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First published online 16 November 2005
doi: 10.1242/dev.02080

Development 132, 5399-5409 (2005)
Published by The Company of Biologists 2005
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The roles of FGF signaling in germ cell migration in the mouse

Yutaka Takeuchi1, Kathleen Molyneaux2, Chris Runyan3, Kyle Schaible3 and Chris Wylie3,*

1 Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan
2 Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA
3 Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati, OH 45229, USA



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  		Fig. 1. Fgfr1-IIIc and Fgfr2-IIIb isoforms are expressed by migrating PGCs. (A) cDNA prepared from FACS-sorted E10.5 PGCs and somatic cells was subjected to PT-PCR. The purity of the preparations was confirmed by expression of the PGC marker gene stella, and the somatic marker genes steel and Twist1. Lane 1, E10.5 PGC cDNA (PGC); Lane 2, E10.5 somatic cell cDNA (SC); Lane 3, E10.5 whole embryo cDNA (WE); Lane 4, RT(-) E10.5 whole embryo cDNA [WE(RT-)]. (B) The expression patterns of all FGFR isoforms were assayed by RT-PCR. Fgfr1-IIIc and Fgfr2-IIIb were found to be expressed by E10.5 PGCs (lane 1; lanes as in A). PGC-derived PCR products were sequenced and verified that these bands truly represented the expression of each FGFR. The illustration shows the potential FGF ligands that could activate each FGFR.

 


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  		Fig. 2. FGFR-ERK1/2 signaling is activated in migrating PGCs. (A) Whole-mount dp-ERK1/2 (red) staining in transverse slices from E9.5 OCT4{Delta}PE:GFP embryos; germ cells are shown in green. To show the specificity of the dp-ERK1/2 antibody, trunk regions of embryos were cultured with (right) or without (center) MEK inhibitor (50 µM of U0120) for 4 hours, fixed, sliced and stained for dp-ERK1/2. Slices in which the primary antibody was left out of the protocol (left) were used as negative controls. (B-D) Higher magnification views of hind gut regions of the embryos shown in A. Confocal sections reveal that cytoplasmic staining observed in PGCs of the control slice (arrowheads in C) was diminished in the U0126-treated slice (D) and the negative control slice (B). (E-H) The percentage of dp-ERK1/2-positive PGCs was dramatically affected by 4-hour treatments of FGFs (FGF7 and FGF2) or FGFR inhibitor (SU5402). (E) Dp-ERK1/2 staining of control embryo cultured with DMSO. The numbers of dp-ERK1/2-positive PGCs (red; arrows) were increased in embryos incubated with (F) 10 ng/ml of FGF7 or (G) 10 ng/ml of FGF2, and decreased in embryos treated with (H) 5 µM of SU5402. (B-H) dp-ERK1/2 images are on the right; GFP overlaid image, left. (I) The percentage of dp-ERK1/2-positive PGCs obtained from each treatment is summarized. Error bars show the s.e.m. Asterisks indicate the degree of statistical significance (P<0.01), of increase (*) and decrease (**) compared with the controls. Scale bars: in A, 200 µm; in B, 20 µm for B-D; in E, 40 µm for E-H.

 


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  		Fig. 3. Effects of adding FGFs or FGFR inhibitor on the numbers of PGCs in E9.5 slice cultures. (A) `Relative PGC number' represents the number of PGCs at the end of the culture period (18 hours) expressed as a percentage of the number at the start of the culture period in the same slice. Relative PGC number is significantly increased by 100 ng/ml of FGF7 treatment, but significantly reduced by 10 µM of SU5402. FGF2 treatments do not affect PGC numbers. (B) The proliferation of PGCs was quantified by BrdU incorporation assay in cultured slices. Slices, 6-hour pre-cultured with FGF or FGFR inhibitor, were exposed to BrdU for 2 hours. PGCs in the FGF7-, FGF2- and SU5402-treated slices incorporate BrdU at rates comparable with those in the control slices. Each bar shown in A and B shows the mean±s.e.m. obtained from 14-18 slices from triplicate experiments. Asterisks show the degree of statistical significance (P<0.05) of increase (*) and decrease (**) compared with the controls.

 


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  		Fig. 4. Fragmentation of migrating PGCs is more frequently observed in the presence of FGFR inhibitor. (A) Fragmentation of a PGC (arrowed) is shown from a time-lapse movie of an E9.5 OCT4{Delta}PE:GFP embryo slice treated with 1 µM SU5402. The GFP-labeled PGC (arrow), started to fragment at frame 62, corresponding to 434 minutes from the beginning of movie. This time of onset of fragmentation is represented by the single dot with the red circle in D. (B) A fragmented PGC is stained by the apoptotic cell marker anti-cleaved caspase-3. (C) Pictures from frame 20 of a control and FGFR inhibitor-treated slice. Fragments of PGCs are observed in the dorsal body wall of the 1 µM FGFR inhibitor-treated slice (dashed circle). No fragmented PGCs are observed in the control slice. (D) Timing of onset of fragmentation of PGCs, taken from a total of seven movies for each treatment. The onset of fragmentation of each PGC is represented as a dot. Fragmentation of the arrowed PGC in A started at frame 62, and is represented by the single dot with the red circle. Inset shows the average number of fragmentations in a single movie (fragmented germ cells/movie). Data are means±s.e.m. from three separate experiments (n=7). Means with different letters were significantly different (P<0.05) from each other. Scale bars: 10 µm for A,B; 100 µm for C.

 


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  		Fig. 5. Adding FGF2 to slices increased motility of migrating PGCs. The reverse effect was seen in slices cultured with the FGFR inhibitor SU5402. Time-lapse movies are available in the supplementary material. (A) The first (frame 1), middle (frame 50) and last (frame 100) frames from a single movie are shown as an example of cell tracing. Numbered germ cells were traced. Germ cells marked `X' moved out of the plane of focus during filming, and were not traced. When PGCs divided, one of the siblings was traced. (B) Typical trajectories of migrating PGCs in controls, 10 ng/ml FGF2-treated slices, 100 ng/ml FGF7-treated slices and 10 µM SU5402-treated slices. Trajectories of PGCs were acquired using NIH image software. The green lines, connecting notochord (n) and the midline of hind gut (g), indicate the dorsoventral axis. Red arrows show the direction of PGC migration. In all treatments, PGCs showed directional movement towards the genital ridges. Trajectories were extended by FGF2 treatment, and shortened by SU5402 treatment. (C) Summary of PGC velocity data from six control, three FGF2- and FGF7-, and four SU5402-treated slices. Six to eight cells in each slice were analyzed. Mean, mean velocity for the whole culture period; Max., maximum velocity in any 35-minute period of culture. Error bars show the s.e.m. (D) PGCs exhibited exaggerated processes when exposed to FGF2. Every 10 frames, the percentage of PGCs showing processes was scored. In contrast to FGF-treatment, the formation of processes was inhibited in SU5402-treated slices. Pictures of PGCs taken from frame 40 of each treatment are shown in D. Asterisks in C and D indicate a degree of statistical significance (P<0.05) of increase (*) and decrease (**) compared with controls. Scale bars: 100 µm for A,B; 50 µm for D.

 


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  		Fig. 6. Phenotypes of the FGFR2-IIIb-/- embryo. (A) Heterozygous and homozygous littermates at E11.5. Null embryos grow to the same size as heterozygotes at this stage, and are relatively normal, but they lack both fore and hind limbs. (B) Genotyping of embryos by genomic PCR. (C) Alkaline phosphatase (AP)-stained genital ridge and midline preparations are compared in E11.5 embryos. The numbers of ectopic PGCs were drastically reduced in null embryos. (D) Transverse section of AP-stained genital ridges showing a reduction of gonadal germ cells. (E) The targeted FGFR2-IIIb mutation was bred into the OCT4{Delta}PE:GFP mouse line. The same effect, reduction of ectopic germ cells, was seen in null embryos. (F) Transverse sections of genital ridges shown in E. Sections were stained with OCT3/4 antibody to analyze the density of gonadal germ cells. (G) Sagittal sections of genital ridges were stained for the apoptosis marker cleaved caspase-3 (red). Cleaved caspase-3-positive germ cells are compared in genital ridges at E11.5. (H) BrdU-positive germ cells (arrows) in E11.5 genital ridges of heterozygous and null embryos. (I) The number of ectopic germ cells in heterozygous and null embryos at E11.5. (J) Quantitation of germ cell density in genital rides at E11.5. Because the density of germ cells varied between littermates, the data obtained from null embryos were normalized to the heterozygous littermates. (K) Quantitation of germ cell death in genital ridges at E11.5. (L) Percentage of BrdU-positive germ cells in genital ridges at E11.5. There is no statistically significant difference between the percentage of BrdU-positive germ cells in null gonads and heterozygous gonads. Asterisks in I-K indicate the degree of statistical significance (P<0.05) of increase (*) and decrease (**) compared with heterzygotes. Scale bars: 1 mm for A; 200 µm for C,E; 50 µm for D,F,G; 20 µm for H.

 

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