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Disruption of mesodermal enhancers for Igf2 in the minute mutant

Karen Davies¹, Lucy Bowden^1,*, Paul Smith¹, Wendy Dean¹, David Hill², Hiroyasu Furuumi³, Hiroyuki Sasaki³, Bruce Cattanach⁴ and Wolf Reik^1,

¹ Laboratory of Developmental Genetics and Imprinting, Developmental Genetics Programme, Babraham Institute, Cambridge CB2 4AT, UK
² Lawson Health Research Institute, St. Joseph’s Health Care, 268 Grosvenor Street, London, Ontario N6A4V2, Canada
³ Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan
⁴ Medical Research Council, Mammalian Genetics Unit, Harwell, Didcot OX11 0RD, UK
^* Present address: Gardiner-Caldwell Communications, The Towers, Park Green, Macclesfield SK11 7NG, UK

View larger version (10K): [in a new window]   Fig. 1. Foetal and plactental weights of Mnt mutants. Weights following paternal (blue circles) and maternal (red circles) transmission of the Mnt mutation, and their corresponding wild-type littermates (black squares) are shown in grams. (A) Foetal weights at embryonic (E) and postnatal (P) stages following paternal transmission of the Mnt mutation. (B) Placental weights with paternal transmission of Mnt (blue circles). (C) Foetal weights at embryonic (E) and postnatal (P) stages following maternal transmission of the Mnt mutation. (D) Placental weights with maternal transmission of Mnt (red circles).

View larger version (17K): [in a new window]   Fig. 2. Expression analysis of Igf2 and H19 in Mnt. (A) Northern blot analysis of total RNA prepared from the different classes of E14 intercross embryo and placenta samples; MntP n=4; MntM n=4; wild type n=4; homozygous Mnt (Hom) n=2 (n=sample number analysed). (B) Histogram of Igf2 (3.8 kb transcript) relative expression levels obtained in A normalised against Gapdh for each class of intercross sample, error bars show the standard deviations. (C) Circulating Igf2 serum (ng/ml) levels in neonates (P1) following paternal (n=12) and maternal (n=17) transmission of Mnt, and their corresponding wild-type littermates (n=9 and n=11, respectively). Error bars show the standard deviations.

View larger version (81K): [in a new window]   Fig. 3. Tissue specific analysis of Igf2 expression. (A) Northern analysis of Igf2 and H19 in neonatal (P1) tissues following paternal transmission of Mnt and the corresponding wild-type littermates. (B) Histogram of relative Igf2 expression levels normalised against Gapdh. Error bars show the standard deviations when multiple samples were analysed: tongue 35% (n=2 wild type, n=2 MntP), liver 110% (n=5 wild type, n=6 MntP), intestine 71% (n=4 wild type, n=5 MntP), muscle 62%% (n=1 wild type, n=2 MntP). (C) In situ hybridisation analysis of Igf2 expression in E14 MntP embryos and placentae. Images were captured with standardised exposure times, degree of illumination and level of magnification. Choroid plexus (cp), tongue (t), heart (h), lung (lu), liver (li), intestine (i) and intercostal muscle (im). Note the difference in embryonic and placental sizes between MntP and wild type. (D) Higher magnification of some organs. Note the reduced levels (apparently cell-type specific) observed in the intestine, lung and tongue. (E) Cell-type specific expression within the intestine. Expression in MntP is retained in the endodermal epithelial lining (e) but lost in the mesodermal muscular layer (m) of the intestine. (F) Cell-type specific expression within the lung. Igf2 expression in MntP is only retained in those bronchi with closed lumen (c) while it is lost in the open bronchi (o) and mesenchyme.

View larger version (40K): [in a new window]   Fig. 4. Expression and methylation analysis with maternal transmission of Mnt. (A) Northern analysis of Igf2 and H19 in neonatal (P1) in MntM and the corresponding wild-type littermates. (B) Representation of the Igf2 expression levels normalised against Gapdh (in tissues in which Igf2 expression was detectable after paternal transmission). Error bars show the standard deviations when multiple samples have been analysed. Tongue wild type n=1, MntM n=1; lung wild type n=4, MntM n=3; liver wild type n=5, MntM n=3; intestine wild type n=4, MntM n=4; muscle wild type n=1, MntM n=1. (C) Methylation analysis of H19 DMR in homozygous Mnt foetuses (E17) using a 3.8 kb SacI probe, hybridised to SacI- and AatII-(methylation sensitive) digested genomic DNA (Tremblay et al., 1995). Note the absence of the unmethylated allele in the homozygous Mnt sample, showing methylation of the maternal Mnt allele, while in the wild type the maternal allele is unmethylated. (D) Allele specific expression analysis of Igf2 in MntM neonatal (P1) tissues (MntxSD7) and the corresponding wild-type littermates (F1xSD7) by RT-PCR (Dean et al., 1998) (three individual samples analysed). The 602 bp band corresponds to the maternal allele, while the 473 bp corresponds to the paternal SD7 allele.

View larger version (34K): [in a new window]   Fig. 5. Identification and characterisation of the Mnt mutation. (A) Representation of three cosmids used to screen for alterations in the Igf2-H19 region. The approximate location of the Mnt mutation is marked with #. (B) Hybridisation of cosmid CH to homozygous (Mnt) and wild-type EcoRI and BamHI digested genomic DNA. Note differences in bands between Mnt and wild-type samples are marked with *. (C) Arrangement of the two breakpoint regions on a wild-type chromosome. The breakpoint 1 (BP1) region (downstream of H19) is shown as a red line, while the BP2 region which is 3.5 cM further centromeric is shown as a blue line. The regions conserved between mouse and human (1-10) as identified by Ishihara et al. (Ishihara et al., 2000) are shown (green squares), while the region (+22 to +28 kb from the H19 promoter) identified by Kaffer et al. (Kaffer et al., 2000) as possessing enhancer activity is shown by a black line. (D) The arrangement of the two breakpoint regions on the Mnt chromosome. Note that the rearrangement isolates the conserved elements 9 and 10 from the H19 region. PCR primers (1-4) for the detection of the breakpoints are shown. (E) Relative positions of the two Mnt breakpoints, the surrounding genes and the genetic distance between the two breakpoints. The red and green lines represent the probes used in the FISH analysis. (F) Fluorescence in situ hybridisation (FISH) analysis on heterozygous Mnt/F1 nuclei, verifying that an inversion has occurred on the Mnt chromosome. Red, H19 probe; green, BP2 probe. In the merged image, 1 is the H19 region on the wild-type chromosome; 2 is the BP2 region on the wild-type chromosome; 3 is the 3' part of the BP2 region on the Mnt chromosome (see E); and 4 is the co-localisation of the 5' part of the BP2 region and the H19 region on the Mnt chromosome (yellow).

View larger version (44K): [in a new window]   Fig. 6. Transgene expression analysis to identify mesodermal enhancers for Igf2. (A) The two transgenes microinjected for transient expression analysis. Restriction sites NotI (N) and SalI (S) cut within the bluescript vector to excise +gh transgene. NotI and XhoI (X) digest excises –gh transgene. 5' H19 constitutes nucleotides 4700-5523 (H19 promoter), while 3' H19 constitutes nucleotides 9511-36040 (GenBank Accession Number, AF049091). (B) lacZ expression analysis in +gh-positive E14 embryo. (C) lacZ expression analysis in –gh-positive E14 embryo. Organs indicated are liver (li), intestine (i), tongue (t), heart (h) and lung (lu).

View larger version (8K): [in a new window]   Fig. 7. Summary of the main features of the Mnt mutation. (A) The situation in wild type with the endoderm enhancers (E), the mesoderm enhancers for tongue and skeletal muscle (M1), and the mesodermal enhancers for heart, lung and kidney (M2) activating paternal Igf2 and maternal H19. (B) The Mnt situation with disruption of the M1 enhancers, isolation of the M2 enhancers, maternal methylation at the H19 DMR (hence the absence of a functional boundary on the maternal allele), absence of H19 expression, and subsequent biallelic activation of Igf2 from the remaining active enhancers (E).