Carlos Vicario, CSIC, Spain), neuronal specific -III Tubulin (TUJ-1, mouse monoclonal, 14000, Babco, Richmond, VA), A2B5 (mouse monoclonal, 150, Hybridoma Lender, Iowa, USA), GFP (rabbit polyclonal, 1500, Molecular Probes, Eugene, USA), SNAP-25 (SMI81, mouse monoclonal, 11000, Becton-Dickinson, USA), Syntaxin 1 (mouse monoclonal, 11000, Hybridoma Lender, Iowa, USA), Synaptophysin (mouse monoclonal, 11000, Dako Diagnostics S.A), VAMP-2 (mouse monoclonal 11000, Synaptics System, USA), Synapsin (rabbit polyclonal, 11000, Synaptics System, USA), Neurofilament 200 kD (mouse monoclonal, 1300, Sigma-Aldrich, St Louis, MO), TAG-1 (rabbit polyclonal, 11000, gift from Dr. CA3, pyramidal cell regions of the hippocampus; DG, dentate gyrus; MZ; marginal zone; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone; ML, molecular layer; ICVI, cortical cell layers; IIIV, third ventricle; ON, olfactory nerve; GL, glomerular layer; EPL, external plexiform layer; EGL, external granular layer; P, Centrinone Purkinje cell layer; DCN, deep cerebellar nuclei Centrinone EGL; EPL, external plexiform layer; GL, glomerular cell layer; IGL, internal immature granular layer of the cerebellum. Level bars?=?100 m (ECI); 75 m (L); 50 m (I, O,P); 30 m (J,K,N).(7.45 MB TIF) pone.0012003.s001.tif (7.1M) Rabbit Polyclonal to TF2A1 GUID:?79EEBEBE-B350-4822-BF73-0907AD93AE64 Physique S2: Sialylation of PC protein in brain. (A) Western Blot showing PC and NCAM immunoreactivities in E16 brain lysates in control conditions (UT), and after treatment with EndoN and Neuraminidase. Neuraminidase removes PSA from PC, but not from NCAM; in contrast, EndoN removes PSA from NCAM, but not from PC. (B) Western Blot of brain extracts from different ages treated with neuraminidase shows that PC is usually sialylated in both developing and adult stages.(6.66 MB TIF) pone.0012003.s002.tif (6.3M) GUID:?9AC82090-0F4C-448A-AE51-9B6218D787FE Physique S3: PC-deficient embryos display normal axonal trajectories but abnormal fasciculation. (A, B) Low-power views of L1-immunolabeled forebrain sections showing a normal distribution of fibers in podx(?/?) embryos, compared to wt brains at E18. (CCF), shape and size of TAG-1 immunoreactive axonal fascicles are altered in PC-deficient hippocampus. Note that in the podx(?/?) hippocampus (E, F) axonal bundles of the white matter were smaller, less compacted and occupied a wider zone in the adjacent stratum oriens, compared to wt littermates (C, D). (D, F) high magnification of C and E, respectively. (G, H) TAG1-immunoreacted sections showing increased defasciculation in the habenulo-peduncular tract of podx(?/?) embryos. Cx, cerebral cortex; CPu, caudate-putamen of the striatum; Hip, hippocampus; hpt, habenulo-peduncular tract; T, thalamus. Level bars?=?400 m (A, B), 200 m (C, E); 50 m (D, F), 75 m (G, H).(10.07 MB TIF) pone.0012003.s003.tif (9.6M) GUID:?86926967-CCD2-4F76-97E3-0A27849C0056 Physique S4: PC embryos display normal axonal projection patterns. Pattern of DiI labeling in wt (A,B,C) and podx(?/?) embryos (C,D,F) following DiI injections in the cerebral cortex (A,D) and olfactory bulb (E,F). After DiI injections in the cortex, strong corticothalamic projections invade the thalamus in wt (A,B) and podx(?/?) (D,E) embryos, traversing the striatum (Cpu). Arrowheads point to the anterior commissure. (C,F) Labeling of the Lateral Olfactory Tract after DiI injections in the Centrinone Olfactory Bulb, suggesting normal formation of this tract in podx(?/?) embryos. All Centrinone images correspond to coronal sections. Cpu, caudate-putamen; LOT, lateral olfactory tract; T, thalamus. Level bars?=?500 m (ACD); 100 m (E,F).(3.94 MB TIF) pone.0012003.s004.tif (3.7M) GUID:?E02873CB-0799-47C3-A71B-AF9E68540417 Abstract Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is usually a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is usually widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments and phenotypic analyses of brain (E18), kidney (P4) and embryos. Thus, brain regions, nuclei and layers were clearly recognizable throughout the brains of and and rhombic lip explants co-cultured with aggregates of Netrin-1-expressing cells, common chains of migrating neurons were formed that were chemoattracted by Netrin-1-expressing cells (Fig. 3and than those in littermates (Fig. S3). Similarly, other axonal tracts, such as the habenulo-peduncular tract in the dorsal thalamus (Fig. S3) or the fornix, Centrinone displayed reduced fasciculation. These data show that PC is involved in neuron-to-neuron adhesion and in the fasciculation of developing axonal tracts. Next, we examined the role of PC in axonal growth explants gave rise to numerous axons that fasciculated and grew along straight courses (Fig..