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First published online October 26, 2007
doi: 10.1242/10.1242/dev.009167

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FGF signaling acts upstream of the NOTCH and WNT signaling pathways to control segmentation clock oscillations in mouse somitogenesis

¹ Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
² Genetics of Development and Diseases Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
³ Laboratory of Cancer and Developmental Biology, NCI-Frederick, National Institutes of Health, Frederick, MD 21702, USA.
⁴ Howard Hughes Medical Institute, Kansas City, MO 64110, USA.

View larger version (55K): [in this window] [in a new window]   Fig. 1. Expression of Fgf ligands. In situ hybridization for (A) Fgf3, (B) Fgf4,(C) Fgf8, (D) Fgf17 and (E) Fgfr1, in E9.0 embryos. Fgf3, Fgf8 and Fgf17 are expressed in a gradient in the posterior PSM. Fgf4 expression is restricted to a small cell population in the tail bud, which also expresses the other FGF ligands.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Progressive disruption of segmentation in the Fgfr1f/f;T-Cre mutant embryos. (A,B) Skeletons stained with Alizarin Red (bone) and Alcian Blue (cartilage). (C-H) Uncx4.1 staining in Fgfr1f/+;T-Cre control (C,E,G) and Fgfr1f/f;T-Cre mutant embryos (D,F,H); C and D show whole mounts of E9.5 embryos and E and F are higher magnifications of the somite region at E10.0. (G,H) Sagittal sections through Uncx4.1-stained embryos. The posterior-most Uncx4.1-positive region is shown at the same magnification for both. In the Fgfr1f/f;T-Cre mutant embryos (H), somites fail to separate and a giant somite spanning over the region normally covering two somites is formed. Arrowheads indicate the boundaries between somites.

View larger version (114K): [in this window] [in a new window]   Fig. 3. Expression of different marker genes in E9.0 Fgfr1f/f;T-Cre mutant embryos. Expression of (A,B) Sox2, (C,D) Wnt3a, (E,F) T,(G,H) Uncx4.1/Msgn1,(I,J) Fgf8,(K,L) Dll1,(M,N) Notch1,(O,P) Dll3, (Q,R) Paraxis and (S,T) Mesp2 in Fgfr1f/+;T-Cre control and Fgfr1f/f;T-Cre mutant embryos, respectively.

View larger version (125K): [in this window] [in a new window]   Fig. 4. Progressive downregulation of FGF target genes in Fgfr1f/f;T-Cre mutant embryos after the 6-somite stage. (A-D) Pea3 is normally expressed in control (A) and Fgfr1f/f;T-Cre (B) embryos at the 5-somite stage, and in 7-somite control embryos (C), but it becomes progressively downregulated in the posterior PSM/tail bud of 7-somite Fgfr1f/f;T-Cre mutant embryos (D). (E) Real-time PCR for FGF target genes Erm and Pea3 in the posterior tail of Fgfr1f/f;T-Cre mutant embryos and Fgfr1f/+;T-Cre control embryos at somite stages 5 (n=1/1), 6 (n=3/5), 7 (n=2/2) and 9 (n=1/1). Both genes become progressively downregulated from somite stages 6 onward. Levels of Erm and Pea3 were normalized to the housekeeping gene Hprt and values are given as the mean change in crossing points (CT) in Fgfr1f/f; T-Cre mutant embryos. (F-O) For other target genes (F,G) Erm, (H,I) Gbx2, (J,K) Dusp6, (L,M) Spry2 and (N,O) Sef, the expression in the posterior PSM and the tail bud of E8.75 Fgfr1f/f;T-Cre mutant embryos is lost, whereas the expression in the anterior PSM and the adjacent structures is normal.

View larger version (104K): [in this window] [in a new window]   Fig. 5. FGF signaling is not sufficient to position the RA-responsive domain in the PSM. (A,B) Cyp26 expression in the posterior region of the Fgfr1f/f;T-Cre (B) mutant is downregulated compared with that in Fgfr1f/+;T-Cre control (A) embryos. (C,D) There is a lack of significant change in RA activity, as detected by crossing to RARE-lacZ reporter mice, in Fgfr1f/f;T-Cre mutant (D) compared with the Fgfr1f/+;T-Cre control (C) embryos. (E,F) Expression of Raldh2 is not significantly changed in the PSM of Fgfr1f/f;T-Cre mutant embryos (F) compared with control Fgfr1f/+;T-Cre (E) embryos.

View larger version (115K): [in this window] [in a new window]   Fig. 6. Disruption of cyclic gene expression in the Fgfr1f/f;T-Cre mutant embryos. (A-H) Comparison of expression of the cyclic genes of the Notch (Lfng), FGF (Spry2, Snail1) and Wnt (Axin2) signaling pathways at stages E8.75 (A-F) and E9.0 (G,H). All genes show dynamic expression in heterozygous Fgfr1f/+;T-Cre control embryos (A,C,E,G). Oscillations in Fgfr1f/f;T-Cre mutant embryos are lost (B,D,F,H). Lateral views are shown for Fgfr1f/+;T-Cre in A,E,G and dorsal views are shown in C. For Fgfr1f/f;T-Cre mutant (B,D,F,H), a lateral view is shown in the left panels, whereas dorsal views are shown in the right panels.

View larger version (9K): [in this window] [in a new window]   Fig. 7. Summary of the onset of the phenotypes observed in Fgfr1f/f;T-Cre mutant embryos. FGF target genes become downregulated in the posterior PSM of Fgfr1f/f;T-Cre mutant embryos at the 5- to 7-somite stage, followed by the arrest of cyclic gene expression between somites 8 and 10. Normal somites and corresponding vertebrae elements are observed up to somites 10 to 13; however, abnormal skeletal elements derived from paraxial mesoderm posterior to somite 13 were present.