(A) Schematic representation of the Pcgf6 locus, the targeting vector, and the constitutive mutant allele. To disrupt Pcgf6, most of the exon 2 and the entire exon 3 which code for the RING finger domain were replaced with neo. The neo and HSV-tk cassettes were used for positive and negative selection, respectively. The positions of the restriction sites (XhoI, EcoRI and KpnI), external probe and PCR primers, and sizes of diagnostic fragments are indicated. Coding regions and untranslated regions of Pcgf6 are indicated by closed and open boxes, respectively. (B) Southern (top) and PCR (bottom) analyses for genotyping. For Southern blotting, genomic DNA was digested by KpnI and probed with the 3’ probe, as indicated in A. For PCR, a mixture of three primers (p1, p2 and p3 in A) was used. (C) Northern analysis of Pcgf6 mRNA expression in wild type (+/+) and homozygous (−/−) adult kidney (top). Ethidium bromide (EtBr) staining of the same gel is shown below. (D) Genotype distribution of progeny of Pcgf6 heterozygous intercrosses. *p-value<0.05 (χ2 test), indicating a < 5% probability of conforming to the Mendelian law, which predicts a 1:2:1 ratio between +/+:+/−: −/−. (E) Delayed and/or abnormal development of Pcgf6-KO (−/−) embryos at 10.5 dpc. Wild-type (+/+) embryos are shown as controls. (F) Genotype distribution of ESC lines derived from embryos from intercrosses between Pcgf6+/+ or −/− male and +/− female is shown. Genotypes of 22 ESC lines were determined using genomic PCR. (G) Skeletal alterations in Pcgf6−/− newborn mice. (a, d) Lateral views of the cervical and thoracic regions revealed the prominent spinous process on the 9–10 in Pcgf6−/− (d, indicated by an arrow) but on the nine in the wild type (a, indicated by an arrow). (b, e) Overviews of the seventh vertebrae identified association of an anterior tubercle on seven in Pcgf6−/−, which appears on six in the wild type (e, indicated by an arrow). (c, f) Ventral views of rib cages identified an ectopic sternal rib in Pcgf6−/− (f, labeled 15). (H) Schematic representation summarizing the axial alterations in Pcgf6−/− newborn mice. Each arrow represents the following morphological changes in the vertebrae; (a) 1←2: association of the anterior arch of the atlas with the atlas; (b) 6←7: the anterior tubercle(s) on 7, which are normally on 6; (c) 8←9: fusion of rib(s) on 9 to 8; (d) 9←10: the prominent spinous process on 10, which is normally on 9; (e) 14←15: fusion of ribs on 15 to the sternum. (I) Changes in Hoxb6 expression in Pcgf6-KO mice. Hoxb6 expression in 11.5-dpc wild-type (a) and Pcgf6−/− (c) embryos. Segment boundaries are indicated by red lines and segment numbers of anterior expression domains of Hoxb6 are shown. Bright field views are shown in (b, d). (J) HE sections of 11.5 dpc wild type (+/+) and Pcgf6-deficient (−/−) placentae. Al: allantois, Ch: chorion, La: labyrinth layer, asterisks: enucleated erythrocytes from the mother, arrows: fetus-derived nucleated erythrocytes. Note that fetus-derived nucleated erythrocytes are observed in well-developed labyrinth layer in Wild-type mice but not in Pcgf6-KO mice. Expression of trophoblast lineage-restricted markers was analyzed by RNA in situ hybridization. Pl1 identifies trophoblast giant cells. Tpbp identifies spongiotrophoblasts. (K) Quantification of placental area from 10.5 dpc HE sections of wild type (+/+), Pcgf6 heterozygote (+/−) and Pcgf6-deficient (−/−) embryos.