Embryology of Myelomeningocele and Anencephaly

Mark S. Dias, MD; Michael Partington, MD

Disclosures

Neurosurg Focus. 2004;16(2) 

In This Article

Embryogenesis of Human Myelomeningoceles

There are two fundamental theories regarding the embryogenesis of myelomeningocele, both encompassing a disorder of primary neurulation. In the so-called nonclosure theory initially suggested by von Recklinghausen, it is proposed that neural tube defects represent a primary failure of neural tube closure. In the overdistension theory, introduced in 1769 by Morgagni and popularized by Gardner, it is proposed that NTDs arise through overdistension and rupture of a previously closed neural tube. The nonclosure theory is more widely accepted and certainly accounts for the majority of human NTDs; however, overdistension may contribute to some experimental neural tube defect models, particularly those caused by vitamin A[7] and the T-curtailed mouse mutant.[11]

In addition to the traditional view of NTDs as disorders of neurulation, Dias and Pang[15] and Dias and Walker[17] have more recently proposed that a number of myelomeningoceles (particularly some cervicothoracic myelomeningoceles, hemimyelomeningoceles, and those myelomeningoceles associated with SCMs (diastematomyelia) and other complex dysraphic malformations) arise not as a result of a primary failure of neurulation, but as a result of disordered midline axial integration during gastrulation. The primary embryonic abnormality for these malformations, according to this theory, is the failure of the Hensen node to lay down properly a single notochord flanked by a cohesive surrounding sheet of neuroepithelium. Instead, paired notochordal anlagen develop from each half of the Hensen node during gastrulation and two relatively independent hemineural plates, each developing into a hemicord, arise on either side of the node (Fig. 11). The malformations so induced may secondarily disrupt neurulation and result in either a hemimyelomeningocele (if neurulation fails in only one hemicord) or a myelomeningocele associated with an SCM rostral or caudal to the placode (if primary neurulation fails in both hemicords). Multipotent cells contained within the Hensen node and laid down between the two hemicords may form various normal and abnormal tissue types (such as ovarian and renal tissues, Wilm tumor, or teratoma) and result in a wide variety of malformations such as neurenteric cysts, combined spina bifida (split notochord syndrome), and other entities.[17] Experimental manipulations of the Hensen node in chick embryos can produce malformations that resemble human SCMs and provide some experimental support for such a mechanism.[17]

Embryogenetic theory for the origin of some myelomeningoceles, hemimyelomeningoceles, SCMs, and other complex dysraphic malformations. According to this theory, the Hensen node is altered resulting in disrupted midline axial integration during gastrulation. Two heminotochords are laid down and the surrounding neural plate is induced to form two relatively independent hemineural tubes. The intervening cells derived from multipotent cells in the Hensen node can develop into a variety of both normal and abnormal tissues between the two resultant hemicords such as neurenteric cysts, ectopic ovarian or renal tissues, Wilm tumor or teratomas (all reported in the literature). From Luo and Nye, 2001.

Because early neural embryogenesis in general, and neurulation in particular involve a complex interplay of multiple cellular processes, tissue morphogenesis, and the timely expression of multiple transcription and cell signaling factors, it is not surprising that NTDs may result from a number of embryonic insults. Neural tube defects have been produced experimentally using a number of teratogens,[8] genetic mutations,[10,11,31] and experimental manipulations.[50] Although these all suggest a number of potential mechanisms whereby NTDs might arise, the cause of human malformations remains unknown. Neural tube defects are most likely heterogeneous in origin[8,10,11,17] and represent the end result of a various embryonic disorders.

What is clear, however, is that NTDs represent the intricate interactions between complex genetic and environmental factors. Many lines of evidence point to a genetic component for NTDs. First and most obvious, the incidence of NTDs increases in first-degree relatives of patients with these defects. For example, the risk of the parents of a child with an NTD giving birth to a second child with an NTD is 2 to 3%; after having two children with NTDs, the risk of a third child with an NTD rises to 10% or more.[36] Second, the incidence of NTDs varies widely between different populations, even after accounting for geographic migration and other factors. Third, concordance rates between monozygotic twin pairs vary between 3.7 and 18%.[39] Fourth, NTDs are seen in association with known genetic syndromes (such as Waardenberg syndrome) and chromosomal anomalies (such as trisomy 13 and 18). There are also families in whom Mendelian inheritance is documented (one dominant and the other recessive). Finally, chromosomal rearrangements such as aneuploidy have been reported.

Genetic models of NTDs provide a means of identifying particular gene(s) that might be involved in neural tube closure.[10,11,31] To date, more than 60 genetic mouse mutants with NTDs have been identified.[31] These mutations code for a bewildering variety of molecular species including various transcription factors and coactivators, signal transducers, folate binding proteins, tumor suppressor gene products, cytoskeletal components, DNA methyltransferases, nuclear and cell membrane receptors, chromosomal proteins, gap junction proteins, cell surface receptors, and actin regulators and binding proteins.[31] Many mutants exhibit specific types of NTDs involving disruption of specific waves of neural tube closure and suggest that each wave has specific and discrete elements that contribute to closure and which can be disrupted without interfering with other waves. The looptail mouse mutant, for example, results in disruption of only spinal neurulation without producing a cranial NTD; the mutation causes abnormally broad misexpressions of Sonic hedgehog, Netrin1, and Brachyury. Other mutations appear to disrupt more than one wave of closure suggesting that there may be common pathways as well.[31]

Three examples illustrate the varied ways in which genetic mutations can result in an NTD. The splotch mouse mutants are identified by a peculiar patch of white fur and exhibit both exencephaly and myelomeningoceles. The genetic locus for the splotch mutation is within the Pax-3 gene (as previously discussed), a transcription factor that may be involved in neural fold fusion; how Pax-3 mutation causes NTDs is largely unknown. A second mouse mutant, curly tail, exhibits posterior NTDs and tail deformities associated with a delay in the closure of the caudal neuropore. The delay, however, is not the result of faulty neuroepithelial development, because isolated neuroepithelium from curly tail mutants undergoes normal neurulation. Rather, there is a delay in cell proliferation in the underlying notochord and hindgut endoderm that, in turn, causes an abnormal anterior curvature to the body axis and impedes posterior neuropore closure.[10,11] If the anterior curvature is corrected by splinting the caudal curly tail embryo with an eyelash or by retarding neuroepithelial proliferation by using retinoic acid; the posterior neuropore closes normally and the incidence of NTDs is reduced. More recent evidence points to a deficiency in Wnt5a, RARβ, and RARγ gene expression.[20] A third mouse mutant, T-curtailed produces a lumbosacral myelomeningocele with dorsoventral forking of the caudal neural tube. Rather than delayed or failed neural tube closure, however, the T-curtailed mutant causes rupture of the roof plate and re-opening of a previously closed neural tube.[11] Each of these mouse mutants exhibits an NTD that arises via a wholly different mechanism.

Although responsible for many different mouse animal models of NTDs, whether any of these mutations is responsible for human NTDs is largely unknown. The frustration of trying to isolate candidate genes that are causal in humans is demonstrated by the work of the Neural Tube Defect Collaborative Group whose investigators have undertaken a detailed genetic analysis of patients with myelomeningoceles and their families; they have screened blood samples obtained in a large number of cases for various genetic mutations. These analyses have been more helpful in determining what genes are not involved in human NTDs rather than in finding candidate gene(s). For example, neither the folate uptake genes FRα or FRβ, the BMP antagonist noggin, Pax-3, p53, nor the t-locus (brachyury) appear to be involved in the genesis of human NTDs.[3,39,45,55]

Particular interest has recently focused on genes that control folate metabolism and methyltransferase reactions involving methionine and homocysteine; this approach also provides an opportunity to examine the interactions of both genetic and environmental factors. The role of nutrition in the embryogenesis of NTDs was brought to light very early by a simple observation made by a Dutch midwife that the incidence of NTDs rose in 1722 and 1732 during which there were very poor crop yields in Holland. The incidence again rose abruptly following the so-called Hunger Winter of 1944 to 1945 in the Netherlands.[65] Various studies conducted by Hibbard and Smithels and colleagues in the 1960s and 1970s noting reduced levels of red blood cell folate in mothers of offspring with NTDs spurred research to examine the role of folate in the embryogenesis of NTDs. Maternal administration of folate antagonists such as aminopterin had long been known to produce NTDs.[52] Periconceptional administration of supplemental folate in randomized placebo-controlled studies was shown to reduce the recurrence rate of NTDs among both women with a previously affected pregnancy and the incidence among women who had never had an affected pregnancy.[12,42] Studies of maternal serum and red blood cell folate levels among mothers of infants with myelomeningocele, however, have produced inconsistent results,[53,66] and folate deficiency does not cause NTDs in mice or rat embryos.[21] These observations suggest that NTDs are rarely the result of an isolated absolute folate deficiency.

More recent attention has focused on the possibility that NTDs are caused by abnormalities involving metabolic pathways (in the mother or fetus) that require folate;[51] these would be abnormalities that predispose an individual to NTDs and might therefore be overcome by folate supplementation. A thorough discussion of this area is well beyond the reach of this paper, and the interested reader is referred to an excellent recent monograph by van der Put and colleagues.[65] Folate is a vitamin absorbed in the jejunum as 5-methyltetrahydrofolate monoglutamate. The transport of 5-methyltetrahydrofolate is moderated both by carrier- and receptor-mediated mechanisms. Receptors for folate are membrane bound and include four isoforms: FRα, FRβ, FRγ, and FR`γ. Unfortunately, neither FRα nor FRβ occurs with increased frequency in mothers or offspring having NTDs.

Folate and its metabolites (particularly tetrahydrofolate and 5-methyltetrahydrofolate) are vital for various mammallian metabolic reactions, including purine and pyrimidine (and therefore DNA) synthesis, and in the transfer of methyl groups during the metabolism of methionine and homocysteine (Fig. 12). In particular, the role of folate in methionine and homocysteine metabolism has generated considerable interest recently.[5,6,27,40,47,57,59,63,67] The metabolism of homocysteine follows one of two pathways. One involves a transsulphuration to cystathionine, catalyzed by the enzyme cystathionine synthase; cystothianine is subsequently converted to cysteine. The second metabolic pathway for homocysteine is the remethylation of homocysteine to methionine, catalyzed by the enzyme methionine synthase and requiring the donation of a methyl group from the folate metabolite 5-methyltetrahydrofolate (which, in turn, is converted to tetrahydrofolate), using vitamin B12 as a cofactor. Methionine is activated by adenosine 5'-triphosphate to produce S-adenosylmethionine, a high-energy compound that donates methyl groups for over 100 cellular methyltransferase reactions involving protein, lipid, DNA, and RNA metabolism. This critical reaction is controlled by methionine synthase that in turn is controlled by the soluble cytochrome b5 and methionine synthase reductase. Tetrahydrofolate is converted back to 5-methyltetrahydrofolate by the enzyme 5,10-methylenetetrahydrofolate reductase (Fig. 12).

The homocysteine/methionine cycle. The conversion of homocysteine to methionine is mediated by the donation of a methyl group from 5-methyltetrahydrofolate (5-Me tetrahydrofolate); in the process, tetrahydrofolate is formed. This reaction is catalyzed by the enzyme methionine synthase and uses cobalamin (vitamin B12) as a coenzyme. The 5-methyletrahydrofolate is regenerated from tetrahydrofolate by using 5,10-methylenetetrahydrofolate (5–10 Me Tetrahydrofolate) as an intermediary, completing a folate cycle. Methionine is subsequently used as a methyl (CH3) donor in various important metabolic reactions and is converted to homocysteine in the process. SAH = S-adenosylhomocysteine; SAM = S-adenosylmethionine.

There are therefore a large number of potential candidate genes that code either for enzymes involved in the methylation cycle or gene products that control it, and considerable research has focused on identifying which of these might be responsible for the embryogenesis of NTDs. One hypothesis is that maternal or fetal mutations in either methionine synthase or 5,10-methylenetetrahydrofolate reductase may slow down this methylation cycle, drive the conversion of methionine to homocysteine, and lead to methionine deficiency and/or homocysteine excess. Ingestion of higher doses of folate may overcome this relative deficiency by restoring more normal homocysteine and methionine levels.[51,57,58] Numerous observations support this view. For example, elevated maternal homocysteine levels have been identified in women carrying a fetus that harbors an NTD, both in serum[40] and in amnionic fluid.[57] Disordered methionine metabolism has been demonstrated in nonpregnant women who previously had given birth to a child with an NTD.[57,58] Methionine supplementation can reduce the risk of NTDs in rats and possibly in humans.[65] Finally, polymorphisms involving the 5,10-methylenetetrahydrofolate reductase gene have been demonstrated in one study in 18% of individuals with NTDs, and in 13% of parents of children with NTDs, compared with 6% in controls.[28,67] The risks of NTD are additive if both mother and fetus are homozygous for an methylenetetrahydrofolate polymorphism.[65] Other authors have failed to find evidence of such polymorphism.[39] Moreover, there is no specific pattern of genetic polymorphisms that appears to be consistently responsible for the embryogenesis of even a majority of NTDs. Given the complexities of the methylation cycle and the many enzymatic controls, it may be that NTDs reflect complex interactions between multiple enzyme systems that, with the proper combination of polymorphisms in either mother or fetus, result in failure of neural tube closure.

A number of teratogens are known to act through various cellular mechanisms to cause NTDs in animals and/or humans.[11] One important teratogen, valproic acid, produces NTDs in both animal models and in humans, probably by inhibiting neural fold fusion.[11] Although the exact mechanism is not known, valproic acid appears to disrupt folate metabolic pathways, perhaps by interfering with the conversion of tetrahydrofolate to 5-formyltetrahydrofolate.[57] Maternal folate administration reduces the incidence of valproic acid–associated NTDs in some, but not all, studies, and mouse strains that are susceptible to valproate acid-induced NTDs have significantly lower levels of 5,10-methylenetetrahydrofolate following valproic acid administration compared with resistant strains. In addition, a number of developmental regulatory genes (such as transcription factors and cell cycle checkpoint genes) may be altered in these susceptible strains. One hypothesis is that valproic acid may act by changing folate-dependent methylation of regulatory proteins such as transcription factors.

Finally, as many as 30 to 50% of NTDs appear to be caused by factors that are not responsive to folate administration and likely involve folate-independent mechanisms. Given the heterogeneity of factors that can potentially disrupt the developing neural tube, it is unlikely that a single agent will explain the embryogenesis of myelomeningocele.

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