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,
,
,1
* Department of Biology, School of Education and
Integrative Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, Tokyo, Japan;
Section for Studies on Metastasis,
Genetics Division, National Cancer Center Research Institute, Tokyo, Japan;
|| First Department of Surgery, Nara Medical University, Nara, Japan
1 Correspondence: Section for Studies on Metastasis, National Cancer Center Research Institute, 5-1-1, Chuo-ku, Tsukiji, Tokyo 104-0045 Japan. E-mail: tochiya{at}ncc.go.jp
SPECIFIC AIMS
The FGF-4 (fibroblast growth factor 4, known as HST-1) protein, is an important mitogen for a variety of cell types. However, only limited information is available concerning tissue distribution and the biological role of FGF-4 in the brain. To clarify the exact role of FGF-4 in neuronal development, we have examined here in detail its tissue distribution in the neonatal mouse brain and its function in neurosphere development.
PRINCIPAL FINDINGS
1. Fgf-4 mRNA is expressed in neural progenitor cells and neurospheres
To detect the localization of Fgf-4 mRNA expression in the mouse brain, we performed in situ hybridization (Fig. 1
). As illustrated in Fig. 1A-D
, Fgf-4 expressing cells were observed in the subventricular zone (SVZ), hippocampus, and rostral migratory stream (RMS), regions where adult neurogenesis is continuously occurring. This is emphasized by the presence of many bromodeoxyuridine (BrdU)-positive nuclei in areas such as the SVZ, hippocampus, and RMS (Figs. 1A-C
, respectively), which are positive for Fgf-4 expression. The results in Fig. 1A-C
suggest that FGF-4 may have the ability to promote NSC (neural stem cell) proliferation or differentiation. Therefore, we further explored the functional role of FGF-4 using a neurosphere culture system. First, we used RT-polymerase chain reaction (PCR) analysis to quantitate Fgf-4 mRNA in neurospheres (Fig. 1D
). The results show the detection of Fgf-4 mRNA in the whole brain, from embryos at 14.5E, ganglionic eminence, and neurospheres. Next, we examined the expression of FGFR-1 and FGFR-2, high-affinity receptors for FGF-4 and found that they were expressed in neurospheres (Fig. 1E
). These results suggest the existence of relationships between FGF-4 and neural stem cell behavior in vivo and in vitro.
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2. FGF-4, like FGF-2, induces neurosphere proliferation
To further explore the role of FGF-4 in NSC proliferation, we investigated whether recombinant FGF-4 could stimulate proliferation in the neurosphere model. Primary germinal zone cells from the ganglionic eminence of E14 mouse embryo were plated into single wells of a 96-well plate in the presence of growth factors. FGF-4 increased the neurosphere numbers, as did FGF-2. To test the self-renewal capacity of cells proliferating in the presence of FGF-4, the spheres were dissociated individually and plated in the same initial growth medium. The result indicated that almost all FGF-4-generated spheres can generate spheres after dissociation. Consistent with a role in stem cell regulation, FGF-4 generated neurospheres exhibited enhanced self-renewal capacity compared with that of FGF-2 generated neurospheres. Specifically, FGF-4-generated primary neurospheres produced 25 ± 4/500 cells as secondary neurospheres in contrast to 17 ± 4/500 cells for FGF-2 generated neurospheres. We also found that this effect of FGF-4 was dose-dependent. Moreover, FGF-4-generated spheres could produce neurons, astrocytes, and oligodendrocytes. Thus, FGF-4 is sufficient to maintain self-renewal as well as the multipotentiality of striatal NSCs. These results indicate that FGF-4 appears to be sufficient for renewal of an NSC that has the ability to differentiate into neurons, astrocytes, and oligodendrocytes.
3. FGF-4 induces stem cell differentiation to neurons
Our in situ hybridization analysis reveals Fgf-4 transcripts in the murine SVZ, hippocampus, and RMS, regions where adult neurogenesis is continuously occurring. These results suggest that FGF-4 may also have a role in stimulating neuronal differentiation. Therefore, we examined whether the addition of FGF-4 could increase the number of differentiated neurons produced by stem cells in culture. As shown in Fig. 2
A and B, recombinant FGF-4 induced a significant increase in neuron numbers (MAP2-immunoreactive cells with neuronal morphology). This effect was comparable to that of FGF-2 at 2–20 ng/ml. These results suggest that FGF-4 preferentially induces neuronal differentiation in epidermal growth factor (EGF)-generated neurospheres.
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CONCLUSION AND SIGNIFICANCE
To our knowledge, this paper is the first direct demonstration of: 1) spatial localization of Fgf-4 in the mouse brain; 2) FGF-4 involvement in the proliferation of neural progenitor cells; and 3) FGF-4 dependent differentiation of neural precursor cells along neuronal lineages. These results strongly suggest the possibility of physiological FGF-4 functions in the central nervous system (Fig. 3
). In light of the many recent reports related to applying neural stem cells to neurological disorders, understanding in detail the roles of various growth factors and cytokines in stem cell biology will be critical. In this respect, we propose that FGF-4 offers a new strategy for restoring neurogenesis in a clinical setting.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5293fje
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  M. Timmer, K. Cesnulevicius, C. Winkler, J. Kolb, E. Lipokatic-Takacs, J. Jungnickel, and C. Grothe Fibroblast Growth Factor (FGF)-2 and FGF Receptor 3 Are Required for the Development of the Substantia Nigra, and FGF-2 Plays a Crucial Role for the Rescue of Dopaminergic Neurons after 6-Hydroxydopamine Lesion J. Neurosci., January 17, 2007; 27(3): 459 - 471. [Abstract] [Full Text] [PDF]
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