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First published online 6 October 2004
doi: 10.1242/dev.01405

Development 131, 5469-5480 (2004)
Published by The Company of Biologists 2004
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Skeletal defects in ringelschwanz mutant mice reveal that Lrp6 is required for proper somitogenesis and osteogenesis

Chikara Kokubu1,*,{dagger}, Ulrich Heinzmann2,*, Tomoko Kokubu1, Norio Sakai3, Takuo Kubota3, Masanobu Kawai3, Matthias B. Wahl1, Juan Galceran4, Rudolf Grosschedl4, Keiichi Ozono3 and Kenji Imai1,{ddagger}

1 Institute of Developmental Genetics, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
2 Institute of Pathology, GSF-National Research Center for Environment and Health, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
3 Department of Developmental Medicine (Pediatrics), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
4 Gene Center and Institute of Biochemistry, Ludwig Maximilians University, Feodor Lynenstrasse 25, 81377 Munich, Germany



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  		Fig. 10. Osteoporosis in rs/rs adult mice. (A-F) X-ray radiography (A,B) and histology (C-F) of the lumbo-sacral vertebrae were carried out in rs/rs (B,D,F) and control (A,C,E) animals. The boxed areas in C and D are shown at a higher magnification in E and F, respectively. The arrowhead in F points to a group of chondrocytes inside the trabecule. Scale bars: 50 µm. (G-I) Low bone mass phenotype in rs mutants demonstrated by pQCT. Bone density and the thickness of cortical bones in the metaphysis of the tibia were measured in rs/rs, rs/+ and +/+ animals (14 month-old females in triplicate for each genotype), and results are graphically depicted for bone density measures from total tibial metaphysis (G) and the cortex part (H), and for the cortical bone thickness (I). Values represent the means + standard errors. Single and double asterisks indicate statistically significant reductions (P<0.05 and P<0.01, respectively) in rs/rs compared with +/+.

 


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  		Fig. 9. Delayed ossification in rs/rs. (A,B) Skeletal analysis at the newborn stage revealed delayed ossification in metatarsal and phalangeal bones of the hindlimb (dorsal view, right side) from wild-type (A) and rs/rs (B). (C-F) Histology of non-decalcified bones from the contralateral (left) side of the hindlimbs from the same newborn animals. The corresponding areas is indicated by the boxes in A and B (the proximal phalanx of the second toe). In C and D, sections are stained by Alcian Blue, while sections are stained Alizarin Red and hematoxilin in E,F. Note the absence of Alizarin Red staining in the region of chondrocyte hypertrophy. The zone of prehypertrophic chondrocytes starting to undergo hypertrophy (brackets with ph in E,F) seems reduced in rs/rs. Scale bars: 100 µm.

 


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  		Fig. 1. Dysmorphologies in rs mutants. (A) A typical rs/rs animal with the original BALB/c background and (B) rs/rs and rs/+ animals with BALB/c-C57BL/6 mixed backgrounds. (C-F) Vertebrae from newborn rs/rs viewed from the ventral (C), dorsal (D,E), or right-lateral (F) sides. (G,H) Alcian Blue-stained E14 skeletons (dorsal view) from control (G) and rs/rs (H) show vertebral segmentation defects. Note neural arch fusion (black arrowheads in H) and its loss (yellow arrowheads in H). (I,J) SEM of control (I) and rs/rs (J) embryos at E11 shows neural tube closure defects in the lumbo-sacral region (the arrowhead in J). (K) Oligodactyly (arrowhead) is occasionally observed in rs/rs. Scale bars: 100 µm.

 


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  		Fig. 2. Histology of the lumbar region of rs embryos at E11. (A,B,D,E) On the sections (A,D, near midsagittal; B,E, parasagittal; the rostral side is to the left of each panel), somite borders demarcated by intersegmental vessels (arrowheads in A,B,D,E) are clearly visible in the control (A,B), whereas they are unclear in rs/rs (D,E). DRGs (d in B,E) are fused in rs/rs (E), while they are clearly segmented in controls (B). (C,F) Transverse sections at a proximal tail region from control (C) and rs/rs (F) embryos reveal malformations of the neural tube in rs/rs. The epithelial organization of dermomyotome (dm in C) is disturbed in rs/rs (F). da, dorsal aorta; hg, hindgut; nc, notochord; nt, neural tube. Scale bars: 100 µm.

 


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  		Fig. 3. Mutation detection in Lrp6. (A) Chromatograms show the C to T transition in rs. (B) PCR-based genotyping with HpaII digestion. In wild-type (BALB/c and C57BL/6 in lanes 1 and 2, respectively), the 458-base product from wild-type is cut into three fragments (205, 154 and 99 bases), while one from rs into only two fragments (359 and 99 bases) (Lane 4). Heterozygous specimens show four fragments (359, 205, 154 and 99 bases) (lane 3). (C) A schematic drawing of the Lrp6 protein structure with designations for known functional domains. The rs mutation resides right in front of the third EGF-like repeat. (D) The Arg (R) residue at the corresponding positions is highly conserved among various Lrp proteins. Designations after Lrp: M, mouse; H, human; D, Drosophila; F, Xenopus; C, C. elegans.

 


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  		Fig. 4. Non-complementation between the rs mutation and a null allele of Lrp6. The Lrp6-null allele is designated as null. (A) Compound heterozygotes (rs/null), obtained from mating between rs/+ and null/+ at the newborn stage, exhibit a rudimentary tail (red arrowheads) and spina bifida aperta (yellow arrowheads) (4-6). (1-3) are either of +/+, rs/+, or null/+. (B-I) E14 fetuses of rs/rs (C,G), rs/null (D,H), or null/null (E,I) genotypes exhibit a clear gradient in the severity of the dysmorphology phenotypes, both externally (B-E) and internally (F-I). Note the presence of a variety of morphological defects in null/null (E), including micropthalmia (mo), micrognathia (mg), oligodactyly (od), hypomelia (hm), gastroschisis (gs), spina bifida aperta (sba) and axial truncation (at). In the skeletal samples (F-I), note the difference in the levels of axial truncation (arrowheads).

 


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  		Fig. 5. Transcriptional activation by LEF1 in response to Wnt1 signaling in primary-cultured fibroblasts from newborns. Fibroblasts of rs5 (+/+), rs7 (rs/+), rs3 (rs/rs) and rs8 (rs/rs) were transfected with a LEF-LUC reporter construct (500 ng), in combination with expression plasmids encoding LEF1 (50 ng), ß-catenin (300 ng) or Wnt1 (300 or 1000 ng). The levels of luciferase activity were normalized for the expression of a co-transfected Rous sarcoma virus-ß-galactosidase expression plasmid (50 ng). The normalized activity was quantitated relative to the level of activity (100%) from cells transfected with LEF1 and ß-catenin expression plasmids. Means with error bars representing standard errors. Experiments were repeated at least three times.

 


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  		Fig. 6. Disturbance in the AP polarity of somites and preserved expression of paraxis in rs mutants. Whole-mount in situ hybridization of rs/rs embryos (B,D,F,H,J) compared with stage-matched control embryos (A,C,E,G,I) at early E10 (A-F) and late E10 (G-J) with probes for Sox10 (A,B) Tbx18 (C,D), Uncx4.1 (E,F) and paraxis (G-J). (A-F) Sox10-positive domains show the status of segmentation of the DRGs. In rs/rs, DRG segmentation in the caudal region is disturbed (yellow arrowheads in B). Expression patterns of Tbx18 and Uncx4.1 in rs/rs in somites rostral to the hindlimb bud are fairly normal and are restricted to the anterior and posterior compartments, respectively. In more caudal regions, their expression patterns become diffuse in rs/rs. (G-J) Expression of paraxis is well maintained in rs/rs (overviews in G,H; magnified views in I,J). Both the initiation of paraxis expression in the PSM (red arrowheads in I,J) and its strong upregulation in the rostral PSM (black arrowheads in G-J) take place normally in rs/rs. FL, forelimb buds; HL, hindlimb buds.

 


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  		Fig. 7. Disturbances in expression of notch-delta pathway genes in rs mutants. Whole-mount in situ hybridization of rs/rs embryos (B,D,F,H,I,K,M,O) compared with stage-matched control embryos (A,C,E,G,J,L,N) at carefully selected stages of E9 (A-F) and of E10 (G-O) with probes for Dll1 (A-I), Mesp2 (J-M) and Lfng (N and O). Expression of Dll1 in rs/rs appears fairly normal up to E9.5 (B). However, stripe-like expression of Dll1 in the rostral PSM and in newly formed somites (black arrowheads in C-F) becomes unclear and less intense (red arrowheads in D,F) from E9.5 on (D), while strong Dll1 expression in the caudal two-thirds of the PSM is still maintained during late E9 (F), starts to reduce from early E10 (H), and diminishes by mid E10 (I). Surprisingly, Mesp2 expression in a stripe pattern in the rostral PSM continues, though significantly reduced in intensity, until late E10 in rs/rs (K,M). Dynamic and oscillating expression of Lfng in the PSM is also observed until early E10, but appears to cease by mid E10 in rs/rs (O). FL, forelimb buds; HL, hindlimb buds.

 


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  		Fig. 8. Cell proliferation and programmed cell death in the PSM of rs/rs. BrdU incorporation (A,B) and TUNEL (C,D) assays were performed on serial frozen sections of E10.5 control (A,C) and rs/rs (B,D) embryos. Note that a number of BrdU-positive cells are present in the PSM (psm) as well as in somites (so) of both rs/rs (B) and control (A). The numbers of apoptotic cells detected by TUNEL in the PSM (examples indicated by the black arrowheads in C,D) are comparable between control and rs/rs. Tb, tail bud. Scale bars: 100 µm.

 

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© The Company of Biologists Ltd 2004