Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Analysis of neurogenesis using transgenic mice expressing GFP with nestin gene regulatory regions
Department of Physiology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Correspondence to be sent to: Masahiro Yamaguchi, e-mail: yamaguti{at}m.u-tokyo.ac.jp
Key words: dentate gyrus, granule cell, neural stem cell, olfactory bulb, periglomerular cell, progenitor
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Introduction |
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 Top Introduction Embryonic periodNeonate and adultImplications of GFP expression...ConclusionReferences |
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The complex structure of mammalian central nervous system (CNS) originates from multipotent neural stem cells. Even in the adult brain neural stem cells persist and neurogenesis continues in some regions (Alvarez-Buylla and Garcia-Verdugo, 2002
;
Kempermann et al., 2004).
Furthermore, recent observations are revealing a potential of non-neurogenic CNS regions
to generate neurons in pathological conditions (Lie et al., 2004). Unraveling the mechanisms of
neurogenesis is therefore important to understand the development, remodeling, and
restoration of CNS structure and function.
To facilitate experimental analysis of neurogenesis in the mammalian CNS, effective
identification of stem or progenitor cells is crucial. Several groups have generated
transgenic mice expressing a marker protein such as green fluorescent protein (GFP) under
the control of nestin gene regulatory regions (Yamaguchi et al., 2000;
Kawaguchi et al., 2001;
Beech et al., 2004;
Mignone et al., 2004).
Nestin is a class IV intermediate filament expressed in stem cells and progenitor cells
of the nervous system (Lendahl et al.,
1990). This review aims to summarize how the transgenic mice have been
utilized and discuss potentials and reservations of using this animal system.
For the transgene construction, we utilized the second intron of nestin gene, which
was known to drive the expression in neural stem and progenitor cells (Zimmerman et al., 1994). We also
included the 5' upstream region (promoter region) in the transgene construct, whose
regulatory function is still unclear.
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Embryonic period |
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TopIntroductionEmbryonic periodNeonate and adultImplications of GFP expression...ConclusionReferences |
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At embryonic day 11.5, GFP expression was observed in the neuroepithelium, with the strong expression in the ventricular zone. Kawaguchi et al. (2001
)
generated transgenic mice that express GFP under the control of the second intron of
nestin gene and Hsp 68 minimal promoter, in which strong GFP expression was similarly
observed in the ventricular zone. By sorting GFP-fluorescent cells, they successfully
enriched the stem cell population from embryonic forebrain as well as from the
periventricular area of adult forebrain. Likewise, GFP-fluorescent cells were isolated
from the mesencephalic region of embryos and expanded in vitro. These cells were
transplanted to Parkinson’s disease model mice, which restored the animals from the
abnormal movement (Sawamoto et al.,
2001).
Yoshida et al. (2003) sorted
GFP-fluorescent progenitor cells from telencephalic regions and showed the correlation
between their differentiation and integrin expression. These results indicate the
usefulness of the animal system for the isolation of a stem cell population that is
applicable to functional restoration of CNS diseases.
Importantly, GFP in the transgenic mice could be observed not only in authentic
neuroepithelial cells but also in radial glia (Yamaguchi et al., 2000;
Kawaguchi et al., 2001).
Radial glia is now considered to be the neural stem cell in various brain regions
(Anthony et al., 2004). GFP
expression in radial glia would be useful for analyzing the most initial events of
neurogenesis.
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Neonate and adult |
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TopIntroductionEmbryonic periodNeonate and adultImplications of GFP expression...ConclusionReferences |
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At postnatal day 7, strong GFP expression was observed in the dentate gyrus of hippocampus and olfactory system that extends from periventricular zone to the olfactory bulb (OB) via the specific pathway called rostral migratory stream (RMS) (Figure 1A). In addition, GFP expression was observed in the cerebellum, where massive neurogenesis occurs in postnatal period. In the adult animals (Figure 1B), strong GFP expression continued in the two neurogenic regions, the dentate gyrus of hippocampus and the olfactory system, but extinguished from the cerebellum. These results indicate that GFP expression generally occurs when and where neurogenesis is undergoing.
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Stem cells in the RMS are considered to be GFAP positive (Alvarez-Buylla and Garcia-Verdugo, 2002
). In the
RMS of our transgenic mice, GFAP-expressing cells were GFP-positive, suggesting that RMS
stem cells were visualized by GFP fluorescence. Migrating neuroblasts that express
PSA-NCAM (Alvarez-Buylla and Garcia-Verdugo,
2002) were also GFP-positive in our transgenic mice. In the OB, GFP-positive
cells were scattered in the granule cell layer and glomerular layer (Figure
1C). BrdU labeling showed that some of
newly generated neurons that migrate into the OB were GFP-positive (Figure
1D). Some GFP-expressing cells in the
granule cell layer extended radial processes, which might correspond to radially
migrating ‘class 2’ granule cells (Petreanu and Alvarez-Buylla, 2002). Thus GFP expression
appears to visualize cells from their multipotent precursor stage to the early stage of
the maturation into neuronal cells.
In the hippocampal dentate gyrus, GFP-expressing cells were mostly confined to the
subgranular layer. Detailed morphological, molecular, and electrophysiological analysis
revealed that there were two types of GFP-expressing cells (Filippov et al., 2003;
Fukuda et al., 2003). One
type of cells had astrocytic features, while the other type of cells showed phenotypes of
neuronal lineage. It was proposed that the latter cells were produced from the former
ones. Interestingly, exposure of animals to environmental complexity and voluntary
physical activity differentially affected the population of GFP-expressing cells
(Kronenberg et al., 2003).
These results shed light on the mechanisms of morphological and functional
differentiation during early steps of neurogenesis.
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Implications of GFP expression in various transgenic mouse lines |
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TopIntroductionEmbryonic periodNeonate and adultImplications of GFP expression...ConclusionReferences |
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Mignone et al. (2004
)
generated transgenic mice expressing GFP using the second intron of nestin gene and the
5' promoter region (5.8 kb) which is longer than that in our mice (2.5 kb). GFP
expression pattern in the mice appeared quite similar to our mice. In the adult animals
they divided GFP-expressing cells into GFP-bright and GFP-dim cells, and showed that
GFP-bright cells express GFAP while GFP-dim cells express a neuronal marker,
ßIII-tubulin.
Kawaguchi et al. (2001)
divided GFP-expressing cells into GFP weakly-positive and strongly-positive cells, and
demonstrated that cells with stem cell-like properties were enriched in the strongly
positive cell population of embryos. These results indicate that GFP fluorescence level
can be utilized to segregate distinct populations of precursor cells.
It should be cared that GFP expression might not faithfully reproduce endogenous
nestin expression. In the mice raised by
Mignone et al. (2004), most
GFP-positive periglomerular cells in the OB are negative for nestin expression. In our
transgenic mice also, some GFP-expressing cells in the OB are negative for nestin protein
(unpublished observation). Implication of GFP expression should be confirmed in
individual types of cells and brain regions, in individual animal lines using different
transgene constructs.
Beech et al. (2004) have
generated transgenic mice expressing a marker protein using the 5.8 kb 5' promoter
region and the 5.4 kb downstream region that contains all three introns and adjacent exon
sequences of the nestin gene. Intriguingly, the marker expression in the OB of the mice
was observed only in periglomerular cells but not in granule cells. They suggest that the
transgene expression represents a subset of cells which differentiates exclusively into
periglomerular cells.
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Conclusion |
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TopIntroductionEmbryonic periodNeonate and adultImplications of GFP expression...ConclusionReferences |
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Utilizing GFP fluorescence in the nestin gene regulatory region–GFP transgenic mice, isolation, morphological analysis and functional analysis for neural stem and precursor cells have been facilitated. However, the profile of GFP expression might not always be faithful to the endogenous nestin expression, and the expression appears to vary among animal lines with different transgene constructs. Thus the phenotypes of GFP-expressing cells need to be carefully examined in every case of analysis. Detailed analysis would make such heterogeneity advantageous for dissecting the mechanisms of neurogenesis.
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References |
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TopIntroductionEmbryonic periodNeonate and adultImplications of GFP expression...ConclusionReferences |
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Alvarez-Buylla, A. and Garcia-Verdugo, J. (2002) Neurogenesis in adult subventricular zone. J. Neurosci., 22, 629–634.
Anthony, T.E., Klein, C., Fishell, G. and Heintz, N. (2004) Radial glia serve as neuronal progenitors in all regions of the central nervous system. Neuron, 41, 881–890.[CrossRef][Web of Science][Medline]
Beech, R.D., Cleary, M.A., Treloar, H.B., Eisch, A.J., Harrist, A.V., Zhong, W., Greer, C.A., Duman, R.S. and Picciotto, M.R. (2004) Nestin promoter/enhancer directs transgene expression to precursors of adult generated periglomerular neurons. J. Comp. Neurol., 475, 128–141.[CrossRef][Web of Science][Medline]
Filippov, V., Kronenberg, G., Pivneva, T., Reuter, K., Steiner, B., Wang, L.P., Yamaguchi, M., Kettenmann, H. and Kempermann, G. (2003) Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes. Mol. Cell. Neurosci., 23, 373–382.[CrossRef][Web of Science][Medline]
Fukuda, S., Kato, F., Tozuka, Y., Yamaguchi, M., Miyamoto, Y. and Hisatsune, T. (2003) Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus. J. Neurosci., 23, 9357–9366.
Kawaguchi, A., Miyata, T., Sawamoto, K., Takashita, N., Murayama, A., Akamatsu, W., Ogawa, M., Okabe, M., Tano, Y., Goldman, S.A. and Okano, H. (2001) Nestin-EGFP transgenic mice: visualization of the self-renewal and multipotency of CNS stem cells. Mol. Cell. Neurosci., 17, 259–273.[CrossRef][Web of Science][Medline]
Kempermann, G., Wiskott, L. and Gage, F.H. (2004) Functional significance of adult neurogenesis. Curr. Opin. Neurobiol., 14, 186–191.[CrossRef][Web of Science][Medline]
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Lendahl, U., Zimmerman, L.B. and McKay, R.D.G. (1990) CNS stem cells express a new class of intermediate filament protein. Cell, 60, 585–595.[CrossRef][Web of Science][Medline]
Lie, D.C., Song, H., Colamarino, S.A., Ming, G.L. and Gage, F.H. (2004) Neurogenesis in the adult brain: new strategies for central nervous system diseases. Annu. Rev. Pharmacol. Toxicol., 44, 399–421.[CrossRef][Web of Science][Medline]
Mignone, J.L., Kukekov, V., Chiang, A.S., Steindler, D. and Enikolopov, G. (2004) Neural stem and progenitor cells in nestin-GFP transgenic mice. J. Comp. Neurol., 469, 311–324.[CrossRef][Web of Science][Medline]
Petreanu, L. and Alvarez-Buylla, A. (2002) Maturation and death of adult-born olfactory bulb granule neurons: role of olfaction. J. Neurosci., 22, 6106–6113.
Sawamoto, K., Nakao, N., Kakishita, K., Ogawa, Y., Toyama, Y., Yamamoto, A., Yamaguchi, M., Mori, K., Goldman, S.A., Itakura, T. and Okano, H. (2001) Generation of dopaminergic neurons in the adult brain from mesencephalic precursor cells labeled with a nestin-GFP transgene. J. Neurosci., 21, 3895–3903.
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Yoshida, N., Hishiyama, S., Yamaguchi, M., Hashiguchi, M., Miyamoto, Y., Kaminogawa, S. and Hisatsune, T. (2003) Decrease in expression of alpha 5 beta 1 integrin during neuronal differentiation of cortical progenitor cells. Exp. Cell Res., 287, 262–271.[CrossRef][Web of Science][Medline]
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