Research Article 1071 The SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons

Fabien Pujol, Patrick Kitabgi and Hélène Boudin*,‡ INSERM E0350, Hospital St Antoine, 184 rue du Fg St Antoine,75571 Paris CEDEX 12, France *Present address: INSERM U643, 30 Bld Jean Monnet, 44093 Nantes CEDEX 01, France ‡Author for correspondence (e-mail: [email protected])

Accepted 30 December 2004 Journal of Cell Science 118, 1071-1080 Published by The Company of Biologists 2005 doi:10.1242/jcs.01694

Summary Recent data have shown that the chemokine SDF-1 plays a the cells mature, staining declines at the tip of the processes critical role in several aspects of development such as and becomes more broadly distributed along and, to cell migration and pathfinding. However, its potential a lesser extent, dendrites. SDF-1 stimulation of neurons at function in the generation of axons and dendrites is poorly day 1-2 in culture triggers several effects on neuronal characterized. In order to better understand the role of morphogenesis. SDF-1 reduces growth cone number and SDF-1 in the development of central neurons, we studied axonal outgrowth but stimulates axonal branching. These the cellular distribution of the SDF-1 CXCR4 by latter two effects are not observed in other neurites. This immunocytochemistry of developing hippocampal neurons study unravels a new role for SDF-1/CXCR4 in specifying and tested the effect of SDF-1 in process patterning at hippocampal neuron morphology by regulating axonal the early stages of neuronal development. We found patterning at an early stage of neuronal development. that CXCR4 immunoreactivity undergoes a striking redistribution during development. At the early stages, from day 2 to day 4 in culture, CXCR4 is particularly Key words: Hippocampal neuron, Axon, Development, Chemokine, concentrated at the leading edge of growing neurites. As Cell culture

Introduction which SDF-1 represents to date the only known ligand. Early The development of neuronal circuits is supported by a fine evidence of the central role of SDF-1 in brain development Journal of Cell Science spatial and temporal orchestration of several cellular came from the striking abnormality of the cerebellum of events, including cell migration, process formation and knockout mice lacking either SDF-1 or its receptor CXCR4 synaptogenesis. The generation of elaborate dendritic trees and (Ma et al., 1998; Zou et al., 1998). Further examination of these axonal arborization is controlled by a repertoire of extracellular mouse lines indicated additional defects in other brain regions, molecules acting in a coordinated manner on guidance, such as the hippocampus and the neocortex (Lu et al., 2002; elongation and branching. Accumulating evidence indicates Stumm et al., 2003). The reported alterations in the brain that molecules involved in axon guidance are also implicated architecture could originate from dysfunctions in cell in process growth and branching. For instance, 3A migration, axonal guidance or process formation. All of these known to repel axons in the cortex (Bagnard et al., 1998; cellular events are regulated by SDF-1. Thus, SDF-1 has been Polleux et al., 1998; Polleux et al., 2000) has been shown to shown to be involved in migration of hippocampal, cerebellar reduce axon branching (Bagnard et al., 1998; Bagri et al., and cortical neurons (Bagri et al., 2002; Zhu et al., 2002; 2003). Similarly, Slit1, which acts as a chemorepellant for Stumm et al., 2003), axon guidance (Lu et al., 2001; Xiang cortical axons, promotes the growth and branching of both et al., 2002; Chalasani et al., 2003) and axon elongation axons and dendrites (Nguyen Ba-Charvet et al., 1999; Wang et (Arakawa et al., 2003). However, the question whether SDF-1 al., 1999; Whitford et al., 2002). In the same way, recent data could differentially influence the development of the axon and suggest that the chemokine SDF-1 is a bifunctional factor the other neurites remains open. In addition, the possibility that regulating both axon pathfinding and elongation (Xiang et al., SDF-1 could selectively affect elongation versus branching is 2002; Arakawa et al., 2003; Chalasani et al., 2003). SDF-1, still unexplored. Moreover, there is no data on the cellular originally isolated in the for its role in distribution of CXCR4 in developing neurons although this leukocyte chemoattraction (Tashiro et al., 1993; Nagasawa et would represent an important step in elucidating the potential al., 1996), has subsequently been shown to play important functions of SDF-1 at different stages of neuron development. functions in many organs including the haematopoietic system, We thus studied the distribution of CXCR4 during the cardiovascular system and brain (Nagasawa et al., 1996; development of cultured hippocampal neurons by Tachibana et al., 1998; Zou et al., 1998). A particularity of immunofluorescence, and tested the effect of SDF-1 in the SDF-1 among the large family of is that it binds patterning of the axon and the other neurites. We used a low- to only one receptor, CXCR4, a G protein-coupled receptor for density hippocampal neuron culture system because the axon 1072 Journal of Cell Science 118 (5)

and the other neurites emerge simultaneously from the cell 5 mM EDTA and a cocktail of protease inhibitors. The resulting body allowing us to compare the effects of SDF-1 on these two suspension was centrifuged at 3500 g for 10 minutes. The supernatant classes of processes. We found that CXCR4 immunoreactivity was collected, centrifuged at 20,000 g for 20 minutes and the pellet is strikingly redistributed during development from the tip was resuspended in 10 mM Tris-HCl containing 5 mM EDTA. The of nerve processes at early stages to a more widespread membrane preparation was solubilized in Laemmli buffer and samples localization along the processes, predominantly along axons, were resolved by SDS-PAGE, and processed as described above. as the cells mature. In addition, our data showed that SDF-1 regulates axonal elongation and branching, without affecting Immunostaining the other neurites. This study defines SDF-1 as a new Neurons were fixed in PFA for 20 minutes and permeabilized for 5 extracellular signal involved in the axonal patterning of minutes in 0.25% Triton X-100. The cells were blocked for 30 minutes hippocampal neurons, which suggests that this chemokine in 10% bovine serum albumin (BSA) in PBS and incubated for 2 hours might play a critical role on neuronal connectivity during at 37°C in primary antibodies diluted in PBS containing 3% BSA. The development. primary antibodies used were as follows: mouse monoclonal anti- MAP2 antibody clone AP20 shown to react with MAP2-A, MAP2-B but not with MAP2-C or MAP1 (1:400; Sigma), rabbit anti- synaptophysin (1:1000, Sigma), mouse anti-SV2 (1:50; Materials and Methods Developmental Studies Hybridoma Bank), goat anti-CXCR4 (1:100; Neuron culture and transfection Santa Cruz Biotechnology) and goat anti-SDF-1 (1:100; Santa Cruz Rat hippocampal cultures were prepared from 18-day-old rat embryos Biotechnology). Specificity of CXCR4 and SDF-1 antibodies has by previously described methods (Goslin et al., 1998). All the previously been characterized in non-neuronal as well as neuronal protocols were carried out in accordance with French standard ethical cells (Hasegawa et al., 2001; Banisadr et al., 2002; Banisadr et al., guidelines for laboratory animals (Agreement number 75-669). 2003). Cells were then incubated with appropriate Texas Red- or Briefly, hippocampi were dissected and dissociated by trypsin and FITC-conjugated secondary antibodies (Jackson Laboratories), and trituration and were plated at low density (4000 cells/cm2) on glass the coverslips were mounted on glass slides for analysis on a BX 61 coverslips coated with poly-L-lysine in MEM with 10% calf serum. microscope (Olympus). Pictures were acquired with a ×63/1.4 After cells were allowed to attach on coverslips, they were transferred objective using a digital camera (DP 50, Olympus) driven by Analysis to a dish containing a glial feeder layer cultured from newborn rat image-acquisition software. For experiments involving transfection, forebrain and were maintained for up to 2 weeks in serum-free MEM each coverslip was systematically scanned with a ×25 lens and images with N2 supplements. The neurons were used at 1, 2, 4, 7, 10 and 15 of each transfected cell acquired with a ×63/1.4 objective. Images for days after plating for immunocytochemical staining and morphology presentation were prepared for printing with Adobe Photoshop. analysis. For pharmacological treatments, neurons were incubated at day 1 with 50 nM SDF-1 (AbCys) or 10 µM bicyclam (custom synthesized Calcium imaging by Orgalink, Gif sur Yvette, France), or both, or corresponding Neurons at day 10 were incubated at 37°C for 1 hour with 5 µM Fura- vehicle (control cells) for 16 hours and were fixed at day 2 in 4% 2/AM in PBS pH 7.4 supplemented with 0.8 mM MgCl2, 1.3 mM paraformaldehyde, 4% sucrose in phosphate-buffered saline (PFA) for CaCl2, 20 mM HEPES, 5 mM glucose and 0.2% pluronic F-127. 15 minutes at room temperature for quantification analysis. Before analysis, the coverslips were inserted into a temperature-

Journal of Cell Science Transfection experiments of green fluorescent protein (GFP) and controlled chamber at 35°C and examined with an inverted CXCR4-GFP were performed at plating with either GFP or CXCR4- epifluorescence microscope (Nikon, Japan). Images were captured GFP expression vectors using the Lipofectamine 2000 reagents every 2 seconds and the ratio of fluorescence intensities at 340 and (Invitrogen) essentially according to the manufacturer’s instructions. 380 nm were recorded using the Ca2+ imaging system Quanticell 700 The CXCR4-GFP expression plasmid was a gift of M. Alizon (Institut (Visitech, UK). Cochin, Paris, France). Transfected neurons were plated at 14,000 cells/cm2 and processed as for untransfected cells. When neurons were co-cultured with COS cells, SDF-1-transfected COS cells were plated COS-7 cell culture and transfection at a density of 10,000 cells/cm2 on day 1 cultured neurons previously COS-7 cells were grown in DMEM containing 10% calf serum. transfected with CXCR4-GFP and were placed in the incubator for Transfections with CXCR4-GFP and SDF-1 cDNAs were performed 16-20 hours without the glial feeder layer. Cells were fixed in PFA at using the Lipofectamine kit (Qiagen). Cells transfected with CXCR4- day 2 and immunostained for SDF-1. GFP were used for internalization experiments. After 24 hours of transfection, cells were incubated for 2 hours with or without 100 nM SDF-1, fixed in PFA and the distribution of CXCR4-GFP was Western blot analysis examined. To evaluate the distance of diffusion of SDF-1 released For western blot analysis of cultured neurons, ~500,000 cells at day from a transfected cell, SDF-1 and CXCR4-GFP were expressed 7-10 were scraped into PBS, pelleted and resuspended in Laemmli separately in COS cells and subsequently co-cultured. The pattern of buffer. Proteins were separated by SDS-PAGE (12% acrylamide), CXCR4-GFP was analysed in correlation with the distance separating transferred to a nitrocellulose membrane, blocked in 20 mM Tris-HCl, the CXCR4-GFP-expressing cells from the SDF-1-transfected cells. pH 7.4 containing 0.45 M NaCl, 0.1% Tween 20 (TBST) and 10% We defined two groups of CXCR4-GFP-transfected cells based on the dried milk. The membranes were incubated overnight in polyclonal occurrence of receptor internalization (see Results). Cells exhibiting goat anti-CXCR4 antibody (1:2000; Santa Cruz Biotechnology) a pattern of diffuse CXCR4-GFP signal were classified as the control diluted in TBST containing 8% dehydrated milk, washed with TBST, group and were consistently found at least 50 µm from a SDF-1- incubated for 1 hour in HRP-conjugated secondary goat anti-mouse expressing COS cell. Cells exhibiting a CXCR4-GFP internalization antibody (1:20,000; Jackson ImmunoResearch) and visualized using pattern characterized by the presence of intracellular clusters were chemiluminescent substrate (Amersham) and exposure to X-ray film. classified as the ‘SDF-1-stimulated’ group and were consistently For western blot analysis of rat brain homogenate, rat forebrains were found at a distance between 0 and 5 µm from an SDF-1-transfected homogenized using 10 strokes with a motor driven Dounce COS cell. CXCR4-GFP-transfected cells found between 5 and 50 µm homogenizer in 10 mM Tris-HCl, pH 7.4 containing 320 mM sucrose, showed variation in receptor pattern distribution and were excluded SDF-1 regulates axonal development 1073

from the analysis. For neuron and COS cell co-culture experiments, COS cells were transfected with SDF-1, collected 24 hours later and plated over a day 1 neuronal culture previously transfected with CXCR4-GFP. Cells were fixed 16 hours later with PFA and the immunostaining procedure for SDF-1 was performed essentially as described above.

Morphological analysis Phase-contrast images of 30-40 neurons per coverslip were randomly acquired with a microscope as described above. Two coverslips per Fig. 1. Western blot analysis of CXCR4 in cultured hippocampal condition were analysed from two to three independent experiments. neurons. Cultured neurons and rat brain membrane homogenates Images projected on the computer screen were traced with a mouse were immunoblotted with anti-CXCR4 antibody. The antibody using the Analysis software program and number and length of revealed a 45 kDa band in both cultured neurons and rat brain processes, number of primary branch points, number of growth cones, samples, corresponding to the expected molecular mass for CXCR4. area of growth cones and cell bodies were scored. For analysis of Molecular weight markers are indicated on the right. neurons transfected with CXCR4-GFP and co-cultured with SDF-1- expressing COS cells, the former were classified into two groups depending on the distance separating the transfected neuron from an receptors on cultured hippocampal neurons, intracellular Ca2+ SDF-1-transfected COS cell (see above). For a distance between 0 and 2+ 5 µm, neurons were classified as the ‘SDF-1 stimulated’ group, and mobilization was measured by fluorescent Ca imaging on live for a distance above 50 µm, neurons were pooled into the control neurons following CXCR4 activation by 20 nM SDF-1. It is group. Neurons between 5 µm and 50 µm from a COS cell were not important to note that these experiments were carried out in the taken into account. For both groups, length and number of primary absence of the glial feeder layer, thus allowing us to measure processes as well as number of growth cones per process were scored direct effects of SDF-1 on hippocampal neurons. Indeed, the as described above. low-density culture system we used represents a virtually pure neuron culture model (Goslin et al., 1998). About 70% of 2+ hippocampal neurons gave rise to an [Ca ]i increase upon Results SDF-1 stimulation both in soma and processes (Fig. 3). The 2+ Cultured hippocampal neurons express functional increase of [Ca ]i was detected within 3 seconds on average CXCR4 receptors in the somatodendritic and axonal after SDF-1 exposure, peaked between 7 and 12 seconds, and compartments. usually recovered to near basal levels within 1 minute. As Primary cultures of hippocampal neurons were grown at low CXCR4 immunoreactivity was distributed in both dendrites 2+ density to allow the study of CXCR4 cellular distribution in and axons, we examined whether the SDF-1-induced [Ca ]i individual neurons and to perform subsequent morphometric increase was detectable in both processes. Based on different analysis. CXCR4 protein expression in this neuronal model morphological features between axons and dendrites, we was first examined by western blot analysis. The anti-CXCR4 distinguished the axons as thin processes often far removed Journal of Cell Science antibody revealed a band at 45 kDa, as observed for rat from any cell body and the dendrites as much thicker processes forebrain homogenate (Fig. 1), and was consistent with the generally forming an elaborate tree around a cell body. SDF-1 2+ reported CXCR4 molecular mass (Stumm et al., 2002). To stimulation triggered [Ca ]i rises in both dendrites and axons study the cellular distribution of CXCR4, neurons cultured for within the same time frame (Fig. 3). Taken together, these data 10-15 days were immunostained by double labelling with the suggest that CXCR4 was expressed on the neuronal cell CXCR4 antibody and the microtubule-associated protein 2 membrane of somatodendritic and axonal domains, where it (MAP2) antibody, shown to be a dendritic marker in was functionally coupled to a second messenger cascade 2+ hippocampal neurons as early as day 4 of culture (Mandell and leading to an increase in [Ca ]i. Banker, 1995). CXCR4 immunoreactivity, detected in about 50% of neurons, was mainly observed along MAP2-negative axons and, to a lesser extent, within MAP2-positive soma and Developmentally regulated distribution of CXCR4 dendrites (Fig. 2). Not all processes were immunostained, but receptors. a subset of processes originating from one neuron was, CXCR4 distribution was studied by immunofluorescence suggesting a preferential targeting of CXCR4 to specific during development of cultured hippocampal neurons. At day branches. Within nerve cell bodies, CXCR4 immunoreactivity 2, CXCR4 immunoreactivity was strikingly concentrated at the predominated at the periphery of the perikarya, sparing the tip of neuronal processes and branches, independently of cell- nucleus and most of the cytoplasm. Along the axonal and cell contact (Fig. 4A). Critical developmental stages of these somatodendritic domains, CXCR4 immunoreactivity was not low-density hippocampal cultures have been well characterized distributed uniformly, but was present in a pattern of stretches (Dotti et al., 1988) and day 2 has been defined as a selective and large puncta (Fig. 2A insets). To determine whether axon elongation step. Thus, according to published work (Dotti CXCR4 clusters were associated with synapses, neurons were et al., 1988), we defined the longer process as the growing axon simultaneously labelled for CXCR4 and the synaptic marker and the remaining short processes as minor neurites. CXCR4 synaptophysin. There was no overlap between synaptophysin accumulation was equally observed at the endings of growing and CXCR4 clusters, indicating that CXCR4 clusters were axons and minor neurites and showed no correlation with the localized at extrasynaptic sites (Fig. 2B). presence of growth cones. Some labelling was also seen To determine whether CXCR4 was expressed as functional throughout some processes but was mostly distributed in 1074 Journal of Cell Science 118 (5)

Fig. 2. CXCR4 is distributed in a non-uniform manner both in the somatodendritic and axonal domains, but is preferentially associated with axons. Hippocampal neurons at day 10 in culture were fixed and immunostained for CXCR4 (A1,B1) and either the dendritic marker MAP2 (A2) or the synaptic marker SV2 (B2). CXCR4 immunoreactivity was detected within neuronal cell bodies and a subset of nerve cell processes identified as MAP2-positive and -negative processes, indicating a somatodendritic and axonal localization of CXCR4 (A1-A3). The extent of axonal CXCR4 immunostaining was however more prominent than the dendritic labelling. CXCR4 immunoreactivity was not uniformly distributed but was often observed in large clusters along the cell body and processes (arrows in insets corresponding to higher magnification of boxed regions in A3). Double labelling with SV2 (B3) shows that these CXCR4 clusters did not overlap with the synaptic marker, indicating that CXCR4 clusters were localized at nonsynaptic sites. Bar, 20 µm.

Fig. 3. The somatodendritic and axonal CXCR4 receptors are functionally coupled to intracellular Ca2+ mobilization. (A) Phase contrast and pseudocolour images of a field containing dendrites and axons. Neurons were loaded with Fura-2 AM and exposed to 20 nM SDF-1. Pseudocolour images were taken 5 seconds before SDF-1 stimulation (–5 sec), 7 seconds and 40 seconds after SDF-1 stimulation. 2+ Most processes displayed SDF-1-induced [Ca ]i increase. The colour calibration bar shows pseudocolour mapping of the ratio of fluorescence emission at 340 and 380 nm. (B) Higher magnification of the boxed regions shown in A show a dendrite and an axon based on morphological features analysed by 2+ phase-contrast microscopy. (C) Traces of variations of [Ca ]i 2+ recorded in the boxed regions depicted in A. A rise in [Ca ]i levels was observed with a similar time course for dendrites and axons, 3 seconds after SDF-1 exposure as symbolized by Journal of Cell Science asterisks, and recovered to near basal levels within 1 minute. Bar, 10 µm.

patches. Nerve cell bodies also exhibited CXCR4 taking place at some specific sites could impede the recognition immunostaining localized both at the periphery and throughout of CXCR4 receptors by the antibodies. To discriminate the cytoplasm. Day 4 is characterized by continued axonal between these two possibilities, we analysed the distribution of growth and elongation of the remaining minor processes CXCR4-GFP transfected into neurons, which was then directly that will acquire dendritic morphological features. CXCR4 visualized without the use of antibodies (Fig. 4D). As observed distribution at this stage of culture was similar to that observed for endogenous immunolabelled CXCR4, transfected CXCR4- at day 2, consisting of a further specific enrichment of CXCR4 GFP was predominantly localized at the tips of the neurites immunoreactivity at the growing regions of both dendrites and from neurons cultured for 2 days. By contrast, the GFP signal axons, which, at this stage, often contacted neighbouring resulting from the transfection of the parent GFP control vector neurons (Fig. 4B). By day 7, which corresponds to a stage of was diffusely distributed throughout the cell with, however, a continued maturation of axonal and dendritic arborization and higher fluorescence intensity in the cell body probably due to synaptogenesis, the pattern of CXCR4 distribution resembled the greater thickness of the soma compared with neuronal that observed at day 10-15. CXCR4 immunoreactivity was no processes (Fig. 4E). These data further confirm that at early longer enriched at growing tips, but was more widely stages of neuronal development CXCR4 was selectively distributed along neuronal processes (Fig. 4C). The stronger targeted to the growing regions of neurites. CXCR4 immunoreactivity in growing regions of the processes observed at early developmental stages could result either from a preferential receptor targeting to these sites or from Regulation of axonal patterning by SDF-1 differences in CXCR4 immunoreactivity towards the Based on our finding that CXCR4 receptors were located at antibodies at and out of the growing regions. For instance, strategic sites to regulate neurite outgrowth, we examined biochemical modifications or protein-protein interactions whether the CXCR4 agonist SDF-1 could affect neuronal SDF-1 regulates axonal development 1075 development. Neurons at day 1 were incubated for 16 hours with 50 nM SDF-1, fixed and phase-contrast images of randomly chosen neurons were acquired for quantification of several morphological parameters using a computer-assisted image analysis system. Neurite morphology of SDF-1-treated neurons showed numerous alterations compared to control cells (Fig. 5). The number of primary processes originating from the neuronal soma was slightly increased in SDF-1- treated cells (Fig. 5F). Among the primary processes, we distinguished between the axon, defined as the longest process and the other neurites, which will differentiate into dendrites later on in development (Dotti et al., 1988). Treatment of neurons with the CXCR4 agonist induced a 32% decrease in the axonal length of SDF-1-treated cells compared to control cells, whereas the length of the other neurites remained unchanged (Fig. 5C). The number of branch points per 100 µm of process was selectively increased by 45% along the axon of SDF-1-treated cells compared to control cells, whereas no difference was observed along the other neurites (Fig. 5G). As another index of neuronal development, we quantified the number of growth cones per primary process. Neurons incubated with SDF-1 showed a lower number of growth cones per primary process than that observed in control neurons (Fig. 5H). However, no change either in the growth cone area nor in the soma area were detectable after treatment with SDF-1 (Fig. 5D,E). To confirm that the modifications in neuron morphology induced by SDF-1 treatment were mediated through CXCR4, neurons were treated with 50 nM SDF-1 for 16 hours in the presence of 10 µM bicyclam, a CXCR4 antagonist (Hatse et Fig. 4. Developmental distribution of CXCR4 in hippocampal al., 2002). In the presence of bicyclam alone or mixed with neurons from day 2 to day 7. Hippocampal cultures were fixed at day SDF-1, the axonal length, the number of axonal branch points 2 (A1,A2), day 4 (B1-B4) and day 7 (C1,C2) and immunostained for and the number of growth cones per process were all similar endogenous CXCR4 at all stages and for MAP2 at day 4 (B3,B4) or to values in the control, suggesting that the effects of SDF-1 were transfected at the time of plating with CXCR4-GFP (D1,D2) or on neuronal morphology were indeed mediated through SDF- the parent vector GFP (E1,E2). At day 2 and day 4, the tips of the 1 binding to CXCR4 (Fig. 5C,G,H). Importantly, neuronal axon and the other neurites visualized in the phase-contrast images Journal of Cell Science survival as determined by the density of untransfected as well (A1,B1) were frequently enriched in CXCR4 immunoreactivity as seen in the paired immunolabelled images (A2,B2). This CXCR4 as CXCR4-GFP-transfected neurons was unaffected by SDF- accumulation at the leading edge of processes was observed in the 1 treatment. Thus, in neurons at day 1-2 corresponding to a absence (A1-A2, arrows) and in the presence of contacts with other stage of axonal elongation in this hippocampal culture model, neurons (B1-B4, arrows). At day 4, the tips of two processes the chemokine SDF-1 affected axonal development by originating from the same cell body were highly immunopositive for decreasing the elongation of the primary axon, and by CXCR4 at the site of contact with neighbouring neurons (B1-B4, promoting the emergence of axonal branches. Moreover, these arrows). At day 7 (C1,C2), CXCR4 immunoreactivity was no longer effects were associated with a reduction in the number of enriched at growing tips, but was rather detected along a subset of growth cones at the tip of the neurites. processes. At day 2, in neurons transfected with CXCR4-GFP cDNA To confirm the regulatory role of SDF-1 in axonal the fluorescence was preferentially concentrated at the tips of development, we examined whether secreted, instead of processes (D1,D2), whereas in neurons transfected with the parent GFP vector the fluorescence was diffusely distributed throughout the exogenously added SDF-1 could act as an extracellular factor cell (E1,E2). Bar, 20 µm. controlling axon patterning through CXCR4. The experiments consisted of co-culturing non-neuronal cells expressing high levels of SDF-1 with neurons transfected with CXCR4-GFP to subsequently co-cultured. A typical endosomal pattern of analyse the morphology of the transfected neurons in the internalized CXCR4-GFP receptor was noticed in cells located vicinity of SDF-1-expressing cells. We first ensured that the in close proximity to an SDF-1-transfected COS cell (Fig. 6C). GFP-tagged receptor was functional as assessed by Ca2+ This pattern was similar to that observed in CXCR4-GFP- imaging (data not shown). Although several cell systems transfected COS cells incubated for 2 hours with 100 nM SDF- transfected with SDF-1 cDNA were shown to produce and 1 (Fig. 6B), conditions previously reported to induce receptor release SDF-1 in a biologically active form, we performed internalization (Amara et al., 1997; Signoret et al., 1998; Orsini initial experiments to demonstrate it in our system. We used et al., 1999). By contrast, in cells located at a greater distance the reported property of CXCR4 to internalize upon agonist from an SDF-1-expressing cell, CXCR4-GFP was activation as a marker of SDF-1 activity (Amara et al., 1997; homogeneously distributed throughout the cell (Fig. 6C), as Signoret et al., 1998; Orsini et al., 1999). SDF-1 and CXCR4- observed in control untreated CXCR4-GFP-expressing cells GFP were expressed separately in COS-7 cells and (Fig. 6A). This suggests that only CXCR4-GFP-expressing 1076 Journal of Cell Science 118 (5) cells that were located close to SDF-1-transfected COS cells found a receptor internalization pattern for a distance less than were activated by secreted SDF-1. We measured the distances 5 µm, and conversely, a control pattern for a distance greater between SDF-1- and CXCR4-transfected cells and consistently than 50 µm. Based on these observations, CXCR4-GFP- expressing neurons co-cultured with SDF- 1-transfected COS cells were divided into two groups: the SDF-1-stimulated group corresponding to neurons located up to 5 µm from SDF-1-expressing cells and the control group corresponding to neurons located at a distance greater than 50 µm from SDF-1-transfected COS cells (Fig. 6D,E). Neurons 5 to 50 µm distant from a SDF-1-transfected cell were ignored for this analysis. Measurements of the number of primary processes, their length, number of branching points and the density of growth cones were recorded for the two groups (Fig. 7). Although the number of primary processes originating from the transfected cell body was greater in the SDF-1-stimulated group than in the control group, no statistical differences were observed between the two groups (Fig. 7A). The average axonal length was lower for the SDF-1-stimulated groups than for the control group (Fig. 7B). As shown for non- transfected neurons, the reduction in process length was unique to axons and was not observed for the other neurites. The number of branch points per 100 µm of process was increased more than twofold along the axon of SDF-1-stimulated cells compared to control cells, whereas no difference was observed along the other neurites (Fig. 7C). In addition, a marked Journal of Cell Science reduction in the number of growth cones per process was seen in the SDF-1- stimulated group compared to that in the control group (Fig. 7D).

Discussion This study reveals several novel features on the localization and function of the chemokine SDF-1 and its receptor CXCR4 in cultured hippocampal neurons. First, CXCR4 immunoreactivity exhibited a striking developmental change in its cellular distribution. Early in development, CXCR4 was particularly concentrated at Fig. 5. SDF-1 regulates axonal patterning without influencing the other neurites. the leading edge of growing neurites. As Hippocampal neurons were treated at day 1 for 16 hours with (B) or without (A) 50 nM the cells matured, staining at the tip of the SDF-1 and were fixed at day 2 for analysis by phase-contrast microscopy. Quantification of processes declined and became more the length of the axon and the other neurites in the presence and absence of the glial feeder broadly distributed along axons and, to a layer (C), the growth cone area (D), the soma area (E), the number of processes emerging 2+ µ lesser extent, dendrites. Ca imaging upon from the cell body (F), the number of primary branch points per 100 m of axon and other SDF-1 stimulation indicated that both the neurites (G) and the number of growth cones per process (H) was carried out. The analysis was performed on control cells, cells treated with 50 nM SDF-1, cells treated with 50 nM axonal and somatodendritic CXCR4 SDF-1 in the presence of 1 µM bicyclam, a CXCR4 antagonist, and cells treated with responded to SDF-1 and were thus both bicyclam alone. Data are presented as the mean±s.e.m. of two to three independent membrane-associated and functionally experiments. **P<0.01, ***P<0.001 (Student’s t-test) compared to corresponding coupled to the intracellular signalling measurements in control cells. Bar, 12 µm. cascade. In accordance with the SDF-1 regulates axonal development 1077

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formation of CXCR4-GFP-containing intracellular clusters (B). n (C) COS cells were separately transfected with either CXCR4-GFP 0 0 (green) or SDF-1 (red, identified with an anti-SDF-1 antibody), and axon other neurites Journal of Cell Science were subsequently co-cultured. CXCR4-GFP distribution exhibits a typical internalization pattern only if an SDF-1-expressing cell was located in the proximity of the former. By contrast, in cells distant Fig. 7. Quantitative analysis of the effect of SDF-1-expressing COS from any SDF-1-transfected cells, CXCR4-GFP was distributed cells on the morphology of CXCR4-GFP-transfected hippocampal diffusely over the cell. (D,E) Hippocampal neurons were transfected neurons. Hippocampal neurons transfected with CXCR4-GFP were at the time of plating with CXCR4-GFP. COS cells previously co-cultured for 16-20 hours with SDF-1-transfected COS cells. For transfected with SDF-1 were added to the neuron culture at day 1 morphometric analysis, CXCR4-GFP-expressing neurons were and the co-culture was fixed at day 2. CXCR4-GFP-expressing classified into the control group or the SDF-1-stimulated group neurons in contact with SDF-1-expressing COS cells showed a depending on their distance from a SDF-1-expressing COS cell. reduced axonal length (E1,E2) compared to neurons distant from any µ Quantification for the number of processes emerging from the cell SDF-1-transfected cells (D1,D2). Bar, 20 m. body (A), the length of the axon and the other neurites (B), the number of branch points per 100 µm of axon and other neurites (C), localization of CXCR4 at the growing region of neurites, we the number of growth cones per process (D) were performed for the two groups. Data are presented as the mean±s.e.m. of two showed that SDF-1 triggered several effects on neuronal independent experiments. *P<0.05, **P<0.01 (Student’s t-test) morphogenesis. At day 2, SDF-1 reduced growth cone number compared to corresponding measurements in control cells. and axonal outgrowth but stimulated axonal branching. These latter two effects were not observed within the other neurites. This study unravels a new role for SDF-1/CXCR4 in specifying the tip of neuronal processes to a broader localization along hippocampal neuron morphology by regulating axonal axons and, to a lesser extent, along the somatodendritic patterning at early stage of neuronal development. domain. This switch in receptor distribution occurred after the initial period of axonal and dendritic differentiation, but preceded the synaptogenesis and the extensive growth and Developmentally regulated CXCR4 distribution in branching of nerve processes generating the elaborate hippocampal neurons arborization of mature neurons. Previous studies on the We found a marked modification in the pattern of CXCR4 developmental profile of the expression of cerebral CXCR4 distribution during development of hippocampal neurons in and SDF-1 proteins and mRNAs have shown a particularly culture. Between day 4 and day 7, CXCR4 redistributed from high expression during the embryonic stage and at the early 1078 Journal of Cell Science 118 (5) postnatal period followed by a progressive decrease thereafter Our findings show that SDF-1 influences several parameters until adulthood (Westmoreland et al., 1998; McGrath et al., of hippocampal neuron development in different ways. 1999; Tham et al., 2001). Differences in the level of expression Whereas the growth cone number and axon elongation were of CXCR4 depending on the brain area have also been reported both reduced upon SDF-1 treatment, the number of processes during ontogeny, with high levels in the ventricular zone of cell emerging from the cell body and that of primary axonal proliferation at the embryonic stage followed at the early branches were increased. Process elaboration involves a postnatal stage by a progressive increase in other brain areas coordinated and complex repertoire of cellular systems. A such as the hippocampus, cerebral cortex, thalamus and highly controlled balance between elongation and branching is cerebellum (Westmoreland et al., 1998; Tissir et al., 2004). Our critical to achieve the ultimate shape of a neuron, which results extend these previous data by showing the existence at underlies its ability to connect to multiple targets properly. the cellular level of a differential localization of CXCR4 in Elongation and branching of neurites may operate through hippocampal neurons during development. This modification similar mechanisms as reported for tumour necrosis factor-α, in the compartmentalization of CXCR4 suggests that this which affects both events through a RhoA-mediated pathway chemokine receptor may play different functional roles within (Neumann et al., 2002), or through distinct albeit related the same neuron over the course of its development. Moreover, mechanisms as reported for δ-catenin (Martinez et al., 2003). this observation implies that there are different cellular For the latter, inhibition of Src family enables δ-catenin mechanisms governing CXCR4 targeting in neurons depending to promote neurite outgrowth whereas inhibition of the RhoA on the stage of neuron development. One of the factors pathway associated with δ-catenin expression leads to the responsible for the redistribution of CXCR4 could be the level formation of branched secondary neurites. Alternatively, some of SDF-1 stimulation. There are a large number of studies molecules, such as -1 and semaphorin-3A, independently demonstrating the activity dependency of the cellular affect elongation and branching events (Dent et al., 2004). Our localization of several receptors, including synaptic (Craig, results showing that SDF-1 reduces axon elongation but 1998; Bredt and Nicoll, 2003) and non-synaptic (Boudin et al., promotes axon branching suggest that different mechanisms 2000; Dumartin et al., 2000; Csaba et al., 2001) receptors. As underlie each of these effects. Interestingly, it has been shown the expression of neuronal and glial SDF-1 was strongly that different effectors of Ras signalling play distinct roles in regulated during development (Tham et al., 2001), variations axonal patterning. Activated Raf-1 causes axon lengthening, in the intensity of CXCR4 stimulation by its endogenous ligand whereas active Akt increases axon branching (Markus et al., could be an important factor in the differential targeting 2002). Along these lines, it has been reported that SDF-1- of CXCR4 during development. Alternatively, selective induced CXCR4 stimulation elicits activation of the Ras-Akt interactions during development between CXCR4 and protein pathway in several neuronal models (Floridi et al., 2003; Peng partners implicated in anchoring, transport and signalling could et al., 2004), whereas no study has reported the activation of account for differential CXCR4 targeting. Raf-1 by SDF-1. One can hypothesize that SDF-1-induced axonal branching might involve the Ras-Akt pathway and that of axon growth inhibition could involve the Rho/ROCK SDF-1/CXCR4 regulates axonal development pathway as previously reported in cerebellar granule cells Journal of Cell Science The transient enrichment of CXCR4 at growing regions of (Arakawa et al., 2003). neuronal processes in immature neurons led us to investigate Our data and previous studies point to the critical role of the role of SDF-1/CXCR4 in neuronal development. We found SDF-1 and its receptor CXCR4 in several aspects of neuron that exogenous as well as cell-produced SDF-1 influenced and brain development. Concerning patterning of neuronal neuronal development by acting on axonal patterning. These processes, SDF-1 has been previously shown to differentially actions were probably mediated by neuronal CXCR4 and not affect axon elongation in cerebellar granule cells according to by an indirect event through glial-released molecules. the concentration range (Arakawa et al., 2003). A low Although CXCR4 has been described in astrocytes (Dorf et al., concentration of SDF-1 stimulated axon elongation whereas a 2000), microglia (Lavi et al., 1997; Albright et al., 1999) and higher concentration (from 30 to 125 nM) repressed axon neurons (Bajetto et al., 1999; Banisadr et al., 2002; Stumm et formation. Our study extends these data by demonstrating that al., 2002), several lines of evidence suggest that SDF-1 acts SDF-1 not only regulates axon elongation, but also regulates directly on neuronal CXCR4 receptors to modulate axonal branching and that these effects were selectively observed with morphology rather than indirectly via glial cells. First, the axons and not detected within the other neurites, at least with concentration of CXCR4 receptors at growing regions of the SDF-1 concentration used in the present study. In addition, neuronal processes makes them the probable molecular targets SDF-1 acts as a chemotactic molecule that influences axonal of SDF-1 in mediating effects on neuronal development. guidance (Chalasani et al., 2003) and migration of cerebellar, Second, when hippocampal neurons were cultured in the hippocampal and cortical neurons (Lu et al., 2001; Bagri et al., absence of the glial feeder layer, the inhibitory action of SDF- 2002; Lu et al., 2002; Zhu et al., 2002; Stumm et al., 2003). 1 on axonal outgrowth was still observed. Given that the low- The ability of SDF-1 to affect multiple aspects of neuron density culture system we used represents a virtually pure development appears to be shared by other factors such as neuron culture model (Goslin et al., 1998), our data suggest some members of the slit and semaphorin families (Polleux et that the CXCR4 receptors mediating these effects were located al., 1998; Nguyen Ba-Charvet et al., 1999; Wang et al., 1999; on neurons. These observations do not exclude the additional Whitford et al., 2002; Bagri et al., 2003). contribution of glial CXCR4, but strongly suggest a role for Although CXCR4 was detected at the leading edge of both the neuronal CXCR4 receptor in regulating neuronal the axons and the other minor neurites, SDF-1 modelling morphology. activity affected only the former and not the latter. One SDF-1 regulates axonal development 1079

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