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Left-sided embryonic expression of the BCL-6 corepressor, BCOR, is required for vertebrate laterality determination.
Hilton EN
,
Manson FD
,
Urquhart JE
,
Johnston JJ
,
Slavotinek AM
,
Hedera P
,
Stattin EL
,
Nordgren A
,
Biesecker LG
,
Black GC
.
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Oculofaciocardiodental (OFCD) syndrome is an X-linked male lethal condition encompassing cardiac septal defects, as well as ocular and dental anomalies. The gene mutated in OFCD syndrome, the BCL-6 corepressor (BCOR), is part of a transcriptional repression complex whose transcriptional targets remain largely unknown. We reviewed cases of OFCD syndrome and identified patients exhibiting defective lateralization including dextrocardia, asplenia and intestinal malrotation, suggesting that BCOR is required in normal laterality determination. To study the function of BCOR, we used morpholino oligonucleotides (MOs) to knockdown expression of xtBcor in Xenopus tropicalis, thus creating an animal model for OFCD syndrome. The resulting tadpoles had cardiac and ocular features characteristic of OFCD syndrome. Reversed cardiac orientation and disorganized gut patterning were seen when MOs were injected into the left side of embryos, demonstrating a left-sided requirement for xtBcor in lateral determination in Xenopus. Ocular defects displayed no left-right bias and included anterior and posterior segment disorders such as microphthalmia and coloboma. Expression of xtPitx2c was shown to be downregulated when xtBcor was depleted. This identifies a pathway in which xtBcor is required for lateral specification, a process intrinsically linked to correct cardiac septal development.
Figure 1. Patients with OFCD syndrome have defects in laterality determination.
A, B. Clinical features of an OFCD syndrome patient with dextrocardia. Characteristic facial (A), and dental (B) phenotypes were observed.
C. Sequence chromatogram of BCOR from the patient shown in (A). A single base pair insertion was observed in exon 5 causing a frame-shift and premature stop (c.1539insG, p.P514AfsX3).
Figure 2. Characterization of a X. tropicalis homologue of BCOR and inhibition of translation by MOs
A. Temporal expression of xtBcor was examined in a cDNA series derived from pools of developmentally-staged embryos. Initially, xtBcor was shown to be expressed as a maternal mRNA. Zygotic expression was observed from stage 9, and there after throughout embryogenesis to ~stage 30.
B. Spatial expression of xtBcor was analyzed in a cDNA tissue panel derived from adult female X. tropicalis. Expression of xtBcor was detected in all tissues assayed except for gall bladder and skin.
C. Two adjacent, non-overlapping, translation-blocking MOs were designed to target the 5’ UTR of xtBcor mRNA. As human anti-BCOR antibodies do not recognize xtBcor (data not shown), blocking of xtBcor mRNA translation was confirmed using an in vitro transcription/translation assay. A 5’ fragment of xtBcor mRNA was generated from a PCR template, which included a T7
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Downloaded from http://hmg.oxfordjournals.org/ at Cincinnati Children's Hospital Library on April 8, 2014
29 promoter and a 3’ FLAG tag. The sequence of xtBcor mRNA around the start codon and the
position of the MO binding sites is shown.
D. The 5’ xtBcor mRNA fragment was used as a template in an in vitro translation reaction using a rabbit reticulocyte lysate kit. The reaction was supplemented with xtBcor MO1 or MO2 as appropriate, at 1 μg of MO per 1 μg mRNA template. Using an anti-FLAG antibody, a product of 16 kDa was detected in the presence of xtBcor mRNA. The appearance of this product was completely inhibited in the presence of either xtBcor MO1 or MO2. A control MO had no inhibitory effect. All experiments were carried out using each MO and are derived from at least three independent experiments.
Figure 3. xtBcor MO causes defects in lateral determination. (A) Embryos injected with 8 ng of control MO were normal with only a background level of situs defects, affecting approximately 5% of embryos. (B and E). Embryos injected with 4 ng of xtBcor MO showed situs defects, manifesting as alterations to cardiac orientation and defects of gut origination and coiling (B). At high doses (8 ng), large numbers of embryos displayed a severe intestinal phenotype, with the majority of embryos displaying no anterioposterior patterning of the gut tube (E). (C and D) Unilateral left-sided injection of xtBcor MO caused cardiac and gut defects in over 85% of embryos. Cardiac development and gut patterning were uncoupled in most embryos, leading to randomized phenotypes (C). However, low numbers of embryos were observed with situs inversus phenotypes (D). In (A–D), cardiac outline/outflow tract is marked by red interrupted line. (F) Dose-dependent situs defects were observed in embryos injected with xtBcor MO (left panel). Cardiac orientation was completely randomized when xtBcor MO was injected into the left side of embryos (right panel). (G) Tadpoles injected with 8 ng control MO had no heart defects. Heart chambers were normal, with the outflow tract emerging from the left aspect of the ventricle and looping to the right. (H) Fifty per cent of tadpoles injected with 4 ng xtBcor on the left of the embryo displayed reversed cardiac orientation, with the outflow tract emerging from the right side of the ventricle and looping toward the left. No septal defects were observed in these embryos.
Figure 4. xtBcor MO causes defects in oculogenesis.
A. Embryos injected with 8 ng control MO at the one-cell stage displayed a normal ocular phenotype, with well-formed, symmetrical eyes.
B-F. Embryos injected unilaterally with 4 ng xtBcor MO in either the left or right side displayed defects in ocular specification, most frequently unilateral microphthalmia associated with the side of injection (B, arrow). Colobomas were observed with and without microphthalmia and in both posterior (C) and anterior (F) segments of the eye. Several embryos displayed optic nerve pigmentation that extended into the midbrain and appeared to be continuous with the retinal pigmented epithelium (D, arrow). The degree of microphthalmia was variable (B, E).
Figure 5. xtBcor is required for xtPitx2c expression.
At gastrulation stages, expression of xtPitx2c was found to be significantly down regulated in the absence of xtBcor, while there less effect on xtXnr1. xtPitx2c expression at this stage is required for correct endoderm specification (28). At tailbud stages, expression of xtPitx2c was decreased in the presence of the xtBcor MO. The effect on xtXnr1 was less pronounced. Alterations to xtPitx2c at this stage of development, either to the spatio-temporal pattern or absolute mRNA levels, would be predicted to effect lateral development and the concomitant cardiac development.
Table 1. Embryo injections and numbers—laterality scoring
Table 2. Embryo injections and numbers—eye scoring
