Skip Navigation


Human Molecular Genetics Advance Access originally published online on May 21, 2007
Human Molecular Genetics 2007 16(14):1773-1782; doi:10.1093/hmg/ddm125
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrowOA All Versions of this Article:
16/14/1773    most recent
ddm125v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Google Scholar
Right arrow Articles by Hilton, E. N.
Right arrow Articles by Black, G. C.M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hilton, E. N.
Right arrow Articles by Black, G. C.M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Left-sided embryonic expression of the BCL-6 corepressor, BCOR, is required for vertebrate laterality determination

Emma N. Hilton1,2,{dagger}, Forbes D.C. Manson2,3,{dagger}, Jill E. Urquhart1,2, Jennifer J. Johnston4, Anne M. Slavotinek5, Peter Hedera6, Eva-Lena Stattin7, Ann Nordgren8, Leslie G. Biesecker4 and Graeme C.M. Black1,3,*

1 Academic Unit of Medical Genetics and Regional Genetic Service, St Mary's Hospital, Manchester, UK, 2 Centre for Molecular Medicine, The University of Manchester, Manchester, UK, 3 Manchester Royal Eye Hospital, Central Manchester and Manchester Children's University Hospitals NHS Trust, Oxford Road, Manchester, UK, 4 Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA, 5 Department of Pediatrics, Division of Genetics, University of California, San Francisco, CA, USA, 6 Department of Neurology, Vanderbilt University, Nashville, TN, USA, 7 Department of Clinical Genetics, Umeå University Hospital, Umeå, Sweden and 8 Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Institutet, Stockholm, Sweden

* To whom correspondence should be addressed at: Department of Clinical Genetics, Central Manchester and Manchester Children's University Hospitals NHS Trust, St Mary's Hospital, Hathersage Road, Manchester M13 0JH, UK. Tel: +1 612766269; Fax: +1 612766145; Email: gblack{at}man.ac.uk

Received February 28, 2007; Accepted May 3, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC DATABASE
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC DATABASE
 REFERENCES
 
Congenital heart disease (CHD) is the commonest developmental malformation and is a common cause of death in the first year of life (1). Septation defects represent about half of all CHD and may be associated with monogenic disorders (including both isolated cardiac phenotypes and multisystemic syndromes) whose identification has enabled the study of molecules and pathways critical in the regulation of cardiac morphogenesis. These include transcriptional regulators such as TBX5, mutated in Holt–Oram syndrome, and NKX2.5 and GATA4, which are mutated in non-syndromic forms of CHD (2–4). Further advances have been made through vertebrate model organisms in which the effects of both loss- and gain-of-function mutations can be assessed (5). These suggest that controllers of left–right axis asymmetry are important in the molecular etiology of CHD (6–10).

Oculofaciocardiodental (OFCD) syndrome (syndromic microphthalmia 2/MCOPS2, MIM no. 300166 [OMIM] ) is a pleiotropic developmental anomaly syndrome with a broad phenotypic spectrum. At birth, patients present with cardiac abnormalities (septal defects) and ocular anomalies (congenital cataracts, microphthalmia and coloboma). In young adulthood, characteristic minor facial anomalies (long and narrow face, broad nasal tip with cartilage separation) and dental problems (delayed dentition, radiculomegaly and oligodontia) become apparent and a clinical diagnosis can be made. All reported patients are female, predicting an X-linked pattern of inheritance with presumed male lethality (11). OFCD syndrome is caused by mutations in the BCL-6 corepressor (BCOR) gene at chromosomal location Xp11.4. All described OFCD-associated mutations, which include nonsense, frameshifts, splicing and deletions, predict loss-of-function via nonsense-mediated mRNA decay (11). In addition, a single missense mutation (c.254C>T, p.P85L) has been identified in a family with Lenz microphthalmia syndrome, demonstrating that this disorder can be allelic to OFCD syndrome.

BCOR encodes a 1755 amino acid protein which effects transcriptional repression as part of a larger DNA-binding complex. Binding partners include BCL-6 (B-cell lymphoma 6), AF-9 (ALL1 fused gene from chromosome 9) and members of the PcG (Polycomb group) repression complex (12–14). The target promoters of the BCOR complex are largely unknown, although it is recruited to BCL-6 target genes such as cyclin D2 and P53 in B lymphocytes (14). However, as the phenotype of the Bcl-6 knockout mouse is limited to hematological and inflammatory processes (15), it is probable that BCOR functions with alternative transcription factors at other promoters to regulate gene activity. The mechanism by which BCOR effects repression at target promoters is also unknown. The C-terminus of the protein, which contains three tandem ankyrin repeats, is required for interaction with mediators of histone demethylation and histone ubiquitination (14), suggesting BCOR operates as a transcriptional repressor via epigenetic mechanisms.

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 characterize the vertebrate developmental processes requiring BCOR function, we created an animal model of OFCD syndrome using morpholino oligonucleotides (MOs) designed to inhibit translation of the Xenopus tropicalis ortholog of BCOR, xtBcor. When these were injected into X. tropicalis embryos at the one- and two-cell stage, lateral defects of cardiac orientation and intestinal rotation were observed. These effects were limited to left-sided MO injection, defining a left-sided requirement for xtBcor in laterality determination. The effects of xtBcor expression knockdown on possible downstream gene targets were examined and the expression of xtPitx2c was found to be downregulated, providing a possible mechanism for the observed disrupted lateral development (16).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC DATABASE
 REFERENCES
 
Patients with OFCD syndrome exhibit defects in laterality specification
Developmental cardiac defects are found in ~85% of patients with OFCD syndrome, with >65% having septal defects [(11) and our unpublished data]. However, until now, laterality spectrum defects have not been reported to be associated with OFCD syndrome. We evaluated the clinical findings of patients with OFCD syndrome. We identified two newly diagnosed female patients, as well as a third patient who had previously been reported, all of whom displayed a classical OFCD syndrome phenotype and carried truncating mutations in BCOR. All three patients had additional phenotypic features suggestive of defective lateral patterning, particularly in the observation of dextrocardia in patient 1.

Patient 1
The patient was born by normal delivery at full term, birth weight 4270 g (Fig. 1). Bilateral congenital cataracts with microphthalmia, ptosis and nystagmus were noted. Cataract surgery was performed at 3 months, with subsequent secondary glaucoma developing during childhood. Echocardiogram examination showed dextrocardia, patent ductus arteriosus (PDA), ventricular septal defect (VSD) and mitral incompetence. The PDA and VSD were surgically corrected at 2 years of age.


Figure 1
View larger version (108K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Patients with OFCD syndrome have defects in laterality determination. 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 frameshift and premature stop (c.1539insG, p.P514AfsX3).

 
Dental examination of the patient in young adulthood revealed persistent primary teeth with radiculomegaly and delayed eruption of secondary dentition. Oligodontia was also noted. The patient has distinctive minor facial anomalies with septate cartilage at the nasal tip, a long face and small ears. She has hypoplasia of the left thumb and hammer-type flexion deformity of the toes. Additionally, she developed seizures at 1 year (electroencephalogram results were normal). The patient was found to be a heterozygote for c.1539insG, which predicts p.P514AfsX3.

Patient 2
The proband in the family was born at term, small for gestational age, and found to have a double outlet right ventricle (DORV) with atrial septal defect (ASD) and VSD, bilateral microphthalmia with cataracts, cleft palate and hammer toes. Abdominal ultrasound revealed an asplenia phenotype. She died at 4 years following a febrile illness. The family history revealed that at least six females were affected with OFCD. The other affected women in the family have a spectrum of abnormalities including microphthalmia, cataracts, glaucoma, canine radiculomegaly, retained primary teeth, missing teeth, hammer toes and bifid nasal tip. Addition investigations in several of those affecteds (e.g. echocardiography and abdominal ultrasound) have not identified other defects of situs. The patient was found to be a heterozygote for c.4014delGinsCT, which predicts p.E1339LfsX32.

Patient 3
The phenotype of this patient was previously reported by Hedera and Gorski (17). The patient had the characteristic ocular and cardiac phenotype of OFCD syndrome, including bilateral congenital cataracts and severe microphthalmia, ASD and PDA. Minor facial anomalies included a long and narrow face and a septate nasal tip cartilage. The patient also had a midline cleft palate. During infancy, the patient was hospitalized because of severe feeding difficulties and failure to thrive. Gastroenterological examination revealed intestinal malrotation (complicated by intermittent volvulus and gastroesophageal reflux), a condition strongly associated with heterotaxy disorders (18). The patient's mother displayed similar phenotypic features, accompanied by persistent primary teeth, delayed secondary dentition and canine radiculomegaly. Both affected women in this family were found to be heterozygotes for c.4140delAG, which predicts p.T1380TfsX27. This is identical to a mutation that we have previously reported (11) in an unrelated family from the UK.

A X. tropicalis homolog of BCOR
Using similarity searches, we identified the putative X. tropicalis ortholog (xtBcor) of the human BCOR gene from the X. tropicalis genome project database. The sequences were conserved, with the same exon number, exon/intron boundaries and identically positioned translation initiation sites. The Xenopus ortholog is 1694 amino acids in length and is 53% identical (68% similar) to BCOR. The BCL-6 binding domain shows 97% similarity, the AF-9 binding site 80% and the ankyrin repeats 91%. We found xtBcor to be expressed throughout embryogenesis (Fig. 2A). It is expressed maternally and then continuously throughout embryogenesis and into metamorphosis. Spatial xtBcor expression was analyzed in a panel of cDNAs derived from adult female X. tropicalis and was detected in most tissues, including eye, heart and brain (Fig. 2B). This correlates with human studies where expression of BCOR was ubiquitous, with particularly high levels in heart structures, ovary, lung, spinal chord and glandular organs (12). We note that temporal and spatial expression of xtBcor correlates with the specification and development of organ systems affected in OFCD syndrome.


Figure 2
View larger version (45K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Characterization of a X. tropicalis homolog 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 thereafter 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 promoter and a 3' FLAG tag. The sequence of xtBcor mRNA around the start codon and the position of the MO binding sites are 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.

 
MO knockdown demonstrates a role for xtBcor in laterality determination
Translation-blocking MOs were designed to target the 5'-UTR of xtBcor mRNA (Fig. 2C and D). Antibodies that recognize both the human and murine orthologs fail to recognize xtBcor (data not shown). Therefore, in order to measure the efficacy of these MOs, specific inhibition of xtBcor mRNA translation was demonstrated in an in vitro transcription/translation assay that generated a FLAG-tagged, 16 kDa N-terminal fragment of xtBcor. In the absence of either MO, translation of this protein fragment was visible by immunoblot analysis. In the presence of either MO, translation was inhibited (Fig. 2D), demonstrating that both MOs are effective at blocking translation from the xtBcor mRNA translation initiation codon. All experiments described below were carried out using each MO with reproducible results. Data presented are derived from at least three independent sets with both MOs. Injection of 2–6 ng xtBcor MO at the one-cell stage did not cause gross embryonic defects in either dorsoventral or anterioposterior development to stage 46, compared with either uninjected or control MO-injected siblings. Head development and movement were normal, and the escape reflex was unimpaired. Tadpoles continued to thrive beyond stage 45.

The most striking abnormalities in MO-injected embryos were defects of heart orientation (forward or reversed), gut origin (left or right) and coiling (clockwise or counterclockwise), and these defects were observed at only background frequency in uninjected, water-injected or control MO-injected embryos (Fig. 3A). Embryos were scored according to Branford et al. (19) (Table 1). For injections of MO up to 8 ng, we observed a dose-dependent increase in the frequency of situs randomization (Fig. 3B, E and F). At low doses of xtBcor MO (2 ng), ~70% of embryos had normal organ asymmetry, whereas at high doses (8 ng), ~70% displayed laterality defects in the absence of other embryonic defects (Fig. 3F). Gut patterning appeared more susceptible to MO injection, with only lower doses required for the disruption of gut coiling as the sole defect. As MO dose increased, alterations to gut situs were accompanied by reversals in cardiac orientation, with the incidence of cardiac reversal as the sole defect occurring in ~5% of embryos injected with 8 ng of MO. However, at this dose, the intestinal phenotype was lethal in the majority of embryos, which displayed poor anterioposterior gut patterning and severe ventral edema (Fig. 3E). Overall, these observations suggested that xtBcor is required for correct embryonic laterality determination.


Figure 3
View larger version (65K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		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 ~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.

 


View this table:
[in this window]
[in a new window]

  		Table 1. Embryo injections and numbers—laterality scoringa

 
Left-sided expression of xtBcor is required for correct laterality determination and cardiac orientation
To further investigate the requirement of xtBcor in laterality determination, we generated unilateral knockdowns by injecting 4 ng of xtBcor MO at the two to four cell transition, where partitioning of the embryo into left and right halves is complete. The injection side was identified using an enhanced green fluorescent protein (EGFP) tracer and embryos were scored for situs defects. Right-sided injections had no effect on laterality determination. Gut and heart laterality defects were recorded in 86% of embryos injected with xtBcor MO on the left side (Fig. 3C and D) and the distribution of scoring phenotypes was even across the panel (Table 1), demonstrating that the loss of xtBcor from the left side of the embryo resulted in a randomized and uncoordinated development of heart and gut asymmetry. Strikingly, ~50% of embryos injected on the left side had a reversed cardiac orientation, suggesting a stochastic mechanism for asymmetric specification in the absence of xtBcor (Fig. 3F).

More than 90% of human laterality disease is complicated by CHDs, causing significant morbidity and mortality (20). Cardiac imaging in tadpoles injected on the left side was undertaken in situ by confocal microscopy (Fig. 3G and H) (21). The hearts in tadpoles with a reversed heart orientation showed gross structural integrity of the outflow tract, ventricle and atrium and no septal defects were noted. This is in agreement with Dagle et al. (16), who found a low incidence of septal defects in completely reversed hearts induced by Pitx2c inhibition. However, defective septa in hearts with more subtle changes in asymmetry (e.g. straightened outflow tracts) were noted. Although we infrequently observed straightened outflow tracts, we did not examine these hearts further.

xtBcor is required for correct ocular development
To confirm that loss of xtBcor in embryonic development recapitulates the phenotype observed in OFCD syndrome patients, we searched for eye defects in stage 45 embryos injected with xtBcor MO at the one-cell stage. In uninjected or control MO-injected embryos, no structural eye defects (e.g. coloboma) and <1% microphthalmia (defined as one eye <90% normal eye size) were observed (Fig. 4) (Table 2).


Figure 4
View larger version (101K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		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).

 


View this table:
[in this window]
[in a new window]

  		Table 2. Embryo injections and numbers—eye scoringa

 
At 2–4 ng of xtBcor MO, eye development was normal except for a delay in eye specification, as judged by optic placode pigmentation. At an 8 ng dose, frequent eye defects were apparent in injected embryos, with unilateral microphthalmia and colobomata of both posterior and anterior segments being the most common abnormalities (Table 2). Unilaterally injected embryos allowed a direct comparison of eyes on injected and uninjected sides and gave a more accurate measure of microphthalmia frequency. We only observed eye defects on the side of MO injection (i.e. xtBcor loss) (Fig. 4B–F). When 4 ng of xtBcor MO was injected unilaterally, the frequency and severity of microphthalmia increased to 66% (left-injected) and 71% (right-injected), with >5% of affected embryos displaying severe microphthalmia on the injected side (Fig. 4E) (Table 2). Colobomas were observed in 3% of all unilaterally injected embryos (Fig. 4C and F) (Table 2). The spectrum of eye defects (microphthalmia and coloboma) observed in MO-injected embryos parallels those observed in OFCD syndrome patients and suggests that there is no left–right bias to the development of ocular defects.

xtBcor is required for the expression of xtPitx2c at gastrulation and tailbud stages
The generation of asymmetric left and right sides in vertebrates focuses on a conserved inductive signaling network functioning in the left side of the embryo at post-neurula stages. One of the earliest events in this pathway is the expression of nodal, a TGFß-like extracellular signaling molecule. In Xenopus, the nodal ortholog Xnr1 is expressed in the left lateral plate mesoderm at early tailbud stages (stage 18+). Nodal signaling acts via a receptor complex comprising a type I activin receptor, a type II activin receptor and an epidermal growth factor (EGF)-CFC cofactor. This receptor complex phosphorylates the cytoplasmic signaling molecules Smad2 and Smad3, either of which then interacts with Smad4 to form an ‘activated Smad’ complex. This complex becomes sequestered to the nucleus and is then recruited to promoters by DNA-binding transcriptional activators. These include the FoxH1 transcription factor that is able to activate Pitx2c expression via an intronic asymmetric enhancer element. Expression of Pitx2c in the left side of the embryo is necessary to affect left-sided morphogenesis (22).

To investigate the mechanism by which xtBcor-mediated repression is required on the left side for cardiac development and on both sides for ocular development, we assessed the effects of xtBcor knockdown on candidate genes involved in left–right asymmetry. RT–PCR analysis of embryos injected with 8 ng xtBcor MO at the one-cell stage showed an alteration of xtPitx2c expression (Fig. 5). In the absence of xtBcor, xtPitx2c was lost from gastrulation stage embryos (stage 10–12) and was downregulated in later stages (stage 16–22). Expression of Xnr1 was downregulated at stage 12 (Fig. 5), but there was no concomitant change in either Lefty or FoxH1 expression levels (data not shown).


Figure 5
View larger version (33K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. xtBcor is required for xtPitx2c expression. At gastrulation stages, expression of xtPitx2c was found to be significantly downregulated in the absence of xtBcor, whereas there is 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 spatiotemporal pattern or absolute mRNA levels, would be predicted to affect lateral development and the concomitant cardiac development.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 ELECTRONIC DATABASE
 REFERENCES
 
We have described three patients with a loss-of-function mutation in BCOR, who have the classic features of OFCD syndrome (congenital cataracts, microphthalmia, radiculomegaly and characteristic facial features) and additional clinical manifestations consistent with abnormal laterality determination. Both dextrocardia and asplenia are characteristic of right isomerism, where left-sided structures take on characteristics of right-sided structures (23). In patient 1, dextrocardia was also accompanied by VSD and mitral incompetence but with normal intracardiac anatomy. Cardiac function has been investigated in patients with OFCD and developmental defects found in over 85%. The most common heart defects are septal defects with over 65% of patients diagnosed with an ASD and 30% having a VSD. Septal defects have also been associated with enlarged or stenosed pulmonary arteries, thickened pulmonary valves, enlarged right ventricle/atrium and floppy mitral valve, whereas a single patient was diagnosed with ASD, VSD, DORV and hypoplasia of the aortic arch. These anomalies, although not specific to laterality disorders, are frequently found in patients with such disorders (24). Furthermore, as well as displaying characteristic laterality defects and associated CHDs, patients with OFCD syndrome have other defects characteristic of midline defects such as septate cartilage at the nasal tip, cleft or high-arched palate, bifid tongue and bifid uvula.

In order to study the role of BCOR in development, we have used X. tropicalis as a model organism. We identified the Xenopus ortholog of BCOR and showed it has structural conservation at both mRNA and protein levels. We also demonstrated that xtBcor has a similar broad spatio-temporal expression to BCOR. As OFCD syndrome is presumed to result from reduced or absent expression of BCOR, we then created an animal model of the condition using X. tropicalis MOs designed to inhibit the translation of xtBcor.

We injected MOs at the one- and two-cell stage and demonstrated effects upon cardiac orientation and intestinal rotation. As these effects were limited to left-sided MO injection, we have defined a left-sided requirement for xtBcor in laterality determination. The most significant cardiac finding was a randomization of cardiac orientation in embryos subjected to injection of xtBcor MO on the left side. Importantly, the eye phenotypes we observed, including microphthalmia and coloboma, confirm the fidelity of our animal model to the human condition. There was no lateral bias of eye phenotype, suggesting that the requirement for xtBcor in ocular specification is developmentally uncoupled from the requirement in the left side of the embryo for correct lateral determination.

Vertebrate laterality signaling, midline integrity and cardiac development are complex and linked developmental processes that involve at least 80 genes (25). These genes, or their encoded proteins, have either asymmetrical expression patterns or a differential function on the left and right sides of the embryo. In either case, the functional requirement for a molecule on the correct side of the embryo can be tested by generating unilateral overexpression or knockout embryos. For example, injection of xtPitx2c mRNA into the dorsal right blastomeres of developing embryos causes situs disturbances including heart and gut defects (26). Localization data for BCOR suggested that expression is ubiquitous with no left–right bias (12). However, as evidenced by our unilateral knockout in Xenopus, left-sided expression of xtBcor is necessary for laterality determination. Right-sided xtBcor is not required for correct left–right specification, but is required for normal oculogenesis. Thus, xtBcor, although involved in left–right signaling, has alternative roles in embryonic development.

We have examined the effects of xtBcor expression knockdown on possible downstream gene targets. We have shown that Xenopus embryos lacking xtBcor fail to express xtPitx2c at gastrula stages and downregulate xtPitx2c at tailbud stages, suggesting that xtBcor is an upstream regulator of xtPitx2c. In Xenopus, xtPitx2c is required throughout cardiac development to direct left-sided cardiac morphogenesis, and its expression is dependent on multiple upstream members of the left–right signaling cascade (16,19,22,27). We propose that downregulation of xtPitx2c in the left lateral plate mesoderm at tailbud stages would lead to bilateral right-sidedness and explains the right isomeric characteristics observed in OFCD syndrome. The high frequency of CHDs in OFCD syndrome patients may also derive from changes to PITX2C expression. Alterations in left-sided xtPitx2c expression induced by constitutively active Alk4 do not always cause heterotaxy in Xenopus, but do cause widespread disruption to cardiac lineage identity (27). This demonstrates that isolated heart defects may result from subtle alterations of PITX2C expression and underline an etiological link between left–right patterning and cardiac defects.

A lack of zygotic xtPitx2c expression probably accounts for the severe endodermal defects observed in embryos injected with a high dose of MO (28). We observed that correct gut rotation was susceptible to lower doses of xtBcor MO, whereas cardiac morphology was unaffected until higher doses of MO were applied, suggesting that xtPitx2c expression levels have different organ-specific effects. This correlates with data from a mouse Pitx2c allelic series (29). The mechanism by which left-sided expression of xtBcor is required for xtPitx2c expression is unknown, but as xtBcor is predicted to function as a transcriptional repressor, the loss of xtPitx2c expression is presumed to be mediated by at least one intermediate. This will be the focus of future studies.

PITX2 is a bicoid-type homeodomain containing transcription factor. The gene produces three protein isoforms (PITX2A, B and C) because of a combination of different promoters and alternative splicing. The PITX2A/B isoforms carry a common N-terminus that is different from that of PITX2C and are required in mammals for the correct development of the major systems affected in OFCD syndrome. Its role in eye and dental development is evidenced by the PITX2 mutations associated with Axenfeld–Rieger syndrome in which patients have abnormal ocular anterior segment mesenchymal development, dental hypoplasia and umbilical abnormalities (30,31). We hypothesize that the phenotype observed in OFCD syndrome is caused by a loss of BCOR function and, as BCOR may be required for the expression of PITX2A/B as well as that of PITX2C, concomitant deregulation of PITX2 gene expression.

The left–right signaling pathway has been studied in humans, and pathological mutations have been described in several genes involved in non-ciliary signaling. These include ZIC3 (10), LEFTY A (6), CFC1 (7) and ACVR2B (8). In all cases, there is a wide range of phenotypic variability. For example, in five unrelated patients with a common ancestral mutation in CFC1 (p.R75W), laterality phenotypes varied from complex CHDs with visceral heterotaxy to isolated VSD with cyclopia and other midline defects. In three patients with p.G174delfsx55 CFC1 mutation, one displayed right isomerism, second left isomerism and third had isolated CHD (VSD, DORV and aortic arch hypoplasia), but no further manifestations of heterotaxy (32). In each study, the spectrum of phenotypes observed in human patients included classic laterality defects, midline defects and CHDs, with no apparent genotype–phenotype correlation. These data are mirrored by our own findings.

Population studies estimate the prevalence of laterality defects at 1–1.5 in 10 000 live births, with a 2:1 male predominance (33). ZIC3 is the only X chromosome gene known to be mutated in human heterotaxy disorders and has been shown to account for the majority of X-linked familial human heterotaxy phenotypes (it is likely to be responsible for ~1% of affected individuals) as well as a small number of sporadic CHD cases. Importantly, mutations within the ZIC3 coding sequence did not underlie the male predominance (34). This raises the possibility that altered expression of other X-linked genes, such as BCOR, may contribute to human laterality disorders.

We conclude that defects of laterality determination are part of the phenotypic spectrum in OFCD syndrome and that the overlap of the present findings in Xenopus with those in human OFCD syndrome patients demonstrates that BCOR is a regulator of vertebrate laterality. In addition to laterality defects, OFCD syndrome patients also display CHDs and classic midline defects (cleft palate), both of which can be etiologically linked to defective laterality determination. The OFCD syndrome should therefore be included in the group of genetic disorders associated with altered left–right patterning.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
ELECTRONIC DATABASE
 REFERENCES
 
Patient sequencing
The BCOR gene was amplified and sequenced as described previously (11). The human subjects research described here was reviewed and approved by Ethics Committees at the National Institutes of Health and at St Mary's Hospital.

Embryo culture and injection
X. tropicalis embryos were obtained by standard methods (35). Untreated embryos were cultured in standard conditions and staged according to Nieuwkkop and Faber (36). MO injections were performed using a Picospritzer III microinjector (Intracel) to inject 1 nl volumes. Injections were performed at the one-cell stage or into one blastomere at the two to four cell transition as indicated in the text. Where required, 16 pg EGFP plasmid was co-injected with the MO and the injection side identified using the appropriate fluorescent filter. During injection and for subsequent incubation, embryos were maintained in 0.1 x MMR/3% Ficoll at 22°C. Prior to gastrulation, embryos were removed to 0.05 x MMR/1 x gentamicin at 22°C. At stage 17, embryos were removed to 0.05 x MMR at 22°C for the remainder of culture.

Heart and intestinal development were scored at stage 46. Laterality scoring was carried out using the system proposed by Branford et al. (19).

Morpholino oligonucleotides
Two non-overlapping MOs were designed against the 5'-UTR of xtBcor around the start codon following the manufacturer's guidelines (Gene Tools LLC). The sequences were xtBcor ATG1 5'aag cat ttt ctc tcc tta cgc ggc t 3' and xtBcor ATG2 5'gcc tcc ccc ttc tta taa atc tgt c 3'. The standard Gene Tools control MO was used. All MOs were resuspended in DEPC-treated dH2O to a stock concentration of 10 ng nl–1. All experiments were repeated with both MOs.

RT–PCR analysis of gene expression
Total RNA was extracted from pools of five embryos (or up to 50 mg of tissue) using Trizol reagent (Invitrogen) and resuspended in DEPC-treated dH2O. RNA (1 µg) was reverse-transcribed using MMLV reverse transcriptase (Bioline) in 20 µl reactions. PCR amplifications were carried out using the following primer oligonucleotides (Invitrogen): xtGapdh f 5' gcc gtg tat gtg gtg gaa tct 3'; xtGapdh r 5' aag ttg tcg ttg atg acc ttt gc 3'; xtBcor f 5' agg gcc tca tat tgc atc ag 3'; xtBcor r 5'ccc ttg gtt gtt tgc tgt tt 3'; xtPitx2c f 5' ttg ttg tac gag taa ctg ggg tac 3'; xtPitx2c r 5' gct ctg ggg agt gta agt caa g 3'. PCR reactions comprised 1 x Custom Reddymix (ABgene), 1 µl cDNA reaction and 2.5 µM each primer and carried out on a DNA Engine (MJ Research) using the following cycling conditions: 94°C for 5 min and then (94°C for 30 s; Y°C for 30 s; 72°C for 30 s) for Z cycles with a final extension at 72°C for 10 min (xtGapdh: annealing at 45°C, 36 cycles; xtBcor: annealing at 54°C, 38 cycles; xtPitx2c: annealing at 45°C, 38 cycles).

In vitro transcription/translation of 5' xtBcor fragment
PCR amplification of a 5' fragment of xtBcor cDNA was achieved using the following primer oligonucleotides (Invitrogen): xtBcor 5' mRNA f 5' taa tac gac tca cta ta g* ggg gac aga ttt ata aga agg 3' (T7 promoter sequence, start of transcription*) and xtBcor 5' mRNA r 5' tca* ctt gtc gtc gtc atc ctt gta gtc ttt cat aga cat gtg aga att 3' (stop codon*, FLAG tag). cDNA derived from stage 20 wild-type embryo RNA was used as a template. PCR reactions were carried out using 1x Custom Reddymix (ABgene), 1 µl cDNA reaction and 2.5 µM each primer, using the following cycling conditions: 94°C for 5 min then (94°C for 30 s; 55°C for 30 s and 72°C for 30 s) for 35 cycles with a final extension at 72°C for 10 min. PCR products were purified by gel extraction (Qiagen Qiaquick kit) and sequenced.

xtBcor mRNA was generated from 1 µg PCR template using a T7 mMessage mMachine kit (Ambion), according to manufacturer's instructions, except reactions were incubated for 16 h. The reaction was stopped and transcripts isolated using the recommended ammonium acetate/phenol–chloroform method. One microgram mRNA template was added to an in vitro translation reaction, carried out using a rabbit reticulocyte lysate kit (Promega) according to the manufacturer's instructions. Translation reactions were supplemented with 1 µg xtBcor ATG1 or xtBcor ATG2 MO as appropriate. Translation products were separated by 15% SDS–PAGE and detected by immunoblot using a mouse anti-FLAG M2 primary antibody (Sigma) and visualized using ECL reagent (Amersham). Immunoblot was performed by standard methods, using a BioRad semi-dry blotter.

Confocal microscopy of X. tropicalis hearts
X. tropicalis hearts were imaged in situ exactly as described by Kolker et al. (21). The mouse anti-bovine cardiac troponin T antibody (CT3) was developed by Jim Jung-Ching Lin and was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa.


   ELECTRONIC DATABASE
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC DATABASE
REFERENCES
 
Xenopus tropicalis genome project: http://genome.jgi-psf.org/xentr4/xentr4.home.html; Expasy Translate tool: http://us.expasy.org/tools/dna.html; ClustalW: http://clustalw.genome.jp; BioEdit Sequence Alignment Editor: http://www.mbio.ncsu.edu/bioedit/bioedit.html.


   ACKNOWLEDGEMENTS
 
The authors thank Julie Sapp and Michael Lyons for assistance with the clinical evaluation of family 2. G.C.M.B. is a Wellcome Senior Research Fellow in Clinical Science. E.N.H. is funded by the Wellcome Trust (ref. GR067443MA). J.J.J. and L.G.B. are supported by intramural funds of the NIH National Human Genome Research Institute.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 ELECTRONIC DATABASE
 REFERENCES
 

  1. Garg V. Insights into the genetic basis of congenital heart disease. Cell. Mol. Life Sci. (2006) 63:1141–1148.[CrossRef][Web of Science][Medline]

  2. Li Q.Y., Newbury-Ecob R.A., Terrett J.A., Wilson D.I., Curtis A.R., Yi C.H., Gebuhr T., Bullen P.J., Robson S.C., Strachan T., et al. Holt–Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nat. Genet. (1997) 15:21–29.[CrossRef][Web of Science][Medline]

  3. Schott J.J., Benson D.W., Basson C.T., Pease W., Silberbach G.M., Moak J.P., Maron B.J., Seidman C.E., Seidman J.G. Congenital heart disease caused by mutations in the transcription factor NKX2–5. Science (1998) 281:108–111.[Abstract/Free Full Text]

  4. Garg V., Kathiriya I.S., Barnes R., Schluterman M.K., King I.N., Butler C.A., Rothrock C.R., Eapen R.S., Hirayama-Yamada K., Joo K., et al. GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature (2003) 424:443–447.[CrossRef][Medline]

  5. Yelon D. Cardiac patterning and morphogenesis in zebrafish. Dev. Dyn. (2001) 222:552–563.[CrossRef][Web of Science][Medline]

  6. Kosaki K., Bassi M.T., Kosaki R., Lewin M., Belmont J., Schauer G., Casey B. Characterization and mutation analysis of human LEFTY A and LEFTY B, homologues of murine genes implicated in left–right axis development. Am. J. Hum. Genet. (1999) 64:712–721.[CrossRef][Web of Science][Medline]

  7. Bamford R.N., Roessler E., Burdine R.D., Saplakoglu U., dela Cruz J., Splitt M., Towbin J., Bowers P., Marino B., Schier A.F., et al. Loss-of-function mutations in the EGF-CFC gene CFC1 are associated with human left–right laterality defects. Nat. Genet. (2000) 26:365–369.[CrossRef][Web of Science][Medline]

  8. Kosaki R., Gebbia M., Kosaki K., Lewin M., Bowers P., Towbin J.A., Casey B. Left–right axis malformations associated with mutations in ACVR2B, the gene for human activin receptor type IIB. Am. J. Med. Genet. (1999) 82:70–76.[CrossRef][Web of Science][Medline]

  9. Ozcelik C., Bit-Avragim N., Panek A., Gaio U., Geier C., Lange P.E., Dietz R., Posch M.G., Perrot A., Stiller B. Mutations in the EGF-CFC gene cryptic are an infrequent cause of congenital heart disease. Pediatr. Cardiol. (2006) 27:695–698.[CrossRef][Web of Science][Medline]

  10. Gebbia M., Ferrero G.B., Pilia G., Bassi M.T., Aylsworth A.S., PenmanSplitt M., Bird L.M., Bamforth J.S., Burn J., Schlessinger D., et al. X-linked situs abnormalities result from mutations in ZIC3. Nat. Genet. (1997) 17:305–308.[CrossRef][Web of Science][Medline]

  11. Ng D., Thakker N., Corcoran C.M., Donnai D., Perveen R., Schneider A., Hadley D.W., Tifft C., Zhang L.Q., Wilkie A.O.M., et al. Oculofaciocardiodental and Lenz microphthalmia syndromes result from distinct classes of mutations in BCOR. Nat. Genet. (2004) 36:411–416.[CrossRef][Web of Science][Medline]

  12. Huynh K.D., Fischle W., Verdin E., Bardwell V.J. BCoR, a novel corepressor involved in BCL-6 repression. Genes Dev. (2000) 14:1810–1823.[Abstract/Free Full Text]

  13. Srinivasan R.S., de Erkenez A.C., Hemenway C.S. The mixed lineage leukemia fusion partner AF9 binds specific isoforms of the BCL-6 corepressor. Oncogene (2003) 22:3395–3406.[CrossRef][Web of Science][Medline]

  14. Gearhart M.D., Corcoran C.M., Wamstad J.A., Bardwell V.J. Polycomb group and SCF ubiquitin ligases are found in a novel BCOR complex that is recruited to BCL6 targets. Mol. Cell. Biol. (2006) 26:6880–6889.[Abstract/Free Full Text]

  15. Dent A.L., Shaffer A.L., Yu X., Allman D., Staudt L.M. Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science (1997) 276:589–592.[Abstract/Free Full Text]

  16. Dagle J.M., Sabel J.L., Littig J.L., Sutherland L.B., Kolker S.J., Weeks D.L. Pitx2c attenuation results in cardiac defects and abnormalities of intestinal orientation in developing Xenopus laevis. Dev. Biol. (2003) 262:268–281.[CrossRef][Web of Science][Medline]

  17. Hedera P., Gorski J.L. Oculo-facio-cardio-dental syndrome: skewed X chromosome inactivation in mother and daughter suggest X-linked dominant inheritance. Am. J. Med. Genet. (2003) 123A:261–266.

  18. Borenstein S.H., Langer J.C. Heterotaxia syndromes and their abdominal manifestations. Curr. Opin. Pediatr. (2006) 18:294–297.[CrossRef][Medline]

  19. Branford W.W., Essner J.J., Yost H.J. Regulation of gut and heart left–right asymmetry by context-dependent interactions between Xenopus Lefty and BMP4 signaling. Dev. Biol. (2000) 223:291–306.[CrossRef][Web of Science][Medline]

  20. Nugent E.W., Plauth W.H., Edwards J.E., Williams W.H. The Pathology, Pathophysiology, Recognition and Treatment of Congenital Heart Disease (1994) New York: McGraw-Hill.

  21. Kolker S.J., Tajchman U., Weeks D.L. Confocal imaging of early heart development in Xenopus laevis. Dev. Biol. (2000) 218:64–73.[CrossRef][Web of Science][Medline]

  22. Raya A., Belmonte J.C.I. Left–right asymmetry in the vertebrate embryo: from early information to higher-level integration. Nat. Rev. Genet. (2006) 7:283–293.[CrossRef][Web of Science][Medline]

  23. Bartram U., Wirbelauer J., Speer C.P. Heterotaxy syndrome—asplenia and polysplenia as indicators of visceral malposition and complex congenital heart disease. Biol. Neonate (2005) 88:278–290.[CrossRef][Web of Science][Medline]

  24. Phoon C.K., Neill C.A. Asplenia syndrome—insight into embryology through an analysis of cardiac and extracardiac anomalies. Am. J. Cardiol. (1994) 73:581–587.[CrossRef][Web of Science][Medline]

  25. Levin M. Left–right asymmetry in embryonic development: a comprehensive review. Mech. Dev. (2005) 122:621–621.[CrossRef][Web of Science]

  26. Campione M., Steinbeisser H., Schweickert A., Deissler K., van Bebber F., Lowe L.A., Nowotschin S., Viebahn C., Haffter P., Kuehn M.R., et al. The homeobox gene Pitx2: mediator of asymmetric left–right signaling in vertebrate heart and gut looping. Development (1999) 126:1225–1234.[Abstract]

  27. Ramsdell A.F., Bernanke J.M., Trusk T.C. Left–right lineage analysis of the embryonic Xenopus heart reveals a novel framework linking congenital cardiac defects and laterality disease. Development (2006) 133:1399–1410.[Abstract/Free Full Text]

  28. Faucourt M., Houliston E., Besnardeau L., Kimelman D., Lepage T. The pitx2 homeobox protein is required early for endoderm formation and nodal signaling. Dev. Biol. (2001) 229:287–306.[CrossRef][Web of Science][Medline]

  29. Liu C., Liu W., Lu M.F., Brown N.A., Martin J.F. Regulation of left–right asymmetry by thresholds of pitx2c activity. Dev. Biol. (2001) 235:244–245.

  30. Amendt B.A., Semina E.V., Alward W.L.M. Rieger syndrome: a clinical, molecular, and biochemical analysis. Cell. Mol. Life Sci. (2000) 57:1652–1666.[CrossRef][Web of Science][Medline]

  31. Lin C.R., Kioussi C., O'Connell S., Briata P., Szeto D., Liu F., Izpisua-Belmonte J.C., Rosenfeld M.G. Pitx2 regulates lung asymmetry, cardiac positioning and pituitary and tooth morphogenesis. Nature (1999) 401:279–282.[CrossRef][Medline]

  32. Goldmuntz E., Bamford R., Karkera J.D., dela Cruz J., Roessler E., Muenke M. CFC1 mutations in patients with transposition of the great arteries and double-outlet right ventricle. Am. J. Hum. Genet. (2002) 70:776–780.[CrossRef][Web of Science][Medline]

  33. Lin A.E., Ticho B.S., Houde K., Westgate M.N., Holmes L.B. Heterotaxy: associated conditions and hospital-based prevalence in newborns. Genet. Med. (2000) 2:157–172.[Web of Science][Medline]

  34. Ware S.M., Peng J.L., Zhu L.R., Fernbach S., Colicos S., Casey B., Towbin J., Belmont J.W. Identification and functional analysis of ZIC3 mutations in heterotaxy and related congenital heart defects. Am. J. Hum. Genet. (2004) 74:93–105.[CrossRef][Web of Science][Medline]

  35. Kenwrick S., Amaya E., Papalopulu N. Pilot morpholino screen in Xenopus tropicalis identifies a novel gene involved in head development. Dev. Dyn. (2004) 229:289–299.[CrossRef][Web of Science][Medline]

  36. Nieuwkkop P.D., Faber J. Normal Table of Xenopus laevis (Daudin). (1967) 2nd edn. Amsterdam: North Holland Publishing Company.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrowOA All Versions of this Article:
16/14/1773    most recent
ddm125v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Google Scholar
Right arrow Articles by Hilton, E. N.
Right arrow Articles by Black, G. C.M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hilton, E. N.
Right arrow Articles by Black, G. C.M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?