,c, Amar Kara,c, Kun-Liang Guanb,2,3, Yi Raoa,d,3, and Jane Y. Wua,c,3

aDepartment of Neurology and cRobert H. Lurie Comprehensive Cancer Center, Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611; bLife Sciences Institute, Department of Biological Chemistry, and Institute of Gerontology, University of Michigan, Ann Arbor, MI 48109; and dSchool of Life Sciences, Peking University, Beijing 100871, China

Edited by Lily Y. Jan, University of California School of Medicine, San Francisco, CA, and approved December 3, 2008 (received for review November 4, 2008) Down syndrome cell adhesion molecule (DSCAM) is required for axon with netrin-1. DSCAM is expressed on the spinal commissural as guidance and dendrite arborization. How DSCAM functions in verte- they extend to and across the floor plate. In vitro knockdown of brates is not well understood. Here we show that DSCAM is ex- DSCAM inhibits netrin-induced axon outgrowth and commissural pressed on commissural axons and interacts with Netrin-1, a proto- axon attraction. Knockdown of DSCAM in ovo causes defects in typical guidance cue for commissural axons. The knockdown of commissural axon pathfinding in chick neural tube. These results DSCAM by specific siRNA or blockage of DSCAM signaling by over- indicate that DSCAM is a receptor required for netrin-dependent expression of a mutant lacking its intracellular domain inhibits netrin- commissural axon outgrowth and pathfinding. While our work was induced axon outgrowth and commissural axon turning in vitro. under review, a study was published indicating that DSCAM functions SiRNA-mediated knockdown of DSCAM in ovo causes defects in as a netrin receptor in collaboration with DCC (29). commissural axon projection and pathfinding. In transfected cells, DSCAM by itself, in the absence of DCC, is capable of mediating netrin Results signaling in activating phosphorylation of Fyn and Pak1. These find- Expression of DSCAM in Commissural Neurons of the Neural Tube. In ings demonstrate an essential role of vertebrate DSCAM in axon situ hybridization studies have shown that DSCAM is expressed guidance, indicating that DSCAM functions as a receptor of netrin-1. throughout the developing mammalian nervous system (16, 30). To Our data suggest previously unexpected complexity in receptors that examine the expression of DSCAM protein, we carried out immu-

mediate vertebrate netrin signaling. nostaining of mouse spinal cords using a specific antibody against NEUROSCIENCE DSCAM (27) and compared the results with the staining pattern of neurite outgrowth ͉ commissural ͉ TAG-1, an antibody specifically recognizing commissural axons neuronal guidance receptor ͉ signal transduction (Fig. 1A, D, and G). At E11.5, DSCAM is expressed in the spinal cord, including the motor columns, motor axons, dorsal root etrins, a conserved family of secreted proteins, can promote ganglions, commissural axons, and ventral funiculus [Fig. 1B, C, E, Naxon outgrowth and guide navigation in species F, and H; supporting information (SI) Fig. S1D]. In dissociated ranging from Caenorhabditis elegans to mammals (1–5, 37). Recep- E11.5 commissural neurons, DSCAM was detected in the soma and tors for UNC-6/netrin have been identified in C. elegans as UNC-40 on the cell membrane, axon, and growth cone (Fig. 1H, K and N; and UNC-5 (1, 6, 7). The mammalian homologs of UNC-40 are Fig. S1 A–C). A similar expression pattern was found in E15 cortical Deleted in Colorectal Cancer (DCC) and neogenin (8, 9). neurons (Fig. S1 J and M). Co-localization with TAG-1 indicated that can act as either axon attractants or axon repellents. DCC/UNC40 DSCAM is expressed in commissural axons (Fig. 1C, F, and I). can mediate both attractive and repulsive responses, whereas Confocal immunofluorescent microscopy showed that DCC and UNC-5 mediates repulsion (6, 8–13). In the embryonic spinal cord, DSCAM are partially co-localized in commissural neurons (Fig. 1J–O) DCC mediates the attractive effect of netrin in commissural axons and cortical neurons (Fig. S1 I–N). DSCAM expression in the embry- (3, 9). DCC-deficient mice exhibit defects in commissural axon onic spinal cord in chicken is similar to that in mice (Fig. S1 E–H). projections that are similar to those seen in netrin-1–deficient mice, with reduction in, shortening of, and misguidance of commissural Biochemical Characterization of DSCAM Interaction With Netrin-1. To axons (14, 15). Some commissural axons in DCC knockout mice still examine a potential interaction between netrin-1 and DSCAM, we project to and cross the floor plate, however, suggesting that other transfected HEK293 cells using plasmids expressing netrin-1 tagged guidance receptors may be involved in this process (14, 15). with HA (netrin-HA) and Flag-tagged full-length human DSCAM The human DSCAM gene was originally identified as a gene (DSCAM-Flag), DSCAM mutants [lacking the extracellular do- ⌬ ⌬ associated with mental retardation (16). It encodes a protein of the main (DSCAM N) or the intracellular domain (DSCAM C), as Ig superfamily containing 10 Ig domains, 6 fibronectin type III (Fn shown in Fig. 2A]), or DCC. As expected, netrin-HA was co- III) domains, 1 transmembrane, and 1 intracellular domain (16). immunoprecipitated by DCC (Fig. 2B, lane 4). Either the full- ⌬ ⌬ The Drosophila homolog of human DSCAM has been isolated in a length DSCAM or DSCAM C, but not DSCAM N, co- screen for tyrosine-phosphorylated proteins interacting with Dock, an intracellular adaptor protein homologous to mammalian Nck Author contributions: K.-L.G., Y.R., and J.Y.W. designed research; G.L., W.L., L.W., and A.K. (17). The fly Dscam gene has an amazing molecular diversity, with performed research; G.L., W.L., K.-L.G., Y.R., and J.Y.W. analyzed data; and G.L., Y.R., and 38,016 potential alternative splicing isoforms, and is required for J.Y.W. wrote the paper. neuronal wiring (17–21; reviewed in ref. 22); however, the verte- The authors declare no conflict of interest. brate DSCAM gene encodes only a few splicing isoforms (23). This article is a PNAS Direct Submission. Mouse DSCAM is expressed widely in the developing nervous 1G.L. and W.L. contributed equally to this work. system (16, 24). Recent studies indicate that DSCAM plays an 2Current address: Department of Pharmacology and Moore’s Cancer Center, University of important role in neurite arborization, cell body spacing, and California at San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093. lamina-specific synaptic targeting in vertebrate retina (25, 26). 3To whom correspondence may be addressed. E-mail: [email protected], In a previous study, we found that human DSCAM can bind to [email protected], or [email protected]. p21-activated kinase 1 (Pak1) and stimulate Pak1 activity (27). Netrin-1 This article contains supporting information online at www.pnas.org/cgi/content/full/ also activates Pak1 (28). These observations prompted us to test the role 0811083106/DCSupplemental. of DSCAM in netrin signaling. Here we report that DSCAM interacts © 2009 by The National Academy of Sciences of the USA

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Fig. 2. Netrin-1 interacts with DSCAM and activates protein phophorylation in DSCAM-expressing cells. The control vector, or DSCAM (wild-type or mutant), Fig. 1. DSCAM expression in the developing spinal cord. (A–F) Expression of together other plasmids, as indicated, were transfected into HEK293 cells, then TAG-1 (A, C, D, and F) and DSCAM (B, C, E, and F) in transverse sections of the E11.5 treated with netrin or the control vehicle. Immunoprecipation (IP) and Western mouse spinal cord was detected by confocal microscopy after immunostaining blot (WB) experiments were carried out using specific antibodies. (A) A diagram with anti-TAG-1 and anti-DSCAM antibodies, respectively. Panels C, F, and I are illustrating the full length and the mutant DSCAM proteins. (B) DSCAM co- overlay images of panels A with B, D with E, and G with H, respectively. Panels D, E, immunoprecipitated with netrin-1, similar to DCC. (C) The full-length DSCAM or ⌬ ⌬ and F are higher-magnification images of the ventral spinal cord of A–C. (Scale bar: DSCAM C, but not DSCAM N, interacted with netrin-1 in co-immunoprecipita- 100 ␮m.) (G–N) Expression of TAG-1 (G and I) and DSCAM (H and I) and of DCC (J and tion. (D) Netrin-1 induced Pak1 phosphorylation in DSCAM-expressing cells. After M) and DSCAM (K and N) in dissociated E11.5 spinal cord neurons. Panels L and O are transfection of HEK293 cells with DSCAM-Flag or/and HA-Pak1 plasmids, Pak1 the overlay images of panels J with K and M with N, respectively. (Scale bars: 10 ␮m.) protein was immunoprecipitated using HA antibody. Phospho-Pak1 and the total Pak1 protein were detected by Western blot analysis using anti–phospho-Pak1 (S423) and anti-HA antibodies, respectively. (E) Netrin-1 enhanced tyrosine phos- immunoprecipitated with netrin, demonstrating that the phorylation of DSCAM. HEK293 cells transfected with DSCAM-Flag were treated with netrin for 5 minutes (lane 2) or 20 minutes (lane 3). DSCAM protein was extracellular domain of DSCAM is required for its interaction with immunoprecipitated using anti-Flag antibody, followed by Western blot analysis netrin [Fig. 2B (lane 3) and C]. with phospho-tyrosine antibody (4G10). (F) DSCAM or DCC alone was sufficient to DSCAM interacts with Pak1, a serine/threonine kinase (27). To mediate netrin-induced phosphorylation of Fyn. DCC-myc or DSCAM-flag plasmids investigate whether DSCAM is involved in netrin-induced Pak1 were transfected into HEK293 cells individually or in combination. After netrin phosphorylation, we co-transfected DSCAM with Pak1 into stimulation, Fyn phosphorylation was detected by Western blot analysis using anti– HEK293 cells. DSCAM expression alone was sufficient to activate phospho-tyrosine after immunoprecipitation using a specific anti-Fyn antibody. Pak1 phosphorylation (Fig. 2D; compare lanes 4 and 1), consistent with findings in our previous study (27). Addition of netrin-1 further increased phospho-Pak1 in the presence of DSCAM (Fig. 2D, lane on the commissural axons (32–34), did not bind to netrin (Fig. 3 3), whereas netrin-1 alone in the absence of DCC or DSCAM did F–H). Co-immunoprecipitation experiments also demonstrated not stimulate Pak1 phosphorylation (Fig. 2D; compare lanes 2 and that Robo1 did not bind to netrin (Fig. S2A). Similarly, no 1). Netrin treatment also stimulated tyrosine phosphorylation of interaction was detected between DSCAM and Semaphorin3A, DSCAM (Fig. 2E). In addition, phosphorylation of Fyn, a Src another neuronal guidance molecule (Fig. S2C). We further char- family kinase required for netrin signaling (31), was increased in the acterized the netrin–DSCAM interaction using an assay used presence of either DCC or DSCAM after netrin stimulation (Fig. previously to examine netrin–DCC and –Robo interactions (9, 2F; compare lanes 5 and 6 and lanes 3 and 4). In multiple 32). DSCAM-expressing cells were incubated with netrin-1 tagged experiments, we found no further increase in netrin-induced Fyn with alkaline phosphatase (netrin-AP), and netrin binding to DSCAM phosphorylation when both DSCAM and DCC were expressed in was determined by measuring AP activity bound to the cells. An HEK293 cells (Fig. 2F; compare lanes 1 and 3). These findings indicate apparent dissociation constant (Kd) of approximately 12.6 nM was that DSCAM expression alone in HEK293 cells is sufficient to mediate obtained for netrin–DSCAM interaction from the binding curve (Fig. netrin-induced phosphorylation of Pak1 and Fyn (Fig. 2). 3L and M), comparable to that in netrin–DCC and Slit–Robo inter- actions (9, 32). Together, our findings indicate that the interaction Binding of Netrin-1 to DSCAM Expressed on the Cell Surface. To between DSCAM and netrin-1 is specific and that DSCAM does not examine whether netrin-1 binds to DSCAM protein expressed on interact promiscuously with other neuronal guidance molecules. the cell surface, we transfected the DSCAM cDNA plasmid (Fig. 3 B and I) into HEK293 cells, incubated these cells with netrin-1 Involvement of DSCAM in Netrin-Induced Axon Outgrowth. To exam- protein, and then examined netrin-1 binding by immunostaining ine the function of DSCAM, we designed small interfering RNAs using a specific anti–netrin-1 antibody (Fig. 3C and J). HEK293 cells (siRNA) targeting the mouse and chicken DSCAM genes. After expressing Robo1, another member of the Ig superfamily expressed testing several siRNAs, we identified one siRNA that significantly

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Fig. 3. Binding of netrin to DSCAM-expressing cells. HEK293 cells were trans- fected with Robo-GFP or DSCAM plasmid and then incubated with netrin protein. Netrin bound to the cell surface was detected by the anti-netrin antibody and NEUROSCIENCE Cy3-conjugated secondary antibody. DSCAM expression was detected by the I J anti-DSCAM with Cy2 conjugated secondary antibody. Expression of Robo-GFP was visualized by GFP fluorescence. (A–D) Netrin binding to DSCAM-expressing cells: (A) bright field image; (B) DSCAM expression; (C) netrin-1 binding; (D) overlay of panels B and C.(E–H) The absence of netrin binding on Robo-GFP cells: (E) the bright field image; (F) Robo-GFP expression; (G) no binding of netrin; (H) overlay of panels F and G. (Scale bar, 100 ␮m.) (I–K) High-magnification confocal images (Z stacks) showing the binding of netrin to DSCAM-expressing cells: (I) expression of DSCAM; (J) netrin binding; (K) overlay of panels I and J.(L and M) Netrin-AP– containing medium was diluted to different concentrations and incubated with either DSCAM-expressing cells or control cells. Free and bound AP activities were Fig. 4. Inhibition of netrin-induced axon outgrowth by DSCAM siRNA. (A–F) Axon outgrowth from YFP-positive dorsal spinal cord neurons transfected with measured as described in SI Materials. The apparent Kd was estimated as 12.6 nM. YFP only (A and B), with YFP plus the control siRNA (C), with YFP plus DSCAM siRNA (D and E), or with YFP plus DSCAM-siRNA and wild-type human DSCAM (F) reduced the expression of endogenous DSCAM in mouse and chicken in the presence of netrin (B, C, E, and F) or the control (A and D). Netrin-induced axon outgrowth was inhibited by DSCAM-siRNA (E). The expression of wild-type neurons without affecting other proteins, such as DCC (Figs. S2D and human DSCAM plasmid reversed the inhibition of netrin-induced axon out- S3). We used this DSCAM siRNA in subsequent experiments. Co- growth by DSCAM siRNA (F). (G and H) Axon outgrowth from explants trans- transfection of the wild-type human DSCAM with DSCAM siRNA fected with YFP plus control siRNA (G) or YFP plus DSCAM siRNA in the presence restored DSCAM expression level (Fig. S3; data not shown), indicating of anti-DCC antibody (H). (Scale bar: 100 ␮m.) (I and J) Quantification of the that human DSCAM is resistant to the DSCAM siRNA. number of axon bundles and the total length of axons per explant for different groups: YFP plus control siRNA without netrin [5.66 Ϯ 0.65 (0.60 Ϯ 0.09 mm)], YFP To evaluate whether DSCAM is involved in axon outgrowth, we Ϯ Ϯ Ϯ co-transfected a Venus YFP plasmid with either the control siRNA only or YFP plus control siRNA with netrin [30.63 1.70 or 34.2 3.5 (3.97 0.30 mm or 3.14 Ϯ 0.54 mm)], DSCAM siRNA [5.76 Ϯ 0.62 (0.59 Ϯ 0.07 mm) without (Fig. S4 A, B, and E) or DSCAM siRNA (Fig. S4 C, D, and F) into netrin and 16.99 Ϯ 1.42 (1.73 Ϯ 0.20 mm) with netrin], DSCAM siRNA plus wild-type E15 cortical neurons and then treated the neurons with netrin-1 or human DSCAM with netrin treatment [26.89 Ϯ2.31 (3.30 Ϯ0.41 mm)], anti-DCC with the control. Transfection of DSCAM siRNA, but not the control netrin [13.40 Ϯ 1.56 (1.33 Ϯ 0.17 mm)], and DSCAM siRNA plus anti-DCC with netrin siRNA, inhibited netrin-induced neurite outgrowth without affect- [8.88 Ϯ 1.06 (0.75 Ϯ 0.10 mm)]. Data are presented as mean Ϯ SEM. Con, the control ing the basal level of axon outgrowth (Fig. S4 A–D). In contrast, treated; Net, the netrin-1–treated group; WT, wild-type human DSCAM; Ab, anti- DSCAM siRNA did not inhibit BDNF-induced neurite outgrowth DCC antibody; siRNA, DSCAM siRNA; Ctl siRNA, control siRNA. The P values for the differences are Ͻ .001 between groups I and II (for both I and J), Ͻ .001 between (Fig. S4 E and F), indicating a specific effect of DSCAM siRNA. groups I and III (for both I and J), Ͻ .001 between groups II and VI (for both I and J), These results demonstrate that DSCAM plays a role in netrin- Ͻ .001 between groups III and VI (for both I and J), .0025 (I) and .0048 (J) between induced neurite outgrowth in cortical neurons. groups VI and VII, and .02 (I) and .007 (J) between groups IV and VIII. To examine the role of DSCAM in commissural axon outgrowth, we used chick dorsal spinal cord explant cultures. DSCAM siRNA or control siRNA together with YFP plasmid were introduced in ovo into of wild-type human DSCAM, which is resistant to DSCAM siRNA, chicken neural tubes at stage 12–15, and the YFP-labeled segments rescued the effect of DSCAM siRNA on netrin-induced axon out- were dissected at stage 18–20. Axon outgrowth was quantified by growth (Fig. 4 F, I, and J). These results indicate that DSCAM is measuring the numbers of axon bundles and the total axon length per required for netrin-induced commissural axon outgrowth in vitro. explant. In explants transfected with YFP only or with YFP plus control To explore the possibility that DSCAM may function together with siRNA, netrin-1 significantly induced axon outgrowth (Fig. 4 A, B, C, DCC in netrin signaling, we used a DCC antibody capable of blocking I, and J). DSCAM siRNA, but not control siRNA, significantly inhib- DCC function (11). Anti-DCC antibody significantly reduced, but did ited netrin-induced axon outgrowth (Fig. 4 B–D, I, and J). Expression not completely eliminate, netrin-induced axon outgrowth (Fig. 4C and

Liu et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 24, 2021 A co-cultured along with an aggregate of HEK cells. More than 90% of the axons projecting from the dorsal spinal cord electroporated at these stages were commissural axons, as demonstrated by im- munostaining with the anti–axonin-1 antibody (35). With YFP expression alone, commissural axons turned toward the netrin- secreting cell aggregate (Fig. 5 A, C, and G), whereas most axons projected straight toward the floor plate when co-cultured with the B C control cell aggregate not secreting netrin (Fig. 5 B and G). Co-transfection of YFP with the wild-type DSCAM plasmid did not affect netrin-induced axon attraction (Fig. 5G; microphotograph not shown); however, DSCAM⌬C (the mutant lacking the intracellular domain) significantly reduced netrin-induced axon attraction (Fig. 5 D and G). Electroporation of DSCAM siRNA, but not the control siRNA (data not shown), significantly inhibited axon turning toward netrin- secreting cells (Fig. 5 E and G). Furthermore, the expression of D E wild-type human DSCAM rescued the defects in axon turning caused by DSCAM knockdown (Fig. 5 F and G). In contrast, DSCAM siRNA had no impact on the repulsive effect of Sema3A on dorsal root ganglion axons (data not shown), suggesting that DSCAM is not a nonspecific receptor for different neuronal guidance cues. Taken together, these findings indicate that DSCAM is required for netrin-mediated commissural axon attraction.

The Requirement of DSCAM for Commissural Axon Projection. We F G examined the effects of the DSCAM mutant and DSCAM siRNA on commissural axon projection in chick embryos in ovo. The YFP plasmid was electroporated into the neural tube along with wild- type DSCAM or mutant DSCAM⌬C. Open-book preparations of spinal cords at stage 23 were immunostained with the anti–axonin-1 antibody (35, 36). More than 90% of the axons expressing YFP stained positive for axonin-1 (Fig. 6 A–O). By stage 23, most commissural axons expressing YFP had reached the floor plate (Fig. 6 G, H, I, P, and Q). The expression of wild type DSCAM did Fig. 5. DSCAM plays a role in netrin-induced attractive turning of spinal cord not affect commissural axon projection (Fig. 6 A, B, C, P, and Q); axons. Electroporation of the YFP plasmid into chick neural tubes allows visual- however, expression of DSCAM⌬C significantly inhibited commis- ization of axons. (A) A schematic diagram of the open-book preparation of the sural axon projection (Fig. 6 D, E, F, P, and Q). Most of the spinal cord co-cultured with cell aggregates after electroporation. Netrin- commissural axons in the spinal cord transfected with YFP and induced axon attraction is illustrated by the turning of axons toward cell aggre- control siRNA reached the floor plate (data not shown). In gates secreting netrin protein. (B) YFP-labeled axons projected straight toward the floor plate when co-cultured with an aggregate of the control HEK cells. (C) contrast, only a small fraction of commissural axons expressing Netrin-secreting HEK cells attracted the commissural axons transfected with YFP. DSCAM siRNA reached the floor plate (Fig. 6 J, K, L, P, and Q). The red arrowheads indicate the turning axons. (D) Axons co-expressing YFP and The effect of DSCAM siRNA on commissural axon projection was DSCAM⌬C projected straight toward the floor plate and were not attracted by reversed by co-transfecting wild-type human DSCAM plasmid (Fig. the aggregate of netrin-secreting cells. (E) DSCAM siRNA inhibited nerin- 6 M–Q). mediated attraction of the commissural axons. The red arrows designate the To further characterize the phenotype of DSCAM knockdown in representative axons projecting straight toward the floor plate. (F) Co- vivo, we examined transverse sections of the chick spinal cords at transfection of DSCAM siRNA with wild-type human DSCAM plasmid rescued ␮ stage 23 after electroporation (Fig. 7). When transfected with YFP netrin attractive response. (Scale bar: 300 m.) (G) Quantification of axon turning alone or YFP with wild-type DSCAM, commissural axons pro- as described in Materials and Methods. The numbers on the top of each bar (n) jected normally toward the floor plate (Fig. 7 B and C); however, indicate the numbers of explants tested in the corresponding groups. Data are ⌬ presented as mean Ϯ SEM. The percentages of turning axons were 5.7% Ϯ 0.9% expression of DSCAM C or DSCAM siRNA led to misguidance for group I (B), 90.7% Ϯ 1.4% for group II (C), 87.2% Ϯ 2.1% for group III (not and shortening of commissural axons (Fig. 7 D, F, and H). The shown), 43.6% Ϯ 4.7% for group IV (D), 34.7% Ϯ 3.2% for group V (E), and 72.2% mutant lacking the extracellular domain, DSCAM⌬N, did not Ϯ 3.2% for group VI (F). Con, control cell aggregates; Net, netrin-1–secreting cell affect commissural axon projection (Fig. 7E). Expression of wild- aggregates. P values (Student t-test) are all Ͻ .0001 between groups I and II, type human DSCAM, but not DSCAM⌬N, rescued the defects in groups III and IV, groups II and V, and groups V and VI. commissural axon projection caused by the DSCAM siRNA (Fig. 7 G and H). These findings demonstrate that DSCAM is required for commissural axon projection and pathfinding. G; group IV in Fig. 4 I and J). Knockdown of DSCAM by siRNA in the presence of anti-DCC antibody further reduced the netrin-induced Discussion axon outgrowth (Fig. 4 E and H; group VIII in Fig. 4 I and J). These Our experiments provide several lines of evidence supporting the results demonstrate that both DCC and DSCAM are required for ligand–receptor relationship between netrin-1 and DSCAM. First, netrin signaling in stimulating axon outgrowth. DSCAM is expressed on the developing commissural axons when they project toward and across the floor plate (Fig. 1). Second, netrin-1 The Role of DSCAM in Axon Attraction by Netrin-1. To examine the interacts with the full-length or the extracellular domain of DSCAM role of DSCAM in axon attraction by netrin-1, we performed a (Fig. 2). Soluble netrin-1 binds to the surface of DSCAM-expressing commissural axon turning assay as described previously (31, 35). As cells with an apparent Kd similar to that of DCC–netrin interaction (Fig. illustrated in Fig. 5A, the YFP plasmid was electroporated into the 3). Third, DSCAM expression is sufficient to mediate netrin-induced chick neural tube at stage 12–15. The YFP-labeled neural tube was phosphorylation of Pak1 and Fyn (Fig. 2), important signaling events isolated, and the spinal cord was laid out as an ‘‘open book’’ and for netrin function in neurons. Fourth, expression of the DSCAM

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Fig. 7. DSCAM is essential for commissural axon pathfinding in vivo. (A)A diagram illustrating the commissural axon projection in a transverse section of M N O the chick spinal cord. The chick neural tube was electroporated with YFP only (B); with YFP plus wild-type DSCAM (C); with YFP plus DSCAM⌬C(D); with YFP plus DSCAM⌬N(E); with YFP plus DSCAM siRNA (F); with YFP, DSCAM siRNA, and wild-type DSCAM (G); or with YFP with DSCAM-siRNA and DSCAM⌬N(H). DSCAM siRNA not only inhibited the commissural axon extension, but also caused aber- P Q rant axon turning (F and H). The arrow in panel F and the arrowhead in panel H indicate the misguided commissural axons. Expression of wild-type human DSCAM rescued the defects caused by DSCAM siRNA in commissural axon pro- jection and turning (G). DSCAM⌬N expression did not reverse the effects of DSCAM siRNA (H). (Scale bar: 100 ␮m.) NEUROSCIENCE

axons to the floor plate (14, 15). Our data suggest that DSCAM may Fig. 6. DSCAM is required for commissural axon projection in vivo. Different contribute to such a DCC-independent mechanism. combinations of plasmids and siRNAs were electroporated into the chick neural Consistent with our findings, Ly et al. (29) recently reported that ⌬ tube. (A–C) YFP with wild-type DSCAM plasmid. (D–F) YFP with DSCAM C. (G–I) DSCAM is a receptor of netrin-1 and that DSCAM is required for YFP only. (J–L) DSCAM siRNA with YFP. (M–O) DSCAM-siRNA with human DSCAM and YFP plasmids. Panels A, D, G, J, and M show YFP images; panels B, E, H, K, and netrin-induced commissural axon outgrowth (29). According to N show anti–axonin-1 antibody immunostaining; and panels C, F, I, L, and O are these authors, DSCAM siRNA did not affect axon turning, how- merged images. (Scale bar: 100 ␮m.) (P) Quantification of the percentage of ever. Our data demonstrate that DSCAM is required for both axons reaching the floor plate for different groups: 81.8% Ϯ 4.0% for wild-type netrin-induced axon outgrowth and axon attraction in embryonic DSCAM, 31.9% Ϯ 3.7% for DSCAM⌬C, 79.1% Ϯ 2.3% for YFP only, 36.9% Ϯ 4.7% chick spinal cord (Figs. 4–7). Furthermore, DSCAM alone in the Ϯ for YFP and DSCAM siRNA, and 76.1% 3.4% for DSCAM siRNA and wild-type absence of DCC is sufficient to mediate netrin-induced phosphor- DSCAM. P values are Ͻ .001 between the groups compared: wild-type and DSCAM⌬C, YFP and DSCAM siRNA, and DSCAM-siRNA and DSCAM-siRNA plus ylation of Fyn and Pak1 in transfected cells (Fig. 2). The results of human DSCAM plasmid (Student t-test). (Q) Quantification of the average dis- our immunostaining experiments suggest that DCC and DSCAM tance of axons from the floor plate in different groups: 7.39 Ϯ 1.43 ␮m for do not have completely overlapping expression, and that different wild-type DSCAM, 55.24 Ϯ 7.72 ␮m for DSCAM⌬C, 9.38 Ϯ 1.26 ␮m for YFP, 49.25 Ϯ subsets of commissural neurons may exhibit differential DCC and 8.93 ␮m for DSCAM siRNA, and 16.31 Ϯ 3.51 ␮m for DSCAM siRNA plus wild-type DSCAM expression. Moreover, both DCC and DSCAM have Ͻ DSCAM. P values between groups compared are .0001 for wild-type DSCAM different splicing isoforms that may exhibit differential expression and DSCAM⌬C, Ͻ .01 for YFP and DSCAM-siRNA, and Ͻ .01 for DSCAM-siRNA and DSCAM siRNA plus wild-type DSCAM plasmid. The numbers on the top of each bar among different subpopulations of neurons. It also is possible that (n) indicate the numbers of embryos tested in the corresponding groups. The purple the difference between these 2 studies reflects species differences arrowheads indicate the commissural axons stalled before reaching the floor plate. between the rodents and chicks. Further investigation is needed to identify the factors contributing to the differences between our findings and those of Ly et al. (29). mutant lacking its intracellular domain reduces the effect of Whether DSCAM functions alone or as a component of a multi- netrin in inducing commissural axon turning and midline cross- subunit receptor complex remains unclear. Our data indicate that ing (Figs. 5, 6, and 7). Fifth, knockdown of DSCAM by siRNA DSCAM is involved in netrin-induced phosphorylation of Pak1, Fyn, inhibits netrin-induced commissural axon outgrowth (Fig. 4), and DSCAM itself (Fig. 2). DCC also plays a role in Pak1 phosphor- turning (Fig. 5), and midline crossing (Figs. 6 and 7). ylation (28). The findings from our co-immunoprecipitation experi- In a cell surface binding assay, it is difficult to rule out the ment suggests that DSCAM does not interact with DCC (Fig. S2B). possibility that netrin-1 binds to a cell surface component induced Immunostaining revealed that DSCAM partially co-localized with by DSCAM expression rather than binding directly to DSCAM DCC in dissociated cortical and spinal cord neurons (Fig. 1 J–O). Either itself. Other adaptors or cofactors possibly may play a role in netrin DCC antibody or DSCAM siRNA alone was not sufficient to com- signaling mediated by DSCAM. Nonetheless, our data support the pletely eliminate netrin responses. Blocking of DCC function by the need for a specific interaction between DSCAM and netrin for specific DCC antibody, in conjunction with DSCAM siRNA, almost netrin signaling in commissural neurons. The overall phenotypic completely abolished netrin signaling in spinal cord axon outgrowth similarities in the DCC knockout and netrin-1–deficient mice (Fig. 4). These findings suggest that both DCC and DSCAM play a role suggest that DCC is important for netrin-1 signaling; however, in in netrin signaling. Further studies are needed to gain insight into the DCCϪ/Ϫ mice, some commissural axons still cross the floor plate, relationship between DSCAM and DCC in guiding commissural axon suggesting a DCC-independent mechanism guiding commissural projection during neural development.

Liu et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 24, 2021 Signal transduction mechanisms downstream of DSCAM in the mouse cortical neurons were cultured and transfected as described previously vertebrate nervous system remain poorly understood. In Drosoph- (31). For details, see SI Materials and Methods. ila, Dock adaptor interacts with the cytoplasmic domain of DSCAM and activates Pak1 (37), and the cytoplasmic domain of Cell Surface Binding. HEK293 cells grown in 12-well dishes were transfected with DSCAM, Robo-GFP, or DsRed plasmids using the calcium phosphate method as DSCAM is required to convert attachment to repulsion between described previously (32). Approximately 40 h after transfection, cells were incu- sister axon branches (38). Unlike Drosophila Dscam, human bated with netrin-1– or Sema3A-containing media. Double staining of DSCAM DSCAM directly binds to and activates Pak1 (27). DSCAM also and bound netrin-1 or Sema3A was carried out using the affinity-purified anti- activates both JNK and p38MAP kinases (27). Our data indicate DSCAM antibody and an anti–netrin-1 antibody or anti-myc antibody. The bound that DSCAM⌬C (the mutant lacking the intracellular domain) netrin-1 or Sema3A was visualized using a Cy3- or Cy2-labeled secondary anti- inhibits commissural axon outgrowth, axon turning, and pathfind- body. Netrin-AP was used for equilibrium binding to cells expressing DSCAM or ing in vitro and in vivo (Figs. 5–7), suggesting that the cytoplasmic the vector control, and Scatchard analysis was carried out as described previously (9, 32). For details, see SI Materials and Methods. domain of DSCAM is important for mediating netrin signaling. CAS Tyrosine phosphorylation of FAK, Fyn, and p130 is required for Chick Spinal Cord Explant Culture, Axon Outgrowth Assay, Axon Turning Assay, netrin-induced axon outgrowth and turning (31, 35, 39, 40). The and Commissural Axon Projection In Vivo. White leghorn chicken embryos were netrin–DCC signaling complex includes Cdc42, Rac1, Pak1, and collected and staged according to methods outlined by Hamburger and Hamilton N-WASP (28). Our findings indicate that DSCAM is involved in (41). Chick spinal cord explant culture, electroporation, and analysis of axon netrin-induced phosphorylation of Pak1 and Fyn. More research is outgrowth were performed as described previously (31, 35). The numbers of needed to investigate whether other signaling molecules, such as axons and total axon length were measured using the National Institutes of CAS Health Image J software. Electroporation, open-book preparation, co-culture FAK, p130 , Rac, Cdc42, or N-WASP, also act downstream of with control or netrin-secreting cell aggregates, immunostaining, and quantita- DSCAM, and also how the molecules downstream of different tive analyses of axon turning were performed as described previously (31, 35, 42). netrin receptors coordinate with one another to mediate netrin The percentage of turning axons was calculated from the numbers of YFP expressing signaling in axon outgrowth and pathfinding. axons turning toward the HEK cell aggregate divided by the total numbers of YFP expressing axons within 300 ␮m of the HEK cell aggregates. Transverse sections of Materials and Methods spinal cords were immunostained to visualize commissural axons by confocal fluo- rescent microscopy. For details, see SI Materials and Methods. Antibodies, Plasmids, and and siRNAs. see SI Text.

ACKNOWLEDGMENTS. We thank Drs. J. Chernoff, E. Fearon, and E. T. Stoeckli for Cell Culture, Transfection, Immunoprecipitation, and Immunostaining. Cell cul- generously providing plasmids or antibodies for Pak1, netrin and axonin, and ture, transfection, and immunoprecipitation were performed as described pre- Xiaoping Chen and Justin Meyer for providing technical assistance. This work was viously (32). Double staining of DSCAM with axonin-1 or TAG-1 or DCC was carried supported by National Institutes of Health Grants CA107193 and CA114197 (to out using the same protocol as described in our previous study (35). Dissociated Y.R., K.G., and J.W.).

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