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First published online 13 December 2006
doi: 10.1242/dev.02726

Development 134, 417-426 (2007)
Published by The Company of Biologists 2007
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Differential regulation of imprinting in the murine embryo and placenta by the Dlk1-Dio3 imprinting control region

Shau-Ping Lin1,2, Phil Coan1, Simao Teixeira da Rocha1, Herve Seitz3,*, Jerome Cavaille3, Pi-Wen Teng1, Shuji Takada1,{dagger} and Anne C. Ferguson-Smith1,{ddagger}

1 Department of Physiology, Development and Neuroscience, University of Cambridge, Anatomy Building, Downing Street, Cambridge CB2 3DY, UK.
2 Institute of Biotechnology, College of Bioresources and Agriculture, National Taiwan University, Taipei, 106, Taiwan.
3 LBME-CNRS, UMR 5099, IFR, Toulouse, France.


Figure 1
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  		Fig. 1. Gross phenotype of E18 {Delta}IG-DMR/+ embryos. Comparison of (A) wild-type littermate (+/+) with (B) an embryo with the maternally inherited IG-DMR deletion (KO/+). Scale bar: 1 mm.

 

Figure 2
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  		Fig. 2. Skeletal defects of E19 {Delta}IG-DMR/+ embryos. Comparison of skeletal defects between E19 maternally transmitted IG-DMR knockout embryos (B,D; KO/+) and wild-type littermates (A,C; +/+). Preparations are stained with Alcian Blue for cartilage and Alizarin Red for bone. An upward slanting thoracic cage, with wider angulation of the ribs relative to the sternum, is observed in B as compared with A. Eight ribs (instead of 7) were attached to the sternum for IG-DMR knockout embryos (compare D with C). The green arrow indicates the extraossification observed in KO/+ embryos at the site where the 6th to 8th ribs attach to the sternum (D). The numbers in C and D refer to the costal cartilages, where the anterior-most rib is defined as 1. c, costal cartilage; ste, ossification centre of one of the sternebrae; xp, xiphoid process. Scale bar: 1 mm.

 

Figure 3
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  		Fig. 3. Malformed thoracic cage and abdominal distension of {Delta}IG-DMR/+ embryos. Comparable sections of E19 wild-type (A) and {Delta}IG-DMR/+ (B) embryos, illustrating the malformed thoracic cage and abdominal distension of {Delta}IG-DMR/+ embryos, which is also associated with protrusion of the abdominal organs (B). Embryos were stained with Haematoxylin and Eosin. Scale bar: 1 mm.

 

Figure 4
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  		Fig. 4. The immature and hypertrophic muscle phenotypes observed in {Delta}IG-DMR/+ embryos. Muscle morphology of normal (A,C,E) and {Delta}IG-DMR/+ (B,D,F) embryos at E19, stained with the myofibre-specific antibody MY-32. (A,B) Cross-sections of the forelimb. (C,D) High power views of the extensor carpi radialis longus. (E,F) High power views of the extensor pollicis longus muscle. (G,H) Morphometric analyses show that there are statistically significant differences, both in the proportion of myofibres with centrally-located nuclei (G; P<0.0001; Mann-Whitney U test), and in the mean myofibre cross-sectional area (H; P<0.0001, unpaired t-test). epl, extensor pollucis longus; ecrl, extensor carpi radialis longus; r, radius; u, ulna.

 

Figure 5
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  		Fig. 5. Expression of imprinted genes in placentas as a consequence of the IG-DMR deletion. (A) Northern blot analysis for imprinted genes in the Dlk1-Dio3 domain (Gtl2, Dlk1, Rtl1, Dio3), and primer extension assay (for micro-RNAs miR-127 and miR-410). MBII-48, MBII-49 and MBII-78 are snoRNA genes. Gapdh and U3 (Rnu3 - Mouse Genome Informatics) are controls. Gene symbols in blue and red denote those expressed from the paternally and maternally inherited chromosomes, respectively. Each genotype is represented by duplicate tracks of RNA isolated from different placentas. (B) Bar chart comparing expression levels between E16 placentas carrying the maternally-derived IG-DMR deletion (red; KO/+), with the paternally derived deletion (blue; +/KO), and wild-type littermates (yellow; +/+). Error bars represent s.e.m. Values were calculated from data generated for each gene using control (n=5-8) and mutant (n=2-5) placentas from different litters (n=2-4). Statistically significant differences from normal are: *, P<0.05; **, P<0.005; ***, P<0.0005; ANOVA + Fisher's PLSD test. Values were normalised against Gapdh except the small RNAs which were normalised against the unlinked, non-imprinted snoRNA U3. For comparison, the inserted panel shows the equivalent expression analysis in E16 embryos (see Lin et al., 2003Go). (C) Biallelic expression of Dlk1 in placentas of conceptuses with a maternally transmitted IG-DMR deletion. Sequence analysis of RT-PCR products from control littermate and heterozygote placenta upon maternal transmission of the IG-DMR deletion. Activation of Dlk1 from the normally repressed maternally inherited allele is only observed when the IG-DMR deletion is maternally transmitted. The Dlk1 expression in the normal placenta is exclusively from the paternal allele.

 

Figure 6
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  		Fig. 6. Methylation studies of the Dlk1-Gtl2 imprinted domain in placenta. (A) The IG-DMR region is fully methylated on the paternal chromosome and unmethylated in the maternal chromosome in placenta. The Dlk1-DMR (B) and Gtl2-DMR (C) are partially methylated on both parental alleles in the placentas, with methylation level being slightly higher on the paternal allele. (D) A placenta-specific differentially methylated region, Dlk1-DMR0, is identified ~7.4 kb upstream of the Dlk1 transcriptional start site. (E) Summary of the methylation status of the Dlk1-Gtl2 domain in embryos, sperm and placentas. White and black circles represent unmethylated and fully methylated regions, respectively. One, two and three quarters-filled circles represent alleles methylated by approximately 25%, 50% and 75%, respectively. M, maternal allele; P, paternal allele

 

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© The Company of Biologists Ltd 2007