N reported to be associated with aggressive tumor growth and proliferation [34,35]. Hence, CD44 might be an important molecule for proliferation of cerebellar progenitor cells. Notably, OPCs also expressed CD44, and the timing of CD44 expression in these cells was restricted. CD44 expression disappeared during oligodendrocyte maturation (Fig. 7). CD44 expression was also observed in subpopulations of immature neurons (Fig. 8). CD44 was expressed in immature Purkinje neurons at P7, after which expression of CD44 disappeared from the PCL (Fig. 8). In contrast, granule neurons expressed CD44 more strongly in the adult than during developmental stages (Fig. 8). Thus, the expression of CD44 was restricted to granule neurons and had a regulated time course during development.CD44 is expressed by hematopoietic cells, including stem and progenitor cells [10,11,36] and cancer stem cells [16]. In the central nervous system, it was reported that a subset of CD44+/ CD90+ cells in the cortex was capable of neurosphere initiation [37]. In this study, we indicated that CD44 was expressed in the ventricular zone of the cerebellum at early embryonic stages (Fig. 2) and in Sox2-positive cells at early postnatal stages (Fig. 5). A part of CD44-positive cells yield neurospheres (Fig. 1). Based on these results, we hypothesize that CD44 is expressed in neural progenitor cells, including neural stem cells, in both the embryonic ventricular zone and postnatal WM. Therefore, further careful analysis will be required to determine whether CD44-positive cells give rise to only astrocytes, or give rise to not only astrocytes but also other types of cells in vivo. In addition to fibrous astrocytes, we found that CD44 was also transiently expressed in Bergmann glia (Fig. 6). Hyaluronic acid, which is one of the ligands of CD44, is strongly expressed in the WM at P7 and P15 in the cerebellum [38]. The interaction of hyaluronic acid and CD44 in astrocytes stimulates Rac1 signaling, leading to cytoskeletal effects and cell migration [39], suggesting that Bergmann glia and fibrous astrocytes might require CD44 for their migration. Recently, it was reported that cleavage of CD44 generates a CD44 intracellular domain (ICD) that functions as a Fosamprenavir (Calcium Salt) transcription factor [15]. This transcriptional cascade might be important for cerebellar development. On the other hand, differentiating protoplasmic astrocytes were CD44 negative,CD44 Expression in Developing CerebellumFigure 5. CD44 expression in neural stem/progenitor cells during postnatal development. A : Double RG-7604 biological activity immunostaining of CD44 and Sox2 in the cerebellum at P3 (A ), P7 (D ) and P14 (G ). Nucleus was counterstained with TO-PRO-3 (blue). J: Quantitative analysis of the number of CD44-positive neural stem/progenitor cells by FACS at P3, P7 and P10. *p,0.05, 1527786 **p,0.005, ***p,0.001. Scale bars, 20 mm. doi:10.1371/journal.pone.0053109.galthough most of astrocyte precursors seemed to be CD44-positive (Fig. 6). 11967625 Thus there is a possibility that CD44 expression must be shut off in astrocyte precursor cells, if they differentiate into protoplasmic astrocytes. Alternatively, generation of protoplasmic astrocytes might be independent from CD44-positive astrocyte precursors. So far we cannot address these questions, since we have never done the lineage analysis. Fine lineage analysis andfurther functional analysis is necessary to determine the roles of CD44 in the developing cerebellum. The expression of CD44 in OPCs was transient and.N reported to be associated with aggressive tumor growth and proliferation [34,35]. Hence, CD44 might be an important molecule for proliferation of cerebellar progenitor cells. Notably, OPCs also expressed CD44, and the timing of CD44 expression in these cells was restricted. CD44 expression disappeared during oligodendrocyte maturation (Fig. 7). CD44 expression was also observed in subpopulations of immature neurons (Fig. 8). CD44 was expressed in immature Purkinje neurons at P7, after which expression of CD44 disappeared from the PCL (Fig. 8). In contrast, granule neurons expressed CD44 more strongly in the adult than during developmental stages (Fig. 8). Thus, the expression of CD44 was restricted to granule neurons and had a regulated time course during development.CD44 is expressed by hematopoietic cells, including stem and progenitor cells [10,11,36] and cancer stem cells [16]. In the central nervous system, it was reported that a subset of CD44+/ CD90+ cells in the cortex was capable of neurosphere initiation [37]. In this study, we indicated that CD44 was expressed in the ventricular zone of the cerebellum at early embryonic stages (Fig. 2) and in Sox2-positive cells at early postnatal stages (Fig. 5). A part of CD44-positive cells yield neurospheres (Fig. 1). Based on these results, we hypothesize that CD44 is expressed in neural progenitor cells, including neural stem cells, in both the embryonic ventricular zone and postnatal WM. Therefore, further careful analysis will be required to determine whether CD44-positive cells give rise to only astrocytes, or give rise to not only astrocytes but also other types of cells in vivo. In addition to fibrous astrocytes, we found that CD44 was also transiently expressed in Bergmann glia (Fig. 6). Hyaluronic acid, which is one of the ligands of CD44, is strongly expressed in the WM at P7 and P15 in the cerebellum [38]. The interaction of hyaluronic acid and CD44 in astrocytes stimulates Rac1 signaling, leading to cytoskeletal effects and cell migration [39], suggesting that Bergmann glia and fibrous astrocytes might require CD44 for their migration. Recently, it was reported that cleavage of CD44 generates a CD44 intracellular domain (ICD) that functions as a transcription factor [15]. This transcriptional cascade might be important for cerebellar development. On the other hand, differentiating protoplasmic astrocytes were CD44 negative,CD44 Expression in Developing CerebellumFigure 5. CD44 expression in neural stem/progenitor cells during postnatal development. A : Double immunostaining of CD44 and Sox2 in the cerebellum at P3 (A ), P7 (D ) and P14 (G ). Nucleus was counterstained with TO-PRO-3 (blue). J: Quantitative analysis of the number of CD44-positive neural stem/progenitor cells by FACS at P3, P7 and P10. *p,0.05, 1527786 **p,0.005, ***p,0.001. Scale bars, 20 mm. doi:10.1371/journal.pone.0053109.galthough most of astrocyte precursors seemed to be CD44-positive (Fig. 6). 11967625 Thus there is a possibility that CD44 expression must be shut off in astrocyte precursor cells, if they differentiate into protoplasmic astrocytes. Alternatively, generation of protoplasmic astrocytes might be independent from CD44-positive astrocyte precursors. So far we cannot address these questions, since we have never done the lineage analysis. Fine lineage analysis andfurther functional analysis is necessary to determine the roles of CD44 in the developing cerebellum. The expression of CD44 in OPCs was transient and.
