OFCD syndrome and extraembryonic defects are revealed by conditional mutation of the Polycomb-group repressive complex 1.1 (PRC1.1) gene BCOR

Abstract

BCOR is a critical regulator of human development. Heterozygous mutations of BCOR in females cause the X-linked developmental disorder Oculofaciocardiodental syndrome (OFCD), and hemizygous mutations of BCOR in males cause gestational lethality. BCOR associates with Polycomb group proteins to form one subfamily of the diverse Polycomb repressive complex 1 (PRC1) complexes, designated PRC1.1. Currently there is limited understanding of differing developmental roles of the various PRC1 complexes. We therefore generated a conditional exon 9-10 knockout Bcor allele and a transgenic conditional Bcor expression allele and used these to define multiple roles of Bcor, and by implication PRC1.1, in mouse development. Females heterozygous for Bcor exhibiting mosaic expression due to the X-linkage of the gene showed reduced postnatal viability and had OFCD-like defects. By contrast, Bcor hemizygosity in the entire male embryo resulted in embryonic lethality by E9.5. We further dissected the roles of Bcor, focusing on some of the tissues affected in OFCD through use of cell type specific Cre alleles. Mutation of Bcor in neural crest cells caused cleft palate, shortening of the mandible and tympanic bone, ectopic salivary glands and abnormal tongue musculature. We found that defects in the mandibular region, rather than in the palate itself, led to palatal clefting. Mutation of Bcor in hindlimb progenitor cells of the lateral mesoderm resulted in 2/3 syndactyly. Mutation of Bcor in Isl1-expressing lineages that contribute to the heart caused defects including persistent truncus arteriosus, ventricular septal defect and fetal lethality. Mutation of Bcor in extraembryonic lineages resulted in placental defects and midgestation lethality. Ubiquitous over expression of transgenic Bcor isoform A during development resulted in embryonic defects and midgestation lethality. The defects we have found in Bcor mutants provide insights into the etiology of the OFCD syndrome and how BCOR-containing PRC1 complexes function in development.

Keywords: Cardiac; Craniofacial; Oculofaciocardiodental syndrome; Placenta; Salivary glands; X-linked developmental disorder.

Figure 1:. Generation of conditional Bcor allele.

(A) Diagram of human BCOR locus showing position of OFCD deletions (Δ) and mutations that generate frame shifts (fs) or stop codons (red triangles), and a splice site mutation (sm) leading to an in-frame exon 10 deletion (green triangle). (Note: splice site mutation sequence is shown in Figure 1d of (Ng et al., 2004). This mutation corresponds to family OFCD1 with IVS9G->T mutation (not IVS8G->T as stated), while family OFCD2 is actually IVS10G->A; L. Biesecker pers. comm.). (B) Diagram of Bcor gene targeting strategy. Homologous recombination in embryonic stem (ES) cells generated the Bcor ^Neo2 allele, in which exons 9 and 10 of Bcor are flanked by loxP sites. Immediately downstream of exon 10, Bcor ^Neo2 also contains a neomycin resistance cassette (Pgk-Em7-Neo-bGH1pA, shortened to Pgk-Neo) flanked by recognition sites for the FLP recombinase (frt sites), allowing its excision. Bcor ^Fl allele was generated by deleting Pgk-Neo cassette in mice expressing FLPe. CRE-mediated recombination excises exons 9 and 10, generating a premature stop codon in the now out-of-frame exon 11. Genotyping PCR primers and Southern probes are indicated. (C) Southern blot hybridization of the wild type and targeted ES cell genomic DNA, digested with ApaI, reveals successful homologous recombination of both the 3’ and 5’ homology arms. The ApaI site introduced immediately upstream of the 5’ frt site in the targeting vector results in a 7.3 kb band recognized by the 5’ probe and an 8.8 kb band recognized by the 3’ probe. (D) Western blot analysis of wild-type, Bcor ^Neo2, and Bcor ^ΔE9−10/Y ES cell protein extracts shows that the targeted Bcor ^Neo2 cells display wild-type levels of full length BCOR and the Bcor ^ΔE9−10/Y cells lack full-length BCOR and contain reduced amounts of a C-terminally truncated version of BCOR (BCORΔE9–10), which is unable to interact with KDM2B, PCGF1/3, and other PRC1 proteins and is thus predicted to be non-functional. ß-ACTIN levels were used as a loading control (bottom panel). (E) Multiplex PCR genotyping reaction of genomic DNA, using primers C, G and J, identifies all three alleles: wild type (424 bp), Bcor ^Fl (570 bp), and Bcor ^ΔE9−10 (519 bp). (F) Breeding strategies with β-actin-Cre and summary of outcomes. (G) E18.5 gross morphology of the maxillary region (mandibles removed) highlighting cleft palate (arrowhead) in OFCD but not control animal. Representative images of control and OFCD mice showing (H) kinked tails in adults and adult skeletal preparations, and (I) size at weaning.

Figure 2.. Ocular defects and kyphosis in OFCD mice.

(A) Bar plot of lens opacification in animals ages 3–28 weeks. (B) Boxplots of eye globe widths including all globes or separated by wider and narrower eye in each animal. (C) Boxplots of the eye globe width differences within each animal. (D-F) Images of paired littermate adult control and OFCD heads, note visible lens opacification in E and ptosis in E and F. Dorsal (left panel) and lateral (right panel) views of the same animals are shown in F. (G) Images of dermestid skeletal preparations of adult control and OFCD animals. Note kyphosis in OFCD skeletons. (H) Kyphosis index (KI) in control and OFCD animals. Distance AB is from the posterior edge of vertebrae C7 to the posterior edge of L6. CD is the distance from line AB to the dorsal border of the vertebral body farthest from that line (Laws and Hoey, 2004). p values are from one-sided t tests for B and H and Welsh one-sided t-tests for C.

Figure 3.. Early embryonic lethality in Bcor ^ΔE9−10/Y males.

(A-L) Bcor in situ analysis using probe sequences absent (exon 9–10, A-H) or present (exon 4, I-L) in the deleted allele. The exon 4 probe is more robust and is able to maintain hybridization with the exon 9–10 deleted transcript. Arrowheads indicate extra-embryonic ectoderm/chorionic region; arrow indicates escape from paternal XCI in P-TGCs. (M-W) LacZ staining in Flk1 expressing lineages of early embryonic development for control (M, N, Q,R, U) and Bcor ^ΔE9−10/Y (O, P, S, T, V, W) embryos. (X-II) In situ analysis in somite-number matched control (X, Z, BB, DD, FF, HH, JJ, LL, NN and PP) and Bcor ^ΔE9−10/Y embryos (Y, AA, CC, EE, GG, II, KK, MM, OO and QQ ) showing Uncx2.1, a marker of somites (X-EE), Otx2, a marker of forebrain and midbrain (BB-II), Fgf8 a marker of the anterior neural ridge (JJ-MM, arrowhead) and Foxg1 a marker of forebrain (NN-QQ).

Figure 4.. Bcor is required in neural crest lineages outside the palate for normal palate formation.

(A, B) E18.5 gross morphology of the maxillary region (mandibles removed) in control Bcor ^Fl/Y (A) and mutant, Bcor ^Fl/Y;Pax3-Cre (B), which has cleft palate (white arrow head). Clefting was observed in all mutants (n>10). (C-F) H & E staining of frontal sections through E18.5 heads confirms the cleft palate in mutant (D, expanded in F) but not in control (C, expanded in E). In addition, mutants had ectopic salivary glands in the submandibular region (D, black arrows) and disorganized tongue morphology (F, black arrowhead). Both phenotypes were present in all mutants examined (n>4). P = palatal shelves, T = tongue. (G-H) Skeletal preparations of E18.5 control Bcor ^Fl/Y (G), and mutant Bcor ^Fl/Y;Pax3-Cre, (H) heads, mandibles removed, confirm cleft palate defect. Cartilage is stained with Alcian blue, while bone is stained with Alizarin red. These phenotypes were observed in all mutant skeletal preparations (n=3). (I-N) Time course of palatal shelf elevation. H & E staining of frontal sections through Bcor ^Fl/Y control (I, K, M) and Bcor ^Fl/Y;Pax3-Cre mutant (J, L, N) palates at indicated ages reveals a failure in palatal shelf elevation that became evident at E14.5 and persisted throughout development. Scale bars, 0.5 mm. These phenotypes were observed in all mutants (n=2 for each time point). (O,P) Bcor mutant palatal shelves can elevate and fuse in culture. Alcian blue staining of cartilaginous structures in Bcor ^Fl/Y control (O) and Bcor ^Fl/Y;Pax3-Cre mutant (P) palatal shelves isolated at E12.5, cultured for 5 days and cartilage stained with Alcian blue. Dashed white lines outline the elevated and fused palate in both the control and the mutant tissue.

Figure 5.. Bcor is required in neural crest lineages to suppress ectopic salivary gland formation and for normal craniofacial development.

(A-F) H & E stained frontal (A-D) and sagittal (E, F) sections through control Bcor ^FL/Y (A, C), Bcor ^FL/Y;Wnt1-Cre (B), and Bcor ^FL/Y;Pax3-Cre (D, E, F) E18.5 heads. (A) In control animals, two pairs of, submandibular and sublingual salivary glands (arrows) are found below the lower jaw in control animals. (B) In the mutant, in addition to the normal pairs of SM and SL glands (arrows), an ectopic pair of salivary glands (black arrowheads) is found between the mylohyoid, hyoglossus, and genioglossus muscles. Main excretory ducts of the SM and SL glands also run through this area on the buccal side (white arrowheads). (A) and (B) are sectioned at the level of the first molars (M). (C) In control animals two excretory ducts are found on each side of the lower jaw, the SL duct on the buccal side and the SM duct on the lingual side. (D) In mutant animals, excretory ducts of ectopic SGs run alongside the ones of the SL and SM glands, on the lingual side. They are either completely separate ducts (right hand side) or join with the SM duct shortly before it opens in the mouth (left hand side). (E) At the midline, ectopic SGs (arrowhead) abut the lingual frenulum. (F) Further from the midline, on the buccal side of the tongue, ectopic (arrowhead) and regular (arrow) SGs come closer to each other. (F) Ectopic SGs (arrowhead) are positioned more distally along the proximo-distal axis of the lower jaw than the SM and SL glands (arrow). (G, H) Trichrome-stained sagittal sections through Bcor ^FL/Y (G) and Bcor ^FL/Y;Pax3-Cre (H) E18.5 heads. Groups of mucous acini stained in blue (black, arrow) are found on the buccal side of ectopic SGs (H), reminiscent of the SL gland in control animals (G). Ec = ectopic salivary gland, Gen = genioglossus muscle, Hy = hyoglossus muscle, I = lower incisor, M = first molar, My = mylohyoid muscle, SL = sublingual salivary gland, SM = submandibular salivary gland, T = tongue. (I-N) Skeletal preparations of E18.5 control Bcor ^Fl/Y (I, K, M) and mutant Bcor ^Fl/Y;Pax3-Cre (J, L, N) heads reveal shortening of the tympanic ring bone (white arrows indicate the normal position of the dorsal end; I, K vs. J, L) and mandible (black arrowheads, I, M vs. J, N). Cartilage is stained with Alcian blue and bone is stained with Alizarin red. These phenotypes were observed in all mutants (n=7). (O-R) Alcian blue staining of cartilaginous structures in E14.5 Bcor ^Fl/Y control (O, Q) and Bcor ^Fl/Y;Pax3-Cre mutant (P, R) mice reveals defective Meckel’s cartilage patterning in the mutant animals, in side (O, P) and frontal (Q, R) views (black arrowheads). Note tongue protrudes from mouth in control (asterisk) but not mutant. These phenotypes were observed in all mutants (n=9).

Figure 6.. Bcor is required in Isl1-expressing cell lineages for normal cardiac outflow tract formation and ventricular septation and hindlimb patterning.

(A) Survival/Lethality of E13.5 (n=263), E14.5 (n=30), E15.5 (n=42), E16.5 (n=26), E18.5 (n=35) and P21 (n=39) animals. Cardiac analysis (B-L): Summary of visible cardiac defects at E13.5 (B). (C-F) Scanning electron microscopy of Bcor ^Fl/Y (C, D) and Bcor ^Fl/Y;Isl1-Cre (E, F) hearts at E13.5 reveals persistent truncus arteriosus (PTA) in mutant hearts. (G - L) H & E staining of Bcor ^Fl/Y (G, I, K), and Bcor ^Fl/Y; Isl1-Cre (H, J, L) hearts at E13.5 (G-J) and E14.5 (K-L) sections confirm PTA in the mutant hearts (H, L) and illustrate ventricular septal defect (J) compared to control (G, I, K) Ao = aorta, PA = pulmonary artery, RA = right atrium, LA = left atrium, RV = right ventricle, LV = left ventricle, DAo = descending aorta, AR = aortic root, PR = pulmonary root, RAVC = right atrioventricular canal, PTA = persistent truncus arteriosus, PT = truncal valve, VSD = ventricular septal defect. (M-T) Hindlimb analysis: Gross morphology (M, N) and Alcian blue staining (O-R) of hindlimbs at E14.5 in Bcor ^Fl/Y (M, O, Q) and Bcor ^Fl/Y; Isl1-Cre (N, P, R) and H & E staining of Bcor ^Fl/Y (S) and Bcor ^Fl/Y; Isl1-Cre (T) E15.5 section showing mutants with second-third digit syndactyly (2/3 arrow heads).

Figure 7:. Bcor is required in extraembryonic lineages for placental development and consequently embryo survival.

(A,B) Freshly dissected E10.5 placentae and embryos illustrating IUGR and excess blood in Bcor ^{ΔE9−10/+(p)} concepti (B) vs. Bcor ^Fl/+(p) control (A). Midpoint sections of E10.5 concepti (C-H) and E11.5 placentae (I, J) H & E (C,D) or lectin staining (E-J) of Bcor ^Fl/+(p) controls (C, E, G and I) vs. Bcor ^{ΔE9−10/+(p)} (D,F,H, and J) showing P-TGC and Sp-T (blue line) cell expansion, reduced labyrinth (black line) and excess blood (bright pink) in mutants. Boxes in E and F indicate region expanded in G and H respectively.

Figure 8:. Generation and lethality of a conditional Bcor expression allele.

(A) Diagram of the targeting strategy. Homologous recombination in embryonic stem cells (ES cells) generated the Rosa26^LsLmBcorA allele, in which a splice acceptor (SA) site precedes a LoxP-flanked transcriptional “4xpA stop” sequence (4 copies of a polyadenylation signal) that is followed by a myc-tagged (m) murine BcorA (isoform A) coding sequence. CRE-mediated recombination excises the LoxP-flanked “stop” sequence, allowing expression of myc-tagged BcorA driven by the Rosa26 promoter. (B) Southern blot hybridization of the wild type and targeted ES cell genomic DNA digested with EcoRV reveals successful homologous recombination of the 5’ homology arm. The introduced EcoRV site generates a 4.1 kb fragment in addition to the 11.5 kb band from the wild type allele (3’ homology arm not shown). * = partially digested DNA. (C) In situ analysis of Bcor expression in Rosa26^LsLmBcorA/+ vs. Rosa26^mBcorA/+ concepti at E8.5. (D) quantitative RT-PCR comparing total Bcor transcripts (endogenous plus transgene, left) vs. endogenous (right) in Rosa26^LsLmBcorA/+ and Rosa26^mBcorA/+ placenta and embryos at E9.5. (E) Western blots of total BCOR levels vs. transgene expressed mBCORA in individual E9.5 fetuses (#1–4) and their placenta. In the color panel a yellow band corresponds to a protein stained with both BCOR and myc antibodies. (F) Survival of Rosa26^LsLmBcorA/+ vs. Rosa26^mBcorA/+ embryos from E10.5 (n=54), E11.5 (n=86), E12.5 (n=27), E13.5 (n=18) to E14.5 (n=9). (G-N) Images of representative Rosa26^LsLmBcorA/+ (G, I, K, M) and Rosa26^mBcorA/+ (H, J, L, N) placentae and embryos from E10.5 (G, H), E11.5 (I, J), E12.5 (K, L) and E13.5 (M, N) illustrating embryo phenotypes.