Experimental Neurology 201 (2006) 515 – 518 www.elsevier.com/locate/yexnr
Brief Communication
Musashi1 antigen expression in human fetal germinal matrix development Charles Chan a , Brian E. Moore a , Carl W. Cotman b , Hideyuki Okano c , Rosemarie Tavares a , Virginia Hovanesian a , Halit Pinar a , Conrad E. Johanson a , Clive N. Svendsen d , Edward G. Stopa a,⁎ b
a Brown Medical School, Providence, RI 02903, USA Institute for Brain Aging and Dementia, University of California, Irvine, CA 92697, USA c Department of Physiology, Keio University School of Medicine, Tokyo, Japan d The Waisman Center, University of Wisconsin-Madison, Madison, WI 53706, USA
Received 4 May 2004; revised 7 February 2006; accepted 10 April 2006 Available online 14 June 2006
Abstract Musashi1 is a highly conserved protein found in neural progenitor cells. We examined the expression dynamics of Musashi1 in conjunction with other representative neural progenitor antigenic determinants (Ki-67 and nestin) during 8 different stages of the developing human fetal germinal matrix. Our results indicate that Musashi1 is a useful marker for immature cells in periventricular areas inhabited by stem cells, progenitor cells, and differentiating cells. © 2006 Elsevier Inc. All rights reserved. Keywords: Germinal matrix; GFAP; Ki-67; Musashi1; Nestin; Neural progenitor cell; Subventricular zone
Neurons in the adult mammalian brain were postulated to be irreplaceable if lost through disease. Recent evidence from studies utilizing Bromodeoxyuridine (BrdU) has demonstrated the continued presence of adult neurogenesis in the mammalian brain (Eriksson et al., 1998). BrdU-positive hippocampal cells of adult macaque monkeys have been shown to co-label with either neuronal or glial lineage identifying markers (Kornack et al., 1999), suggesting the presence of multipotent CNS stem cells in the adult primate hippocampus. BrdU incorporation is currently the method of choice for demonstrating the presence of CNS progenitor cells due to the lack of specific protein markers. However, BrdU incorporation imposes its own set of limitations, mainly the requirement of live rather than post-mortem tissue. Here, we examine the protein Musashi1 as a potential CNS progenitor cell marker. Musashi1 is an RNA-binding protein required for two successive asymmetric divisions of sensory organ precursor cells in Drosophila (Okabe ⁎ Corresponding author. Rhode Island Hospital, APC, 12-219, Providence, RI 02903, USA. E-mail address:
[email protected] (E.G. Stopa). 0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2006.04.023
et al., 2001). Immunohistochemical and in-situ hybridization studies in mice have demonstrated that Musashi1 expression is highly enriched in the developing CNS (Kaneko et al., 2000). Musashi1 is also expressed by human neurospheres. In-vitro experiments show that neurospheres lacking expression of both the Musashi1 and Musashi2 genes are unable to maintain proliferative activity, while deletion of Musashi1 alone resulted in reduced multipotency (Sakakibara et al., 2002). To this extent, Musashi1 seems to be a potential marker for identifying neural and glial progenitor cells in post-mortem tissue (Kanemura et al., 2001; Keyoung et al., 2001). To further characterize Musashi1(+) cells in the human fetal CNS, we examine Musashi1 expression during 8 stages of human fetal development. We demonstrate that Musashi1 colocalizes with nestin and Ki-67 in immature cells and that Musashi1 expression correlates in time and proximity with the transient expression of the human fetal germinal matrix. Normal brain samples were obtained from archival tissue specimens that had been previously sanctioned by the Institutional Human Subjects Committee for teaching and investigative purposes. These tissues were originally fixed in
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10% neutral buffered formalin and subjected to a thorough neuropathologic examination. All studies were performed using coronal sections at the mid-infundibular level. The specificity of the Musashi1 antibody 14H1 has been extensively characterized in previous studies (Sakakibara et al., 2002; Kanemura et al., 2002). In addition to 14H1, the following commercial antibodies were used: glial fibrillary acidic protein (Boehringer Mannheim), Mib-1 (Dako) and nestin (Chemicon). Five micron paraffin-embedded tissue sections were deparaffinized and rehydrated through xylene and graded alcohols then pre-treated with 30% H2O2. Tissue sections probed for Ki67 or Musashi1 underwent an additional step of antigen retrieval using citrate buffer. Sections were blocked with 5% normal horse serum and incubated in primary antibodies for 1 h. Visualization was achieved using a commercial biotin–streptavidin system and then reacted with diaminobenzidine (Vector Labs). For double immunofluorescent labeling, samples underwent identical fixation, rehydration, and antigen retrieval steps
as described in DAB staining methods. Samples were blocked in both 5% normal horse and goat sera and incubated for 1 h with 14H1 plus Mib-1 or 14H1 plus anti-nestin antibody. Sections were incubated in secondary antibodies (Cy3-conjugated goat anti-rabbit and Cy2-conjugated goat anti-mouse; Jackson Laboratory) for 2 h at 4°C. To eliminate lipofuscin autofluorescence, sections were post-treated with 0.3% Sudan Black B. At 12-week gestation, a significant level of Musashi1 expression was observed in tightly packed, round cells in the subventricular zone of the lateral ventricles (Fig. 1A). The pattern of expression corresponds to the well described developing germinal matrix which is several layers thick and lines the ventricular wall at this stage of gestation. There is early evidence of migration of Musashi1(+) cells to the developing cerebral cortex. The ependyma has not formed yet, except in the immediate vicinity of the choroid plexus, where ependymal differentiation first appears as the distinct staining of a monolayer of cells.
Fig. 1. A, D, G, and J are the fetal ependyma/germinal matrix stained for Musashi1. B, E, H, and K are the fetal ependyma/germinal matrix stained for GFAP. C, F, I, and L are the fetal ependyma/germinal matrix stained for Ki-67. The dark arrows demarcate the border of the fetal ependyma. GM indicates the developing germinal matrix. All images are viewed at 100×.
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At 20-week gestation, Musashi1 immunoreactivity has become more extensive on the tissue plane corresponding to the proliferation of the germinal matrix (Fig. 1D). The immunoreactivity is most intense near the ventricular border illustrating the formation of the ependyma that is now 2 to 3 cell layers thick. There is again widespread evidence of cell migration, although cells adjacent to the germinal matrix appear to stain more robustly than cells further away from the ependyma. At 24-week gestation, the intensity of Musashi1 immunoreactivity is diminished, though the zone containing Musashi1(+) cells has enlarged, suggesting continued outward migration of Musashi1(+) cells (Fig. 1G). The trend of increasing Musashi1 immunoreactivity continues until 27-week gestation when there is evidence of diminishing immunoreactivity within both the ependyma and germinal matrix. By 40-week gestation, the number of Musashi1(+) cells has markedly decreased in contrast to 12week gestation, corresponding to the disappearance of the germinal matrix (Fig. 1J). In contrast to the Musashi1 staining, there is little evidence of GFAP immunoreactivity within the germinal matrix or elsewhere in the CNS at 12 weeks of gestation (Fig. 1B). GFAP staining is minimal in the ventricular wall at 16-week gestation (Fig. 1E) From 20-week gestation onward, there is an increase in GFAP immunoreactivity within the primitive ependymal layer (Fig. 1H). There is also evidence of GFAP (+) cells in areas of the brain distant from the germinal matrix. Robust GFAP expression is not observed in the proximity of the germinal matrix until 34 weeks gestation, corresponding with the disappearance of the germinal matrix and suggesting maturation of the progenitor cells to glia (Fig. 1K). The spatiotemporal expression of Ki-67 approximately follows the trend seen in Musashi1 expression in the fetal CNS
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until 34-week gestation at which they diverge. At 12-week gestation, a significant level of Ki-67 expression is found in tightly packed round cells in the subventricular zone of the lateral ventricles similar to the population expressing Musashi1 (Fig. 1C). Ki-67 expression is robust in both the developing ependyma and germinal matrix at 16-week gestation. There is also increasing evidence of Ki-67 expressing cells migrating towards the developing cortex. This trend of immunoreactivity is observed until 24-week gestation when Ki-67 expression is clearly diminished in the ependyma and germinal matrix in contrast to earlier stages of gestation (Fig. 1F). By 27-week gestation, Ki-67 immunoreactivity is weakly present within the ependyma and (Fig. 1I) all but disappeared by 34-week gestation (Fig. 1L). To characterize the replicative potential of Musashi1(+) cells, a double immunofluorescent co-localization studies of Musashi1 with nestin and Ki-67 were performed. Musashi1 and nestin, both cytoplasmic elements, demonstrated co-localization (Fig. 2A). Cells expressing both Ki-67 and Musashi1 were found within the ependyma and germinal matrix (Fig. 2B). Our results demonstrate an increase in GFAP expression and a concomitant decrease in both Musashi1 and Ki-67 with advancing gestational age. Although the cellular proliferation marker Ki-67 is not a specific indicator of progenitor phenotype, high proliferation rate is a characteristic likely to be seen in progenitor cells. Furthermore, Musashi1 expression is pronounced in the germinal matrix and persists at lower levels in the subventricular zone after germinal matrix regression following a pattern parallel to that of immature neural cells. Finally, confocal immunofluorescent microscopy demonstrates Musashi1 co-localization with Ki-67 and nestin. The presence of Musashi1(+)/Ki-67(−) and Musashi1(+)/nestin(−) cells suggests the presence of quiescent progenitor cells. These observations taken together suggest that Musashi1 is an antigen
Fig. 2. (A and B) Confocal microscopy. A1 is fetal ependyma at 22 weeks gestation stained for Musashi1 with Cy3 (red). A2 shows Cy2 (green) nuclear Ki-67 immunoreactivity. A3 is a merged view demonstrating cytoplasmic Musashi1 (red) and nuclear Ki-67 (green) staining respectively. B1 is fetal ependyma at 22 weeks gestation stained with Musashi1 Cy3 (red). B2 shows nestin Cy2 (green) immunoreactivity. B3 is a merged view demonstrating the co-localization (yellow) of Musashi1 and nestin immunoreactivity.
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expressed by immature cells in areas inhabited by progenitor cells, differentiating neurons, and glia. Musashi1 expression is not limited to the putative CNS progenitor cells but persists in the processes of astrocytes in brain tissue at 27-week gestation. These Musashi1 (+), GFAP (+) cells could represent a transitional state from progenitor to astrocyte. Alternatively, this observation may indicate that astrocytes can serve as a reserve of multipotent progenitor cells in the mature brain. Doetsch et al. (Doetsch et al., 1997, 1999) have presented evidence that reactive astrocytes at the site of ischemic and mechanical subventricular zone lesions in the adult rodent brain begin to express nestin and are capable of converting to multipotent neural stem cells. Other groups, however, have variously described neural stem cells as having astrocytic (Bernier et al., 2000), neuronal (Nakamura et al., 2003), or hematopoietic lineages (Hess et al., 2002). The determination of neural stem cell derivation is further complicated by lack of agreement regarding the defining characteristics of these cells (Moore and Quesenberry, 2003). As research extends to the therapeutic use of stem cells in such conditions as neurodegenerative disease, the identification of endogenous neural progenitor populations becomes increasingly important. Musashi1 will prove useful in mapping the location of immature neural cells within the human CNS. Additional studies will be necessary to further characterize the antigenic profile of neural progenitor cells as new potential markers are discovered. Acknowledgments This study was supported in part by grants RR-00-003 and NIH NS27601. References Bernier, P.J., Vinet, J., Cossette, M., Parent, A., 2000. Characterization of the subventricular zone of the adult human brain: evidence for involvement of bcl-2. Neurosci. Res. 37, 67–68. Doetsch, F., Garcia-Verdugo, J.M., Alvarez-Buylla, A., 1997. Cellular composition and three-dimensional organization of the subventricular
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