Author contributions: S.S., A.v.H.: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; A. Weber, A. Wizenmann: Collection of data; U.T.: Collection and assembly of data; M.G.: Conception and design, financial support, administrative support; data analysis and interpretation, manuscript writing, final approval of the manuscript; A.F.: Conception and design, financial support, administrative support; manuscript writing, data interpretation, final approval of the manuscript.
First published online in STEM CELLS EXPRESS December 23, 2009.
Disclosure of potential conflicts of interest is found at the end of this article.
The neural stem cell niche of the embryonic and adult forebrain is rich in chondroitin sulfate glycosaminoglycans (CS-GAGs) that represent complex linear carbohydrate structures on the cell surface of neural stem/progenitor cells or in their intimate environment. We reported earlier that the removal of CS-GAGs with the bacterial enzyme chondroitinase ABC (ChABC) reduced neural stem/progenitor cell proliferation and self-renewal, whereas this treatment favored astroglia formation at the expense of neurogenesis. Here, we studied the consequences of CS-deglycanation further and revealed that CS-GAGs are selectively required for neurosphere formation, proliferation, and self-renewal of embryonic cortical neural stem/progenitor cells in response to fibroblast growth factor (FGF)-2. Consistently, the FGF-2-dependent activation of the MAPKinase in neural stem/progenitor cells was diminished after ChABC treatment, but unaltered after epidermal growth factor (EGF) stimulation. Upon EGF treatment, fewer radial glia were brain lipid-binding protein (BLBP)-positive, whereas more were glutamate aspartate transporter (GLAST)-positive after CS-GAG removal. Only in this latter situation, GLAST-positive radial glia cells extended processes that supported neuronal migration from differentiating neurospheres. CS-deglycanation also selectively increased astrocyte numbers and their migration in response to EGF. Thus, our approach revealed that CS-GAGs are essential for FGF-2-mediated proliferation and maintenance of neuron-generating neural stem/progenitor cells. Simultaneously, CS-GAGs act as a brake on the EGF-dependent maturation, migration, and gliogenesis of neural stem/progenitor cells. We conclude that neural stem/progenitor cell subpopulations reside in neurospheres that are distinguishable by their responsiveness to FGF-2 and EGF which is differentially regulated by CS-carbohydrate structures. STEM CELLS 2010;28:775–787
The central nervous system develops through the controlled proliferation of neural stem cells that initially increase in number. In the forebrain, the expansion phase is followed by neurogenesis when neurons differentiate as progeny of radial glia cells (RGCs) [1, 2] and intermediate basal progenitors [3, 4] at midgestation. Toward the end of embryogenesis and during early postnatal life, astrocytes and oligodendrocytes are generated during gliogenesis when neurogenesis has largely ceased, with the marked exception of the adult neurogenic niches .
Two views on how the timely differentiation of neural stem cells into neurons and glia occurs have been proposed. One is based on time-lapse video microscopy studies of single cells that generated mixed clones of neuronal and glia progeny in a comparable order as observed in vivo . This is interpreted as neural stem cells that undergo a switch from a neurogenic to a gliogenic mode of cell division [7, 8]. The alternative view stems from retroviral cell-lineage tracing experiments in vivo where most clones are either neuronal or glial [9, 10]. The rare observation of few multipotent clonal progeny of single infected cells also holds true for early neuroepithelial stages of forebrain development before the onset of neurogenesis . These latter findings suggest that different neural stem/progenitor cell (NSPC) populations that preferentially generate neurons or glia reside side by side in the neuroepithelium. This is in line with the observed heterogeneity of RGCs [12, 13] that can be prospectively isolated and faithfully predicted in their cellular fate .
In both views it remains unknown how the timing of cell fate and the later onset of gliogenesis are governed, but changes in growth factor responsiveness have been proposed . Cells isolated from the developing forebrain can be clonally expanded in defined media as free-floating cellular aggregates called neurospheres  or, alternatively, under adherent growth conditions [17, 18]. These cells behave as neural stem cells because they self-renew in response to basic fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) and give rise to neurons, astrocytes, and oligodendrocytes upon differentiation [19, 20]. Early on, NSPCs proliferate only in response to FGF-2 and preferentially generate neuronal progeny [17, 21]. This is in accordance with the forebrain phenotype of FGF-2-deficient mice that display a smaller cortex because of reduced proliferation and neurogenesis [22–24]. At later stages, NSPCs become responsive to EGF in addition to FGF-2 [19, 25, 26]. EGF signaling supports their proliferation and has been associated with enhanced gliogenesis , although EGF-expanded cells generate neurons . The timed acquisition of EGF-responsiveness is associated with increased expression levels of the receptor tyrosine kinase EGFR [26, 28], which allows for the isolation of late NSPCs . The existence of separate FGF-2- and EGF-responsive NSPCs during forebrain development [30, 31] is in accordance with different radial glia subtypes [14, 32]. Alternatively, FGF-2-responsive NSPCs may acquire EGF-responsiveness over time and so represent different stages of the same lineage [27, 31, 33].
Telencephalic NSPCs can be immunoselected with the 473HD monoclonal antibody  that detects defined chondroitin sulfate glycosaminoglycan (CS-GAG) structures . These complex carbohydrates are crucial for NSPCs of the embryonic forebrain because their digestion in culture or in utero suppressed neurosphere formation, self-renewal, and proliferation . The degradation of CS-GAGs in vitro and in vivo also reduces the number of newborn neurons and generated glial progeny at the peak of neurogenesis . Despite showing the definite importance of chondroitin sulfates for NSPCs, it remained unknown whether CS-deglycanation affected the response to FGF-2 and EGF and whether a single or separate populations of NSPCs were affected.
Here we show that digestion of CS-GAGs selectively reduced neurosphere formation, self-renewal, and proliferation of cortical NSPCs in response to FGF-2, but not in response to EGF. This effect was accompanied by reduced BLBP-positive RGC numbers and diminished activation of the MAPKinase signaling pathway. In parallel, we recorded enhanced EGF-responsiveness that resulted in maturation of GLAST-positive RGCs and increased generation and migration of astrocytes. We conclude that CS-deglycanation influences NSPCs with different growth factor requirements that represent preferentially neurogenic or gliogenic radial glia lineages. Therefore, the presence of defined CS structures accounts in part for the correct timing of FGF-2-dependent neurogenesis and EGF-mediated gliogenesis during NSPC development.
MATERIALS AND METHODS
All experiments were performed with embryos (E13, Theiler stage 21) derived from timed pregnancies of the NMRI or C57/Bl6 mouse strains obtained from Charles River Laboratories (Sulzfeld, Germany, http://www.criver.de). Embryonic cerebral cortices were isolated and enzymatically digested, and dissociated cortical cells were allowed to form free-floating spheres in the presence of 20 ng/ml EGF or FGF-2 with or without 50 mU/ml chondroitinase ABC (ChABC; EC 188.8.131.52; Sigma-Aldrich, Schnelldorf, Germany http://www.sigmaaldrich. com/germany.html) following the neurosphere culture protocol described previously [34, 36] and detailed in supporting information data 1. For differentiation and migration assays, individual neurospheres were washed three times in neurosphere medium before transfer onto polyornithine-coated wells and incubated for an additional 5 or 7 days.
The differentiated cell types were identified by immunostaining with the commercially obtained primary antibodies anti-Nestin, anti-βIII-tubulin, anti-BLBP, anti-GLAST, and anti-glial fibrillary acidic protein (GFAP), as described previously [34, 37] and detailed in supporting information online data 1. To detect the various primary antibodies, we used specific secondary antibodies (all from Dianova, Hamburg, Germany http://www.dianova.de/).
Intracerebroventricular Injections in Utero.
All experimental procedures in vivo were done in accordance with the Society for Neuroscience and European Union guidelines and were approved by the institutional animal care at the Helmholtz Center (License: 55.2-1-24-2531-044-07) (Munich, Germany). Intracerebroventricular injections (ICVI) into telencephalic ventricles of E13 embryos in utero were performed as described previously .
BrdU Pulse Labeling.
Labeling of cycling cells was performed by intraperitoneal injection of bromodeoxyuridine (BrdU) (10 mg/100 g body weight; Sigma) 1 hour before removal of the litter or by addition of 10 μM BrdU to culture medium for 12 or 24 hours. The number of BrdU-incorporating cells was determined by immunostaining according to the supplier's protocol (BrdU Labeling and Detection Kit I, Roche Applied Science, Mannheim, Germany).
Reverse Transcription Polymerase Chain Reaction.
Total RNA was isolated and reverse transcribed as previously described . The polymerase chain reaction (PCR) reactions were performed as reported earlier [34, 39] and detailed in supporting information online data 1.
The total protein content was harvested by cell lysis or directly from the conditioned medium as described previously [38, 40]. The samples were fractionated and transferred to polyvinylidene fluoride membranes, which were then incubated with anti-phosphorylated mitogen-activated protein kinase (pMAPK) and antitubulin antibodies or with mab 473HD (1:600) and pk-anti-phosphacan, batch KAF13 (1:2,000) overnight at 4°C (details given in supporting information online data 1). The band intensities were quantified using the freely available ImageJ software (NIH, http://rsb.info.nih.gov/ij/) on digital images of the scanned films.
Statistical significance of differences observed between distinct experimental groups was assessed using Student's t test and is given as p values in the figures and legends with * p < .05; ** p < .01; and *** p < .001.
The Removal of CS-GAGs Selectively Affects Neurosphere Formation of Neural Stem/progenitor Cells in Response to FGF-2
We have previously shown that the deglycanation of CS-GAGs by injection of the bacterial enzyme ChABC into the lateral ventricle of mid-gestation embryos at E13.5 followed by dissection and seeding single cells 1 day later reduced the number of cortical and striatal neurospheres obtained in the presence of EGF and FGF-2 . In the light of the heterogeneity of the neural stem/progenitor cell (NSPC) population  and their ability to grow as neurospheres in response to FGF-2 and/or EGF , it remained unanswered whether deglycanation of CS-GAGs affected the responsiveness of NSPCs toward FGF-2, EGF, or both growth factors. Therefore, we monitored neurosphere formation in response to FGF-2 or EGF in cortical cell suspensions that were obtained from E 14.5 forebrains 1 day after intracerebroventricular (ICVI) of ChABC, keratanase, or artificial cerebrospinal fluid (ACSF) into the lateral ventricles (Fig. 1A). Cortical cell suspensions obtained from ChABC-injected forebrains generated significantly lower numbers of neurospheres in the presence of FGF-2 compared with parallel seeded cultures from ACSF-injected or keratanase-injected control embryos (Fig. 1B). The number of neurospheres generated in response to EGF was threefold lower compared with the FGF-2 condition. Also, similar quantities of EGF-grown neurospheres emerged under all three conditions, showing that the selective removal of CS-GAGs affected neurosphere formation of cortical NSPCs in response to FGF-2, but not in response to EGF (Fig. 1B). To confirm the requirement of CS-GAGs for FGF-2-dependent neurosphere formation, cortical cell suspensions from non-injected control embryos were grown in the presence of ChABC and compared to keratanase-treated or control sister cultures. In line with the results from the ICVI injection experiments, removal of CS-GAGs by ChABC selectively reduced the number of primary neurospheres in the presence of FGF-2 by half compared with the control cultures, whereas the number of neurospheres produced in the presence of EGF and ChABC was not significantly altered (Fig. 1C, 1D). Thus, CS-GAGs are required for FGF-2-dependent neurosphere formation of embryonic cortical NSPCs.
FGF-2-Sensitive Neural Stem/progenitor Cells Require Intact CS-GAGs for Proliferation
The formation of neurospheres depends on the addition of mitogenic growth factors , and our previous study showed that ChABC treatment affected proliferation when neurospheres were grown in FGF-2 in combination with EGF . To assess the treatment in the presence of singular mitogens, the relative number of actively cycling cells was measured by BrdU incorporation. Approximately half of the cells in FGF-2- and EGF-grown neurospheres were identified as actively cycling after an overnight pulse of BrdU (Fig. 2A). Quantification of BrdU-positive cells in ChABC-exposed neurospheres revealed a significant reduction to almost half the number of cells undergoing S-phase in the presence of FGF-2 compared with control cultures (Fig. 2B). Also, in EGF-grown neurospheres, fewer cells incorporated BrdU upon exposure to ChABC, but the reduction was less extensive compared with the situation with FGF-2 (Fig. 2B). This latter finding was unexpected because ChABC-treatment did not alter the number of neurospheres obtained in response to EGF. However, it may indicate that endogenously expressed FGF-2 contributes to proliferation within neurospheres growing in response to EGF. Therefore, we plated acutely dissociated cortical cells from E13 embryos on polyornithine-coated dishes, added FGF-2 or EGF, and assessed BrdU-incorporation after 12 and 24 hours in the presence or absence of ChABC (Fig. 2). The removal of CS-GAGs significantly suppressed the increase in BrdU-positive cells in response to FGF-2 observed after a 12 and 24-hour pulse, but the number of BrdU-positive cells undergoing S-phase in EGF was unaltered by ChABC treatment (Fig. 2C). The reduced number of cells in S-phase in FGF-2 cultures exposed to ChABC could be caused by an increased fraction of cells leaving the cell cycle or by an impaired cell survival. We consider alterations in cell survival an unlikely explanation for the reduced proliferation in response to FGF-2 after CS-GAG removal because the number of cells undergoing apoptosis as determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was low and not significantly increased by treatment with ChABC (supporting information Table 1). The documented propagation of BrdU-incorporated cells argues, however, for a delayed S-phase entry but otherwise similar cell cycle kinetics. In fact, the initial number of BrdU-positive cortical cells (after a 1-hour pulse) was 7.4 ± 1.7 (n = 3), and we observed a near doubling of BrdU-positive cells between 12 and 24 hours in the presence of FGF-2. In the same time window, a similar doubling of cortical cells undergoing S-phase occurred in the presence of FGF-2 and ChABC, whereas during the first 12 hours, the pool of S-phase cells remains unchanged when compared with the initial number of BrdU-positive cells. Taken together, CS-GAGs are crucial for FGF-2-mediated proliferation of cortical NSPCs.
CS-GAGs Are Required for FGF-2-Dependent Self-Renewal of Neural Stem/Progenitor Cells
The observed reduction in cell proliferation of NSPCs in response to FGF-2 upon ablation of CS-GAGs would have predicted smaller neurospheres in FGF-2 containing media, but not necessarily a reduced number of primary neurospheres as described above. This suggested that ChABC-treatment could compromise the self-renewal and maintenance of FGF-2-sensitive NSPCs. To probe this assumption, an equal number of cells derived from different primary neurospheres grown in FGF-2 or EGF in the presence or absence of ChABC were allowed to form secondary neurospheres in response to FGF-2 or EGF (Fig. 3A). Under all employed combinations, we observed the generation of secondary neurospheres, indicating that subpopulations of NSPCs exist in primary FGF-2- or EGF-grown neurospheres, which can be distinguished by their responsiveness to either FGF-2 or EGF in secondary neurosphere assays in the absence of ChABC. The removal of CS-GAGs in primary FGF-2-grown neurospheres severely compromised the ability of neurosphere-derived cells to generate secondary neurospheres in response to FGF-2 (Fig. 3B). This clearly indicated that FGF-2-mediated self-renewal depends on CS-GAGs as suspected. Regardless of the primary growth factor condition, when cortical neurospheres were initially exposed to ChABC, only about 10% of the cells formed secondary neurospheres in response to FGF-2 in comparison with control cultures (Fig. 3C, 3D). Indeed, ChABC-treatment of primary FGF-2-grown neurospheres also significantly reduced the number of secondary neurospheres in response to EGF (Fig. 3C), which indicates that half of the EGF-responsive NSPCs depends on FGF-2 for maintenance. Similarly, the compromised response of ChABC-exposed primary EGF-grown neurospheres to FGF-2 (Fig. 3D), which was not seen when secondary neurospheres were allowed to form in EGF (Fig. 3D), suggests the presence of an FGF-2-dependent subpopulation of NSPCs in EGF-grown neurospheres. Thus, the complex protocol of alternating the growth factor conditions after CS-GAG removal revealed that the FGF-2-responsive neurosphere-forming cells requires intact CS-GAGs for maintenance and self-renewal.
The Removal of CS-GAGs Biased Subsets of Radial Glia Cells
The diversity of NSPCs observed during normal forebrain development [12, 14, 41] is reflected by the known heterogeneity of cell types within neurospheres [34, 42, 43]. Our observation of selectively affected subpopulations of NSPCs and their differential responsiveness in the secondary neurosphere assay after CS-GAG removal led us to examine the relative number of Nestin-positive, BLBP-positive, and GLAST-positive radial glia cells (RGCs) within neurospheres. As suspected because of the above findings and from our previous work , ChABC-treatment reduced the fraction of Nestin-positive cells by one third and led to a two-fold reduction of BLBP-positive cells, independent of the growth factor condition (Fig. 4B). This was confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis of neurospheres grown for 7 days with or without ChABC in that BLBP mRNA levels were significantly reduced in response to CS-GAG degradation in both FGF-2- and EGF-expanded neurospheres (Fig. 4C). Interestingly, BLBP is present in both the cytoplasm and nucleus of RGCs and its expression level is dynamically modulated by environmental cues during corticogenesis . We further noticed a significant doubling of GLAST-positive cells after ChABC-exposure that occurred selectively in EGF-grown neurospheres, but not in response to FGF-2 (Fig. 4B).
To confirm these observations, we also performed an analysis of acutely dissociated cortical cells plated on polyornithine (Fig. 5). The adherent cortical cells were grown for 24 hours in neurosphere medium with growth factors and immunostained with antibodies against Nestin, BLBP, and GLAST (Fig. 5A). Nearly a third of FGF-2-grown and EGF-grown cells were Nestin-positive and coexpressed the radial glia markers GLAST and/or BLBP. Only a small fraction of FGF-2-stimulated cells expressed GLAST, but were negative for Nestin and BLBP (Fig. 5B). This cell fraction was selectively expanded in the EGF-containing cultures. This trend in phenotypic differences between the FGF-2- and EGF-grown precursor cell pools was most extensive in cultures supplemented with ChABC (Fig. 5B). Moreover, stimulation with EGF also supported morphologic differentiation of GLAST-positive cells (Fig. 5C). When EGF was applied in conjunction with ChABC, the outgrowth of GLAST-labeled processes was even more accentuated. In this regard, the morphologic maturation of GLAST-positive cells, sometimes referred to as astroglial precursors , was most pronounced in response to EGF after deglycanation of CS-GAGs.
Removal of CS-GAGs Selectively Reduces the Activation of the MAPK by FGF-2
ChABC-treatment reduced FGF-2-dependent proliferation, neurosphere formation, and self-renewal, and we wondered which signaling pathway could be perturbed. Since both growth factors, FGF-2 and EGF, act as canonical activators of the mitogen-activated protein kinase (MAPK) cascade  and since the MAPK pathway has been implicated in glial differentiation of NSPCs [47, 48], we hypothesized that ChABC treatment could interfere with the activation of the MAPK signaling pathway in response to FGF-2 and/or EGF. To test this idea, neurospheres obtained in the presence of EGF plus FGF-2 were dissociated. The neurosphere-derived cells were plated on a laminin-1 substrate and starved from the two growth factors in the absence or presence of ChABC. The cultures were next stimulated with low amounts of FGF-2 or EGF for 15 minutes, and the level of phosphorylated MAPKinase (pMAPK) was determined by Western blotting. As expected, the levels of phosphorylated mitogen-activated protein kinase (pMAPK) were low in control cultures and increased in response to FGF-2 or EGF (Fig. 4D). The stimulation of ChABC-treated cultures with FGF-2 resulted in a significantly decreased level of pMAPK (values, n = 3; p <.05), whereas CS-deglycanation did not alter pMAPK activation in response to EGF (Fig. 4D). The differential stimulation of the MAPK pathway by EGF and FGF-2 after ChABC treatment in this short-term assay correlates well with the distinguished long-term response of NSPCs to EGF and FGF-2 in the neurosphere assay. Taken together, CS-GAGs are crucial for activation of the MAPKinase pathway in response to FGF-2 but not to EGF.
Removal of CS-GAGs Supports EGF-Mediated Differentiation of Radial Glia Cell Processes
Because MAPK-signaling in response to FGF-2 was reduced and MAPK-signaling was proposed to be important for functional maturation of RGC in vitro , we explored this approach further by seeding primary EGF- or FGF-2-initiated neurospheres that were expanded for 5 days in the presence or absence of ChABC on polyornithine-coated dishes. The individual primary neurospheres continued to grow in serum-free medium in either the same (homotypic) or switched growth factor (heterotypic) conditions without further addition of ChABC (Fig. 6A). Regardless of the initial growth conditions, almost no neurospheres gave rise to elongated cell processes in the presence of FGF-2 (Fig. 6B). The extension of elongated RGC processes from neurosphere cells was exclusively observed in EGF-containing cultures (Fig. 6C). To distinguish radial glia cell processes from axons, the expression of the neuronal marker βIII-tubulin and the RGC marker GLAST was examined. Indeed, adherent neurospheres growing in FGF-2-containing media did not display GLAST-positive cell processes, whereas the thin, elongated processes that extended in response to EGF stained positive for GLAST and were negative for βIII-tubulin (Fig. 6B, 6C). The digestion of CS-GAGs during the first culture period caused a substantial increase in the number of RGC fibers in response to EGF. These GLAST-expressing fibers were decorated with immature neurons, suggesting that they supported neuronal migration. In contrast, the addition of FGF-2 did not evoke a comparable morphologic evolution accompanied by neuron-glia interactions, although many GLAST-positive cells appeared and migrated out of neurospheres after CS-GAG removal. Thus, consistent with the selective effect of EGF for differentiation of functional RGCs reported previously , the removal of CS-GAGs supports EGF-mediated maturation of RGCs.
Removal of CS-GAGs Enhances Astrogliogenesis and Cell Migration in Response to EGF
We immediately noted the increased cell migration from adherent neurospheres after CS-deglycanation and therefore determined the maximal distance that cells migrated from the neurosphere core (Fig. 6A). Neurosphere-derived cells from both initially FGF-2- as well as EGF-grown neurospheres migrated over significantly longer distances in response to EGF compared with FGF-2-treated sister cultures (Fig. 6D). However, independent of the applied growth factors, a massive increase in cell migration was visible after removal of CS-GAGs (Fig. 6D). Remarkably, after removal of CS-GAGs, the maximal distance of cell migration in response to FGF-2 or EGF was comparable for all culture conditions, which suggests that general inhibitory activities are relieved rather than the migration rate of individual cells.
Because neurospheres also contain a minority of neurons and astrocytes [36, 43], it remained open whether their migration would also be affected by CS-deglycanation. As expected from the phase contrast images under control conditions, most of the βIII-tubulin-positive neurons and GFAP-positive astrocytes remained confined to neurospheres in the presence of FGF-2, and only a limited number of differentiated cells had migrated in the presence of EGF (Fig. 7, upper panels). In contrast, ChABC treatment during the initial culture period resulted in a dramatic increase in the migration of neurons and astrocytes in response to FGF-2 (Fig. 7A). Similarly, the migration of astrocytes was larger in response to EGF after CS-GAG removal (Fig. 7B). In line with earlier reports that FGF-2 promotes neurogenesis  and EGF favors an astroglial fate [26, 27] and our previous observations that ChABC treatment reduced neurogenesis , not many neurons were expected to differentiate under the chosen culture conditions, and, indeed, very few neurons migrated from ChABC-exposed neurospheres in response to EGF.
The recovery of CS-GAGs on cell surfaces was examined by immunochytochemistry. Mab 473HD specifically recognizes a CS-dependent GAG epitope on the large form of DSD-1-PG/receptor protein tyrosine phosphatase-β/ζ, and decorates the surface of both embryonic and adult neural stem/progenitor cells [34, 36]. After 5 days in the migration assay under control condition, 10.6 ± 2.2% of cells showed a strong expression of CS-GAGs on their cell membrane. Although the fraction of 473HD-positive cells which originated from initially ChABC-treated neurospheres was diminished by half compared with control cultures (5.8 ± 1.4%, n = 3), there were still twice as many as in the case of neurospheres that had been continuously exposed to ChABC (2.8 ± 0.9%, n = 3) (data not shown). The propagation of the 473HD-positive cell population and, hence, the presence of the CS-specific GAG structure on the cells obtained from ChABC-pretreated neurospheres argue against the persistence of enzyme activity in the migration culture system. The presence of CS-GAGs in the cell culture supernatant in the different culture conditions was examined by Western blotting (Fig. 7C). Taken together, CS-GAGs differentially fine tune the response to FGF-2 and EGF in subpopulations of NSPCs and appear to balance their maturation and differentiation along the neuronal and astroglial lineages.
In the present study we revealed that chondroitin sulfates are essential for the proliferation, self-renewal, and maintenance of FGF-2, rather than of EGF-responsive NSPCs. The removal of CS-GAGs by ChABC treatment reduced FGF-2-mediated MAPKinase phosphorylation and resulted in fewer BLBP-positive RGCs. In parallel, CS-GAG removal allowed maturation of GLAST-positive RGC processes that supported neuronal migration in the presence of EGF. CS-deglycanation selectively increased EGF-dependent generation and migration of GLAST-expressing RGCs and GFAP-positive astrocytes. Our data imply that CS-GAGs constitute an important part of the environment that orchestrates growth factor responsiveness of neurogenic and gliogenic neural stem/progenitor cell populations.
These observations are in line with our previous work in which we reported a shift from neurogenesis to gliogenesis for FGF-2 plus EGF grown neurospheres upon CS-deglycanation . However, with the results obtained here, we interpret our data in the following way: treatment with ChABC affects two separate NSPC populations. CS-removal suppressed the expansion of FGF-2-responsive, BLBP-positive, and preferentially neurogenic NSPCs, which is not entirely surprising when one considers that FGF-2 in conjunction with Notch signaling regulates BLBP expression [50, 51]. These NSPCs may correspond to those RGCs that directly generate neurons [4, 13, 14, 52]. In line with the forebrain phenotype of FGF-2-deficient mice that have a smaller cortex caused by reduced proliferation and neurogenesis [22–24], we propose that intact CS-GAG structures are required for normal proliferation and neurogenesis of BLBP-expression in RGCs in response to FGF-2. Indeed, CS-GAG preparations purified from the embryonic rat telencephalon changed in their composition during development and supported proliferation of neurosphere-derived cells in response to FGF-2 . At the same time, CS-deglycanation favored the increase of GLAST-positive, EGF-responsive progenitors that preferentially generated astroglia. This preferential expansion of GLAST-positive RGCs could reflect an enhanced propensity of dividing cortical progenitors to generate two GLAST-positive daughters in response to EGF. Notably, the treatment with ChABC seems to favor this type of cell division and, consequently, the generation of glial-restricted progenitors, whereas the generation of BLBP-expressing progenitors simultaneously diminished (supporting information Fig. 1). This switch in phenotype of neural progenitor cells mostly likely corresponds to RGC subtypes that appear at later developmental stages [12, 14, 27]. In this way, a selective expression of CSPGs in subsets of cells may account for the generation of NSPC diversity. The digestion of CS-GAGs from the cell surface of progenitors limited not only their cell proliferation, but also the process of cell division itself (supporting information Fig. 1). CS-GAGs are required for the interaction between the receptor protein tyrosine phosphatase-β/ζ (RPTPβ/ζ) long  and its ligand pleiotrophin . Therefore, it is conceivable that the digestion of CS-GAGs impairs the activity of RTPTβ/ζ to dephosphorylate p190RhoGAP, a negative regulator of Rho which also interacts with GRB2, a molecule involved in the MAPKinase pathway. Interestingly, other GRB2-binding proteins, such as FRS2 or sprouty1, differentially influence the FGF-2 and the EGF signaling pathways .
Moreover, the GLAST-positive cells displayed enhanced EGF-dependent migration after ChABC treatment. Remarkably, EGFR-expressing neural stem cells fluorescence-activated cell sorted with Alexa-EGF migrate on cortical tissue sections in an EGF-dependent fashion . However, not only the migration of GLAST-positive RGCs themselves, but also their ability to support neuronal migration along elongated RGC processes, was marked in response to EGF, but not FGF-2, after the digestion of CS-GAGs. Indeed, EGF-mediated MAPKinase signaling has been implicated in this particular differentiation of RGCs . With the later onset of GLAST-expression in RGC subtypes [12, 45, 57, 58], we suggest that a gradually declining or changing composition of CS-GAGs during forebrain development  would contribute to an environment permissive for the differentiation of NSPCs into the astroglial lineage and emigration of astroglial progeny in response to EGF. Taken together, defined CS structures could control the growth factor responsiveness of separate NSPCs and so balance neurogenesis and gliogenesis—at least in vitro.
Chondroitin sulfates and their carrier proteoglycans display an enormous structural diversity , and this diversity is the basis for their wide variety of functions during development . With regard to proliferation, the interactions of CS chains with some FGF-family members have been described  and can potentiate the mitogenic activity of FGF-2 in NIH/3T3  or neurosphere-derived cells . In fact, the biologic activity of FGF-2 depends on the ability to bind cell surface or extracellular matrix heparan sulfate proteoglycans (HSPGs), and cells that express FGF-receptors but lack these proteoglycans neither bind nor respond to their ligands [63, 64]. Based on our data, CSPGs serve as essential cofactors for FGF-2-mediated MAPKinase signaling and proliferation of primary NSPCs. This role of CSPGs in regulating the MAPK signaling pathway by FGF-2 is novel, since previous studies have solely focused on HSPGs in mammary fibroblasts [65, 66]. This implies a selective activity of CSPGs because the presence of heparin did not rescue the loss of FGF-2-responsiveness after removal of CS-GAGs. Notably, defined glycosaminglycan (GAG)-dependent responses have been reported for semaphorin5A-mediated axon guidance, which is attractive in the presence of HS-GAGs and repulsive in the presence of CS-GAGs .
The molecular interactions that lead to the increased EGF-responsiveness after CS-deglycanation are less obvious because EGF has not been reported to bind to or to require CS-GAGs or HS-GAGs for signaling. However, CS-GAGs interact with heparin binding epidermal growth factor (HB-EGF) , which is expressed at lower levels during early stages of corticogenesis and acts as bona fide ligand for the EGFR, causing proliferation  or cell migration . HB-EGF could be a shared ligand between CS-GAGs and the EGFR, which activates the EGFR directly and not competitively when CS-GAGs are removed. It is noteworthy that cell migration is orchestrated by multiple molecular events that involve ECM molecules and cell surface receptors, and EGFR-dependent migration of late telencephalic NSPCs has been described . Of particular interest is the localization of the two alternatively spliced proteoglycan gene products of the Ptprz1 gene, DSD-1-PG/phosphacan and RPTPβ long  and their ligand pleiotrophin  along forebrain RGC fibers during neurogenesis . Pleiotrophin/HB-GAM not only interacts with RPTPβ, but was shown to promote NSPC differentiation  and EGFR-mediated neuronal migration . The latter depended on the HSPG N-syndecan and thus renders a direct competition between growth factors bound to the different proteoglycan families for receptor tyrosine kinases plausible. In this scenario, a competitive ligand-receptor mechanism between RPTPβ, pleiotrophin/HB-GAM, and HB-EGF would play a role in the EGF-mediated cell migration upon CS-GAGs digestion. Alternatively, the selective expression of CS-GAGs and their interaction partners within cortical germinal regions during neurogenesis could act as a barrier for the EGF-mediated long-distance migration , thereby preventing the emigration of neural stem/progenitor cells from the stem cell niche.
CSPGs become upregulated in the glial scar of CNS lesions, interact with a variety of ECM constituents and cell adhesion molecules, such as F3/contactin, N-CAM, L1-CAM, TAG1, or tenascin-C, and represent the major inhibitory entity with regard to axon regeneration . Regarding the ability of isolated core proteins of CSPGs and CS-GAGs by themselves to inhibit cell migration [73–75], the digestion of GS-GAGs entirely coincided with a supportive effect on EGF-mediated radial glia cell migration. However, the importance of CS structures for NSPCs to undergo neurogenesis in response to FGF-2 may be a good reason to caution when inhibitory activities are removed to enhance regeneration of the lesioned CNS.
We are very grateful to Anke Baar, Andrea Steiner-Mezzadri, Marie-Therese Schmid, and Sören Moritz for excellent technical support and helpful assistance. We thank Michael Karus for comments on earlier versions of the manuscript. This work was supported by the Federal Ministry of Research and Technology (Bundesministerium für Bildung und Forschung (BMBF), Stem Cells for Therapies of the Central Nervous System, 01GN0504), the German Research Council (D.F.G.) and the Ministerium für Innovation, Wissenschaft, Forschung und Technologie (MIWFT) of Nordrhein-Westfalen (NRW) (to A.F.). M.G. was supported by grants from the DFG, European union (EU), BMBF, and the Bavarian Ministry of Science and Arts.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.