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Involvement of the Polycomb-group gene Ring1B in the specification of the anterior-posterior axis in mice

Maki Suzuki^1,2,*, Yoko Mizutani-Koseki^1,*, Yu-ichi Fujimura^1,*, Hiro Miyagishima¹, Tomomi Kaneko¹, Yuki Takada¹, Takeshi Akasaka¹, Hideki Tanzawa², Yoshihiro Takihara³, Megumi Nakano⁴, Hiroshi Masumoto⁴, Miguel Vidal⁵, Kyo-ichi Isono¹ and Haruhiko Koseki^1,6,

¹ Department of Molecular Embryology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
² Department of Oral Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan
³ Department of Develomental Biology and Medicine, Osaka Medical Center for Cancer and Cardiovascular Diseases, Nakamiti 1-3-3, Tousei-ku, Osaka, 537-8511, Japan
⁴ Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
⁵ Centro de Investigaciones Biologicas, Department of Developmental and Cell Biology, Velazquez 144, 28006 Madrid, Spain
⁶ RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan
^* These authors contributed equally to this work

View larger version (25K): [in a new window]   Fig. 1. Interactions between Ring1B and Mel18. (A) Interaction in yeast cells indicated by the ß-galactosidase activity. Yeast cellswere transformed with the indicated plasmids. Lamin was a negative control. (B) GST pull-down assays. (Part a) Binding of 35S-labeled full-length Mel18 protein (Mel18fl) to GST or GST fused to either full-length or truncated Ring1B (GST-Ring1Bfl, GST-Ring1BC or GST-Ring1BN, respectively). (Part b) Binding of 35S-labeled full-length Ring1B protein (Ring1Bfl) to GST or GST fused to truncated Mel18 (GST-Mel18PEST) (amino acids 1-233). (Part c) Binding of 35S-labeled truncated Ring1B proteins (Ring1BC and Ring1BN) to GST or GST-Mel18PEST.

View larger version (63K): [in a new window]   Fig. 2. In vivo association of Ring1B with mammalian PcG proteins. (A) Specific co-immunoprecipitation of Ring1B from 11.5 dpc mouse embryos using antibodies against Mel18 and Rae28/Mph1. The signals corresponding to the immunoglobulin heavy chains are indicated by arrowheads (Ig). (B-F) Comparative analysis of the subnuclear distributions of endogenous RING1B and MEL18 (B), BMI1 (C), RING1A (D), RAE28/MPH1 (E) and exogenous Mph2 (F) in normal or transfected human osteosarcoma U2-OS cells using indirect immunofluorescence. (Parts a) The signal of RING1B (green). (Parts b) The signals of endogenous MEL18 (B), BMI1 (C), RING1A (D), RAE28/MPH1 (E) and exogenous Mph2 (F) (red). (Parts c) Merged images. (Parts d) Phase contrast views. Monoclonal and polyclonal antibodies are indicated as M and P, respectively, in parentheses.

View larger version (47K): [in a new window]   Fig. 3. Association of Ring1B, M33 and Rae28/Mph1 with chromosomal DNA. Protein/DNA complex was concentrated by in vivo formaldehyde crosslinking and subsequent cesium chloride isopyknic centrifugation. (A) Chromosomal DNA in each fraction was visualized by Ethidium Bromide staining. DNA digested with StyI was used as molecular weight markers. (B) Protein in each fraction was visualized by Coomassie Brilliant Blue (CBB) staining. Localization by western blot analysis of an ER luminal protein, Bip (C), dimethyl-Histone H3 (D), Ring1B (E), M33 (F) and Rae28/Mph1 (G).

View larger version (89K): [in a new window]   Fig. 4. Subcellular localization of RING1B in interphase and mitotic nuclei. (A,B) Distribution of RING1B and CENPB (A) and centromeric antigens (B) in interphase nucleus of U2-OS cells. (Parts a) Localization of endogenous RING1B (green). (Parts b) Localization of endogenous CENPB (A) and centromeric antigens (B; ACA) (red). (Parts c) DAPI (blue). (Parts d) Merged images. (Parts e) Higher magnification views of the boxed regions in parts d. (C) Distribution of RING1B (part a, green) and CENPB (part b, red) in prophase nucleus of U2-OS cells. (part c) DAPI (blue). (Part d) Merged images. (D) Distribution of RING1B (part a; green) and centromeric antigens (part b; red) in prometaphase nucleus of U2-OS cells. (Part c) DAPI (blue). (Part d) Merged images. (Part E) Distribution of RING1B (part a; green) and CENPB (part b; red) in anaphase nucleus of U2-OS cells. (Part c) DAPI (blue). (Part d) Merged images.

View larger version (28K): [in a new window]   Fig. 5. Disruption of the Ring1B gene in mice. (A) Diagram of the Ring1B locus, the targeting vector and of the modified allele. The Ring1B-coding exons are indicated by black boxes. The PGKneo and pMC1-tk expression cassettes were used for positive and negative selection, respectively. Position of relevant restriction sites (EcoRI, E; BamHI, B; HindIII, H; SalI, S; XhoI, X), location of probe and PCR primers, and sizes of diagnostic fragments are indicated. (B) Southern analysis of genomic DNA isolated from offspring of heterozygote matings after digestion with BamHI + HindIII and probed with the indicated 3' probe shown in A. (C) PCR analysis of tail genomic DNA of the offspring of a heterozygous intercross. Primers RB22 and RB18 amplify a 1.4 kb of the wild-type allele, whereas primers Neo2 and RB11 amplify a 3.0 kb of the mutated allele. (D) Semi-quantitative analysis of Ring1B by western blot analysis of total proteins extracted from 11.5 dpc mouse embryos of the indicated genotypes probed with antibodies against Ring1B. To correct for loading differences, anti-lamin B antibodies and Coomassie Brilliant Blue (CBB) staining of the same gel were used.

View larger version (55K): [in a new window]   Fig. 6. Skeletal alterations of Ring1Bred/red, Mel18–/– and Ring1Bred/redMel18–/– mice. (A) The genotypes are indicated at the top. (Parts a-d) Lateral views of the upper part of the vertebral column. Yellow stars in parts c and d indicate a ectopic arch of the occipital bone. Black arrows in parts b-d indicate ectopic ribs associated with the 7th cervical vertebra. Red arrows indicate the prominent spinous process. Blue arrows in parts b and d indicate the additional ossification center of the sternum implying the anterior shift of the sternum. (Parts e-h) Ventral views of the rib cages. Black arrows in parts f and h indicate ectopic ribs associated with 7th cervical vertebra. (B) Various posterior transformations of the axial skeleton and their penetrance (indicated in parenthesis): (1) Supraoccipital boneC1, appearance of the ectopic bones seen in the craniodorsal region of the C1 vertebra or ectopic arch of the occipital bones; (2) C1C2, presence of the odontoid process on the C1 vertebra; (3) C2C3, lack of the odontoid process from the C2 vertebra; (4) C7T1, appearance of cervical ribs on C7; (5) T1T2, prominent spinous process on T1; (6) T7T8, dissociation of 7th rib from the sternum; (7) T13L1, loss of the rib in 20th vertebra; (8) L5 or 6S1, formation of the sacro-iliac joint in 25th or 26th vertebra; (9) S4Ca1, appearance of the first caudal vertebra in 29th or 30th vertebra; (10) anterior shift of the sternum as revealed by an ectopic ossification.

View larger version (81K): [in a new window]   Fig. 7. Expression of Hoxb genes in 11.5 dpc wild-type and Ring1Bred/red mutant embryos. Lateral view of sagittal sections showing the expression of Hoxb4 (A,B), Hoxb6 (C,D) and Hoxb8 (E,F) in Ring1Bred/red (A,C,E) and wild type embryos (B,D,F). The number of prevertebrae starting at the proatlas are shown and segment boundaries are indicated by bars.

View larger version (67K): [in a new window]   Fig. 8. Skeletal alterations seen in Ring1Bred/red, Mel18–/– and Ring1Bred/redMel18–/– mice on the compound genetic background. (A) Respective genotype of Ring1B and Mel18 loci are indicated at the top. For Ring1Bred/redMel18–/–, three specimens are shown. (Parts a-f) Lateral views of the upper part of the vertebral column are shown. Stars in parts c and e indicate ectopic floating bone or ectopic arch of the occipital bone, respectively. Arrows in parts b, c, e and f indicate ectopic ribs associated with C7. (Parts g-l) Ventral views of the rib cages are shown. In part k, ectopic ossification center of the sternum as a consequence of complete anterior shift of the sternum and complete ectopic ribs are indicated by a blue arrow and black arrows, respectively. Note 7th ribs are articulated with the sternum in Ring1Bred/redMel18–/– mice. (Parts m-r) Ventral views of the thoracolumbar region are shown. Prospective T12 and T13 are indicated by respective numbers. Prospective lumbar vertebrae are indicated by red stars individually. (B) The frequency of the posterior transformations of the axial skeleton is schematically represented. The following parameters are scored and frequency of each alteration is indicated in parentheses: (1) supraoccipital boneC1, appearance of the ectopic bones seen in the craniodorsal region of the C1 vertebra or ectopic arch of the occipital bones; (2) C1C2, presence of the odontoid process on the C1 vertebra; (3) C2C3, lack of the odontoid process from the C2 vertebra; (4) C7T1, appearance of cervical ribs on C7; (5) T1T2, prominent spinous process on T1; (6) T7T8, dissociation of 7th rib from the sternum; (7) T13L1, loss of the rib on 20th vertebra; (8) L1T13, appearance of the rib on 21st vertebra; (9) L5 or 6S1, formation of the sacro-iliac joint in 25th or 26th vertebra; and (10) S4Ca1, appearance of the first caudal vertebra in 29th or 30th vertebra.

View larger version (54K): [in a new window]   Fig. 9. Repression of chicken Hoxb9 expression by overexpression of mouse Ring1B and Mel18. (A, part a) In ovo electroporation. A pair of electrodes held by a manipulator is inserted through a window and placed over the vitelline membrane overlying the embryo. Plasmid solution colored by Nile Blue is injected into the developing spinal cord as indicated by a yellow bracket. (parts b,c) Hoxb9 expression in seven-somite (part b) and 15-somite (part c) stage embryos. Somite boundaries are shown. (B, part a) Expression of mRing1B 24 hours after electroporation. (parts b-d) Repression of Hoxb9 expression observed 48 hours after electroporation. The anterior boundaries of Hoxb9 expression in the control sides are indicated by arrows. Downregulation of Hoxb9 expression by exogenous Ring1B are indicated by brackets. (C, part a) Expression of mMel18 24 hours after electroporation. (part b) Repression of cHoxb9 expression 48 hours after electroporation. The anterior boundary of Hoxb9 expression in the control side is indicated by an arrow. Downregulation of Hoxb9 expression by mouse Mel18 is indicated by a bracket. (D) Ring1B- and Mel18-dependent repression of Hoxb9 expression is influenced by the developmental stage. Frequency of affected embryos by transfection of mouse Ring1B, Mel18 and empty vectors were represented by closed, shaded and open bars, respectively. Mel18-dependent repression is less efficient than that of Ring1B and is similarly influenced by developmental stage. (E) Ring1B-dependent repression of Hoxb9 expression becomes obvious at least 36 hours after the electroporation. Asterisks in D and E indicate results from the identical series of experiments.