Glycobiology Advance Access originally published online on May 14, 2008
Glycobiology 2008 18(8):602-614; doi:10.1093/glycob/cwn040
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Spatiotemporal expression of chondroitin sulfate sulfotransferases in the postnatal developing mouse cerebellum
Department of Developmental Neuroscience, Tokyo Metropolitan Institute for Neuroscience, Musashidai, Fuchu, Tokyo 183-8526, Japan
1 To whom correspondence should be addressed: Tel: +81-42-325-3881; Fax: +81-42-321-8678; e-mail: maedan{at}tmin.ac.jp
Received on April 3, 2008; revised on May 9, 2008; accepted on May 10, 2008
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Chondroitin sulfate (CS) proteoglycans are major components of the cell surface and the extracellular matrix in the developing brain and bind to various proteins via CS chains in a CS structure-dependent manner. This study demonstrated the expression pattern of three CS sulfotransferase genes, dermatan 4-O-sulfotransferase (D4ST), uronyl 2-O-sulfotransferase (UST), and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), in the mouse postnatal cerebellum. These sulfotransferases are responsible for the biosynthesis of oversulfated structures in CS chains such as B, D, and E units, which constitute the binding sites for various heparin-binding proteins. Real-time reverse transcription-polymerase chain reaction analysis indicated that the expression of UST increased remarkably during cerebellar development. The amounts of B and D units, which are generated by UST activity, in the cerebellar CS chains also increased during development. In contrast, the expression of GalNAc4S-6ST and its biosynthetic product, E unit, decreased during postnatal development. In situ hybridization experiments revealed the levels of UST and GalNAc4S-6ST mRNAs to correlate inversely in many cells including Purkinje cells, granule cells in the external granular layer, and inhibitory interneurons. In these neurons, the expression of UST increased and that of GalNAc4S-6ST decreased during development and/or maturation. D4ST was also expressed by many neurons, but its expression was not simply correlated with development, which might contribute to the diversification of CS structures expressed by distinct neurons. These results suggest that the CS structures of various cerebellar neurons change during development and such changes of CS are involved in the regulation of various signaling pathways.
Key words: cerebellum / chondroitin sulfate / dermatan 4-O-sulfotransferase / N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase / uronyl 2-O-sulfotransferase
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Chondroitin sulfate proteoglycans (CS-PGs) play important roles in the developing and adult central nervous system as major components of the cell surface and extracellular matrix (Bandtlow and Zimmermann 2000
; Schwartz and Domowicz 2004). It has been proposed that CS-PGs regulate cell proliferation, differentiation (Tanaka et al. 2003; Ida et al. 2006; Sirko et al. 2007), neuronal migration (Maeda and Noda et al. 1998), neurite extension (Nadanaka et al. 1998; Hikino et al. 2003; Nandini et al. 2004), and neural plasticity (Pizzorusso et al. 2002). Furthermore, CS-PGs are considered to be a major inhibitor of axonal regeneration after injury to the central nervous system (Moon et al. 2001; Bradbury et al. 2002). CS-PGs bind with various growth factors, chemokines, cell adhesion molecules, and extracellular matrix molecules via chondroitin sulfate (CS) and core protein portions (Herndon et al. 1999). Notably, the CS portion is thought to be the critical determinant of CS-PG functions because chondroitinase ABC (CHase ABC), which degrades CS, destroys many of the CS-PG activities (Bradbury et al. 2002; Pizzorusso et al. 2002; Tanaka et al. 2003; Corvetti and Rossi 2005). Although it has long been believed that CS chains are negatively charged simple polysaccharides, recent studies revealed that CS chains bind with various proteins including pleiotrophin, midkine, FGF-2, IL-6, and PDGF in a structure-dependent manner (Fager et al. 1995; Maeda et al. 1996, 1999, 2003, 2006; Mummery and Rider 2000; Deepa et al. 2002; Zou et al. 2003; Fthenou et al. 2007). That is, structurally different CS chains display distinct affinities for various proteins, and therefore, differentially regulate their functions.
CS chains are biosynthesized in the Golgi apparatus by sequential modifications after the polymerization of repeating disaccharide units of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) (O unit), leading to the diverse structural heterogeneity in these polysaccharides (Kusche-Gullberg and Kjellén 2003) (Figure 1A). Many of the GalNAc residues in CS chains are 4-O-sulfated by chondroitin 4-O-sulfotransferases or 6-O-sulfated by chondroitin 6-O-sulfotransferases. The resultant GlcAβ1-3GalNAc(4S) and GlcAβ1-3GalNAc(6S) disaccharide units are called A and C units, respectively (Figure 1A). Although A and C units are the major components of CS chains, a portion of disaccharide units have two sulfate residues, which are called oversulfated structures: GlcA(2S)β1-3GalNAc(6S) (D unit) and GlcAβ1-3GalNAc(4,6diS) (E unit). D units are synthesized by uronyl 2-O-sulfotransferase (UST) from C units, and E units are generated through sulfation of A units by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) (Figure 1A). Furthermore, some of the GlcA residues are converted to iduronic acid (IdoA) by C5-epimerase, which leads to the generation of IdoA
1-3GalNAc(4S) (iA unit) and IdoA(2S)1-3GalNAc(4S) (iB unit) structures through sequential sulfation by dermatan 4-O-sulfotransferase (D4ST) and UST (Figure 1A). These complex biosynthetic processes result in the highly diverse structural heterogeneity in CS chains.
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We recently found that CS plays important roles in the development of cerebellar Purkinje cells using an organotypic slice culture system of the rat cerebellum (Tanaka et al. 2003
). CHase ABC treatment of the cerebellar slices resulted in the aberrant morphogenesis of Purkinje cell dendrites such as multiple and disoriented primary dendrites. The addition of various CS preparations caused a similar malformation of Purkinje cell dendrites. It is noteworthy that the effects of the CS preparations were structure-dependent. Although A unit-rich CS-A had no influence, D unit-rich CS-D and E unit-rich CS-E preparations exerted strong effects on Purkinje cells, suggesting that oversulfated CS structures play important roles in the dendrite formation of this type of neuron. From these findings, we hypothesized that the oversulfated structures regulate the developmental processes of the cerebellum by modulating various signaling pathways. Cerebellum shows rather simple architecture, the development of which is relatively well described (Figure 1B). Thus, this tissue is suitable for the expression analysis of CS and CS sulfotransferases. In this study, we examined the structural changes of CS and the expression of UST, GalNAc4S-6ST, and D4ST mRNAs during cerebellar development. We demonstrate here that CS structures and the expression of these sulfotransferases show spatiotemporally dynamic changes, suggesting that specific CS structures are involved in distinct developmental events in the cerebellum. |
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Microanalysis of CS chains in the developing cerebellum
To analyze the quantitative and structural changes of CS chains in the developing cerebellum, frozen cerebellar sections on glass slides were treated with CHase ABC, and the degradation products were fluorescently labeled with 2-aminobenzamide and separated by anion-exchange high performance liquid chromatography (HPLC) (Koshiishi et al. 1999
). As shown in Figure 2, the disaccharide composition of CS displayed a dynamic change during postnatal cerebellar development (Figure 2 and Table I). At P1, the major components of CS were A (49.5%), C (35.4%), and O (9.1%) units, the amounts of which were stable from P1 to P7. However, after P7, the amount of A unit drastically increased, and the amounts of C and O units remarkably decreased (Figure 2A). At P20, CS chains were composed of 84.3% A units, and only 5.3% C units and 3.1% O units (Table I). On the other hand, the levels of disulfated disaccharide units showed characteristic changes (Figure 2B). The amount of D unit increased from P1 (3.2%) to P10 (6.8%), and then decreased remarkably until P20 (3.2%) (Table I). The amount of E unit gradually decreased from P1 (2.6%) to P20 (0.82%). At P1, few B units were detected (0.15%); however, after that, the amount drastically increased till P20 (3.2%). Because of the decrease in the amount of unsulfated O units and the increase in the amount of disulfated disaccharide units during development, the sulfation degree (average number of sulfate groups per disaccharide) of CS chains increased from 0.97 at P1 to 1.04 after P10 (Table I).
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Mitsunaga et al. (2006
) also reported the disaccharide composition of CS from cerebella at the later developmental stages although they did not quantify O and T units. The structural changes they reported roughly coincided with the present results.
Quantitative analyses of CS sulfotransferase mRNAs in the developing cerebellum
Because disulfated disaccharide units displayed characteristic changes of expression during cerebellar development, we analyzed the expression levels of three CS sulfotransferase genes (UST, GalNAc4S-6ST, and D4ST), which are involved in the synthesis of these structures (Figure 1A). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of UST mRNA indicated that the expression of this gene gradually increased from postnatal day 1 (P1) to P14 (Figure 3A). In contrast, the expression levels of GalNAc4S-6ST and D4ST showed a downward trend until P14 (Figure 3B, C).UST is responsible for the 2-O-sulfation of iB and D units (Figure 1A). The changes in the expression levels of UST during cerebellar development correlated well with the changes in the amounts of B plus D units in CS chains (Figures 2B and 3A). The expression of the GalNAc4S-6ST gene also correlated well with that of E unit in CS chains (Figures 2B and 3B).
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It has been considered that B unit is exclusively present as an iB (IdoA(2S)
1-3GalNAc(4S)) structure in CS chains (Mitsunaga et al. 2006) although our HPLC analysis shown in Figure 2 cannot distinguish between B (GlcA(2S)β1-3GalNAc(4S)) and iB structures in CS because CHase ABC generates common degradation products from these structures. The iB structure is thought to be synthesized through sequential sulfation processes by D4ST and UST after the C5-epimerization of GlcA by C5-epimerase (Figure 1A). However, the expression of the D4ST gene showed no correlation with the expression of B unit revealed by HPLC analysis (Figures 2B and 3C).
Mitsunaga et al. (2006) also quantified the expression of UST, GalNAc4S-6ST, and D4ST mRNAs in the cerebella at later developmental stages. Although their results for GalNAc4S-6ST and UST roughly coincided with ours, those for D4ST did not. Although we do not know the cause of this discrepancy, our results are highly reproducible and we believe that they are correct.
Distribution of CS sulfotransferase mRNAs in the Purkinje cell layer
At P1, Purkinje cells are clustered in the Purkinje cell layer (PCL) with almost no dendritic processes (Shimazaki et al. 2005). After P7, Purkinje cell dendrites vigorously grow, branch, and form synapses with parallel fibers until P20 (Figure 1B(a)). The growing Purkinje cell dendrites closely associate with the processes of Bergmann glia, and this interaction has been suggested to play important roles in the development of Purkinje cells (Yamada et al. 2000) (Figure 1B(b)).
To determine the spatiotemporal expression patterns of UST, GalNAc4S-6ST, and D4ST in the developing PCL, we performed in situ hybridization experiments. The signals of the mRNAs for UST, GalNAc4S-6ST, and D4ST were clearly detected in the PCL throughout development (Figures 4–Figure 6). The signals of the mRNA for UST was low at P1 (Figure 4A), but increased at the later stages (Figure 4B–E). On the other hand, the signals of the mRNAs for D4ST and GalNAc4S-6ST were strongly detected in the PCL at P1 (Figures 5A and 6A). While the expression of D4ST mRNA was strong in the PCL throughout development (Figure 6B–E), that of GalNAc4S-6ST mRNA became remarkably weak after P7 (Figure 5B–E).
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Higher magnification views clearly showed that Purkinje cells expressed UST and D4ST (Figure 7A–D, I–L). Purkinje cells also expressed GalNAc4S-6ST although the signals were weak (Figure 7E–H). Small cells closely associated with Purkinje cells showed significant signals of the mRNA for UST (Figure 7A–D, small arrowheads). They showed only faint signals for GalNAc4S-6ST and D4ST mRNAs (Figure 7E–L, small arrowheads). The double labeling in situ hybridization experiments indicated that these small cells expressed both UST and GLAST mRNAs, indicating that they were Bergmann glia (Figure 7M–O, arrowheads). Immunoreactivity to the MO-225 monoclonal antibody was observed surrounding Purkinje cell bodies (Figure 11A and B; arrowheads) and along the Bergmann glial fibers (Figure 11A, arrows). MO-225 recognizes CS chains rich in the A–D sequence (Yamagata et al. 1987
), which is consistent with the observation that both Purkinje cells and Bergmann glia expressed UST.
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Expression of CS sulfotransferase mRNAs in granule cells
Next, we observed the expression pattern of CS sulfotransferase genes in the developing granular layer. During postnatal cerebellar development, granule cell precursors proliferate in the outermost region of the external granular layer (EGL), and then migrate along Bergmann glial fibers toward the internal granular layer (IGL) (Figure 1B[c]). During migration, granule cells begin to extend parallel fibers.
In the EGL, the signals of the mRNAs for UST, GalNAc4S-6ST, and D4ST were observed throughout (Figures 4–6A–C). Higher magnification views indicated that the cells located in the outermost region of the EGL strongly expressed UST and D4ST mRNAs at P7 and P10 (Figure 8A, C, D and F, arrowheads). In contrast, the signals for GalNAc4S-6ST mRNA were rather uniformly distributed in the EGL (Figure 8B and E). Double fluorescent labeling experiments of in situ hybridization with the D4ST probe and immunohistochemical detection of BrdU incorporation indicated that BrdU-incorporated proliferating granule cells showed stronger signals for D4ST mRNA than the inner postmitotic granule cells (Figure 8G–I).
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In the IGL, the granule cells expressed weak to moderate levels of the mRNAs for UST, D4ST, and GalNAc4S-6ST through development (Figure 7A–L).
Distribution of CS sulfotransferase mRNAs in the molecular layer and the Golgi cells
Small cells in the molecular layer (ML), presumably inhibitory interneurons, displayed signals of the mRNAs for GalNAc4S-6ST and D4ST at P10 (Figure 7F and J, arrows), but the signal for UST mRNA was very weak (Figure 7B). While the signals for UST and D4ST mRNAs increased after P14 (Figure 7B–D and J–L, arrows), those for GalNAc4S-6ST became weak after P14 (Figure 7F–H).
The large neurons in the IGL, presumably Golgi cells, displayed signals for three CS sulfotransferase mRNAs. From P10 to P21, the signals for UST and D4ST mRNAs were constantly detected in Golgi cells (Figure 9B–D, J–L, arrows), but those for GalNAc4S-6ST mRNA were very weak (Figure 9F–H, arrows). In the IGL, Golgi cell axon terminals, granule cell dendrites, and mossy fiber rosettes form complex structures called glomeruli (Figure 1B). Our immunohistochemical analysis revealed that D unit-rich CS epitopes recognized by MO-225 were highly accumulated in the surroundings of Golgi cells (Figure 11B, arrow) and glomeruli (Figure 11B, white asterisks), consistent with the findings that Golgi cells express high levels of UST.
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Distribution of CS sulfotransferase mRNAs in white matter and deep cerebellar nuclei
Several studies have revealed that large amounts of CSPGs, such as phosphacan, neurocan, aggrecan, and versican, were distributed in the developing white matter (WM) (Meyer-Puttlitz et al. 1996
; Popp et al. 2003). Our immunohistochemical staining with MO-225 demonstrated that D unit-rich CS epitopes were accumulated around the elongated cells in WM at P5 (Figure 11D, arrows) and along myelinating axons at P20 (Figure 11E, arrows). The elongated cells in the WM are presumably dividing progenitor cells, which then migrate to their final destination and differentiate into Bergmann glia, interneurons in ML and oligodendrocytes (Zhang and Goldman 1996) (Figure 1B[d]). We next examined whether CS sulfotransferase genes were expressed in the WM cells. The signals for UST and D4ST mRNAs were detected on the elongated shape cells in the WM from P7 to P21 (Figure 9A–D and I–L, arrowheads) although the signals for GalNAc4S-6ST mRNA remained very weak (Figure 9E–H, arrowheads).
In the deep cerebellar nuclei, the extracellular matrix surrounding large neurons was strongly stained with MO-225 (Figure 11C, asterisks). Recently, it was revealed that neurons in the developing deep cerebellar nuclei synthesized CSPGs such as aggrecan, neurocan and phasphacan, which started to form perineuronal nets between P7 and P14 (Carulli et al. 2007). So, we checked the expression of CS sulfotransferase genes in deep cerebellar nuclei at P14 and P21. The signals for UST, D4ST, and GalNAc4S-6ST mRNAs were clearly observed on large neurons (Figure 10A–F, arrowheads). Many small cells in the deep cerebellar nuclei also showed moderate signals for UST and D4ST mRNAs although their identity remains to be determined.
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Changes of the CS structure and expression of CS sulfotransferase genes
During cerebellar development, the disaccharide composition of CS showed dynamic changes. Notably, the oversulfated B, D, and E structures showed a characteristic expression pattern. While the expression of E unit decreased gradually from P1 to P20, that of B unit increased gradually from P1 to P20. On the other hand, the expression of D unit increased rapidly from P1 to P10 and decreased thereafter. Thus, the expression of E, D, and B units seems to be correlated with the early, middle, and later stages of postnatal cerebellar development, respectively. RT-PCR analysis indicated that the expression of E and D/B units is highly correlated with the expression of GalNAc4S-6ST and UST mRNAs, respectively, suggesting that the expression of these CS structures are transcriptionally regulated by these sulfotransferases. In situ hybridization experiments indicated that the GalNAc4S-6ST mRNA was highly expressed in the EGL and PCL at the early stages, but decreased later. On the other hand, the expression of UST mRNA increased at the later stages in the EGL, interneurons, and Purkinje cells (Figure 12). It is noteworthy that the levels of GalNAc4S-6ST and UST mRNAs showed a negative correlation during development in several cell types including EGL cells, interneurons in ML, Purkinje cells, and Golgi cells. This suggests that E unit and 2-O-sulfated D/B units have distinct or opposite functions during cerebellar development. In contrast to the apparent inverse correlation between the expression of UST and GalNAc4S-6ST, the expression of D4ST was not simply correlated with those of the other genes (Figure 12). This suggests that the amounts of iA and/or iB structures increase, or decrease during development depending on the cell type, which might lead to the diversification of CS structures expressed by cerebellar cells.
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B unit is believed to be present exclusively as the iB structure in CS chains (Mitsunaga et al. 2006
), which is generated by sequential sulfation processes by D4ST and UST after the C5-epimerization of GlcA by C5-epimerase (Figure 1A). Therefore, we anticipated that D4ST determines whether B or D unit is generated in the CS chains. However, the expression of the D4ST gene showed no correlation with the expression of B unit as revealed by HPLC analysis (Figures 2B and 3C). Thus, it might be that the expression of the iB structure is critically regulated by C5-epimerase. Alternatively, B unit might be present as mainly the B structure with the little iB structure in the CS of the cerebellum. Our HPLC analysis could not distinguish between B and iB structures in CS chains and so they are collectively quantified as B unit, because CHase ABC degrades both structures producing common degradation products. A future fine chemical analysis is necessary to clarify this point.
Expression of CS sulfotransferases in Purkinje cells and Bergmann glia
We previously demonstrated that D unit-rich CS chains are richly deposited in the extracellular space between Purkinje cells and the lamellate processes of Bergmann glia. (Shimazaki et al. 2005). These CS chains are mainly attached to phosphacan/PTP
, which uses pleiotrophin as a ligand (Shimazaki et al. 2005). Phosphacan/PTP-pleiotrophin signaling is involved in the dendrite formation of Purkinje cells probably through the regulation of Bergmann glia–Purkinje cell interaction (Tanaka et al. 2003). The CS portion of phosphacan/PTP determines the affinity for pleiotrophin, and the phosphacan with D unit-rich CS displays higher affinity for pleiotrophin than that without this structure (Maeda et al. 2003). Further studies indicated that the E structure also contributes to the high-affinity binding to pleiotrophin (Bao et al. 2005; Maeda et al. 2006). It has been suggested that D unit-rich and E unit-rich CS domains have distinct functions; the former constitutes the moderate affinity, and the latter the high-affinity binding sites for pleiotrophin (Deepa et al. 2002; Bao et al. 2004, 2005). Thus, the changes in the contents of D and E structures in CS chains on phosphacan/PTP are presumed to change the affinity for pleiotrophin and therefore regulate the signal strength of this pathway.
PTP/phosphacan was expressed by both Purkinje cells and Bergmann glia (Snyder et al. 1996; Tanaka et al. 2003), but CS sulfotransferase genes were expressed by these cells in a distinct manner (Figure 12). Purkinje cells expressed high levels of UST and D4ST during postnatal development. They also expressed moderate levels of GalNAc4S-6ST during the early developmental period, but its expression was downregulated after P14. On the other hand, Bergmann glia expressed high levels of UST, but the levels of GalNAc4S-6ST and D4ST were low. This suggests that Purkinje cells synthesize D, iA, and iB unit-rich CS chains during development. Furthermore, it is plausible that their CS chains contain a substantial amount of E unit during the early developmental period. On the other hand, Bergmann glia would express a rather simple D unit-rich CS through development. Such differential modification of CS chains on PTP/phosphacan might finely regulate the bi-directional signaling of the PTP-pleiotrophin pathway in Purkinje cells and Bergmann glia.
Expression of CS in developing granule cells
Granule cell precursors proliferate at the outermost layer of the EGL and migrate to the IGL through the ML and PCL during the first 2 weeks after birth (Fujita 1967; Komuro et al. 2001). We here found that the proliferating granule cells in the outermost region of the EGL strongly expressed UST and D4ST mRNAs although the signal for GalNAc4S-6ST mRNA was distributed rather uniformly in the EGL. Recent studies demonstrated that oversulfated and iA structures in CS play important roles in the proliferation and differentiation of neural precursor cells (Holst et al. 2006; Ida et al. 2006; Sirko et al. 2007). Thus, D and iA structures generated by UST and/or D4ST might contribute to the proliferation of granule cells at the EGL. In the EGL, immunoreactivity to MO-225 was observed at the pial surface and along the Bergmann glial fibers (Figure 11). It seems that the granule cells in the outermost layer of the EGL were surrounded by the immunoreactivity, suggesting that the D structure generated by UST is involved in their proliferation.
In contrast to UST and D4ST, GalNAc4S-6ST was expressed by cells all over the EGL although its signal intensity was moderate. A recent study by Purushothaman et al. (2007) indicated that a new anti-CS/DS antibody, GD3G7, which recognizes E and iE (IdoA1-3GalNAc(4,6diS)) structures, stained strongly the developing molecular layer at its border with the EGL at P7. The CS-E structure generated by GalNAc4S-6ST might be involved in the later developmental events of granule cells such as neuronal migration and extension of parallel fibers rather than proliferation.
Expression of CS in the inhibitory interneurons and the cells in white matter
Early in the postnatal period, there are many dividing progenitor cells in the WM of the cerebellum, which actively migrate to their final destination and differentiate into inhibitory interneurons in ML, Bergmann glia, astrocytes, and oligodendrocytes (Zhang and Goldman 1996; Weisheit et al. 2006). In the developing WM, elongated cells with simple processes, characteristic of progenitor cells, were stained with MO-225, indicating that these cells express D unit-rich CS (Figure 11D). Although levels of UST and D4ST expression were low in these elongated cells at P7, they increased significantly after P10, suggesting that these sulfotransferases are involved mainly in the differentiation rather than proliferation because the cell division of neuronal progenitors in WM occurs mostly prior to P7 (Weisheit et al. 2006). Furthermore, the low level of GalNAc4S-6ST expression was observed through development in these cells.
The differentiation and survival of progenitor cells are considered to be regulated by multiple factors including PDGF, FGF-2, IL-6, and IGF I, the activities of which are regulated by cell surface and extracellular matrix-bound CS-PGs (Fager et al. 1995; Mummery and Rider 2000; Deepa et al. 2002; Milosevic and Goldman 2004; Russo et al. 2005; Ida et al. 2006; Kuang et al. 2006; Fthenou et al. 2007). Changes of CS expression in the progenitor cells should finely regulate the differentiation of these cells by modulating the signaling of such proteins.
Some populations of neuronal progenitors generated in the white matter migrate to the ML and differentiate into basket and stellate cells (Zhang and Goldman 1996). These interneurons expressed high levels of UST and D4ST after P14, suggesting that they synthesize D and/or iB unit-rich CS chains. Golgi cells and neurons in the deep cerebellar nuclei also expressed high levels of these sulfotransferases after P10 and P7, respectively. The deep cerebellar neurons express multiple CS-PGs forming perineuronal nets after P7 (Carulli et al. 2007). Thus, the CS chains of these CS-PGs are expected to be rich in D and/or iB units. Perineuronal nets are considered to contribute to the maturation, stabilization, and plasticity of synapses (Carulli et al. 2007), and these CS structures might be involved in such processes.
We can conclude that cerebellar neurons and glial cells synthesize CS with distinct structures depending on the developmental stage. This differential expression of CS probably regulates the multiple signaling pathways of various growth factors, contributing to the differentiation of each cell type in the cerebellum. Further studies are necessary to reveal the signaling molecules coupled with each CS structure.
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Animals
Pregnant ICR mice were purchased from Japan SLC Inc. (Shizuoka, Japan), and the male and female pups were used for experiments. All the animal experiments were performed with the approval by the Animal Use and Care Committee of the Tokyo Metropolitan Institute for Neuroscience.
Quantitative real-time RT-PCR
After ether anesthesia, the brains were dissected out from ICR mice. Total RNA was purified from cerebella of P1, P7, P10, P14, and P21 mice using an RNeasy Mini Kit (Qiagen, Hilden, Germany). For the synthesis of first strand cDNA, 2 µg of the total RNA was treated with ReverTra Ace and Oligo(dT)20 RT primer (Toyobo, Osaka, Japan). Primer sequences of CS sulfotransferases are as follows: for UST (forward, 5'-AGACATGTCCACTTCCTCAACTTCT-3' and reverse, 5'-CAAAGCGACGGAAGAAATAGTTAGA-3'); for GalNAc4S-6ST (forward, 5'-ATATGTTTTCTGTAATCCCCAGCAA-3' and reverse, 5'’-AGTAGAGCA CGTAGGAATTGGTCAG-3'); and for D4ST (forward, 5'-GCCTGCTCTAACTGGAAACG-3' and reverse, 5'-CTGCCAGAAACACCAAGTCA-3'). Quantitative real-time RT-PCR was performed using SYBR Green Master Mix (QuantiTect SYBR Green PCR Kit, Qiagen) and ABI PRISM 7500 (Applied Biosystems, CA). The expression levels of the target genes were normalized to that of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (forward, 5'-AGTCTACTGGTGTCTTCACCACCAT-3' and reverse, 5'-AGTTGTCATATTTCTCGTGGTTCAC-3').
Analysis of the disaccharide composition of CS chains
After ether anesthesia, cerebella of P1, P7, P10, P14, and P20 mice were dissected out, embedded in an OCT compound (Sakura, Tokyo, Japan) and frozen in liquid nitrogen. They were cut into 16-µm-thick sagittal sections, which were collected onto glass slides. The cerebellar peduncle and the deep cerebellar nuclei regions were scraped off with a micro surgical blade under a stereomicroscope. The CS composition was analyzed using trimmed sections 40–60 mm2 in total area. The sections on glass slides were fixed with 1 mL of methanol/acetone (1:1, v/v) at room temperature for 5 min. After discarding the fixing solution, the slides were treated twice with 1 mL of the same solution for 5 min. The sections were air-dried for 90 min, and then washed twice with distilled water. The sections were again air-dried, and then treated with 10% bovine serum albumin in distilled water for 15 min. After three washes with distilled water, the sections were pretreated with 100 mM ammonium acetate for 2 min. Then, they were treated with 100 µL of 0.3 U/mL CHase ABC (Seikagaku, Tokyo, Japan) in 100 mM ammonium acetate for 2 h at 37°C. The solutions were collected into 1.5-mL microcentrifuge tubes, and the sections were extracted with 500 µL of distilled water. The combined solutions were centrifuged at 15,000 x g for 15 min, and the supernatants were dried by Speed Vac lyophilization. The dried materials were treated with 5 µL of 0.35 M 2-aminobenzamide/1 M sodium cyanoborohydride in 30% acetic acid/70% dimethyl sulfoxide for 2 h at 65°C. After the removal of excess 2-aminobenzamide by paper chromatography, the fluorescently labeled unsaturated disaccharides were analyzed by anion-exchange HPLC according to the method described by Mitsunaga et al. (2006).
Preparation of in situ hybridization probes
CS sulfotransferase gene fragments were amplified by PCR from a mouse brain cDNA library (Clonetech, Mountain View, CA) using the following primer sets (F, forward and R, reverse): for UST (685bp), 5'-GCTACCCTGGTGGTCTTCTG-3' (F) and 5'-CGAGCAGGAAGTTTTCGTTC-3' (R); for Gal- NAc4S-6ST (681bp), 5'-CTGCCAGGATTGAGTTCACA-3' (F) and 5'-GCTTGGCTTCTGGTTGAAAG-3' (R); and for D4ST (393bp), 5'-TTTCCCCGCCCTCTGACCC-3' (F) and 5'-TCCCACCGGCAAGTCCCA-3' (R). The amplified sulfotransferase gene fragments were inserted into the vector pBluescript II KS(+) (Stratagene, La Jolla, CA). The antisense or sense probes were produced using T7 or T3 RNA polymerase (Roche Diagnostics, Basel, Switzerland) in the presence of DIG RNA Labeling Mix (Roche Diagnostics).
For double labeling in situ hybridization, we used fluorescein-labeled RNA probes, which were prepared using a Fluorescein RNA Labeling Mix kit (Roche Diagnostics) according to the manufacturer's protocol. We used the glial glutamate transporter (GLAST) gene for double labeling in situ hybridization as a marker of Bergmann glia. The GLAST gene fragment (664 bp) was amplified by PCR using the following primers: forward, 5'-TTTCGTGATCGGAAACATGA-3' and reverse, 5'-CAGAAACCAGTCCACTGCAA-3'.
In situ hybridization
After ether anesthesia, P1, P7, P10, P14, and P21 mice were perfused with the Bouin solution. The brains were dissected out, postfixed in the Bouin solution at room temperature for 4 h, and embedded in paraffin after dehydration through a graded alcohol series. The paraffin-embedded brains were cut into 5-µm-thick sagittal sections. The sections were deparaffinized and pretreated with 0.2 N HCl and 20 µg/mL proteinase K. After prehybridizing in 50% formamide-5x SSC, they were hybridized with the riboprobes in the hybridization buffer (50% formamide, 5x SSC, 1x Denhardt's solution containing 100 µg/mL heparin, 10 mM DTT, 10% dextran sulfate, 0.1 mg/mL salmon sperm DNA, and 0.1 mg/mL yeast tRNA) at 70°C for 18 h. The washing step was performed sequentially as follows: (1) 4x SSC, (2) 2x SSC at 65°C for 30 min (twice), (3) 0.1x SSC at 70°C for 1h (twice), and (4) 100 mM Tris–HCl, pH 7.5/150 mM NaCl for 5 min (twice). After incubation in the blocking reagent and alkaline phosphatase-conjugated anti-digoxigenin antibody (1:5000; Roche Diagnostics) at 4°C overnight, the tissue sections were treated with the BCIP/NBT solution (Roche Diagnostics) at room temperature. The sections were observed on a Zeiss Axioskop (Carl Zeiss, Oberkochen, Germany). Digital images were captured using an AxioCAM HRc CCD camera with AxioVision 3.1 software (Carl Zeiss). The images were processed for publication using Adobe Photoshop 7.0 software (Adobe Systems Inc. San Jose, CA) with minimal adjustments of brightness and contrast applied to the whole images.
For double labeling in situ hybridization, the sections were hybridized with a mixture of DIG- and fluorescein-labeled riboprobes in the hybridization buffer. After the washing step, they were incubated with a mixture of a sheep anti-digoxigenin antibody (1:200; Roche Diagnostics) and a mouse anti-fluorescein antibody (1:200; Roche Diagnostics) at 4°C overnight. Then, the sections were incubated with Alexa Fluor 488 anti-mouse IgG and Alexa Fluor 594 anti-sheep IgG (1:200; Molecular Probes, Eugene, OR). The sections were observed using an FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). Digital images were processed for publication as described above.
Immunohistochemistry
After ether anesthesia, P5, P10, P15 and P20 mice were perfused with 4% paraformaldehyde/0.1 M sodium phosphate buffer, pH 7.4. The brains were dissected out, postfixed in 4% paraformaldehyde/PBS at 4°C for 4 h, and embedded in paraffin after dehydration through a graded alcohol series. Paraffin-embedded tissues were cut into 6-µm-thick sections, which were then deparaffinized and equilibrated with PBS. The sections were incubated sequentially in the following solutions: (1) 2.5% hydrogen peroxide/ PBS for 30 min; (2) 2% bovine serum albumin/4% normal goat serum/PBS for 30 min; (3) MO-225 (Seikagaku; 1:200) diluted in 1% bovine serum albumin/PBS for 60 min; (4) biotinylated anti-mouse IgM (1:200) for 30 min; (5) ABC solution for 30 min; and (6) 0.1% diaminobenzidine/0.02% hydrogen peroxide/PBS. The biotinylated antibody and ABC solution were from the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). MO-225 is a mouse IgM monoclonal antibody raised against chick CS-PG, PG-M (Yamagata et al. 1987). This antibody recognizes CS chains containing A–D sequence in Western blotting and immunohistochemistry (Yamagata et al. 1987; Maeda et al. 2003; Shimazaki et al. 2005). The CHase ABC-treated cerebellar sections showed no immunoreactivity to MO-225, indicating that the immunohistochemical stainings shown in Figure 11 are specific.
5-Bromo-2'-deoxyuridine (BrdU) labeling
P4 mice were injected intraperitoneally with BrdU (50 mg/kg body weight) and were perfused with the Bouin solution under ether anesthesia 2 h later. The brains were dissected out, postfixed in the Bouin solution for 4 h, and embedded in paraffin after dehydration through a graded alcohol series. The brains were cut into 5-µm-thick sagittal sections. The sections were subjected to in situ hybridization as described above. After the hybridization, the sections were treated with a mouse anti-BrdU antibody (1:500, Sigma, Saint Louis) and a sheep anti-digoxigenin antibody (1:200) at 4°C overnight. They were incubated with Alexa Fluor 488 anti-mouse IgG and Alexa Fluor 594 anti-sheep IgG (1:200) and observed under an FV1000 confocal microscope.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingReferences |
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Ministry of Education, Science, Sports and Culture of Japan (to N.M.) and the Naito Foundation (to N.M.).
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Acknowledgements |
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We thank Drs. Y. Shimazaki and T. Hata for technical assistance.
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Abbreviations |
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A unit, GlcAβ1-3GalNAc(4S); B structure, GlcA(2S)β1-3GalNAc(4S); CHase ABC, chondroitinase ABC; CN, cerebellar nucleus; CS, chondroitin sulfate; CS-PG, chondroitin sulfate proteoglycan; C unit, GlcAβ1-3GalNAc(6S); D4ST, dermatan 4-O-sulfotransferase; D unit, GlcA(2S)β1-3GalNAc(6S); EGL, external granular layer; E unit, GlcAβ1-3GalNAc(4, 6diS); GalNAc, N-acetylgalactosamine; GalNAc4S-6ST, N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase; GLAST, glial glutamate transporter; GlcA, glucuronic acid; HPLC, high performance liquid chromatography; iA unit, IdoA
1-3GalNAc(4S); iB unit, IdoA(2S)1-3GalNAc(4S); IdoA, iduronic acid; iE structure, IdoA1-3GalNAc(4, 6diS); IGL, internal granular layer; ML, molecular layer; O unit, GlcAβ1-3GalNAc; P, postnatal day; PCL, Purkinje cell layer; RT-PCR, reverse transcriptase-polymerase chain reaction; T unit, GlcA(2S)β1-3GalNAc(4, 6diS); UST, uronyl 2-O-sulfotransferase; WM, white matter. |
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