FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons
2011; Springer Nature; Volume: 30; Issue: 14 Linguagem: Inglês
10.1038/emboj.2011.189
ISSN1460-2075
AutoresSatoru Yamagishi, Falko Hampel, Katsuhiko Hata, Daniel del Toro, Manuela Schwark, Elena Kvachnina, Martin Bastmeyer, Toshihide Yamashita, Victor Tarabykin, Rüdiger Klein, Joaquim Egea,
Tópico(s)Developmental Biology and Gene Regulation
ResumoArticle14 June 2011free access FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons Satoru Yamagishi Satoru Yamagishi Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, GermanyPresent address: Department of Anatomy and Neuroscience, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan Search for more papers by this author Falko Hampel Falko Hampel Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, Germany Search for more papers by this author Katsuhiko Hata Katsuhiko Hata Department of Molecular Neuroscience, Osaka University Graduate School of Medicine, Osaka, Japan Search for more papers by this author Daniel del Toro Daniel del Toro Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, Germany Search for more papers by this author Manuela Schwark Manuela Schwark Max Planck Institute for Experimental Medicine, Hermann-Rein Strasse, Göttingen, Germany Neurocure Exzellenzcluster, Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany Search for more papers by this author Elena Kvachnina Elena Kvachnina Max Planck Institute for Experimental Medicine, Hermann-Rein Strasse, Göttingen, Germany Search for more papers by this author Martin Bastmeyer Martin Bastmeyer Karlsruher Institut für Technologie (KIT), Zoologisches Institut Abteilung für Zell- und Neurobiologie, Haid-und-Neu-Strasse, Karlsruhe, Germany Search for more papers by this author Toshihide Yamashita Toshihide Yamashita Department of Molecular Neuroscience, Osaka University Graduate School of Medicine, Osaka, Japan Search for more papers by this author Victor Tarabykin Victor Tarabykin Max Planck Institute for Experimental Medicine, Hermann-Rein Strasse, Göttingen, Germany Neurocure Exzellenzcluster, Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany Search for more papers by this author Rüdiger Klein Corresponding Author Rüdiger Klein Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, Germany Search for more papers by this author Joaquim Egea Corresponding Author Joaquim Egea Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, Germany Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida/IRBLLEIDA, Spain Search for more papers by this author Satoru Yamagishi Satoru Yamagishi Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, GermanyPresent address: Department of Anatomy and Neuroscience, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan Search for more papers by this author Falko Hampel Falko Hampel Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, Germany Search for more papers by this author Katsuhiko Hata Katsuhiko Hata Department of Molecular Neuroscience, Osaka University Graduate School of Medicine, Osaka, Japan Search for more papers by this author Daniel del Toro Daniel del Toro Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, Germany Search for more papers by this author Manuela Schwark Manuela Schwark Max Planck Institute for Experimental Medicine, Hermann-Rein Strasse, Göttingen, Germany Neurocure Exzellenzcluster, Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany Search for more papers by this author Elena Kvachnina Elena Kvachnina Max Planck Institute for Experimental Medicine, Hermann-Rein Strasse, Göttingen, Germany Search for more papers by this author Martin Bastmeyer Martin Bastmeyer Karlsruher Institut für Technologie (KIT), Zoologisches Institut Abteilung für Zell- und Neurobiologie, Haid-und-Neu-Strasse, Karlsruhe, Germany Search for more papers by this author Toshihide Yamashita Toshihide Yamashita Department of Molecular Neuroscience, Osaka University Graduate School of Medicine, Osaka, Japan Search for more papers by this author Victor Tarabykin Victor Tarabykin Max Planck Institute for Experimental Medicine, Hermann-Rein Strasse, Göttingen, Germany Neurocure Exzellenzcluster, Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany Search for more papers by this author Rüdiger Klein Corresponding Author Rüdiger Klein Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, Germany Search for more papers by this author Joaquim Egea Corresponding Author Joaquim Egea Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, Germany Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida/IRBLLEIDA, Spain Search for more papers by this author Author Information Satoru Yamagishi1,‡, Falko Hampel1,‡, Katsuhiko Hata2, Daniel del Toro1, Manuela Schwark3,4, Elena Kvachnina3, Martin Bastmeyer5, Toshihide Yamashita2, Victor Tarabykin3,4, Rüdiger Klein 1 and Joaquim Egea 1,6 1Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz, Martinsried, Germany 2Department of Molecular Neuroscience, Osaka University Graduate School of Medicine, Osaka, Japan 3Max Planck Institute for Experimental Medicine, Hermann-Rein Strasse, Göttingen, Germany 4Neurocure Exzellenzcluster, Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany 5Karlsruher Institut für Technologie (KIT), Zoologisches Institut Abteilung für Zell- und Neurobiologie, Haid-und-Neu-Strasse, Karlsruhe, Germany 6Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida/IRBLLEIDA, Spain ‡These authors contributed equally to this work *Corresponding authors: Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz 18, Martinsried 82152, Germany. Tel.: +49 898 578 3150; Fax: +49 898 578 3152; E-mail: [email protected] de Ciencies Mediques Basiques, Facultat de Medicina, Universitat de Lleida/IRBLLEIDA, Montserrat Roig 2, Lleida 25198, Spain. Tel.: +34 973 70 22 87; Fax: +34 973 70 24 26; E-mail: [email protected] The EMBO Journal (2011)30:2920-2933https://doi.org/10.1038/emboj.2011.189 Present address: Department of Anatomy and Neuroscience, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Netrin-1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A–Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin-1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine-rich transmembrane protein-3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5-positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2-expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ-derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5-positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration. Introduction Migrating cells and pathfinding axons are guided by molecular cues within the extracellular matrix or on the surface of ambient cells. These cues are interpreted as attractive or repulsive, depending on the set of receptors and signal transducers the cell expresses. The mammalian Unc5 receptor family comprises four orthologues (Unc5A–Unc5D), which function as repulsive axon-guidance receptors for Netrin (Leonardo et al, 1997; Moore et al, 2007) and RGMa (Bradford et al, 2009; Hata et al, 2009). The role of Unc5 protein in axon guidance has been clearly demonstrated in several model organisms (Mehlen and Furne, 2005) and more recent work has implicated Netrin/Unc5 signalling in angiogenesis and cell survival (Mehlen and Furne, 2005; Bradford et al, 2009). Genetic ablation of Unc5C leads to several CNS phenotypes including aberrant migration of cerebellar granule and Purkinje cells, and motor axon guidance defects (Ackerman et al, 1997; Burgess et al, 2006). Interestingly, these functions were not seen in Netrin-1−/− mice, indicating the participation of other Netrins (Moore et al, 2007) or other unrelated ligands for the Unc5 receptor family (Burgess et al, 2006). Unc5B and Unc5D have recently been shown to bind with high affinity to the ectodomain (ECD) of fibronectin and leucine-rich transmembrane protein-3 (FLRT3) (Karaulanov et al, 2009; Sollner and Wright, 2009), a transmembrane protein that is thought to act cell autonomously either as a co-receptor or as a cell adhesion molecule in early embryogenesis (Bottcher et al, 2004; Karaulanov et al, 2006; Egea et al, 2008; Maretto et al, 2008). The Unc5B/FLRT3 complex regulates cell adhesion through the Rho small GTPase Rnd1 (Ogata et al, 2007; Karaulanov et al, 2009), but its role in neural development is unknown. Other ex vivo studies have shown that FLRT3 promotes neurite growth non-cell autonomously (Tsuji et al, 2004) or cell autonomously (Robinson et al, 2004). The related FLRT2 protein is essential for heart development (Muller et al, 2011), while the function of FLRT1 has not been explored. Unc5D has recently been shown to be expressed by multipolar cells, which are the progeny of a population of cortical precursor cells, the so-called intermediate or basal progenitor cells (BPs), which populate the subventricular zone (SVZ) (Sasaki et al, 2008). BPs can be considered to be neurogenic transit amplifying progenitors that expand the pool of differentiated neuronal cells. This process contrasts with neuronal generation directly from apical progenitors, which are located in the ventricular zone (VZ). Both kinds of proliferative progenitors give rise to cortical projection neurons, which form the mammalian cerebral cortex. Projection neurons are produced in a temporal sequence and undergo radial migration to reach their final laminar positions. Radial migration follows an ‘inside–out’ pattern such that later born neurons trespass older neurons and occupy more superficial positions in the neocortex (Rakic, 1988). This ‘inside–out’ rule, however, is not respected by all cortical neurons. Neurons that express Unc5D (Sasaki et al, 2008) and the SVZ marker Svet1 (Tarabykin et al, 2001), which comprises a non-coding RNA encoded by an intronic region of the unspliced RNA of Unc5D (Sasaki et al, 2008) migrate slowly from the SVZ towards the cortical plate (CP) (Britanova et al, 2008). They begin entering the CP at E18.5 in the mouse and complete their migration by P2 (Tarabykin et al, 2001). Instead, neurons that express the marker Satb2, although born simultaneously or even later than Unc5D+ neurons, do not linger in the SVZ and arrive in the CP as early as E14.5. The molecular mechanisms underlying this different migratory behaviour are not understood. The functional significance of Unc5D expression in BPs is currently unclear, but it raises the possibility that Unc5D is involved in controlling or modulating radial migration towards the CP. Since FLRT2 is expressed in the developing neocortex, we investigated the possibility that FLRT2/Unc5D signalling provides guidance to migrating cells and/or pathfinding axons. Here, we show that all three FLRT ECDs undergo cleavage by metalloproteases and are shed from cultured neurons, suggesting non-cell autonomous functions. Our results revealed specific and high-affinity interactions between FLRT2 and Unc5D (and to a lesser extent Unc5B) and between FLRT3 and Unc5B receptors. Further, we found that soluble FLRT ECDs activate Unc5 repulsive signalling inducing growth cone collapse and cell sorting. In vivo, Unc5D and FLRT2 modulate the radial migration of cortical cells. During corticogenesis, FLRT2 is expressed in cells of the CP at the time when Unc5D+ cells in the SVZ display their delayed migratory behaviour. Using a combination of gain- and loss-of-function experiments in the mouse, we show that FLRT2 and Unc5D have a significant impact on the migration of a subset of projection neurons, consistent with FLRT2 acting as a repulsive cue for Unc5D+ cells. Results The ECDs of FLRTs are shed from neurons To investigate posttranslational modifications of FLRT proteins, we transfected HEK293T cells with epitope-tagged constructs and examined the proteins in cell lysates and culture media by western blotting. In cell lysates, antibodies against the individual FLRT1–3 ECDs specifically detected full-length FLRT proteins and showed no crossreactivity with the other FLRTs (Figure 1A). Interestingly, all three FLRT ECDs were detected in conditioned media of the cultures after pull down with a lectin that recognizes glycoproteins (Figure 1A). The sizes of the cleaved ECDs ranged from 65 to 85 kDa, suggesting a cleavage event near the plasma membrane. Whereas FLRT1 and FLRT3 ECDs consisted of a single protein species, the FLRT2 ECD was a doublet of 75 and 85 kDa (Figure 1A). The analysis of total cell lysates from transfected cells confirmed the presence of low molecular weight bands containing the C-terminal FLAG tag (Figure 1B). Cleavage of the FLRT2 ECD was also detected with a FLRT2 deletion mutant lacking the entire intracellular domain (ICD), indicating that the ICD was not required for FLRT2 ECD shedding (Figure 1C). Endogenous FLRT ECDs were also shed from primary mouse neurons (glial cells express low levels of FLRT2; Supplementary Figure S1). We detected FLRT1–3 ECD species in conditioned media of dissociated embryonic cortical neurons and the ECD sizes matched those of transfected FLRTs, including the doublet of the FLRT2 ECD (Figure 1D, top panels). The proteins in the media were not detected by antibodies against the ICD of FLRTs (Figure 1D, lower panels). The amount of shed FLRT2 ECD in the media of hippocampal neurons remained stable for up to 10 DIV, while the full-length expression in the neurons decreased (Figure 1C). The FLRT2 ECD was even detectable in conditioned media from hippocampal explants of subregion CA1 (Figure 1E) where FLRT2 was strongly expressed (Figure 7A). The specificity of the signal in the culture media was confirmed by using the corresponding cells from FLRT2−/− and Nestin-Cre+;FLRT3lx/− mice (Figure 1E and F; Supplementary Figure S1). We then performed immunoblots of developing brain extracts and detected FLRT1 proteins from E13 to P10 at similar levels, while FLRT2 and FLRT3 expression gradually decreased after E15 (Figure 2A–C). Anti-FLRT ECD antibodies detected a 90-kDa species and an additional band in the range of 65–70 kDa in the cases of FLRT1 and FLRT3, or a doublet of 73 and 80 kDa in the case of FLRT2 (Figure 2A–C, upper panels), whereas anti-FLRT ICD antibodies detected only a single protein species of ∼90 kDa (Figure 2A–C, lower panels). None of these proteins were detected in the corresponding knockout brain extracts (Figure 2A–C). Since the entire polypeptide of each of the FLRTs is encoded by a single exon (Lacy et al, 1999), these different protein species could not have resulted from alternative splicing. In addition, glycosidase treatment of brain extracts converted all protein species (full-length and ECDs) to faster migrating ones to similar extents, indicating that the additional bands detected by the anti-FLRT ECD antibodies were not hypoglycosylated forms, but rather proteolytic cleavage products of the mature FLRTs (Figure 2D–F). Figure 1.FLRT ECDs are shed from transfected cells and neurons. (A, B) Western blot analysis of lectin pull-down (glycoprotein enriched) samples from 2 DIV conditioned media (Media) or of corresponding total cell lysates (TCL in A, B) of HEK293T cells transfected with C-terminally FLAG-tagged FLRT1, FLRT2, FLRT3 or Mock transfected. Samples were analysed using antibodies against the ECD of FLRTs or FLAG as indicated. Arrowheads in (A) point to the FLRT ECDs (one species for FLRT1 or FLRT3, doublet for FLRT2). Various protein species in the range of 18–35 kDa were found in the TCL (arrowheads in B). (C) Same analysis as in (A) of dissociated E17.5 hippocampal neurons kept in culture for the indicated DIV or HeLa cells transiently transfected with C-terminally FLAG-tagged full-length FLRT2 (FL) and FLRT2 lacking the intracellular domain (ΔICD). Anti-tubulin levels in TCLs are shown as loading controls. (D) Detection of shed FLRT1–3 ECDs in 6 DIV conditioned media of E16.5 cortical neurons. FLRT1–3 ECDs are visible in blots probed with anti-ECD antibodies (upper panels) but not with anti-ICD antibodies (lower panels). The positions of full-length FLRT2 (arrow) and of a non-specific band (asterisk) are indicated (see FLRT2–/– control in Figure 2B). (E, F) Mouse mutants demonstrate specificity of the ECD signals. (Upper panels) Western blot analysis of glycoprotein enriched samples of 7 DIV conditioned media from CA1 hippocampal explants (E) or dissociated cortical neurons (F) of the indicated genotypes using anti-FLRT2/3 ECD antibodies. (Middle panels) Corresponding glycoprotein enriched TCL probed with anti-FLRT2/3 ECD antibodies. (Lower panel) Corresponding glycoprotein enriched TCL probed with a loading control (anti-FLRT3 ECD in (E) and anti-EphA4 in (F)). Arrowhead and asterisk in (F) indicate FLRT ECD and a non-specific band, respectively (see also Supplementary Figure S1). Download figure Download PowerPoint Figure 2.Cleaved FLRT ECDs in developing brain and involvement of metalloproteases. (A–C) Cleaved FLRT ECDs in brain extracts. Western blot analysis of glycoprotein enriched brain extracts of the indicated developmental stages using antibodies against the FLRT ECDs (upper panels) or FLRT-ICD (lower panels) (first lanes contain extracts from the indicated knockout animals). Arrowheads indicate the positions of the FLRT ECDs. The positions of full-length FLRTs (arrow) are indicated. (D–F) FLRT ECDs are glycosylated. Glycoprotein enriched E13 brain extracts followed by incubation with (+) or without (−) N-glycosidase were analysed as above. Arrows and arrowheads indicate full-length and shed FLRT proteins, respectively. (G–I) Involvement of metalloproteases in FLRT shedding. Dissociated E16.5 cortical neurons were cultured for 2 DIV plus one additional DIV in the presence of 10 μM of TAPI-0, 10 μM (or 50 μM) of TAPI-I and 5 μM of GM6001 or vehicle only (DMSO). Glycoprotein enriched samples from the media and the total cell lysates (TCL) were analysed by western blotting using anti-FLRT ECD antibodies. Arrowheads in panels (G, H) indicate the position of the FLRT3 ECD. Asterisk in (G) indicates the position of a non-specific band (see also (K) and Figure 1F). (J–M) Mapping the cleavage site in FLRT3 and FLRT2 ECDs. (J, L) Scheme of deletion mutants used (all C-terminal FLAG-tagged; FLRT2 constructs also had an N-terminal Myc tag). (J) ΔFNIII lacking the fibronectin type III domain (amino acids 406–501) and ΔJM lacking the JM region (amino acids 502–523) of FLRT3. (L) ΔJM1 lacking amino acids 512–520, ΔJM2 lacking amino acids 524–536 and ΔJM1+2 lacking amino acids 512–536 but keeping the NGS sequence of the N-glycosylation site of the JM region of FLRT2. (K, M) Western blot analysis of glycoprotein enriched media of HEK293T cells transfected with the above constructs or EGFP (GFP). Samples were analysed using antibodies against FLRT3 ECD (K) or Myc (L, M) (upper panels). Expression in total cell lysates (TCL) was analysed using an anti-FLAG antibody (lower panels). Arrowheads indicate the position of the cleaved ECDs and asterisk in (K) indicate the position of a non-specific band (see also Supplementary Figure S2 for protein expression in plasma membrane). Download figure Download PowerPoint To test whether metalloproteases were involved in FLRT ECD shedding, we examined FLRT2 and FLRT3 expression in the presence of the wide spectrum matrix metalloproteinases and ADAM inhibitors TAPI-0, TAPI-I and GM6001 (Budagian et al, 2005). All three inhibitors markedly reduced the release of the FLRT3 ECD into the conditioned media of cultured neurons (Figure 2G and H). TAPI-I, when analysed for its involvement in FLRT2 cleavage, also reduced the release of the FLRT2 ECD, although somewhat higher concentrations were required (Figure 2I). In contrast, DAPT, an inhibitor of the γ-secretase protease complex (Geling et al, 2002) did not affect FLRT2/3 ECD shedding (data not shown). These results suggest that FLRT ECD shedding is an active process that requires metalloprotease activity. To begin mapping the site of FLRT ECD cleavage, we generated different isoforms of FLRT2 and FLRT3 carrying small deletions in the ECD (Figure 2J and L). Expression controls of these constructs by immunofluorescence and in total cell lysates showed that they were expressed at similar levels in the membrane of transfected HEK293T cells (Figure 2K and M; Supplementary Figure S2). Whereas the deletion of the FNIII domain did not change the abundance of the FLRT3 ECD in the conditioned media, the deletion of the juxtamembrane (JM) region completely blocked release of the FLRT3 ECD (Figure 2K). The two cleavage sites of FLRT2 were also mapped to the JM region and flanking a conserved N-glycosylation site, which is required for proper expression in the cell membrane (Figure 2M and data not shown). Soluble FLRT ECDs bind to Unc5 receptors To identify interaction partners for FLRT ECDs, we used soluble FLRT ECDs fused to alkaline phosphatase (AP) as baits in binding assays on transfected HEK293T cells expressing candidate interaction partners. Although FLRT3 promoted homotypic cell aggregation (Karaulanov et al, 2006; Egea et al, 2008), we found that FLRT3/ECD-AP did not bind any of the three FLRT proteins (Supplementary Figure S3). Moreover, FLRT2 ECD equally bound to cultured FLRT2−/− neurons compared with wild-type controls (Supplementary Figure S3), arguing against FLRT proteins interacting with other FLRTs. FLRT3 binding to the Wnt receptor Brother of CDO (BOC), as recently shown for Zebrafish FLRT3 (Sollner and Wright, 2009), could not be confirmed for mouse FLRT3, nor did we detect specific binding to FGFR2 or the Netrin receptor Neogenin (Supplementary Figure S3). In agreement with published reports (Karaulanov et al, 2009; Sollner and Wright, 2009), FLRT3/ECD-AP strongly bound to cells transfected with the Netrin receptor Unc5B (Figure 3B and G; Supplementary Figure S3). A comparison between all three FLRTs and the entire Unc5 protein family (Unc5A–D) revealed that Unc5B was the preferred binding partner for FLRT3, with an equilibrium dissociation constant (Kd) of 66 nM (n=5 experiments; Figure 3B, D, K and L; Supplementary Figure S3). Interestingly, FLRT2 displayed a different binding pattern with strong affinity for Unc5D (Kd of 11 nM, n=4; Figure 3C, G, I and J; Supplementary Figure S3) and weaker binding to Unc5B (Figure 3A, G and I). A naturally occurring splice variant of Unc5D lacking the first thrombospondin-1 domain (Unc5DΔTP1, accession number BC145695), which is predicted to still bind to Netrin (Kruger et al, 2004), failed to bind to FLRT2 (Figure 3E,G). FLRT1 showed only relatively weak binding to Unc5B and none of the FLRTs displayed strong interaction with Unc5A or Unc5C (data not shown). We confirmed the specificity of FLRT–Unc5 interactions by reciprocal binding experiments: HEK293T cells were transfected with full-length FLAG-tagged FLRTs and incubated with soluble Unc5B/ECD-Fc, Unc5D/ECD-Fc or Fc as control. The Unc5D/ECD showed the strongest preference for FLRT2, while Unc5B/ECD preferred FLRT3 over FLRT2 (Figure 3M–T). The interaction between FLRTs and Unc5 proteins appeared to be independent of Netrin-1. Netrin-1 did not bind FLRT2 in the absence of Unc5D and rather competed with FLRT2-AP for binding to Unc5D (Supplementary Figure S3). Figure 3.Soluble FLRT ECDs bind to Unc5 receptors. (A–H) FLRT2 binds best to Unc5D and to a lesser extent to Unc5B, while FLRT3 shows strong preference to Unc5B. Transfected HEK293T cells (MOCK empty vector or C-terminally FLAG-tagged Unc5B, Unc5D or Unc5DΔTP1 were incubated with similar amounts (250 nM) of FLRT2/ECD-AP or FLRT3/ECD-AP fusion proteins, as indicated. AP activity was revealed using Naphtol-Fast Red (A–F) or by absorbance at 405 nm using p-nitrophenylphosphate (G). (G) Representative plot of average activity+s.e.m (n=3) (P 3 independent experiments) of the FLRT2/ECD-Unc5D (J) and FLRT3/ECD-Unc5B (L) interactions. (M–S) Soluble Unc5B/ECD-Fc, but not Unc5D/ECD-Fc, binds HEK293T cells transfected with C-terminally FLAG-tagged FLRT3. Cells were incubated as indicated with control Fc, Unc5B/ECD-Fc or Unc5D/ECD-Fc, fixed and stained by immunofluorescence with anti-hFc (bound protein; green channel in insets in lower row) and anti-FLAG (transfected protein; red channel in insets in lower row). Co-localization is shown in yellow in the overlay (Q, inset). (S) Results of a representative experiment (average+s.e.m., n=2) of the bound fractions of Unc5D/ECD-Fc, Unc5B/ECD-Fc and Fc normalized to the amount of transfected FLRT (**P<0.01; ***P<0.001, t-test). Scale bar in (M): 50 μm. (T) Summary of the binding affinities of FLRTs to Unc5 receptors (see also Supplementary Figure S3). Download figure Download PowerPoint Soluble FLRT2/3 ECDs induce growth cone collapse of cortical neurons via Unc5 receptors Next, we used dissociated neuron cultures of E16.5 rat motor cortices, which endogenously express Unc5B and Unc5D (see mouse expression data in Supplementary Figure S6 and Figure 7B), to assess whether FLRT/ECD binding to Unc5 receptors would be sufficient to activate their repulsive signalling properties. We counted the number of collapsed growth cones after staining with phalloidin to visualize the actin cytoskeleton. All phalloidin-stained neurons were included in the analysis, although only ∼50% of them specifically bound FLRT3-Fc (Figure 4A–C). Stimulation with different concentrations of FLRT3-Fc-induced growth cone collapse comparable to RGMa, a ligand for Unc5B/Neogenin complex (Figure 4D; Hata et al, 2009). Both responses were significantly reduced in the presence of the soluble Unc5B/ECD, which presumably blocked the active sites of FLRT3-Fc or RGM (Figure 4D). We next performed growth cone collapse assays on cortical neurons in which the endogenous Unc5B expression was reduced by siRNA-mediated knockdown and observed that the FLRT3-Fc-induced collapse response was completely abolished (Figure 4E and F). This effect was specific since overexpression of Unc5B together with the siRNA-mediated knockdown, restored the collapse response induced by FLRT3-Fc (Figure 4E). Next, we turned to FLRT2 and its Unc5D receptor and generated Unc5D−/− mice (Supplementary Figure S4). Unc5D−/− mice were viable and did not display obvious behavioural defects. Gross inspection of forebrain anatomy did not reveal major anatomical defects (data not shown). The growth cone collapse response of Unc5D−/− cortical neurons towards FLRT2-Fc, but not FLRT3-Fc, was significantly impaired compared with cultures derived from wild-type littermates, indicating the specific binding to FLRT2 (Figure 4G). Finally, a similar dose- and Unc5D-dependent collapse response was observed using a FLRT2 ECD carrying a small, non-dimerizing epitope tag (FLRT2-His) (Figure 4H and I), providing support for the concept that the native shed FLRT2 ECD is active. Together, these results provide strong evidence that FLRT2/3 ECDs act as repulsive cues for cortical axons in vitro through a mechanism that requires Unc5 receptors. Figure 4.FLRT2/3 ECDs induce growth cone collapse of cortical neurons via Unc5. (A–C) Representative examples of phalloidin-stained rat embryonic cortical neurons stimulated with 1 μg/ml FLRT3-Fc. Anti-Fc antibody staining shows a neuron with a collapsed, phalloidin-negative growth cone (arrow in B, Bi). Two other neurons (in one case only the axon is visible) do not show FLRT3-Fc binding and have intact growth cones (arrowhead in B, Bii). Scale bar: 50 μm. (D) The soluble Unc5B ectodomain (Unc5B-ECD) inhibits growth cone collapse of rat cortical neurons (expressing endogenous Unc5B) induced by FLRT3-Fc (1 and 3 μg/ml)
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