Fibroblast growth factor receptor 1 is required for the proliferation of hippocampal progenitor cells and for hippocampal growth in mouse - PubMed (original) (raw)

Fibroblast growth factor receptor 1 is required for the proliferation of hippocampal progenitor cells and for hippocampal growth in mouse

Yasushi Ohkubo et al. J Neurosci. 2004.

Abstract

Fibroblast growth factor receptor 1 (Fgfr1) is expressed at high levels by progenitor cells of the ventricular zone (VZ) within the hippocampal primordium. To investigate the role of Fgfr1 in these cells, in vivo Cre recombination of "floxed" Fgfr1 alleles was directed to cells of the radial glial lineage by using the human glial fibrillary acidic protein promoter. Radial glial-like cells of the hippocampal VZ are the progenitors of pyramidal neurons and granule cells of hippocampal dentate gyrus (DG). Mice carrying null Fgfr1 alleles (Fgfr1(Deltaflox)) in cells of this lineage showed a dramatic loss of Fgfr1 gene expression throughout the embryonic dorsal telencephalon. These Fgfr1(Deltaflox) mice exhibited a approximately 30% decrease in dividing radial glial progenitor cells in the hippocampal VZ and DG in the late embryonic period, progressing to a approximately 50-60% loss at birth, without any changes in cell survival. In addition, no FGF2-sensitive neural stem cells could be isolated from the Fgfr1(Deltaflox) hippocampal neuroepithelium, whereas epidermal growth factor-sensitive neural stem cells were not affected. The number of hippocampal pyramidal neurons and DG granule cells was approximately 30-50% decreased from the perinatal period through adulthood, and the number of parvalbumin-containing interneurons was similarly decreased in both the DG and pyramidal cell fields. We conclude that Fgfr1 is necessary for hippocampal growth, because it promotes the proliferation of hippocampal progenitors and stem cells during development.

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Figures

Figure 1.

Figure 1.

Fgfr gene expression and region-specific gene expression in Fgfr1 conditional null mice. A, In E16.5 Fgfr1flox/flox mice lacking Cre (Cre-), Fgfr1 mRNA is expressed in the cortical VZ (top arrowhead), hippocampal VZ (bottom arrowhead), and DG. B, hGFAP-Cre/+; Fgfr1flox/flox (Cre+) mutants clearly lack Fgfr1 expression in these areas, although expression is unaltered in the fimbria (top arrow) and VZ of the diencephalon (bottom arrow). Higher magnification images of red frames in A and B are presented in C and D. Very few cells expressing Fgfr1 are remaining in Cre+ mutants in the stratum LM and around the edge of the DG (arrow). E-H show that Fgfr2 and Fgfr3 expression is not changed in the absence of Fgfr1. _I_-L, The expression of Foxg1 (BF-1) (I, J) or KA-1 (K, L) mRNAs is not altered by the lack of a functional Fgfr1 gene. Scale bars: (in A) _A, B, E_-J, 500 μm; C, D, 111 μm; (in K) K, L, 500 μm.

Figure 2.

Figure 2.

FGFR1 expression is detectable in cells of the radial glial lineage as well as immature neurons and astrocytes. Dissociated cell cultures from E14.5 hippocampal tissue were immunostained for FGFR1 using a mouse monoclonal (A, G) or a rabbit polyclonal antibody (D, J, M) and double stained with various antibodies as indicated (B, E, H, K, N). Images in each horizontal column show the same field with FGFR1 immunoreactivity at the left (green), antibody markers in the middle (red), and merged images with DAPI nuclear staining (blue) at the right. White arrow heads indicate the accumulation of FGFR1 in cell bodies. White arrows in _D_-F indicate a cell with weak staining for FGFR1. White asterisks in C and L indicate GLAST or β III-tubulin-positive fibers that are negative for FGFR1. However, GFAP-positive cells express FGFR1 immunoreactivity in their fibers (M, O). Scale bar, 20 μm.

Figure 3.

Figure 3.

Distribution of Cre recombination driven by the hGFAP-Cre transgene. _A, C_-L, Brain sections from hGFAP-Cre; ROSA26R double transgenic mice were assayed for β-gal antibody staining (_A, C_-H, J) or Cre recombinase antibody (I, K, L) at E16.5. A, Coronal sections showing β-gal immunoreactivity (red) in cells of the cortical primordium (Ctx), the developing hippocampus (Hip), and more lightly in the ganglionic eminences (Ge) but not in the thalamus (Th). B, The same section is double stained with the neuroepithelial marker RC2. C_-H and J represent higher magnifications of the corresponding white dashed squares in A. The majority of β-gal immunoreactivity colocalizes with RC2 in the hippocampal VZ (white arrowheads in C and_E), except for some RC2-positive cells lacking β-gal immunoreactivity in and near the DG (C, arrows). Nuclear staining with DAPI in the same sections confirms that almost all cells are positive for β-gal in the hippocampal VZ (D, F, purple cells, arrowheads), and that some cells are not β-gal-positive in the stratum LM and adjacent DG (D, blue cells, arrows). All of the proliferating cells in hippocampal VZ, detected by BrdU, are also positive for β-gal (G); however, some BrdU-positive cells areβ-gal negative in the DG (H, arrows). Most proliferating cells in the migratory stream (MS) between the VZ and DG are β-gal positive (H, small window). J, GFAP/β-gal double immunostaining shows that GFAP-positive cells detectable in the MS and fimbria (f) can be positive or negative for β-gal; inset in J shows β-gal-positive and β-gal-negative cells (DAPI counterstaining verified the presence of nuclei). I, K, and L represent higher magnifications of the corresponding white dashed squares in B. Cre immunoreactivity is most prominent in the VZ, and in almost all cases, Cre-positive cells colocalize RC2 (I), GLAST (K), and BLBP (L). Scale bars: A, B, 200 μm; _C_-L, 50 μm. All inserted small windows show 3× higher magnification.

Figure 4.

Figure 4.

The hippocampus of Fgfr1 mutants is reduced in size but exhibits normal differentiation of neuronal or glial cell types. A, C, E, G, and I are sections from Fgfr1flox/flox mice (Cre-), and B, D, F, H, and J are from Fgfr1_Δ_flox mutants (Cre+) at corresponding anteroposterior level. A, B, Hippocampal morphology in coronal sections stained by cresyl violet. Fgfr1_Δ_flox mutants have decreased hippocampal size, with maintenance of pyramidal (CA1 to CA3) and granular layers (DG). Note the absence of corpus callosum and the unusual morphology of the medial cerebral cortex in the mutants (B, asterisk). C, D, Hippocampal neurons express the GluR1 in both control and mutant mice. E-J, Hippocampal granule cells express calbindin (E, F), and inhibitory interneurons express parvalbumin (G, H) in both control and mutant mice. However, the total number of these cells appears reduced in the mutants. Specific boundaries within the CA region are also distinguishable in Cre- and Cre+ hippocampi (E, F, arrowheads). The density of GFAP-positive cells is similar in control and mutant cre+ mice (I, J); insets show 4× magnification of the white boxed areas in each images. Scale bar, 500 μm.

Figure 5.

Figure 5.

Decreased number and proportion of proliferative cells in the Fgfr1_Δ_flox hippocampus. A_-D, BrdU-labeling index (A, B) and number of BrdU-immunoreactive cells (C, D) in the hippocampal VZ (A, C) and DG (B, D) after 30 min BrdU labeling in vivo. The labeling index (LI) is the ratio between the number of BrdU-labeled cells and total cell number. Cross-hatched bars are hGFAP-Cre/+; Fgfr1flox/flox_ mutants, and black bars are Cre-negative Fgfr1flox/flox control littermates. ANOVA revealed an overall significant effect of genotype on the number of BrdU-labeled cells (F-ratio = 27.9; p < 0.0001) and on labeling index (p < 0.0005). Sheffe post hoc tests indicated significant differences in the number of BrdU labeled cells between genotypes in the hippocampal VZ (p < 0.001) and the DG (p < 0.005) and strong differences in LI in the hippocampal VZ (p < 0.001) but moderate in the DG (p < 0.05). There is no significant effect at E16.5, with significant differences between control and mutants at P0 (p < 0.01) and P7 (p < 0.001); n = 6 animals per genotype.

Figure 6.

Figure 6.

Decreased proliferation but normal survival in the Fgfr1_Δ_flox hippocampus. A-F, Proliferating cells identified by a 2 hr BrdU incorporation at P0. C-F show a 2× higher magnification of blue squares in A and B. Proliferating cells in DG (C, E) and in hippocampal VZ (D, F, black arrowheads) are greatly decreased in hGFAP-Cre/+; Fgfr1flox/flox mutants. G, H, BrdU was injected at E16.5, and embryos were collected at E18.5. The number of cells along the migrating pathway to the DG (black arrowheads) as well as cells that are locally proliferating in the DG (red arrowheads) are clearly decreased in hGFAP-Cre/+; Fgfr1flox/flox mutants. I, J, Apoptotic cells (white arrowheads) are detected by an antibody for cleaved Caspase-3, revealing no major differences between Cre- and Cre+ mice at E18.5. Cre-, Fgfr1flox/flox; Cre+, hGFAP-Cre/+; Fgfr1flox/flox. Scale bar, 200 μm.

Figure 7.

Figure 7.

Reduction in the proliferation and density of radial glia-like cells in hGFAP-Cre/+; Fgfr1flox/flox mice. A-D, DAPI staining showing the general morphology of coronal sections in control (Cre-) and mutant (Cre+) at the indicated ages. White dashed squares in A and C indicate frame positions shown in E-H and Q-T for the hippocampal VZ (hVZ) and in I-L and U-X for the DG. White dashed squares in B and D indicate frame position for the migratory stream (MS) shown in M-P. E-H, Anti-GLAST staining in hVZ is unchanged at E16.5 (E, F) but is decreased in mutants at E18.5 (G, H). BrdU- or PCNA-positive cells colocalize GLAST immunoreactivity, and the number of PCNA-positive cells is decreased in the mutant hVZ at E18.5 (G, H, white arrows). I-L, A similar reduction in BrdU- or PCNA-positive cells double immunoreactive for GLAST is also observed in the DG at E16.5-E18.5. M-P, GFAP immunoreactivity is present in cells within the MS with no apparent change between control (Cre-) and mutant (Cre+) (yellow arrows). _Q_-_R, U_-V, BLBP-immunoreactive cells and fibers are decreased and disorganized in the mutant VZ (Q, R, yellow arrowheads) and in the DG (U, V, white arrowheads). S, T, W, X, Although nestin immunoreactivity is not substantially changed in the DG, there is an obvious reduction in hippocampal hVZ and presumptive pyramidal layer (S, T, open arrowheads). f, Fimbria. Scale bars: _A_-D, 200 μm; _E_-H, 50 μm; _I_-X, 100 μm.

Figure 8.

Figure 8.

Reduction of FGF2-sensitive neurospheres in hGFAP-Cre/+; Fgfr1flox/flox mice. A, The hippocampal primordium from E14.5 mice was dissociated and cultured with or without FGF2 for 10 d. Tissue from Fgfr1flox/flox mice (Cre-) produced a large number of neurospheres when treated with FGF2. Tissue of mice lacking a functional Fgfr-1 gene (Cre+) failed to produce neurospheres under identical conditions (bottom right). The cell aggregates visible in Cre+ mutant mice have a diameter much smaller than the typical neurospheres detected in Cre- control (top right). Scale bar, 2 mm. B, Hippocampal tissue (n = 5 preparations) was dissociated and cultured as above with or without FGF2 or EGF (10 ng/ml). Neurospheres with a diameter >150 μm were counted after 10 d of culture. Each histogram shows the average number of neurospheres grown per well (±SEM).

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