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First published online 8 February 2006
doi: 10.1242/dev.02274

Development 133, 989-999 (2006)
Published by The Company of Biologists 2006
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Sfrp1 and Sfrp2 regulate anteroposterior axis elongation and somite segmentation during mouse embryogenesis

Wataru Satoh1,2,3, Takafumi Gotoh4, Yasuhiko Tsunematsu3, Shinichi Aizawa1,2 and Akihiko Shimono1,*

1 Vertebrate Body Plan, Center for Developmental Biology, RIKEN Kobe, Minatojima-Minami, Chuou-ku, Kobe 650-0047, Japan.
2 The Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101, Japan.
3 Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 641 Yamazaki, Noda-shi, Chiba 278-8510, Japan.
4 Kuju Agricultural Research Center, Kyushu University Graduate School of Agriculture, Naoiri-gun Kuju-cho 878-0201, Japan.


Figure 1
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  		Fig. 1. Sfrp1-/-;Sfrp2-/- embryos display shortening of the thoracic region and limb morphogenesis abnormality. (A) Gross morphology of Sfrp1-/-;Sfrp2-/- mutant (left) and control (right) embryos at E14.5. Arrows indicate the craniofacial abnormality; arrowheads indicate edematous defects. Scale bar: 1 mm. (B-E) Cartilage staining in control (B,D) and Sfrp1-/-;Sfrp2-/- (C,E) embryos at E15.5. The number of thoracic vertebrae was reduced in Sfrp1-/-;Sfrp2-/- embryos. Although vertebrae numbers were normal, the sides of vertebrae were reduced in the lumbar and sacral regions. Arrowhead, C7 vertebra; double arrowhead, T1 vertebra; single arrow, L1 vertebra; double arrow, S1 vertebrae; C1, atlas. Scale bar: 1 mm. (F-I) Extra digits (arrowhead) on the hindlimb of Sfrp1-/-;Sfrp2-/- embryos (G) at E14.5, and the skeletal pattern of the hindlimb in control (H) and Sfrp1-/-;Sfrp2-/- (I) embryos at E15.5. T, tail. Scale bars: 500 µm in F,G; 200 µm in H,I. (J,K) Fgf8 expression in the limb bud of control (J) and Sfrp1-/-;Sfrp2-/- (K) embryos at E10.5. Arrows indicate the extra stripes of Fgf8 expression in the ventral surface of the hindlimb bud. A, anterior; P, posterior. Scale bar: 400 µm.

 

Figure 2
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  		Fig. 2. Hox gene expression in Sfrp1-/-;Sfrp2-/- embryos. (A,B) Hoxb2 expression in control (A) and Sfrp1-/-;Sfrp2-/- (B) embryos at E9.25. An arrow indicates the anterior boundary of Hoxb2 expression at rhombomere 3. ov, otic vesicle. (C,D) Hoxa7 expression in control (C) and Sfrp1-/-;Sfrp2-/- (D) embryos at E9.25. An arrow and an arrowhead indicate the anterior boundary of Hoxa7 expression in the spinal cord and somite, respectively. (E-H) Hoxd10 and myogenin gene expression in control (E,G) and Sfrp1-/-;Sfrp2-/- (F,H) embryos at E10.5. Myogenin expression was visualized by INT/BCIP in double in situ hybridization (red). An arrowhead depicts the anterior boundary of Hoxd10 in the somite. (I) Summary of the skeletal pattern and Hoxb2, Hoxa7 and Hoxd10 expression in control and Sfrp1-/-;Sfrp2-/- embryos. Correlation between vertebrae and somite number (12-17) was estimated from myogenin expression in Sfrp1-/-;Sfrp2-/- embryos (E,F). FL, forelimb; HL, hindlimb. Scale bars: 250 µm.

 

Figure 3
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  		Fig. 3. Somite segmentation abnormality in Sfrp1-/-;Sfrp2-/- embryos. (A,B) Gross morphology of control (Sfrp1-/-;Sfrp2+/+; A) and Sfrp1-/-;Sfrp2-/- (B) embryos at E9.5. Somite size is indicated by a bracket. Inactivation of Sfrp1 and Sfrp2 resulted in reduced length of the posterior region. Scale bar: 500 µm. (C,D) Somite segmentation was aberrant in Sfrp1-/-;Sfrp2-/- embryos. Somite segmentation was examined in para-sagittal sections, which were stained with Hematoxylin and Eosin, of control (C) and Sfrp1-/-;Sfrp2-/- (D) embryos at E9.5. Regular and similarly sized somites (bracket) were observed in control embryos, whereas incomplete segmentation (arrows in a bracket) was apparent in the region between the forelimb and hindlimb of Sfrp1-/-;Sfrp2-/- embryos. Scale bar: 50 µm. (E,F) Immunostaining with 2H3 monoclonal anti-neurofilament antibody (arrowheads) of control (E) and Sfrp1-/-;Sfrp2-/- (F) embryos at E10.5. Scale bar: 500 µm. FL, forelimb; HL, hindlimb; Sc, sclerotome.

 

Figure 4
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  		Fig. 4. Segmentation and cell differentiation in somites of Sfrp1-/-;Sfrp2-/- embryos. (A-N) Somite segmentation was aberrant in the region between the forelimb and hindlimb in Sfrp1-/-;Sfrp2-/- embryos. However, cell differentiation in the somite did occur in that region. (A-D) Expression of Mox1 in control (A,B) and Sfrp1-/-;Sfrp2-/- (C) embryos at E9.5. D shows a higher magnification of the region indicated in C. Arrowheads indicate irregular segmentation of the somite. Pax3 (E-H), Uncx4.1 (I-K) and myogenein (L-N) expression in control (E,F,I,J,L,M) and Sfrp1-/-;Sfrp2-/- (G,K,N) embryos at E9.5. H shows the region indicated in G at higher magnification. Arrowheads indicate irregular segmentation of the somite. Asterisks indicate somite positions in the Sfrp1-/-;Sfrp2-/- embryo (N) corresponding to those of the control embryo (M). (O-Q) Dll1 expression in control (O,P) and Sfrp1-/-;Sfrp2-/- (Q) embryos. Note that the PSM region was severely affected in the double homozygous mutant embryos. (R-T) Myogenein expression in control (R) and Sfrp1-/-;Sfrp2-/- (S) embryos. The number of somites decreased in the region between the forelimb and hindlimb in the Sfrp1-/-;Sfrp2-/- embryo. T displays higher magnification of the region indicated in S. FL, forelimb; HL, hindlimb. Scale bar: 500 µm.

 

Figure 5
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  		Fig. 5. Defect in posterior axis extension in Sfrp1-/-;Sfrp2-/- embryos at E8.5. (A-C) Brachyury (T) expression in control (A,B) and Sfrp1-/-;Sfrp2-/- (C) embryos. PS, primitive streak; N, node. Defective posterior axis extension initially appeared as an increased thickness of the mesoderm layer (bar) in the posterior region of the Sfrp1-/-;Sfrp2-/- embryos when somite formation began. Scale bar: 250 µm. (D-I) Tbx6 expression in control (D,E,G,H) and Sfrp1-/-;Sfrp2-/- (F,I) embryos. D-F, lateral view; G-I, posterior view. An unusually high intensity of staining was detected on both sides of the primitive streak of Sfrp1-/-;Sfrp2-/- embryos. Furthermore, the PSM region (asterisk) was markedly reduced in these embryos. Scale bars: 250 µm in D-I. (J-O) Dll1 expression in control (J,K,L) and Sfrp1-/-;Sfrp2-/- (M,N,O) embryos at the 11 somite stage. K,L,N and O show cross sections of the tail bud region indicated in J and M. Cross sections were generated following in situ hybridization. Scale bars: 500 µm in J,M; 50 µm in K-L,N-O. (P-S) DiI cell-labeling assay of mesoderm cells in control (P,Q) and Sfrp1-/-;Sfrp2-/- (R,S) embryos at several somite stages. DiI was injected into mesoderm cells around the primitive streak (t=0; P,R), followed by a 2-hour culture of the embryos (t=2h; Q,S). TB, tail bud. Scale bar: 250 µm.

 

Figure 6
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  		Fig. 6. Activation of the Wnt/ß-catenin pathway in the tail bud of Sfrp1-/-; Sfrp2-/- embryos at E8.5. (A-F) Cross sections of the posterior region in control (A,B) and Sfrp1-/-;Sfrp2-/- (D,E) embryos stained with anti non-phospho ß-catenin antibody. (C,F) Control staining was performed without the first antibody. hg, hindgut; mp, mesoderm cells beneath the primitive streak; np, neural plate; asterisk, non-specific staining. Higher staining intensity was observed in the hindgut and in the axial mesoderm of control embryos. A similar intensity in staining was observed in the tail bud region of Sfrp1-/-; Sfrp2-/- embryos, including the mesoderm, neural ectoderm and laterally located paraxial mesoderm (inside of arrowheads and in the box labelled K; E), in addition to the hindgut endoderm and mesoderm beneath the primitive streak. Scale bar: 200 µm. (G-O) Higher magnification of the regions (labeled H,K,N) indicated in B and E. The mesoderm beneath the primitive streak is outlined by the broken line in H. (I,L,O) Nuclei were stained with DAPI (4,6-diamino-2-phenylindole; blue). G,J and M show merged images of H and I, K and L, and N and O, respectively. Arrowheads in J (merged image) indicate the positions of cells corresponding to those in original images (K and L). Scale bar: 33 µm.

 

Figure 7
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  		Fig. 7. Perturbed expression of cyclic genes during somitogenesis in Sfrp1-/-;Sfrp2-/- embryos. (A-G) Altered Lfng oscillating cycles in the PSM of Sfrp1-/-;Sfrp2-/- embryos at E8.5. The Lfng cycle was examined in control (A,D) and Sfrp1-/-;Sfrp2-/- (B,C,E,F) embryos by culture of half of the posterior region for different lengths of time [A, B and C, 0 and 60 minutes (t=0', 60'); D, E and F, 0 and 75 minutes (t=0', 75')]. The dagger indicates a strong extra stripe in the anterior-most region of the PSM. Scale bar: 250 µm. (G) Schematic diagram of Lfng expression in control and Sfrp1-/-;Sfrp2-/- explants. Traveling stripes of Lfng expression are indicated by numbers. (H,I) Oscillation pattern of Lfng in control (H) and Sfrp1-/-;Sfrp2-/- (I) explants at E9.5. Half of the posterior region was cultured for either 0 (t=0') or 75 (t=75') minutes. Scale bar: 250 µm. (J-U) Expression of Hes7, Axin2 and Nkx1 in the PSM of Sfrp1-/-;Sfrp2-/- embryos. Half of the posterior region was cultured for either 0 (t=0') or 75 (t=75') minutes. (J-O) Hes7 expression in control (J,M) and Sfrp1-/-;Sfrp2-/- (K,L,N,O) explants at E8.5. (L,N,O) Hes7 expression was perturbed in a large proportion of Sfrp1-/-;Sfrp2-/- explants. (K) Nearly normal Hes7 expression cycles were observed in Sfrp1-/-;Sfrp2-/- explants at lower frequency. The dagger indicates a strong extra stripe in the anterior-most region of the PSM. Scale bar: 250 µm. (P-R) Dynamic expression of Axin2 in control (P) and Sfrp1-/-;Sfrp2-/- (Q,R) explants at E8.5. An arrowhead indicates the higher expression domain of Axin2 in the primitive streak. Stripes of Axin2 expression were difficult to identify even in control explants at earlier embryonic stages. Scale bar: 250 µm. (S-U) Expression of Nkd1 in control (S) and Sfrp1-/-;Sfrp2-/- (T,U) explants at E8.5. Scale bar: 250 µm.

 

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© The Company of Biologists Ltd 2006