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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
1Correspondence: 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
ABSTRACT
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. In situ hybridization analysis revealed localization of mouse Fgf-4 mRNA in the normal postnatal mouse hippocampus, subventricular zone (SVZ), and the rostral migratory stream where new neurons generate, migrate, and become incorporated into the functional circuitry of the brain. We also investigated whether FGF-4 could promote both proliferation and differentiation of the neural progenitor cells by using an in vitro neurosphere assay. The addition of recombinant FGF-4 generated large proliferative spheres that have a multipotent differentiation ability. Furthermore, recombinant FGF-4 significantly promotes neuronal differentiation in attached clonal neurosphere culture. These findings suggest that FGF-4 has an ability to promote neural stem cell proliferation and neuronal differentiation in the postnatal brain.—Kosaka, N., Kodama, M., Sasaki, H., Yamamoto, Y., Takeshita, F., Takahama, Y., Sakamoto, H., Kato, T., Terada, M., Ochiya, T. FGF-4 regulates neural progenitor cell proliferation and neuronal differentiation.
Key Words: FGF-4 • neural progenitor cell • neurogenesis • self-renewal
THE HST-1(fibroblast growth factor 4, FGF4) gene was first cloned in our laboratory as a transforming gene in the NIH3T3 assay system and was subsequently identified as a member of the fibroblast growth factor (FGF) gene family (1
, 2)
. The product, FGF-4 is prominently involved in embryogenesis (3
4
5
6
7
8
9)
. We have previously shown that FGF-4 has potent angiogenic activity (10)
and is a potent inducer of platelet production from megakaryocytes (11
12
13)
. FGF-4 expression has been reported in adult mouse testis, intestine, and brain (14
15
16)
. Previously we have reported that the Fgf-4 gene is expressed in the brain at both neonatal and adult stages (17)
. Fgf-4 mRNA is expressed in the cerebellum at 0, 6, and 10 d postnatally and persists in the adult through weeks 5, 10, and 38. Expression in the cerebrum is high in embryos at E14.5, persists during late embryogenesis and neonatal stages, and gradually decreases thereafter. In the cerebellum, expression of Fgf-4 is localized in the Purkinje cell layer (17)
. The Fgf-4 gene is also expressed in the medulla oblongata, spinal cord, ischiatic nerve, and cerebrum. In combination with Shh, FGF-8 and FGF-4 induce 5HT neurons to develop in the hindbrain (18)
. However, no evidence suggests a FGF-4 function in neuronal commitment. Since FGF family members play an important role in telencephalic development (19
, 20)
, we have speculated that FGF-4 might have the ability to stimulate neural progenitor proliferation and/or promote neuronal differentiation of neural stem cells (NSC). To clarify the exact role of FGF-4 in neuronal development, we have been examined here in detail its tissue distribution and its function in neurosphere development.
We have localized Fgf-4 mRNA in several key areas of the normal postnatal mouse brain, including the hippocampus, the subventricular zone (SVZ), and the rostral migratory stream (RMS). RT-polymerase chain reaction (PCR) analysis detected Fgf-4 expression in embryonic day 14 (E14) mouse brain and ganglionic eminence, and in cultured neurospheres. Exogenous FGF-4 significantly stimulates neurosphere proliferation. Under differentiation conditions, the number of neurons in the neurospheres significantly increased in the presence of FGF-4. These results suggest that FGF-4 is physiologically significant for neural progenitor cell proliferation and for neuronal differentiation.
MATERIALS AND METHODS
Cell culture
Generation and differentiation of spheres from embryonic forebrain were performed as described previously with minor modifications (21)
. Briefly, pregnant ICR mice of gestational age 14.5 d (E14.5) were killed via cervical dislocation, and uteri were aseptically removed and transferred to Petri dishes containing sterile Dulbecco’s PBS with 10% antibiotic antimycotic Solution (Sigma, St. Louis, MO). Each decidua from the uterine sac was dissected out and transferred to a new sterile Petri dish containing PBS in order to rinse away excess blood. Deciduas were then transferred to another sterile Petri dish containing PBS, and the amniotic sac was gently removed. Embryos were removed from the amnion, and the heads were dissected using tweezers. After removal of the overlying meninges and blood vessels, the ganglionic eminence was dissected out and transferred to serum-free media. Cells were mechanically dissociated with a Pasteur pipette. Cells were plated in 96-well (0.2 ml/well), 24-well (0.5 ml/well), 6-well (1.5 ml/well), or 6 cm dish (1.5 ml/well) uncoated plates (Nunclon Copenhagen, Denmark) depending on the experimental design. Dulbecco’s modified Eagle medium/F-12 was supplemented with B-27 (GIBCO Gaithersburg, MD), 1% antibiotic Antimycotic Solution (Sigma), and epidermal growth factor (EGF) (20 ng/ml; R&D Systems, Minneapolis, MN), 20 ng/ml basic fibroblast growth factor (20 ng/ml; R&D Systems) or FGF-4 (20 ng/ml).
The protocol employed in this study meets the Guideline of Animal Experimentation of the Japanese Society for Pharmacology and was approved by the Committee for Ethical Use of Experimental Animals at the National Cancer Center Research Institute. All efforts were made to minimize animal suffering, to reduce the number of animals used and to utilize alternatives to in vivo techniques.
Primary sphere formation assay
Primary neurosphere formation was performed according to the described method with minor modifications (22)
. In brief, mechanically dissociated ganglionic eminence cells were plated at 500 cells/0.2 ml proliferation medium in each well of a 96-well plate. The number of spheres was counted after 8–10 d in vitro (DIV). Six to eight wells per condition tested were counted. For the FGF-4 dose-response curve, cells were incubated with different concentrations of FGF-4. Cell growth was examined by a colorimetric assay. Briefly, cells were incubated at a density of 500 cells/0.2 ml in 96-well plates in IMDM containing 5% FCS in the presence of various concentrations of FGF-4. After 72 h of culture at 37°C, 10 µl of TetraColor ONE (SEIKAGAKU Corporation, Tokyo, Japan) was added to each well. Following 2–4 h incubation at 37°C, the optical density was measured at a wavelength of 450 nM using a microplate reader. All experiments were done in triplicate.
Secondary sphere formation assay
Primary spheres were collected and passaged by mechanically dissociating neurospheres in 0.2 ml serum-free media containing either 20 ng/ml FGF-2 or 20 ng/ml FGF-4, and plated at 500 cells/well in uncoated 96-well plates. The number of spheres was counted after 8–10 d in vitro. Six to eight wells per condition tested were counted.
For an experiment of clonal analysis, the primary neurospheres were dissociated and the cells were plated at a clonal density 
10 cells/well in 96-well uncoated plates in a serum-free media containing 20 ng/ml FGF-2 or FGF-4. The cell number in microwells was scored after 24 h and showed that 4–16 cells per well existed. To examine for self-renewal capacity more stringently, the primary neuropheres were dissociated and transferring single cells to microwells. Neurospheres were scored after 14 d in either FGF-4 or FGF-2.
Antibodies
Primary antibodies for indirect immunocytochemistry included (final dilution, source): mouse IgG monoclonal antibody (mAb) to MAP2 (1:100; Sigma), rabbit polyclonal antibody to GFAP (1:500; Chemicon Temecula, CA), mouse IgM mAb to O4 (1:20; Chemicon). Secondary antibodies were as follows: AlexaFluor594 goat anti-rabbit IgG (1:2000; Invitrogen Carlsbad, CA), AlexaFluor488 goat antimouse IgG (1:1000; Invitrogen), and Coumarin (AMCA)-conjugated affinity-purified goat antibody (Ab) to mouse IgM (1:100, Chemicon). An anti-5-Bromo-2'-deoxyuridine [bromodeoxyuridine (BrdU)] mAb was purchased from Laboratory Vision Corporation (Fremont, CA). The avidin-peroxidase complexes were obtained from Vector laboratories (Burlingame, CA).
Immunocytochemistry on differentiated neurospheres
Neurospheres (10–15) were transferred using a Pasteur pipette onto a 2-well chamber slide (Nunclon) (1 ml/well) coated with poly-L-ortnithine (15 µg/ml; Sigma) and Fibronectin (1 µg/ml; Sigma) in individual wells of a chamber slide in media containing 1% FBS with or without cytokine (FGF-4 or FGF-2) and kept unchanged for the rest of the culture period. Slides were processed 4 d later using immunocytochemistry. Chamber slides were fixed in 4% paraformaldehyde (in PBS, pH 7.2) for 20 min at room temperature followed by 3 (5 min each) washes in PBS (pH 7.2). Cells were then permeabilized for 5 min in PBS containing 3% Triton X-100, rinsed for 5 min (twice) in PBST (PBS plus 0.1% Triton X-100), and blocked for 30 min at room temperature in Image-iT FX (Invitrogen). After blocking, slides were washed with PBST and then incubated in primary Ab in PBST containing 10% NGS (normal goat serum) at 4°C for overnight. Slides were then rinsed three times (5 min each) in PBST. Then the slides were incubated in a secondary Ab in PBST containing 10% NGS at room temperature for 30 min. After rinsing three times (5 min each) in PBST, all slides were incubated in Hoechst 33342 nuclear stain (1 mM) for 15 min at room temperature. After being washed three times in PBST, the slides were then mounted in fluorescent mounting medium (Dako, Corporation, Carpenteria, CA). Fluorescence was visualized using a Nicon ECLIPES 1000 microscope (Nikon). Secondary Ab-only control slides were processed simultaneously using the identical protocol except that the dilution solutions were devoid of primary Ab. All secondary controls were negative for staining.
RT-PCR
Total RNAs were prepared from cells using Isogen solution (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. Total RNA (200 ng) isolated from cells were treated with DNase (DNase 1, amplification grade; Invitrogen). Reverse transcription was performed with 2 µg total RNA using One-Step RT-PCRkit (Qiagen Germantown, MD) with primers 5'-TGGTGTGACCGCAGACACGA-3' and 5'-GGTAAAGAAAGGCACACCGA-3'. After 30 min at 55°C for reverse transcription reaction of 1 µg of total RNA, the reaction was terminated at 94°C for 2 min, then PCR was performed with 35 cycles in a Takara PCR Thermal Cycler SP (TAKARA), using a cycle profile of: 1 min at 97°C, 1 min at 60°C and 40 s at 72°C. After RT-PCR, aliquots were run on 3% agarose gels, stained with ethidium bromide, and then photographed under UV illumination.
In situ hybridization
In situ hybridization experiments using fresh-frozen tissue sections and dioxygenin-labeled riboprobes were performed essentially as described previously (17)
. Adult organs and embryos were fixed with 4% parformaldehyde in PBS for 10 min at 48°C for 12 h, then dehydrated in 30% sucrose in PBS at 48°C for 4 h with gentle agitation and embedded in O.C.T. and sectioned at a thickness of 5 to 10 mM using a cryostat (Sakura Seiki, Tokyo, Japan). The sections were immediately dried at 45°C for 3 h. Before hybridization, slides were soaked in 0.2 N HCl for 20 min to inactivate endogenous alkaline-phosphatase, rinsed with deionized water, treated with proteinase K (1 mg/ml in PBS, Merk KGaA, Darmistodt, Germany) for 7 to 15 min at 37°C, hydrated with ethanol, then air-dried. An appropriate amount of the probe in 50 ml of hybridization buffer consisting of 300 mM NaCl, 30 mM sodium citrate (pH 7.0), 50% v/v deionized-formamide, 1% w/v sodium dodecyl sulfate, 50 mg/ml heparin, and 50 mg/ml yeast RNA was applied to each slide, and hybridization was performed at 65°C for 12 to 16 h. After hybridization, slides were washed with 300 mM NaCl, 30 mM sodium citrate (pH 7.0), 50% deionized-formamide at 52–55°C for 1 h. Detection was performed by antidigoxygenin Ab conjugated with alkaline-phosphatase (1:500, Boehringer Mannheim). Colormetric reaction with nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate (NBT/BCIP) was performed at room temperature: 12 h for control samples such as E9.5 limb and myotome and 96 h for all the samples from the cerebrum were required until significant signals appeared. As references, standard immunostaining and hematoxylin and eosin staining were performed in serial sections.
For BrdU labeling, animals were intraperitoneally (i.p.) injected with BrdU (Sigma) dissolved in PBS at 50 mg/kg every 12 h for 48 h to label newly synthesized DNA.
Statistical Analysis
The results are given as means ± SD Student’s t test or Bonferroni correction was performed for statistical evaluation, with P < 0.05 considered significant.
RESULTS
Fgf-4 mRNA is expressed in neural progenitor cells and neurospheres
To detect the localization of Fgf-4 mRNA expression in the brain, we carried out 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 BrdU-positive nuclei in areas such as the SVZ, hippocampus and RMS (Figs.1 A, B, and 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 proliferation or differentiation. Therefore, we further explored the functional role of FGF-4 using a neurosphere culture system. Murine embryonic striatal NSCs can be isolated in vitro by inducing their proliferation with either EGF (23)
or FGF-2 (20
, 24
, 25)
. The resulting proliferation generates a large cell cluster termed a neurosphere. Each neurosphere originates from one cell, and thus the presence of a sphere attests to the presence of a stem cell (21)
. We used RT-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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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. The number of spheres was then counted after 8–10 DIV. As shown in Fig. 2
A and B, FGF-4 increased the neurosphere numbers, as did FGF-2. No obvious differences between FGF-4- and FGF-2-generated spheres were observed. Two characteristics that distinguish a neural stem cell from a progenitor cell are (a) self-renewal and (b) multipotentiality. Whereas neurospheres may arise from progenitor cells as well as stem cells, progenitor-derived neurospheres fail to self-renew (26)
. 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 data in Fig. 2C
indicate that almost all FGF-4-generated spheres are able to generate spheres after dissociation. Consistent with a role in stem cell regulation, FGF-4 generated neurospheres exhibited enhanced self-renewal capacity compared to 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. This is consistent with a role for FGF-4 in enhancing self-renewing cell division. In these experiments, FGF-4 appeared to be sufficient to maintain NSC renewal. We also found that this effect of FGF-4 was dose-dependent (Fig. 2D
). FGF-4 is a survival factor up to 1 ng/ml, whereas at or >10 ng/ml, it is neurosphere mitogen. The maximum proliferative effect was observed at 20 ng/ml. To determine whether FGF-4 produces secondary neurospheres at clonal density, we plated dissociated cells from primary neurospheres in microwells containing 4–16 cells per well (24 h after plating) and followed by culturing for 10–14 d in the presence of FGFs. Secondary neurospheres were generated in either FGF-4 (7 spheres/713 cells) or FGF-2 (9 spheres/652 cells). To examine for self-renewal capacity more stringently, we dissociated the primary neuropheres and transferring single cells to microwells. From single cells, cell aggregates of typical neurosphere morphology appeared in the cultures of FGF-4 (20 ng/ml) within 2 wk (5 spheres/384 wells). In addition, almost all FGF-4-generated spheres could generate at least one daughter sphere after dissociation in the presence of FGF-2. Thus, FGF-4 appeared to be sufficient to maintain NSC self-renewal in vitro at clonal density. To determine whether FGF-4-generated spheres were multipotent, single spheres were transferred onto coverslips coated with poly-L-ornithine in a medium containing 1% of FBS (no EGF or FGF-2) to promote cell differentiation. After 7 DIV, the spheres were fixed and subjected to immunocytochemical study. The result showed that FGF-4-generated spheres could produce neurons, astrocytes, and oligodendrocytes (Fig. 2E
). Thus, FGF-4 is sufficient to maintain self-renewal as well as the multipotentiality of striatal NSCs.
|
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 occurs continuously. 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. Dissociated cells derived from EGF-responsive stem cell progeny were cultured on poly-L-ornithine/fibronectin-coated coverslips for 4 DIV in the absence or presence of FGF-4. Resultant cultures were analyzed for neuronal numbers by immunohistochemical staining with MAP-2 Ab (Fig. 3
). As shown in Fig. 3A 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. Cells positive for another neuronal marker, type 3 beta-tubulin, were also increased by the presence of FGF-4 (data not shown). In contrast, cells expressing the astrocyte marker GFAP accounted for 46% of the total number of cells either in the presence or absence of 5 ng/ml FGF-4. These results suggest that FGF-4 preferentially induces neuronal differentiation in EGF-generated neurospheres.
|
DISCUSSION
Neuronal development encompasses a variety of events that occur throughout neuronal life, including the cell-fate decision (neuronal commitment), and postmitotic maturation (a narrow definition of neuronal differentiation). However, the molecular mechanisms of neuronal commitment and differentiation are still largely unknown, especially those that pertain to the biology of neural stem cells. To our knowledge, this paper is the first direct demonstration of (a) spatial localization of Fgf-4 in the mouse brain, (b) FGF-4 involvement in the proliferation of neural progenitor cells, and (c) FGF-4 dependent differentiation of neural precursor cells along neuronal lineages. Three key observations support these conclusions.
First, it was previously reported that FGF-4 acts as a mitogen for neural progenitor cells isolated from fetal and adult rat central nervous system (CNS) (27)
. Furthermore, the addition of FGF-4 increased the number of neural precursor cells that were generated from ES cells. In contrast, FGF-2 had no effect in this system (28)
. In our current experiments we show that FGF-4 also stimulates neural progenitor cell proliferation in neurospheres. We suggest that FGF-4 may regulate neural stem cell number by regulating the type of cell division. The formation of secondary neurospheres from a single primary neurosphere is indicative of self-renewing stem cell division (21)
. Higher numbers of secondary neurospheres indicate that FGF-4 generated neural stem cells underwent a higher proportion of self-renewing or symmetric cell divisions, leading to an expansion of the stem cell pool. These published reports and our experiments indicate that FGF-4 is a potent mitogen for neural stem cells. It was also revealed that FGF-4 could maintain trophoblast stem cell proliferation (29)
. In this regard, although further analysis will be required, FGF-4 might have the capability to maintain stemness.
Secondly, results from the neurosphere differentiation study indicate that FGF-4 is involved in the control of NSC differentiation. Previous studies have shown that FGF-4, which is expressed in the primitive streak, defines an inductive center for hindbrain 5-hydroxytryptamine neurons (18)
. In this regard, Shimozaki et al. (30)
reported that sustained up-regulation of Oct3/4 in ES cells led to efficient neuroectoderm formation and subsequent neuronal differentiation. However, Sox2 is expressed in the neural tube from the earliest stage of its formation (31)
. Sox2 is thought to be essential for early neuroectoderm cells to establish their neural identity during the subsequent steps of neural differentiation. Oct3/4 and Sox2 are known to cooperate in activating the transcription of several genes, including FGF-4 (32)
, indicating a role for FGF-4 in neuronal differentiation. We suggest that up-regulation of FGF-4, triggered by binding of the Oct3/4 and Sox2 complex to its enhancer region, causes neural precursor cells to differentiate into neurons by an autocrine or paracrine mechanism. It seems likely that FGF-4 regulates neuronal differentiation in conjunction with other molecules known to be important in this process. For instance, the concerted action of FGFs and Wnts are believed to be important for inducing neural fate decisions (33)
. Wnts are involved in morphogenesis and patterning, and their proliferation-promoting roles are critical for stem cell maintenance and the expansion of progenitor pools (34
, 35)
. Interestingly, it was reported that the Fgf-4 gene is a direct transcriptional target for LEF1, a nuclear mediator of Wnt signaling. In addition, recombinant FGF-4 protein can overcome the developmental arrest of Lef1–/– tooth germs (36)
. Therefore, it will be important to determine the respective roles of FGF-4 and Wnts in controlling the fate of neural stem cells.
Thirdly, previous studies have shown that the Fgf-4 gene is predominantly expressed in the CNS during embryonic development, in the crystalline lens, and in the peripheral nervous system (17)
. Here we show that expression of Fgf-4 mRNA is highly restricted in the subventricular zone, rostral migratory stream, and subgranular region of dentate gyrus, regions where adult neurogenesis is continuously occurring (37)
. By using RT-PCR analysis, we also demonstrate the presence of mouse Fgf-4-specific PCR products in neurosphere cultures. These results strongly suggest the possibility of physiological FGF-4 functions in the CNS. However, further basic studies of FGF-4 gene function through both in vitro studies and targeted deletion in vivo are necessary to establish the details of FGF-4 involvement. Recently, Amoh et al. reported that the hair-follicle bulge-area stem cells carrying nestin-driven GFP could differentiate into neurons (38)
. This system might be interesting to see whether FGF-4 could enhance the clonal growth of stem cells and differentiation into neurons.
These three key observations support the conclusion that FGF-4 is not only a mitogen for neural stem cells, but is also a key inducer of neuronal differentiation. Although we have not established the effects of FGF-4 concentration on proliferation and differentiation, it seems interesting that a single factor can promote both proliferation and differentiation. A precedent for this phenomenon is seen in the case of insulin-like growth factor-I, which not only promotes proliferation, but also promotes neural precursor differentiation (39)
. In light of the many recent reports related to applying neural stem cells to neurological disorders, it will be critical to understand in detail the roles of various growth factors and cytokines in stem cell biology. In this respect, we propose that FGF-4 offers a new strategy for restoring neurogenesis in a clinical setting.
ACKNOWLEDGMENTS
We thank Ms. Kazumi Kimura, Dr. Gary Quinn, Ms. Masako Hosoda, Ms. Nami Nogawa, and Dr. Youichi Aizawa for their kind help, and Dr. William B. Stallcup for his critical reading of the manuscript and helpful discussions. We also thank Dr. Noriko Osumi and Dr. Yasuo Ishii for great technical assistance with in situ hybridization. This work was supported in part by a Grant-Aid for the Third-Term Comprehensive 10-Year Strategy for Cancer Control, Health Science Research Grants for the Research on Human Genome and Gene Therapy from the Ministry of Health, Labor, and Welfare of Japan.
Received for publication October 25, 2005. Accepted for publication March 14, 2006.
REFERENCES
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