Human Molecular Genetics, 2000, Vol. 9, No. 4 575-581
© 2000 Oxford University Press
A genetic risk factor for mouse neural tube defects: defining the embryonic basis
Neural Development Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK
Received 18 October 1999; Revised and Accepted 14 December 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genetic polymorphisms are thought to play an important role in determining susceptibility to neural tube defects (NTDs), for example between different ethnic groups, but the embryonic manifestation of these polymorphic genetic influences is unclear. We have used a mouse model to test experimentally whether polymorphic variations in the pattern of cranial neural tube closure can influence susceptibility to NTDs. The site at which cranial neural tube closure begins (so-called closure 2) is polymorphic between inbred mice. Strains with a caudal location of closure 2 (e.g. DBA/2) are relatively resistant to NTDs, whereas strains with a rostrally positioned closure 2 (e.g. NZW) exhibit increased susceptibility to NTDs. We tested experimentally whether altering the position of closure 2 can affect susceptibility to cranial NTDs, by back- crossing the splotch (Sp2H) mutant gene onto the DBA/2 background. As a control, Sp2H was transferrred onto the NZW background, which resembles splotch mice in its closure pattern. Approximately 80% of Sp2H homozygotes develop NTDs, both cranial (exencephaly) and spinal (spina bifida). After transfer to the DBA/2 background, the frequency of cranial NTDs was reduced significantly in Sp2H homozygotes, confirming a protective effect of caudal closure 2. In contrast, Sp2H homozygotes on the NZW background had a persistently high frequency of cranial NTDs. The frequency of spina bifida was not altered in either backcross, emphasizing the specificity of this genetic effect for cranial neurulation. These findings demonstrate that variation in the pattern of cranial neural tube closure is a genetically determined factor influencing susceptibility to cranial NTDs.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The aetiology of neural tube defects (NTDs) involves both genetic and non-genetic (e.g. nutritional) factors. The importance of a genetic component in the causation of NTDs is indicated, for example, by the elevated recurrence risk observed among first and second degree relatives of NTD cases (1). The identity of genes of major effect in human NTDs is not known, although the likely existence of such genes is indicated by the many genetic loci that can cause NTDs in the mouse (2,3). At present, it is unclear to what extent homologues of the mouse genes are involved in the aetiology of human NTDs.
Genetic polymorphisms also play an important role in determining susceptibility to NTDs. A thermolabile variant of the enzyme 5,10-methylene tetrahydrofolate reductase appears to confer increased risk of NTDs in humans (4), although this factor probably accounts for only a small proportion of the total genetic predisposition to NTDs (5). In the mouse, NTDs resulting from the action of a variety of mutant genes and teratogenic agents vary in frequency and severity depending on the genetic background involved, suggesting a role for genetic loci that are polymorphic between inbred strains. For instance, homozygotes for the mutant genes Opb and Axd (6,7), and for the gene knockouts p53 and Cart-1 (8,9), develop NTDs with strain-dependent variation in penetrance. Similarly, teratogenic agents such as valproic acid, hyperthermia and hypoglycaemia induce differing frequencies of NTDs on different genetic backgrounds (10–13).
A clue to the embryonic basis of these strain differences in NTD susceptibility has come from studies by Juriloff et al. (14) who reported variations in the morphology of cranial neural tube closure between inbred mouse strains (Fig. 1). Neural tube closure begins in the mouse at the boundary between the hindbrain (HB) and the cervical region (closure 1), with closure spreading from this site rostrally into the HB, as well as caudally into the spinal region. A second point of de novo initiation of neural tube closure occurs in the vicinity of the forebrain–midbrain (FB–MB) boundary (closure 2), and a third initiation site is at the rostral extremity of the neural plate (closure 3). Neural tube closure then spreads bidirectionally, between closures 1 and 2, to complete neurulation at the HB neuropore, and between closures 2 and 3 to complete neurulation at the anterior neuropore.
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Although the location of closures 1 and 3 appears to be uniform between mouse strains, the site of closure 2 is polymorphic (14). Most strains exhibit closure 2 at the FB–MB boundary (Fig. 1B), whereas the closure site is positioned more caudally, within the MB, in some strains (Fig. 1A) and positioned more rostrally, within the FB, in others (Fig. 1C). Variation in the position of closure 2 could provide a basis for differences in susceptibility to cranial NTDs. For instance, the LM/Bc strain, which exhibits closure 2 at the FB–MB boundary, is less susceptible to teratogens such as valproic acid and hyperthermia than is the SWV/Bc strain, which has a more rostrally situated closure 2 (10,12,14).
We hypothesized that the position of closure 2 along the rostro-caudal axis may be the embryonic manifestation of the strain-dependent risk factor for cranial NTDs in the mouse. To test this hypothesis, we studied mutant splotch (Sp2H) mice which carry mutations in the transcription factor Pax3, and which develop cranial and/or spinal NTDs, as well as defects of the neural crest and limb/body wall musculature (15–19). Backcrossing the Sp2H mutation onto the DBA/2 background, in which closure 2 is located caudally, conferred resistance to cranial NTDs, whereas the frequency of spina bifida was not affected. A control backcross onto the NZW background, in which a rostral closure 2 site is present, did not alter the frequency of cranial NTDs. These findings demonstrate that the polymorphic position of closure 2 is a genetically determined factor influencing susceptibility to cranial NTDs in the mouse.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We confirmed the findings of Juriloff et al. (14) that the position of closure 2 varies between mouse strains. Closure 2 was observed at a caudal position, within the MB region, in the inbred DBA/2 strain (Fig. 2A). In contrast, closure 2 is located at the FB–MB boundary in CD1 random bred mice (Fig. 3B), and at a rostral position, within the FB region, in the inbred NZW strain (Fig. 2B) and in CBA/Ca mice (data not shown).
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Closure 2 is present, at a rostral position, in splotch embryos developing NTDs
In order to determine whether closure 2 may be absent in embryos developing cranial NTDs, as has been reported for the exencephaly-prone SELH/Bc strain (20), we examined embryos from matings between Sp2H heterozygotes, in which ~80% of Sp2H/Sp2H embryos develop cranial NTDs (21). All embryos studied (n = 85) from matings between Sp2H/+ mice uniformly exhibited closure 2 at the rostral position, within the FB region (Fig. 2C), regardless of genotype. Hence, Sp2H/Sp2H embryos that are destined to develop cranial NTDs exhibit a rostral closure 2 event in a manner that is indistinguishable from their normally developing +/+ and Sp2H/+ littermates. We conclude that failure to close the cranial neural tube in homozygous Sp2H/Sp2H embryos does not result from absence of closure 2 but rather from a later event in cranial neurulation.
Cranial NTDs in the splotch mouse result from failure of the MB neural folds to adopt a concave morphology
During normal cranial neurulation, the MB neural folds elevate with a convex morphology and then undergo a dramatic change in shape such that the folds no longer flare outwards, but become concave and converge towards the midline (Fig. 3A and B). This change in morphology was seen in non-mutant CD1 embryos and in all +/+ and Sp2H/+ embryos from splotch litters. In contrast, a proportion of Sp2H/Sp2H embryos (16 of 21 homozygotes studied) exhibited failure of the MB neural folds to adopt a concave morphology, remaining convex with a flared appearance (Fig. 3C and D). We followed the developmental outcome of individual embryos in whole embryo culture, with subsequent Pax3 genotyping, and found that only embryos with the MB morphogenetic defect go on to develop a persistently open cranial neural tube (76% of Sp2H/Sp2H embryos studied). Embryos that achieve a concave morphology of the MB neural folds (as in Fig. 3B) progress to closure of their cranial neural tube (24% of Sp2H/Sp2H embryos studied).
We conclude that cranial NTDs in the splotch mutant develop following the successful occurrence of closure 2, in contrast to the exencephalic strain SELH/Bc (20). Whereas in normal embryos, cranial closure proceeds in a caudal direction from the site of closure 2 to close the MB region, caudal progression from closure 2 fails in affected Sp2H/Sp2H embryos because of the flaring of the MB neural folds (Fig. 3D). This observation prompted the idea that the rostral position of closure 2 in the splotch genetic background may predispose to the failure of MB neural fold closure, because of the relative lack of mechanical support for the MB neural folds. To test this idea, we backcrossed the Sp2H mutation onto genetic backgrounds that differ in their site of closure 2 (Fig. 2A and B), in order to determine whether the penetrance of the cranial NTD phenotype is affected in a predictable manner. The experimental design is summarized in Table 1.
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Position of closure 2 shifts during backcross of Sp2H onto the DBA/2 background
In DBA/2 embryos, closure 2 occurs at a caudal position, in the MB region (Fig. 2A) whereas, in NZW embryos, closure 2 is rostral, in the FB region (Fig. 2B), as in splotch embryos (Fig. 2C). Embryos obtained from matings between (Sp2H/+ x DBA/2)F1 mice exhibited closure 2, unlike either parent strain, but positioned at the FB–MB junction (Fig. 2D). With each generation of backcrossing to DBA/2, the position of closure 2 shifted more caudally (Fig. 2E and F), reflecting the increasing influence of the DBA/2 background. By the fourth backcross generation (Fig. 2G), closure 2 closely resembled that seen in inbred DBA/2 embryos (Fig. 2A). In contrast to the DBA/2 backcross, the position of closure 2 at each generation of the NZW backcross did not shift, but remained rostral, within the FB region (Fig. 2H), as in the parental NZW strain (Fig. 2B), reflecting the initial similarity between the splotch and NZW strains.
Reduction in frequency of cranial but not spinal NTDs in the DBA/2 backcross
Analysis of embryonic day (E) 12.5 Sp2H homozygous embryos from the fourth backcross generation (Fig. 4) revealed a significant reduction in the frequency of cranial NTDs (i.e. exencephaly) in the DBA/2 backcross (35.7% of embryos affected) compared with the incidence in the splotch parent colony (75.0%, P = 0.017). In contrast, the incidence of cranial NTDs in Sp2H homozygotes in the fourth generation of the NZW backcross (74.3%) did not differ from the frequency in the splotch colony (P = 0.77). Strikingly, the frequency of spinal NTDs (i.e. spina bifida) did not alter significantly in either the DBA/2 backcross (85.7%, P = 0.80) or the NZW backcross (82.9%, P = 0.99) compared with the splotch parent mice (87.5%).
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Hence, significantly more Sp2H homozygotes in the DBA/2 backcross exhibited spina bifida alone and significantly fewer had combined cranial and spinal NTDs than in the splotch strain (Table 2). This reduction in frequency of cranial NTDs among Sp2H homozygotes in the DBA/2 backcross cannot be explained by the early selective loss of affected embryos, since the overall frequency of Sp2H homozygotes at E12.5 was close to the expected 25% in the DBA/2 backcross, and did not differ significantly from the homozygote frequency in the other strains (Table 2). We conclude that breeding the Sp2H mutation onto the DBA/2 genetic background specifically reduces the penetrance of the cranial NTD phenotype, as predicted from the consideration of the position of closure 2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Many mouse models of cranial NTDs have been described, based on genetic mutations or environmental teratogenic influences (2,3), but few have been characterized with respect to the known closure events of cranial neurulation (22). Absence of closure 2 is associated with development of cranial NTDs in the SELH/Bc strain (20) and in homozygotes for a null mutation of the gene encoding MARCKS-related protein (23). In contrast to these systems, we found that homozygous Sp2H embryos, which develop cranial NTDs in ~80% of cases, undergo closure 2 in a manner that does not differ from their wild-type and heterozygous littermates. These mutant embryos fail in a later event of cranial neurulation, i.e. the adoption of a concave morphology by the MB neural folds.
The mechanism underlying the defect of cranial morphogenesis in Sp2H homozygotes is unclear. Faulty release of neural crest cells from the margins of the cranial neural folds and imbalance of extracellular matrix components in the neural folds are both factors that can impair neural fold closure in the cranial region (24–27). Sp2H mice harbour mutations in the homeobox-containing Pax3 gene (16), with homozygotes exhibiting defects of neural crest migration and overexpression of the extracellular matrix proteoglycan versican (28–31). It remains to be determined whether these abnormalities are the critical pathogenetic factors leading to cranial NTDs in Sp2H embryos.
Polymorphism of closure 2 correlates with susceptibility to cranial NTDs
We confirmed the findings of Juriloff et al. (14) that the position of closure 2 along the rostro-caudal axis varies between inbred strains. We were able to predict, from the variation in susceptibility of different inbred strains to spontaneous or teratogen-induced cranial NTDs, which strain is likely to demonstrate a caudal position of closure 2 (NTD resistant) and which should demonstrate rostral closure 2 (NTD susceptible). Thus, we found that closure 2 in the DBA/2 inbred strain is located caudally, within the MB, as predicted from the resistance of this strain to teratogen-induced exencephaly (10,12). Conversely, the NZW strain has been reported to exhibit an increased incidence of spontaneous exencephaly (32), suggesting susceptibility to cranial NTDs. Although we did not observe spontaneous cranial NTDs in our NZW mice, we confirmed that closure 2 is situated rostrally, within the FB, in NZW embryos. Interestingly, closure 2 is also situated rostrally in embryos of the splotch background, irrespective of their genotype at the Sp2H locus, consistent with the high frequency of exencephaly observed in Sp2H homozygotes.
Rostro-caudal position of closure 2 is a risk factor for cranial NTDs
To test experimentally the impact of closure 2 location on susceptibility to cranial NTDs, we backcrossed the Sp2H mutation onto the DBA/2 background. This shifted the position of closure 2 in a caudal direction with successive backcross generations so that, by the fourth generation, closure 2 was located in the MB region, as in the parental DBA/2 strain. The backcross produced a significant reduction in the incidence of cranial NTDs in homozygous Sp2H embryos. Only 36% developed the defect, compared with 75% in the splotch parent colony. The reduction in the frequency of cranial NTDs seems most likely to result from the specific effect of altering the position of closure 2, for two reasons.
First, a control backcross to the inbred NZW strain, while altering the genetic background, maintained the rostral site of closure 2. This backcross did not alter the incidence of exencephaly. Secondly, whereas the incidence of the cranial NTD, exencephaly, was altered in the DBA/2 backcross embryos, strikingly there was no alteration in the incidence of the caudal NTD, spina bifida, which continued to affect >80% of Sp2H homozygotes in both backcrosses. Hence, there is no overall reduction in susceptibility to NTDs by backcrossing onto the DBA/2 background, but rather a specific protection against cranial NTDs. We suggest that this effect is a direct result of the morphological shift in position of closure 2 along the rostro-caudal axis.
Genetic basis of the variation in closure 2
Several genes appear to participate in the control of the position of closure 2. Matings between splotch heterozygotes and DBA/2 inbred mice produced F1 embryos with an intermediate position of closure 2 that was unlike either parent. Moreover, four generations of backcrossing to the DBA/2 strain were necessary to shift the position of closure 2 caudally to that observed in DBA/2 mice. The simplest explanation of these findings is a gradual replacement of splotch alleles by DBA/2 alleles at a number of controlling loci.
Juriloff et al. (33) and Gunn et al. (34) have suggested a similar polygenic model, involving the additive action of 2–3 genes, to explain the inheritance of predisposition to exencephaly in the SELH/Bc strain in which there is apparent absence of closure 2 (20,35). Cranial neurulation spreads caudally from the site of closure 3 to close the cranial neural tube in 83% of SELH/Bc embryos, whereas this process is incomplete, leading to exencephaly, in 17% of embryos. The phenotype seen in SELH/Bc could be interpreted as forming one extreme in a spectrum of cranial neural tube closure patterns (Fig. 1D). According to this view, closure 2 is located even more rostrally in SELH/Bc than in the NZW or splotch strains, occurring so close to closure 3 as to be indistinguishable from it. It is possible that the same set of genes could underlie both variation in the position of closure 2 in ‘normal’ strains and susceptibility to exencephaly in the SELH/Bc strain. Hence, SELH/Bc would possess alleles that predispose to even more rostral closure 2 than other inbred strains. However, genetic crosses of a substrain of SELH/Bc to two other strains, ICR/Bc and SWV/Bc, which themselves differ in closure 2 position, yield a similar frequency of exencephaly in each case. Gunn et al. (34) interpret these data as showing that the genetic basis of the predisposition to exencephaly in SELH/Bc results from exencephaly-causing mutations, not from polymorphic loci, such as are thought to control the position of closure 2.
Relevance of the mouse findings to human NTDs
Susceptibility to NTDs varies among human ethnic groups, with some of this variation appearing to reflect genetic differences (1). Hence, genetically determined variations in the precise pattern of cranial neural tube closure (e.g. the site of closure 2) potentially could explain the differential susceptibility of human populations to cranial NTDs. It is important to ask, therefore, whether multi-site closure of the cranial neural tube actually occurs in humans, as in the mouse.
The piecemeal pattern of neural tube closure is not unique to the mouse embryo but has also been described in the rabbit (36) and chick (37). Moreover, the variety of NTD types in human fetuses has been explained by applying a model of multi-site closure (38–41). Van Allen et al. (39) proposed that all cases of anencephaly, both meroacrania and holoacrania, are associated with failure of closure 2. The present study demonstrates that this is certainly not true in the mouse, however, since Sp2H homozygotes develop exencephaly in the presence of an intact closure 2. A species difference may exist, with absence of closure 2 being obligatory for development of cranial NTDs in humans, but not in the mouse. Alternatively, this apparent contrast between human and mouse may emphasize the potential dangers of using a ‘multi-site closure’ hypothesis to infer the nature of pathological neurulation events, which occur very early in embryogenesis, on the basis of evidence of NTD appearance in late fetuses.
Direct studies of neurulating human embryos are equivocal on the subject of whether a closure 2 event occurs during human neurulation. O’Rahilly and Müller (42) describe closure as proceeding rostrally into the cranial region from the HB–cervical boundary, and caudally from the region of the telencephalon immediately adjacent to the chiasmatic plate (i.e. the rostral extremity of the neural plate). These human closure events bear a very striking resemblance to mouse closures 1 and 3, respectively. Moreover, O’Rahilly and Müller (43) have stated that the ‘dorsal lip’ of the rostral neuropore in human embryos is equivalent to closure 2 in the mouse. On the other hand, a recent study of neurulation-stage human embryos (44), while identifying closures 1 and 3, has failed to demonstrate an event equivalent to closure 2. One possible explanation is that the site of closure 2 is highly variable between human embryos. In some, it may be a distinct closure site, whereas in others it may be more rostrally located, near to the site of closure 3, as in the SELH/Bc mouse (Fig. 1D). Hence, genetically determined polymorphisms in the precise details of cranial neural tube closure may characterize humans, as well as the mouse, and may be implicated in determining susceptibility of individual human embryos to cranial NTDs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mouse strains
Inbred CBA/Ca, DBA/2 and NZW mice (Harlan Olac, Bicester, UK) and outbred CD1 mice (Charles River, Margate, UK) were maintained on a 12 h light–dark cycle (dark from 19:00 to 07:00 h). Experimental litters were produced by overnight matings, with the day of finding of a copulation plug designated as E0.5. Sp2H, a radiation-induced allele at the splotch locus, is maintained on a mixed background originally derived from the C3H/He, 101 and CBA/Ca strains. Matings between heterozygous Sp2H/+ mice were used to produce experimental litters containing Sp2H/Sp2H, Sp2H/+ and +/+ embryos.
Backcrossing the Sp2H mutation onto the DBA/2 and NZW inbred backgrounds
Sp2H/+ females were mated with DBA/2 males to produce F1 progeny which were genotyped at the Pax3 locus by polymerase chain reaction using DNA from tail-tip biopsies (16). Female F1 mice with the Sp2H/+ genotype were then mated with DBA/2 males and this backcross process was continued for four generations (Table 1). A corresponding backcross was performed using the NZW strain. At each generation of the backcrosses, E8.5 embryos from matings between Sp2H/+ mice were cultured to the stage of closure 2 and processed for scanning electron microscopy. At the fourth backcross generation, litters from matings between Sp2H/+ mice were collected at E12.5 and dissected in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, and embryos were inspected for the presence of cranial and spinal NTDs. The number of resorptions in each litter was noted. Each embryo was genotyped at the Pax3 locus using yolk sac DNA (16). In order to compare the incidence of NTDs in the backcrosses and on the original splotch background, contemporaneous Sp2H/+ x Sp2H/+ litters were collected from the parent colony at E12.5. Statistical comparisons between NTD frequencies were performed using 
2 contingency tests, with the significance level set at P = 0.05.
Determining the position of closure 2 in cultured embryos
Embryos were explanted for whole embryo culture (45) at early E8.5, when they had fewer than five somites, and were removed briefly from culture every 3–4 h to determine the progression of cranial neurulation. Transillumination through the yolk sac on the stage of a Zeiss SV6 stereomicroscope was found to enable an accurate assessment of the elevation and fusion status of the cranial neural folds. Repeated observations did not adversely affect the rate of development in vitro, as judged by an increase in somite number (Table 3). Once the stage of closure 2 was reached (~10 somites), cultures were terminated and the yolk sac was removed and used for DNA extraction prior to genotyping of embryos. The amnion was removed and discarded. Embryos were rinsed twice in phosphate-buffered saline, fixed for 24 h at 4°C in half-strength Karnovsky’s fixative (46), rinsed in cacodylate buffer, post-fixed in 4% osmium tetroxide for 1 h, dehydrated through an ethanol series and dried using liquid CO2 in a critical point dryer. Dried embryos were coated under argon vacuum with a 100 nm layer of gold particles and viewed using a JEOL JSM 35 scanning electron microscope. The position of closure 2 was determined in embryos of the inbred strains CBA/Ca (n = 12), DBA/2 (n = 16) and NZW (n = 15), in outbred CD1 embryos (n = 3) and in embryos from matings between Sp2H heterozygotes (n = 85). Closure 2 was also studied in a minimum of five embryos at each generation of the backcrosses to DBA/2 and NZW.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr Henny van Straaten for critical reading of the manuscript, and to the Wellcome Trust and the Child Health Research Appeal Trust for financial support.
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FOOTNOTES |
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+ Present address: Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK
§ To whom correspondence should be addressed. Tel: +44 171 829 8893; Fax: +44 171 831 4366; Email: a.copp@ich.ucl.ac.uk
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