Human Molecular Genetics, 2002, Vol. 11, No. 3 283-293
© 2002 Oxford University Press
Folic acid prevents exencephaly in Cited2 deficient mice
1MRC Centre for Developmental Neurobiology, 4th floor New Hunt’s House, King’s College London, Guy’s Campus, London Bridge, London SE1 1UL, UK, 2National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK, 3Neural Development Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK, 4Department of Anatomy, University of Vienna, Waehringerstrasse 13, A-1090, Vienna, Austria and 5Developmental Biology Unit, Victor Chang Cardiac Research Institute, St Vincent’s Hospital, 384 Victoria Street, Darlinghurst, NSW 2010, Australia
Received October 9, 2001; Revised and Accepted November 29, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cited2 (also Mrg1/p35srj) is a member of a new conserved gene family that is expressed during mouse development and in adult tissues. In order to investigate the function of Cited2 during mouse embryogenesis, we introduced a null mutation into the Cited2 locus. Cited2–/– mutants died at late gestation and exhibited heart defects and exencephaly, arising from defective closure of the midbrain (MB) and hindbrain. Initiation of neural tube closure at the forebrain–midbrain (FB–MB) boundary, an essential step for closure of the cranial neural tube, was impaired in the Cited2–/– mutants. Gene marker analysis using in situ hybridization revealed that the patterning of the anterior neural plate and head mesenchyme was little affected or normal in the Cited2–/– embryos. However, Cited2 was required for the survival of neuroepithelial cells and its absence led to massive apoptosis in dorsal neuroectoderm around the FB–MB boundary and in a restricted transverse domain in the hindbrain. Treatment with folic acid significantly reduced the exencephalic phenotype in the Cited2–/– embryos both in vivo and in vitro. However, assessment of folate metabolism revealed no defect in the Cited2–/– mutants, and the elevated apoptosis observed in the neuroepithelium of the Cited2–/– mutants was apparently not decreased by folic acid supplementation. To our knowledge, the Cited2 mouse represents the first genetic model in which folic acid can prevent a defect in neural tube closure by a mechanism other than the neutralization of a defect in folate homeostasis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The CITED [CBP/p300-Interacting Transactivators with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain] gene family comprises four genes that encode nuclear proteins. It includes: Cited1 (formerly Msg1), Cited2 (formerly Mrg1/p35srj), Cited3 and Cited4 (formerly Mrg2) (1–7). CITED proteins lack sequence identity with known protein domains and therefore represent a novel family of proteins. When tethered to heterologous DNA-binding domains CITED proteins activate transcription and this function is dependent upon a conserved domain (CR2) that binds the transcriptional coactivators CBP/p300 (4,7). CBP and p300 are widely expressed, homologous nuclear proteins which function as transcriptional coactivators, linking signal-responsive DNA-binding proteins to the basal transcriptional machinery. CBP/p300 bind an increasing number of diverse proteins indicating potential participation in a range of functions, which include cell growth, apoptosis, transformation and embryonic development (8–10).
No evidence exists that CITED proteins bind DNA, and the current working hypothesis is that CITED proteins interact with DNA-binding proteins and act as transcriptional cofactors. Evidence in support of this exists as Cited1 binds Smad4, activating Smad-mediated transcription in a CBP/p300-dependent manner (7,11). Cited2 binds the LIM domain of the DNA-binding protein Lhx2 enhancing transcription of the LH/FSH glycoprotein 
-subunit in a Lhx2-dependent manner (12). An additional role has been proposed for Cited2 as a negative regulator of transcription since, in vitro, Cited2 can bind CBP/p300 and thus prevent CBP/p300 from interacting with the DNA-binding protein hypoxia inducible factor-1 (HIF-1). Given that Cited2 expression is induced by HIF-1, it has been suggested that Cited2 may act as a negative regulator of HIF-1 (4). Cited2 expression is also induced by cytokines and has cell transforming activity (13).
The expression patterns of some members of the CITED family have been analysed during vertebrate development (3,6,14). We have shown previously that in mouse, Cited2 transcripts are localized to the anterior visceral endoderm prior to gastrulation. Following the onset of gastrulation [up to 9.0 days post coitum (d.p.c.)] Cited2 is expressed in tissues of mesodermal origin (the blood islands, cardiac crescent, developing myocardium, septum transversum, presomitic mesoderm, somites and cranial mesenchyme) as well as in the cranial neuroectoderm and cranial neural crest. Later, Cited2 is ubiquitously expressed during embryogenesis and in adult tissues (1,3). In order to investigate the function of Cited2 during mouse embryogenesis, we have generated a Cited2 null mutant mouse. Cited2–/– mutants die before birth and show exencephaly. The neural tube closure defects (NTDs) are associated with increased cell death within the cranial neuroepithelium of the Cited2 mutants. We show that exencephaly is preventable by folic acid administration, although no abnormalities in folate metabolism were detected. This makes the Cited2 mouse a unique model for studying the mechanism by which folic acid acts to prevent NTDs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Embryonic lethality and cranial NTDs in Cited2 null mutants
The Cited2 null mutant mouse was generated by homologous recombination in ES cells (Fig. 1). Intercrosses between Cited2+/– mice produced no live Cited2–/– pups at birth, suggesting that the Cited2 mutation is embryonic lethal. This was confirmed by genotypic analysis of live embryos or resorptions from 8.5 to 18.5 d.p.c. As summarized in Table 1, it appeared that Cited2–/– embryos began to die around 13.5 d.p.c. and by 18.5 d.p.c. most of them were dead or moribund.
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Approximately 80% of Cited2–/– embryos analysed between 14.5 and 16.5 d.p.c. showed exencephaly of the entire brain, although it appeared that only the caudal forebrain (FB), midbrain (MB) and hindbrain had failed to fuse in the midline (n = 30; Fig. 2A–F). In these mutants the developing bones of the skull vault were missing while the base of the skull was present but deformed (Fig. 2G). These defects are probably secondary to the exencephalic development of the brain. No differences in the incidence of the exencephalic phenotype were observed between male and female mutant embryos as determined by polymerase chain reaction (PCR) using specific primers (n = 23; data not shown) and no other regions of the neural tube were affected in the Cited2–/– mutants.
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Cited2–/– embryos analysed from 14.5 d.p.c. onwards were frequently observed to be smaller than wild-type or heterozygous littermates (Fig. 2G). They also showed edema in the cervical region and heart abnormalities (multiple severe defects in the ventricular septum and outflow tract; data not shown), which were fully penetrant. Heart defects are the likely reason for the edema and the embryonic death of the Cited2–/– mutants. An independent targeted mutation in Cited2 results in a similar phenotype to the one described here (S.Bhattacharya, personal communication).
Analysis of embryos at successive developmental stages was carried out to determine when defects first become apparent. Normal neurulation is a complex process involving major morphological changes including elevation of the neural folds from a semi-horizontal to a vertical position, and bending from convex to concave, so that their tips come together towards the midline and fuse. In wild-type mouse embryos neural tube closure is initiated between 8.5 and 9.0 d.p.c. at distinct sites along the neural tube: at the boundary between the hindbrain and cervical region, in the vicinity of the forebrain–midbrain (FB–MB) boundary and at the rostral limit of the neural plate (15,16). Prior to 8.5 d.p.c., Cited2 homozygous mutants could not be distinguished morphologically from heterozygous and wild-type littermates. In contrast, between 8.5 and 9.0 d.p.c. (10–15 somites), Cited2–/– embryos could be recognized by the abnormal separation of neural folds at the FB–MB boundary (Fig. 3A and B). At 9.5 d.p.c. (22–24 somites), when the closure of the cranial neural tube was complete in wild-type and Cited2+/– embryos, variable degrees of NTDs were observed in Cited2–/– embryos ranging from none to severe NTDs affecting the FB, MB and hindbrain. In most embryos, the neural folds had acquired a concave morphology and converged towards the midline. The frequency and positioning of the sites of closure appeared normal in Cited2–/– embryos, but the neural tube remained opened along the MB and/or rostral hindbrain region in these embryos (Fig. 4A and B). In the least severely affected embryos, the neural tube remained open just in a small area of the hindbrain while the rest of the neural tube was apparently fused (Fig. 5N and data not shown). In other Cited2–/– embryos, neural folds of the MB and rostral hindbrain appeared horizontal and wide-open (Fig. 5H and J). Overall, 
50 % of Cited2–/– mutants at 9.5 d.p.c. showed some degree of NTD (n = 20). However, at 10.5 d.p.c., 80% of Cited2–/– mutants displayed the outward expansion of cranial neuroectoderm that characterizes the exencephalic phenotype (n = 25; Fig. 3C–F), while the remaining 20% of homozygous mutants showed a closed neural tube. From this analysis we conclude that the NTDs observed in the Cited2–/– embryos result from impaired elevation and bending of the cranial neural folds in the vicinity of the FB–MB boundary. This defect results in exencephaly by either preventing or delaying neural tube closure in the MB and/or hindbrain regions. Most embryos with delayed neural tube closure fail to neurulate properly and, by 10.5 d.p.c., they invariably show an open neural tube from the caudal FB to the hindbrain/cervical border. Notably, this exencephalic phenotype was identical in all Cited2–/– embryos analysed from 10.5 d.p.c. onwards, regardless of the variable degree of neural tube closure at 9.5 d.p.c. This suggests that regions of the neural tube that were apparently fused at 9.5 d.p.c., reopened subsequently in development. However, a proportion of the mutant embryos (20%) is clearly able to complete neural tube closure.
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Expression of Cited2 is consistent with a role in neural tube closure
Cited2 is expressed in restricted domains within the MB and hindbrain, as well as in head mesenchyme and cranial neural crest at 8.5 d.p.c. Later in development, Cited2 transcripts are detected ubiquitously throughout the embryo and adult tissues (1,3). Taking advantage of the presence of the lacZ gene in the disrupted Cited2 allele, we confirmed and extended the Cited2 expression analysis. X-gal staining was carried out for 30–60 min to reveal the tissues with the highest Cited2 expression. Strong expression was observed in the neural ectoderm around the FB–MB boundary, including the roof plate, and in the hindbrain at 9.0–9.5 d.p.c. (Fig. 4). Together with the previous RNA in situ hybridization analysis these data indicate that Cited2 expression is elevated in regions where the cranial folds initially come into contact and subsequently fuse (3).
Patterning of Cited2 null mutant embryos
The expression profile of Cited2 from 8.5 to 9.5 d.p.c. suggested that the lack of Cited2 in the neural ectoderm might be the likely reason for the NTD. However, since the aetiology of NTDs is very complex, we assessed the integrity and patterning of the neural ectoderm, head mesenchyme and cranial neural crest, all tissues of Cited2 expression, from 8.5 to 10.5 d.p.c.
Defects in head mesenchyme have been shown to cause NTDs in Twist and Cart1 mutant mice (17,18). Histological analysis and RNA in situ hybridization for Twist showed no differences between Cited2–/– and wild-type embryos (n = 5; Fig. 5A and B and data not shown). Similarly, Shh was normally expressed in Cited2–/– and wild-type littermates (data not shown). Expression of the neural crest marker Crabp-1 was largely unaffected in Cited2–/– embryos except for a partial reduction of migratory neural crest into the second branchial arch of the Cited2–/– mutants when compared with wild-type littermates (n = 5; Fig. 5C and D). Immunostaining of neural crest derivatives with anti-acetylated tubulin antibody revealed no significant defects either in cranial and dorsal root ganglia (DRG) or in the cranial or spinal nerves of the Cited2–/– mutants at 10.0–10.5 d.p.c. (n = 3; Fig. 5E and F). Histological examination of Cited2–/– embryos at 14.5 d.p.c. revealed the presence of the trigeminal (V), facioacoustic (VII and VIII), glossopharyngeal (IX) and vagus (X) ganglia, but they were reduced in size when compared with those of wild-type littermates. Whether these defects are a consequence of the overall reduced size of the Cited2–/– mutants or reflect a specific requirement for Cited2 in these structures needs further analysis. Defects were also observed in the DRG, namely fusion of DRG in the cervical region, which are likely to be secondary to the exencephaly of the hindbrain.
These findings suggested that the NTDs probably result from a primary defect in the neural ectoderm. Therefore, neural patterning was analysed by in situ hybridization with specific markers for distinct regions of the neural tube. These analyses revealed that expression domains of the markers Otx2, Fgf8, Krox20, Wnt1, Wnt3a, Pax3, Msx1 and Msx2 were little or not altered in the Cited2–/– embryos when compared with Cited2+/– or wild-type littermates (n = 42; Fig. 5G–P). The only notable difference was a reduction in the expression of Wnt1, Wnt3a, Pax3, Msx1 and Msx2 in the roof plate and dorsal aspects of the FB, MB and rostral hindbrain regions. This was thought to be a consequence of the NTDs, as no abnormal expression of Pax3, Wnt1 and Msx2 was observed in Cited2–/– embryos with successful closure at 10.5 d.p.c. (Wnt3a and Msx1 expression was not analysed in these embryos), nor was the expression of Wnt1 and Pax3 affected at the seven to eight somite stage (data not shown). Altogether, this analysis suggests that gross alterations in patterning of the neuroepithelium or the head mesenchyme do not account for the NTDs observed in the Cited2–/– embryos.
Cell death and proliferation in the Cited2–/– embryos
An imbalance between proliferation and cell survival in the neuroepithelium or head mesenchyme is a potential cause of NTDs leading to exencephaly (18–21). Therefore, apoptosis was assessed in Cited2–/– and wild-type embryos by TUNEL assay. At the six to seven somite stage, Cited2–/– embryos showed a significant increase of apoptotic cells in the dorsal neuroepithelium around the FB–MB boundary and in a distinct transverse domain in the hindbrain (around rhombomeres 3–5; n = 4; Fig. 6A–D). This was apparent before any morphological abnormality was observed in the cranial neural folds of Cited2–/– mutants. A few hours later, at the 14–15 somite stage, increased levels of cell death were still evident in the vicinity of the FB–MB boundary, while in the hindbrain region apoptosis levels were comparable between Cited2–/– and wild-type embryos (n = 4; Fig. 6E–G). The apoptotic domain at the FB–MB boundary and in the adjacent roof plate persisted until the 26–28 somite stage (n = 9; Fig. 6H, and 7D and E). At this stage, we could identify Cited2–/– embryos with apparently normal neural tube closure that showed elevated apoptosis at the FB–MB boundary (data not shown).
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TUNEL staining is based on the detection of DNA fragmentation and this represents a late stage of the apoptotic cascade. It has been shown that DNA fragmentation in apoptotic cells is followed by cell death and removal from the tissue, usually within 1–9 h depending on the presence or absence of professional phagocytes (22,23). In the Cited2–/– embryos, the localized focus of cell death at the FB–MB region was detectable for >24 h (from the 6–7 to the 26–28 somite stage). Therefore, it is apparent that there is a continuous initiation of apoptosis among cells at the FB–MB boundary of the Cited2–/– embryos during this period of time, and some of these cells are detected over several hours.
Cell proliferation was assessed in the three genotypes by immunostaining with the anti-phospho-histone H3 antibody at 9.0 d.p.c. From this analysis it appears that total number of mitotic cells was not changed in the Cited2 null mutants when compared with heterozygotes or wild-type littermates (n = 5; Fig. 6I and J). Altogether, this suggests firstly, that Cited2 is required for the survival of neuroectodermal cells in specific regions of the neural tube. Secondly, elevated apoptosis in the FB–MB region and hindbrain is compatible with normal neural tube closure in 20% of Cited2–/– embryos.
Exencephaly in Cited2–/– mutants is reduced by folic acid
Since folic acid supplementation has been shown to decrease the incidence of NTDs in human and mouse (15,24), we tested the effect of exogenous folate on Cited2–/– embryos. When Cited2+/– pregnant females were treated with folic acid daily throughout pregnancy, exencephaly was reduced from 80 to 12.5% in the Cited2–/– embryos analysed at 14.5 d.p.c. (Tables 2 and 3; Fig. 7A–C). A protective effect was also observed when embryos were cultured in the presence of folic acid for 24 h from 8.5 d.p.c., although this is not statistically significant with the sample size used (Tables 2 and 3). In the cultured embryos the apparently low incidence of exencephaly in the control group (two out of six) probably reflects our in vivo observations, in that the incidence of exencephaly is lower, 50%, at 9.5 d.p.c. compared to later stages. However, we cannot formally exclude the possibility that the in vitro conditions per se might reduce the risk of exencephaly in Cited2–/– embryos. The reduction in NTD incidence following treatment in vitro suggests that folic acid may prevent NTDs by a direct effect on the embryo rather than by correcting an adverse maternal environment (see Discussion).
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70% of human NTDs appear preventable by folic acid supplementation, the degree to which abnormalities in folate metabolism contribute to the aetiology of NTDs remains unclear (25). In the splotch mouse, folate-preventable NTDs are associated with an abnormality of folate metabolism detected, in embryo culture, using the ‘deoxyuridine (dU) suppression test’ (26). This test measures the ability of exogenous deoxyuridine monophosphate (dUMP) to suppress incorporation of 3H-thymidine by stimulating the de novo pathway of pyrimidine biosynthesis, which requires normal metabolic cycling of folate intermediates. Using the dU suppression test, we could not detect any difference in 3H-thymidine incorporation or suppression by dUMP between Cited2–/– mutants developing NTDs in culture and heterozygous or wild-type littermates (Fig. 8). Assuming that an embryonic abnormality of folate metabolism present in vivo would also be present in culture, our findings suggest that the protective effect of folic acid does not act to correct an intrinsic defect of folate metabolism in the embryo.
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As the most likely reason for the NTDs in the Cited2–/– mutants was the increased apoptosis within the neuroectoderm at the FB–MB boundary and hindbrain level, we analysed whether administration of folic acid might reduce the cell death in these regions. From these analyses, we could not detect any obvious reduction in cell death in either the FB–MB boundary or the hindbrain region of the Cited2–/– embryos treated with folic acid (n = 8; Fig. 7D–F). This suggests that exogenous folic acid may compensate for the negative effect on neurulation caused by elevated apoptosis rather than inhibiting cell death directly. However, we cannot rule out the possibility that there might be a small reduction in apoptosis which we cannot detect by TUNEL staining.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cranial NTDs in Cited2 null mutants
We have shown that Cited2 is required for normal neurulation in the mouse embryo. The most likely cause of NTDs in the absence of Cited2 is the massive cell death of neuroectodermal cells at the FB–MB boundary and in the hindbrain region (Figs 6 and 7). Although Cited2 is ubiquitously expressed from 9.0 d.p.c. onwards during mouse embryogenesis, the strong Cited2 expression at the FB–MB boundary and hindbrain region supports a role for this gene in the survival of neuroectodermal cells in these locations (Fig. 4).
Increased cell death within the neuroepithelium around the FB–MB boundary and/or hindbrain might well interfere with neurulation either by preventing or imposing a delayed fusion of the cranial neural folds, leading in turn to exencephaly. In fact, elevated apoptosis is thought to cause exencephaly in the Cart1, Tcof1 and Tulp3 mutant mice (18,20,21). Yet 20% of Cited2–/– mutants appear to compensate for the continuous loss of neuroectodermal cells since they are able to complete neural tube closure and show no sign of exencephaly. This is not an unusual finding since mouse embryos subjected to inactivation of a critical gene via homologous recombination rarely show NTDs with complete penetrance, and similarly, teratogens rarely block neurulation in all treated embryos (15,27). It is likely that the elevated cell death in Cited2–/– mutants exerts a negative effect on neurulation that makes them susceptible to failure of neural tube closure, but the stochastic nature and redundancy of neurulation may compensate for this defect in 20% of the Cited2–/– mutants. However, we cannot rule out that elevated apoptosis and NTDs observed in the Cited2–/– mutants are not causally related events, suggesting that Cited2 might have two separate functions: one as a gene required for survival of neuroepithelial cells and another involved in neural tube closure.
What is the link between Cited2 and folic acid?
NTDs, in particular anencephaly and spina bifida, are among the commonest congenital defects in humans, with a combined frequency of approximately 1 in 1000 live births (28). Folic acid supplementation of the maternal diet before conception and during the first weeks of pregnancy has been observed to significantly reduce the risk of NTDs in humans (28,29). Folic acid has been tested in only a few of the mouse NTD models and is effective in some mutants, but not in others (24,26). The mechanisms for such preventive effects are for the most part unknown.
The exencephaly observed in Cited2–/– mutants was consistently rescued by exogenous administration of folic acid in vivo in two independent experiments (Fig. 7 and Table 2). In vitro, folic acid also appears to have a protective effect as none of the Cited2–/– embryos cultured in the presence of folic acid showed NTDs (none out of seven; Table 3). Therefore, we suggest that folic acid probably prevents NTDs by a direct effect on the Cited2–/– embryos. The reduced incidence of exencephaly among control embryos at 9.5 d.p.c. compared to later stages means that statistical analysis of a larger group of embryos is required to provide a definitive answer to this important question.
No abnormality of folate metabolite cycling was detected in Cited2–/– suggesting that folic acid treatment is not compensating for an intrinsic folate deficiency (Fig. 8). This is in sharp contrast to the splotch mouse where the preventive effect of exogenous folate is thought to be mediated by correction of an embryonic defect in folate metabolism (26). To our knowledge, this is the first genetic evidence to suggest that folic acid can decrease the incidence of NTDs by a mechanism other than compensation of an intrinsic folate related defect in the embryo. Therefore, the Cited2 mouse may represent a useful model to investigate how folic acid prevents NTDs.
Inhibition of apoptosis by exogenous folic acid was investigated as a potential mechanism for prevention of exencephaly in the Cited2–/– embryos (Fig. 7). The data obtained from TUNEL staining suggested that folic acid treatment does not substantially reduce the levels of cell death in Cited2–/– embryos because folic acid treated Cited2–/– embryos showed similar patterns of cell death to untreated Cited2–/– controls (Fig. 7). Elevated cell death in the FB–MB boundary and hindbrain is compatible with normal neural tube closure, since 20% of Cited2–/– embryos did not show exencephaly from 10.5 d.p.c. onwards, but all Cited2–/– mutants analysed exhibited increased cell death in these regions of the neural tube at 8.5 and 9.5 d.p.c. (n = 17). Therefore, administration of folic acid appears to compensate for, rather than directly inhibit, cell death. However, the possibility exists that folic acid might lead to a small reduction of cell death, sufficient to improve neurulation, but difficult to be detected by the assay used in this study. It will be interesting to analyse whether cell death was reduced in the head mesenchyme of the Cart1 mutants, a mouse model in which folic acid prevents exencephaly (18).
An alternative possibility to explain its protective effect is that exogenous folic acid could help to replace dying cells within the neuroectoderm by boosting the proliferation of unaffected neighbouring cells during neurulation. Recently, it has been proposed that folic acid might be required for timed proliferative bursts during gestation (25). We did not observe any significant difference in numbers of proliferating cells in Cited2–/– mutants when compared with wild-type littermates and, similarly, between folic acid treated and untreated Cited2–/– embryos (Fig. 6I and J; data not shown). Nevertheless, it is possible that a subtle proliferation defect exists in the Cited2–/– embryos that is technically difficult to identify. In this respect, it is interesting that Cited2 has been shown to physically interact with the transcriptional coactivators and histone acetyltransferases, CBP and p300, and that animals nullizygous for p300 die between days 9 and 11.5 of gestation, exhibiting defects of neurulation, cell proliferation and heart development (4,30). The Cited2–/– mutants represents a mouse model that should contribute to a better understanding of the mechanism by which folic acid administration prevents human NTDs, a question that is still unanswered.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Targeting vector and generation of the Cited2 null mutant mouse
The Cited2 genomic clone was isolated from a 129/Olac genomic library (a gift from Dr A.Smith) using Cited2 cDNA as a probe (3). To construct the targeting vector, 4.0 and 2.0 kb of 5' and 3' homologous regions were cloned on either side of an IRES-lacZ-Neo cassette (courtesy of Dr A.Smith; Fig. 1A). The entire Cited2 coding region from His4 was deleted in the targeting vector, thus creating a null mutation. This vector was linearized with AhdI and electroporated into E14 ES cells (a gift from Dr A.Smith). G418 selection of ES cell clones and generation of chimeric mice were performed using standard methods (31). To screen for homologous recombinants, ES cell DNA was digested with NdeI and hybridized to sequences located 5' and 3' external to the recombination sites (Fig. 1A). Two correctly targeted ES cell clones were identified and used for injection into C57BL6 blastocysts. Chimeric males from the two clones were mated with C57BL6 females but Cited2+/– animals were obtained only from one of them. Heterozygous animals were backcrossed to C57BL6 wild-type animals, and this backcrossing process was continued for two to four generations before setting up Cited2+/– intercrosses. The severity and incidence of the phenotype did not change among Cited2–/– embryos derived from different generations.
Genotyping of the wild-type and mutant Cited2 alleles
Newborn mice and embryos were genotyped by PCR and Southern blot of DNA samples prepared from tails, yolk sacs or whole embryos (Fig. 1B and C). Primers used for PCR were: R1 (5'-ggcaaactgcttaatcttgtgg-3') and R2 (5'-gaaatgtttgccactgacgac-3') for the Cited2 wild-type allele; N1 (5'-ctgtgctcgacgttgtcactgaag-3') and N2 (5'- tattcggcaagcaggcatcgccat-3') for the Neo gene. Primers used to detect the presence of the Y chromosome were: ZFY (5'-gactagacatgtcttaacatctgtcc-3') and YNLS (5'-cctattgcatggacagcagcttatg-3').
Histology, in situ hybridization and X-gal staining
For histology, embryos were fixed in Bouin’s fixative, dehydrated through graded ethanols, embedded in paraffin wax and sectioned. Sections were stained with haematoxylin and eosin as described previously by Kaufman (32). Staining with alcian blue of whole mount embryos was carried out as described by Jegalian and De Robertis (33). In situ hybridization and X-gal staining of whole-mount embryos used standard procedures (31,34). The following probes were used: Crabp-1, Twist, Shh, Krox20, Otx2, Fgf8, Wnt1, Wnt3a, Pax3, Msx1 and Msx2 (17,35–42). A minimum of three embryos were analysed per probe.
Analysis of cell death and immunostaining
Whole-mount TUNEL assay was performed following the manufacturer’s instructions with some modifications (ApoptagDectect; Quantum Appligene). Briefly, embryos were dissected in M2 medium, fixed in 4% PFA, dehydrated through graded methanols and kept at –20°C until use. For staining, embryos were rehydrated in PBS, fixed in ethanol/acetic acid solution (2:1) for 20 min at –20°C, equilibrated and incubated with terminal deoxynucleotidyl transferase overnight at 37°C. The following day the transferase reaction was stopped, embryos were blocked in 10% sheep serum and incubated with anti-digoxigenin-AP-conjugated antibody (1:2000 dilution; Roche) overnight at 4°C. Finally, embryos were washed and stained with NBT/BCIP according to the manufacturer’s instructions (Roche). Inmunostaining using the monoclonal anti-acetylated tubulin (1:1000 dilution; Sigma) was carried out according to standard protocols (31). Proliferation was assessed by whole-mount staining with the anti-phospho-histone H3 antibody (mitosis marker; Upstate Biotechnology). After staining, at least three embryos for each genotype and at a similar stage of development were embedded in wax and sectioned as described by Kaufman (32). For analysis, the total number of mitotic cells in matched sections from the three genotypes was compared. No consistent differences were observed.
Folic acid treatment
Prenatal folic acid treatment began the day on which a vaginal plug was found. Folic acid (3 mg/kg body weight; Sigma) was administered daily by intraperitoneal injection. PBS was used as the vehicle for these injections. Females were killed at 9.5 or 14.5 d.p.c. and embryos were assessed morphologically for the presence or absence of NTDs. In some cases, embryos were allowed to go to term. Genotyping of the embryos was carried out as described above. The incidence of exencephaly in the treated embryos was compared with that of untreated animals (Tables 2 and 3). For in vitro folate treatment, mice were killed at 8.5 d.p.c. by cervical dislocation and the uterus explanted into Dulbecco’s modified Eagle’s medium (Gibco BRL, Life Technologies) containing 10% fetal calf serum. Embryos, contained within the visceral yolk sac, were explanted from the maternal decidua and cultured in immediately centrifuged, heat-inactivated rat serum at 38°C for 24 h as described previously by Fleming and Copp (26). Serum was equilibrated initially with 5% O2, 5% CO2, 90% N2 and then after 18 h with 20% O2, 5% CO2, 75% N2. After 30 min recovery in culture, folic acid (50 mM) or PBS (control group) was added at 10 µl/ml giving a final concentration of 500 µM. Embryos from individual litters were divided between the experimental groups in order to minimize the effect of any litter to litter variation in NTD incidence. After culture, embryos with more than 16 somites and which had completed turning were assessed for the presence of NTDs, and yolk sacs were retained for genotyping. Measurement of the crown–rump length and number of somites after culture revealed no significant differences between genotypes or treatment groups (data not shown).
Deoxyuridine suppression test
Embryos were cultured (as for in vitro treatment) for 24 h from 8.5 d.p.c. in the presence of 3H-thymidine (1 µCi/ml; specific activity 25 Ci/mmol) with or without 500 µM dUMP (Sigma). The incorporation of 3H-thymidine was measured by scintillation counting of potassium acetate-precipitated DNA as described previously by Fleming and Copp (26). The protein content of embryos was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce) and incorporation (c.p.m./µg protein) was calculated. The percentage suppression of incorporation was calculated by comparison of incorporation in the presence and absence of dUMP. The test was carried out twice with a total of 50 embryos (n = 11 +/+, 26 +/–, 13 –/–).
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ACKNOWLEDGEMENTS |
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We are especially grateful to Paul Thomas for help with the initial screen for Cited2 genomic sequences, E.Grigorieva and E.Puelles for help with histology, also K.Mathers, D.Lynch and M.Clements for excellent technical assistance, and S.Hodges and P.Dawson for animal care. We thank S.Bhattacharya for sharing unpublished results, M.Coles and D.Kioussis for their interest in the Cited2 mutants, and A.Graham for discussions. We also thank A.McMahon, G.Martin, D.Wilkinson, P.Gruss, P.Chambon and C.Abate-Chen for probes. This work was supported by the MRC. N.D.E.G. and A.J.C. are supported by the Wellcome Trust.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 207 848 6536; Fax: +44 207 848 6550; Email: juan.martinez-barbera@kcl.ac.uk This article is dedicated to Rosa Beddington, an extraordinary scientist and a wonderful friend
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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