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First published online 27 June 2007
doi: 10.1242/dev.02871

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Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells

Swetlana Sirko^1,*, Alexander von Holst^1,*, Andrea Wizenmann², Magdalena Götz^2,3 and Andreas Faissner^1,

¹ Chair of Cell Morphology and Molecular Neurobiology, Ruhr-University Bochum, Building NDEF 05/339, Universitaetsstrasse 150, D-44780 Bochum, Germany.
² GSF-Institute for Stem Cell Research, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany.
³ Institute of Physiology, University of Munich, Schillerstaße 46, D-80634 München, Germany.

View larger version (57K): [in this window] [in a new window]   Fig. 1. Cleavage of CS-GAGs reduces the proliferation rate of mouse E13 telencephalic neural stem/progenitor cells and interferes with the generation of neurospheres. The efficacy of the CS-GAG removal after ChABC treatment of Nsph cultures was controlled by double immunofluorescence and western blot analyses. (A) Cryosections obtained from Nsphs grown for 7 days in vitro (div) in the absence (w/o, upper panels) or presence (lower panels) of 50 mU ChABC were immunostained with 473HD and pk-anti-phosphacan antibodies (KAF13) that detect the unique DSD-1 epitope and the proteins encoded by the Rptpß gene, respectively. The 473HD epitope was strongly expressed in the outer cell layers of the Nsph under control conditions and was absent after ChABC treatment, which resulted in enhanced reactivity of the polyclonal antibodies with the RPTP-ß protein (KAF13). (B) Western blot analysis confirmed the efficient removal of the 473HD epitope by ChABC, which was not achieved by keratanase (Ker) treatment. (C-E) Phase-contrast micrographs documenting the formation of Nsphs grown in serum-free medium in the presence of EGF and bFGF (C) and upon addition of ChABC (D) or keratanase (E) after 5 div. ChABC treatment caused an almost complete settling of Nsphs on the culture substrate and subsequent cellular outgrowth. (F) Quantification of the numbers of cortical (Cor) and striatal (GE) Nsphs present per visual field in bulk cultures. Note the significant decrease in the number of primary Nsphs grown in the presence of ChABC in comparison with control (w/o) or keratanase-treated cultures. (G) The total number of secondary Nsphs generated from cortical and striatal control or ChABC-treated primary Nsphs was determined in clonal density assays. In comparison with control cultures (w/o), the number of secondary Nsphs obtained from primary ChABC-treated Nsphs appeared significantly reduced. (H) Photomicrographs of dissociated, BrdU-labeled primary control (w/o, upper panels) and ChABC-treated (lower panels) Nsphs that had received a 15-hour BrdU pulse. (I) The fraction of BrdU-incorporating cycling cells in H was determined relative to the total number of cell nuclei (DAPI), expressed as percentage fraction. Note that approximately twice as many actively cycling cells were detected in the control population (w/o), as compared with the cells that had been exposed to ChABC. (A,H) Cell nuclei were counterstained with bisbenzimide and are shown in blue (DAPI). **, P<0.01; ***, P<0.001. Scale bars: 50 µm in A; 150 µm in C-E; 50 µm in H.

View larger version (55K): [in this window] [in a new window]   Fig. 2. The elimination of CS-GAGs modifies the composition of the radial glia precursor cell pool. (A,B) The distribution of precursor cells within mouse Nsphs grown in the absence or presence of ChABC was assessed by double immunofluorescence microscopy in cryosections stained for the precursor marker nestin and the radial glia markers BLBP (A), or the radial glia markers RC2 and GLAST (B). The degradation of CS-GAGs by ChABC reduced the number of nestin- and BLBP-positive precursor cells, whereas the number of RC2- and GLAST-positive radial glia increased. (C,D) These observations were quantified using acutely dissociated suspensions of control (dark gray) and ChABC-treated (light gray) cortical (C) and striatal (D) Nsphs and plotted as percentage fractions. ChABC treatment of Nsphs led to a 3- to 4-fold reduction in the frequency of nestin/BLBP-positive cells in both the cortical and striatal Nsphs that was paralleled by a 2-fold increase in GLAST-positive cells, as compared with the control. *, P<0.05; **, P<0.01; ***, P<0.001. Scale bar: 50 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 3. Interference with CS-GAGs impairs neurogenesis and simultaneously favors gliogenesis in vitro. The distribution of ßIII-tubulin-positive neurons and GFAP-positive astrocytes was assessed in mouse Nsph cryosections by double immunohistochemistry. (A,B) Photomicrographs illustrating cortical (A) or striatal Nsphs (B) grown in the absence (w/o, upper panels) or presence (lower panels) of ChABC. The digestion of CS-GAGs by ChABC reduced ßIII-tubulin immunoreactivity and enhanced GFAP immunoreactivity. (C,D) These observations were quantified using acutely dissociated suspensions of control (dark gray) and ChABC-treated (light gray) cortical (C) and striatal (D) Nsphs and plotted as percentage fractions. An apparent 4-fold reduction in the frequency of ßIII-tubulin-positive neurons was recorded in both the cortical (Cor) and striatal (GE) ChABC-treated Nsphs that was accompanied by a 2-fold increase in GFAP-positive cells. (E,F) An analogous differentiation assay was performed using the 473HD-expressing subpopulation of radial glia immunopanned from acutely dissociated primary Nsphs. When the 473HD-immunoselected cells were cultivated for 2 days in vitro (div) in the presence of ChABC (light gray), the number of ßIII-tubulin-positive neurons diminished 4-fold (E) and GFAP-expressing astrocytes doubled in number both for cortical and striatal Nsphs (F), as compared with control conditions (dark gray). (G,H) The importance of CS-GAGs for fate decision was confirmed in Nsph differentiation assays in which ChABC treatment for 6 div caused a reduction in the number of ßIII-tubulin-positive neurons (G) and an elevation in the number of GFAP-positive astrocytes (H). (A,B,G,H) Cell nuclei were counterstained with bisbenzimide and are shown in blue (DAPI). **, P<0.01; ***, P<0.001. Scale bars: 50 µm in A,B; 100 µm in G,H.

View larger version (77K): [in this window] [in a new window]   Fig. 4. Injection of ChABC modifies the architecture of the ventricular zone. (A) Phase-contrast photomicrographs of E14.5 mouse forebrain cryosections obtained 1 day after intracerebroventricular injection (ICVI) of ACSF (left panels), ChABC (middle panels) or keratanase (right panels). The upper panels show low-power DIC images, and the lower panels are high-power images of histological stainings. Cryosections of control- or keratanase-injected embryos appeared normal with a clearly defined tissue stratification. By contrast, a conspicuous disorganization of the germinal regions (VZ and SVZ) and an accumulation of cells in the normally positioned cortical plate was observed after injection of ChABC (middle panels). Note that the outer layers of the marginal region appeared comparable to those of the control (i.e. after ACSF injection). (B) The efficiency of ChABC injection was controlled by immunostaining with Mab 473HD. Photomicrographs of cryosections from embryos injected with ACSF (upper panels), ChABC (middle panels) or keratanase (lower panels). The 473HD epitope was almost completely absent after ChABC injection, whereas the reaction of the polyclonal antibodies with the RPTP-ß protein cores (KAF13) was unaltered. The 473HD and KAF13 stainings appeared unaltered by keratanase. Cell nuclei counterstained with bisbenzimide are shown in blue (DAPI). Cor, cortex; cp, cortical plate; e, surface ectoderm; ge, ganglionic eminence; imz, intermediate zone; lv, lateral ventricle; m, marginal zone; sep, septum; svz, subventricular zone; vz, ventricular zone. Scale bars: 100 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 5. CS-GAGs are required for proliferation of neural precursor cells during forebrain development. (A,B) Photomicrographs of immunohistochemical stainings of E14.5 mouse forebrain cryosections 1 day after ventricular injection with ACSF control (upper panels) or ChABC (lower panels). Cell nuclei were counterstained with bisbenzimide and are shown in blue (DAPI). (A) The dividing progenitors were identified by PH3 immunoreactivity. Note that ChABC treatment caused a significant reduction in the number of neural precursor cells undergoing mitosis. (B) The actively cycling cell population was labeled with BrdU. Note that ChABC treatment caused a significant reduction in the number of neural precursor cells undergoing S phase. (C) Quantification of the total number of PH3-positive cells in selected cortical and striatal (GE) areas revealed a significant reduction in the ChABC-injected (light gray) in comparison with the control-injected (dark gray) embryos (upper panel). ChABC treatment also altered the distribution of the neural precursor cells (lower panel). Note that significantly more cells were in M phase at the ventricular surface in control-injected brains. (D) Quantification of the total number of BrdU-positive cells in defined cortical and striatal (GE) areas revealed a significant reduction in the ChABC-injected (light gray) in comparison with the control-injected (dark gray) embryos (upper panel). Note that in ChABC-injected forebrains, more BrdU-positive cells were located in layers of the VZ/SVZ more distant to the ventricular surface (lower panels). Cor, cortex; ge, ganglionic eminence; lv, lateral ventricle. *, P<0.05; **, P<0.01; ***, P<0.001. Scale bars: 100 µm.

View larger version (87K): [in this window] [in a new window]   Fig. 6. CS-GAGs are essential components for neurogenic precursor cells in vivo. (A,B) Photomicrographs of immunohistochemical stainings of E14.5 mouse forebrain cryosections 1 day after ventricular injection with ACSF control (upper panels) or ChABC (lower panels). The cortical precursor pool was examined for coexpression of BLBP and nestin (A) and for GLAST expression (B). Following ChABC treatment, immunoreactivities for nestin and BLBP significantly decreased, whereas more cells were GLAST-positive, in comparison with the situation following ACSF injection. Cell nuclei were counterstained with bisbenzimide and are shown in blue (DAPI). (C,D) Single-cell suspensions obtained from embryos injected with ACSF (dark gray) or ChABC (light gray) were plated and immunopositive cells determined after 2 hours. (C) ChABC treatment caused a 2-fold reduction in the number of nestin- and BLBP-positive cells, whereas GLAST-positive cells increased. (D). The relative fractions of cortical (Cor) and striatal (GE) Nsphs grown from single-cell suspension were reduced following ChABC (light gray) injection. Numbers are plotted with reference to the control-injected telencephali, taken as 100% (dark gray). *, P<0.05; **, P<0.01. Scale bar: 100 µm.

View larger version (105K): [in this window] [in a new window]   Fig. 7. CS-GAGs are necessary for neuronal differentiation of precursors in vivo. (A) Photomicrographs of immunohistochemical stainings of E14.5 mouse forebrain cryosections 1 day after ventricular injection of ACSF control (upper panels) or ChABC (lower panels). Cell nuclei were counterstained with bisbenzimide and are shown in blue (DAPI). The prominent presence of the 473HD epitope on cells with radial morphology in the germinal layers of control sections was lost after ChABC injection, as expected. Concomitantly, a substantial reduction in young postmitotic ßIII-tubulin-positive neurons in the germinal layers of the cortex and ganglionic eminence was observed. Note, however, that we observed an accumulation of neurons in the cortical plate (lower right-hand panel). (B) The changes were quantified by counting immunopositive cells 2 hours after plating of single-cell suspensions obtained from embryos injected with ACSF or ChABC. Note that ChABC treatment caused a clear reduction in the number of ßIII-tubulin-positive cells. We also recorded a small increase in the number of GFAP-positive cells. **, P<0.01; ***, P<0.001. Scale bar: 100 µm.