neural tube defects

Advanced Review

Neural tube defectsdisordersof neurulation and relatedembryonic processesAndrew J. Copp and Nicholas D. E. Greene

Neural tube defects (NTDs) are severe congenital malformations affecting 1 in every1000 pregnancies. Open NTDs result from failure of primary neurulation as seenin anencephaly, myelomeningocele (open spina bifida), and craniorachischisis.Degeneration of the persistently open neural tube in utero leads to loss ofneurological function below the lesion level. Closed NTDs are skin-covereddisorders of spinal cord structure, ranging from asymptomatic spina bifida occultato severe spinal cord tethering, and usually traceable to disruption of secondaryneurulation. Herniation NTDs are those in which meninges, with or withoutbrain or spinal cord tissue, become exteriorized through a pathological openingin the skull or vertebral column (e.g., encephalocele and meningocele). NTDshave multifactorial etiology, with genes and environmental factors interacting todetermine individual risk of malformation. While over 200 mutant genes causeopen NTDs in mice, much less is known about the genetic causation of humanNTDs. Recent evidence has implicated genes of the planar cell polarity signalingpathway in a proportion of cases. The embryonic development of NTDs is complex,with diverse cellular and molecular mechanisms operating at different levels of thebody axis. Molecular regulatory events include the bone morphogenetic proteinand Sonic hedgehog pathways which have been implicated in control of neuralplate bending. Primary prevention of NTDs has been implemented clinicallyfollowing the demonstration that folic acid (FA), when taken as a periconceptionalsupplement, can prevent many cases. Not all NTDs respond to FA, however, andadjunct therapies are required for prevention of this FA-resistant category. 2012Wiley Periodicals, Inc.

How to cite this article:WIREs Dev Biol 2013, 2:213227. doi: 10.1002/wdev.71

INTRODUCTION

Congenital malformations (i.e., birth defects)are important causes of infant morbidity andmortality in developed nations, for example, affecting1 in every 40 pregnancies in Europe.1 As perinataland infant mortality has declined progressively, withimprovements in prenatal, intrapartum, and neonatalcare, so congenital defects have come to representan ever more significant proportion of the life-threatening conditions of infancy. While around 20%of individuals with birth defects die in utero, as

Correspondence to: [email protected] Development Unit, Institute of Child Health, UniversityCollege London, London, UK

stillbirths or as therapeutic abortions, the remaindersurvives beyond the first week of life.1 Such infantshave a 15-fold increased risk of death during the firstyear, with 910% dying during this period.2 Thosewho live beyond 1 year of age are often destined fora life time of ill health, with repeated medical andsurgical interventions.

Neural tube defects (NTDs) affect 0.52 per1000 established pregnancies, world wide3 and arethe second commonest group of birth defects, aftercongenital heart defects. There is a huge range of clin-ical severity among the NTDs. At the severe endof the spectrum, open lesions affecting the brain(anencephaly and craniorachischisis) are invariablylethal before or at birth, and encephalocele can be

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lethal depending on the extent of brain damage dur-ing herniation. While open spina bifida is generallycompatible with postnatal survival, it neverthelessresults in neurological impairment below the levelof the lesion which can lead to lack of sensation,inability to walk, and incontinence. Associated con-ditions include hydrocephalus, which often requirescerebrospinal fluid (CSF) shunting, vertebral deformi-ties, and genitourinary and gastrointestinal disorders.Closed spinal lesions are less severe and can beasymptomatic, as with spina bifida occulta, althoughlumbosacral spinal cord tethering can be associatedwith lower limb motor and sensory deficits and a neu-ropathic bladder. The severity of symptoms increaseswith age and surgical untethering of the cord mayprovide some relief from disability.

TYPES OF NTDsA variety of malformations are included under theoverall description of NTD. However, it is important

to examine carefully whether these are abnormalitiesof the embryonic neurulation process itself, oralternatively result from some later developmentaldisturbance that secondarily affects the neural tube.

Exencephaly and AnencephalyFailure of cranial neural tube closure results inNTDs in which the brain neural folds remainopen (Figure 1(a)) and exposed to the environment.With continued growth and differentiation, the neu-roepithelium characteristically appears to protrudefrom the developing brain, termed exencephaly(Figure 1(b)). The skull vault does not form overthe open region and subsequent degeneration ofthe exposed neural tissue gives rise to the typi-cal appearance of anencephaly, as observed in laterhuman pregnancy or at birth in rodents4 (Figure 1(c)).Human anencephaly can be subdivided into thosecases mainly affecting the rostral brain and skull(meroacrania) and those also affecting posterior brain

(a)

(d) (e) (f)

(b)(c)

FIGURE 1 | Natural history of open cranial (ac) and spinal (df) neural tube defects (NTDs) in mice. After an initial failure of neural tube closurein either the midbrain (a) or low spine (d), the neuroepithelium continues to proliferate and undergoes neuronal differentiation, appearing to protrudefrom the surface of the embryo (b,e). This is termed exencephaly in cranial lesions. With continued gestation, the exposed neuroepithelium becomesdamaged by continuous exposure to the amniotic fluid. Apoptosis and necrosis intervene so that, by the time of birth, the neuroepithelium hasdegenerated, yielding the phenotype of anencephaly (c) or myelocele (d). Developmental stages indicated as E (embryonic day) or P (postnatal day).((a) Reprinted with permission from Ref 6. Copyright 2006 Elsevier Ltd; (b) Reprinted with permission from Ref 7. Copyright 2003 Nature PublishingGroup; (c) Reprinted with permission from Ref 8. Copyright 2005 Wiley; (d, e) Reprinted with permission from Ref 9. Copyright 2001 Springer;(f) Reprinted with permission from Ref 10. Copyright 2003 American Association of Neurological Surgeons).

214 2012 Wiley Per iodica ls, Inc. Volume 2, March/Apr i l 2013

WIREs Developmental Biology Neural tube defectsdisorders of neurulation and related embryonic processes

and skull (holoacrania).5 Mouse exencephaly can sim-ilarly affect the forebrain, midbrain, or hindbrain, ora combination of adjacent brain regions, dependingon the precise event(s) of cranial neurulation that aredisturbed.

Myelomeningocele (Open Spina Bifida;Spina Bifida Aperta)If the progression of spinal neurulation along thebody axis is severely delayed or halted, then openspina bifida results. As with cranial NTDs, the mor-phological appearance varies considerably dependingon the developmental stage at which the embryo orfetus is examined (Figure 1(d)(f)). In humans, themajor forms are myelomeningocele (spina bifida cys-tica), in which the neural tissue is contained within ameninges-covered sac, and myelocele, in which neu-ral tissue is exposed directly to the amniotic fluid. Innormal embryos, the vertebral arches develop fromthe sclerotomal component of the axial mesoderm,which migrates dorsally to surround the closed neuraltube before undergoing cartilaginous and bony dif-ferentiation. When the neural folds remain open, thesclerotome is unable to cover the neuroepithelium anda bifid vertebral column is the secondary result.

CraniorachischisisIn addition to lesions that separately affect the cranialor spinal neural tube, around 10% of NTDs comprisea more extensive lesion, termed craniorachischisis,in which the entire neural tube remains open frommidbrain to low spine. Such individuals show acharacteristically short rostrocaudal body axis, aphenomenon that results from a disruption of theembryonic process of convergent extension. Thismorphogenetic event, which depends on signaling viathe planar cell polarity (PCP) pathway, is specificallydefective in craniorachischisis (see below).

Closed Spinal LesionsThis is the least severe, and least well defined, groupof NTDs often referred to as occult spina bifida ordysraphic disorders. Defects of skeletal development,particularly absent neural arches or a midline bonyspur, are associated with abnormalities of the spinalcord including overdistension of the central canal(hydromyelia), longitudinal duplication or splitting(diplomyelia and diastematomyelia), and tetheringof the cords lower end. Disorders are often asso-ciated with lipoma (as in lipomyelomeningocele),and anorectal abnormalities such as anal stenosis oratresia. Embryologically, there is little experimental

evidence (e.g., from animal models) to identify thedevelopmental origin of closed spinal lesions, althoughthe neurosurgical literature abounds with theoreticalspeculation. It seems most likely that abnormalities ofsecondary neurulation (see section below) are respon-sible for closed spina bifida, as: (1) most defects aresacrococcygeal (i.e., at the level of secondary neuru-lation); (2) they do not open to the external environ-ment; (3) the spinal cord is characteristically tetheredto adjacent tissues, as expected of faulty tissue separa-tion during secondary neurulation; and (4) cell typesof multiple germ layers are often present, represent-ing the multipotential nature of the tail bud.

Encephalocele and MeningoceleIn contrast to anencephaly, which results from failureof cranial neural tube closure, encephalocele appearsto be primarily a defect of cranial mesoderm develop-ment. A persistent opening in the skull, at occipital,parietal, or frontoethmoidal locations, allows themeninges to herniate creating an extracranial mass. Insevere cases, brain tissue can also herniate, in whichcase pathological analysis reveals relatively normalmorphology with no evidence of failed neural tubeclosure.11 In recent years, MeckelGruber syndrome,in which occipital encephalocele is a cardinal feature,has been found to be a ciliopathy; that is, several ofthe causative genes have key functions in determin-ing structure and function of primary cilia.12 Whilemany other inherited human disorders also result fromdisturbance of cilia genes, this has not generallybeen reported for open NTDs. Hence, exencephaly/anencephaly appears developmentally and geneticallydistinct from encephalocele which should, therefore,not be considered an NTD in the strict sense of a defectthat has arisen specifically from disturbance of neuru-lation. Spinal meningocele resembles encephalocele incomprising herniation of meningeal tissue througha skeletal opening (i.e., in the vertebral column).However, much less is known about the etiology orpathogenesis of meningocele, and animal models arerequired to advance understanding of this defect.

IniencephalyOften included within the NTD category of malfor-mations, iniencephaly is a severe defect of the cervicalspine, including bifid neural arches, with backwardflexion of the skull and an extremely short neck. Anoccipital encephalocele often coexists13 and there is astrong female preponderance, as in anencephaly. Toolittle is known of the pathogenesis of this disorderto determine whether it arises during neurulation, orlater during cervical skeletogenesis.



HydrocephalusWhile not classified as an NTD per se, hydrocephalusis very commonly associated with open spina bifida.The link is the presence of the Chiari type IImalformation, in which the brain stem descends intothe foramen magnum, blocking circulation of CSFand leading to hydrocephalus. The origin of ChiariII malformation and its association with open spinabifida is controversial. The persistently open neuraltube is physically tethered to adjacent skin10 andcannot ride upwards within the vertebral canal, asoccurs with the normal, mobile spinal cord. Thismay generate a downward pulling force on theupper, closed cord causing the brain stem to descend.However, other explanations have been suggested,including a primary defect of CSF production (as canoccur in the absence of NTDs), or the local effect ofa relatively small posterior skull fossa that may leaddirectly to the Chiari II malformation.

CAUSATION OF NTDs

Evidence for a Significant Genetic EtiologyBoth genetic and non-genetic factors are involved inthe etiology of NTDs, with up to 70% of the variancein NTD prevalence due to genetic factors.14 Evidencefor genetic causation includes the high recurrencerisk for siblings of index cases (25%), approxi-mately 50-fold more than in the general population,together with a gradually decreasing risk in moredistant relatives. Women with two or more affectedpregnancies have a very high risk (10%) of furtherrecurrence.15 NTD prevalence is greater in like-sextwins (which are assumed to include all monozygoticcases) compared with unlike-sex pairs, consistent witha significant genetic component. It is accepted, there-fore, that genetic factors contribute importantly toNTD risk, although the precise nature of this geneticcontribution remains unclear. NTDs occur at high fre-quency across the world, but with a sporadic patternthat rarely involves multigenerational families. Thisevidence is consistent with a multifactorial polygenicor oligogenic pattern of inheritance, rather than amodel based on single dominant or recessive geneswith partial penetrance.

Search for Genes Causing Human NTDsThe analysis of candidate genes in cohorts of patientswith NTDs16,17 has focused particularly on genesparticipating in folate one-carbon metabolism, inview of the preventive action of folic acid (FA),and those involved in glucose metabolism, owing tothe predisposition of diabetic pregnancy to NTDs.

The most robust finding to emerge from thisanalysis has been the C677T and A1298C poly-morphisms of methylenetetrahydrofolate reductase,which encodes a key enzyme of folate metabolismresponsible for homocysteine remethylation. Thesepolymorphisms are associated with approximately1.8-fold increased risk of NTDs, although thepredisposition is detectable only in non-Hispanicpopulations.18 To date, no positive associations withNTD risk have been found for genes regulating glucosemetabolism.

Assessment of Human Orthologuesof Mouse NTD GenesMore than 200 genetic models of NTD have beendescribed in mice, which include examples all themain open NTD phenotypes: anencephaly, open spinabifida, and craniorachischisis.19 These mouse modelshave provided invaluable information on the roleof molecular signaling pathways and cell biologicalprocesses in neurulation (see section Causation ofNTDs). In addition, the human orthologues of someof the mouse genes have been examined as candidatesfor human NTD causation, using either casecontrolassociation studies or direct sequencing in mutationscreens. Apart from recent studies that have identifiedputative human mutations in the PCP pathway (seesection Causation of NTDs), there have been fewother positive findings.17

Modifier Gene FunctionsMany studies have demonstrated inbred strain vari-ation in the penetrance and expressivity of NTDphenotypes in mice, providing evidence of signifi-cant modifier gene function during neurulation. Thislikely reflects the action of loci that are polymorphicbetween strains. For example, the Cecr2 mutation thatcauses exencephaly in mice is strongly affected in itsexpression by one or more modifier genes on chro-mosome 19. These vary between the BALB/c strain,where NTDs occur in 75% of homozygotes, and theFVB/N strain where 0% NTDs are observed.20 Straindifferences have also been described for non-geneticcauses of NTD including hypoglycemia, hyperther-mia, valproic acid (VPA), and cytochalasins.21 Whilefew modifier genes have been definitively identified,a morphogenetic variation in the pattern of cranialneural tube closure was demonstrated to under-lie the propensity of some strains (e.g., NZW) butnot others (e.g., DBA/2) to promote exencephaly.While most strains exhibit closure 2 at the midbrainforebrain boundary (Figure 2(a)), the DBA/2 strainundergoes closure 2 more caudally within the



Anencephaly Anencephaly

Hindbrainneuropore

(a) (b)

(c)

Anteriorneuropore

Anteriorneuropore

Posteriorneuropore

Lumbosacralspina bifida

Lumbosacralspina bifida

Human35 days

1

3

Cranio-rachischisis

Cranio-rachischisis

Posteriorneuropore

Closure 1

Closure 2

Closure 133

FIGURE 2 | Diagrammatic representation of the main events of neural tube closure in mouse (a) and human (b) embryos. The main types ofneural tube defect (NTD) resulting from failure of closure at different levels of the body axis are indicated. The red shading on the tail bud indicatesthe site of secondary neurulation in both species. Disturbance of this process leads to closed spina bifida. In each species, the initial de novo closureevent (closure 1) occurs at the hindbrain/cervical boundary and closure spreads bidirectionally from this site. In the mouse, a second de novo closuresite (closure 2) occurs at the forebrain/midbrain boundary with closure also spreading rostrally and caudally. Closure 2 does not appear to occur inhuman embryos (b). A third de novo initiation event (closure 3) occurs in both species at the rostral extremity of the neural plate, with closurespreading caudally from here. Hence, in mice, closure is completed sequentially at the anterior neuropore, hindbrain neuropore, and posteriorneuropore. In humans, owing to the lack of closure 2, there are likely to be only two neuropores: anterior and posterior. (Reprinted with permissionfrom Ref 8. Copyright 2005 Wiley) (c) Human embryo aged 35 days post-fertilization from the Human Developmental Biology Resource (www.hdbr.org). Neurulation has recently been completed in the low spinal region. The positions of closures 1 and 3, and the directions of closure are marked.The midbrain in this human embryo (red asterisk) is relatively small compared with the corresponding stage of mouse development. This may haverendered closure 2 an unnecessary intermediate step in achieving cranial neural tube closure in humans.

midbrain, whereas the NZW strain exhibits closure2 more rostrally within the forebrain. When thesplotch (Pax3) mutation was bred onto the DBA/2background, midbrain closure was enhanced and thefrequency of exencephaly among homozygous splotchembryos was reduced from 80 to around 40%.

Conversely, breeding splotch onto the NZW strainmaintained a forebrain closure point and the highexencephaly rate persisted.22 Hence, modifier genesmay regulate the expression of major NTD genesby altering the developmental substrate on which thelatter act.



Environmental FactorsNeural tube closure in mice and rats is adverselyaffected by a wide variety of teratogenic agents,21

whereas a much smaller number of non-genetic fac-tors have been definitively associated with humanNTDs (Table 1). VPA, a widely used anticonvulsantagent, increases the risk of NTDs by approximately10-fold, when taken during early in pregnancy.23

The potent histone deacetylase (HDAC) inhibitoryactivity of VPA may disturb the balance of proteinacetylation versus deacetylation, leading to failureof neural tube closure. This parallels the action ofthe HDAC inhibitor, trichostatin-A, which causesNTDs in mice.24 The fungal product fumonisin isa potent NTD-causing teratogen, with marked effectson spingolipid metabolism, that likely disturbs down-stream embryonic gene expression.25 Other teratogenswith a role in human NTDs are agents that dimin-ish folate uptake/metabolism (e.g., the anticonvulsantcarbamazepine and the antibiotic trimethoprim) oravailability (conditions of metabolic folate or vitaminB12 deficiency). In mice, diminished access to folatemetabolically exacerbates genetic risk of NTDs in aconverse way to prevention by exogenous FA.26 Othercritical factors for neural tube closure include inosi-tol (see below), diabetes mellitus which predisposesto a range of birth defects including NTDs, maternalobesity, which may act via glycemic dysregulation,and hyperthermia, with reports of NTDs followinghigh maternal fever or extreme sauna usage in earlypregnancy.

GeneGene and GeneEnvironmentalInteractionsGenegene and geneenvironment interactions arewell documented in mouse models of NTDs. Whilemost predisposing mouse mutations are recessive,with NTDs occurring only in homozygotes,19 NTDsalso occur in compound heterozygotes for two pre-disposing genes, or in single heterozygotes exposedto an adverse environmental influence. For example,NTDs in splotch mice result from homozygosity forPax3 mutations but can also occur in Pax3 het-erozygotes as a result of interaction with mutationsin Nf1 or Grhl3.17 Environmental factors includ-ing folate deficiency and arsenic can also exacer-bate NTDs in Pax3 homozygotes, or induce NTDsin the usually unaffected splotch heterozygotes.28 Itseems likely that the majority of sporadic humanNTDs will also ultimately prove to result from twoor more heterozygous genetic risk factors coexist-ing in individuals who are also exposed to adverseenvironmental influences, like suboptimal folateintake.

DEVELOPMENT OF NTDs

Primary Neurulation Events in MiceIn mammals and birds, unlike amphibia, primaryneural tube closure is initiated at several discrete pointsalong the rostrocaudal axis; closure is therefore adiscontinuous process21,29 (Figure 2(a)). In the mouse,

TABLE 1 Environmental Factors Linked to the Causation of Neural Tube Defects in Human Pregnancy

Category Teratogenic Agent Proposed Teratogenic Mechanism

Folate antagonists Carbamazepine Inhibition of cellular folate uptake

Trimethoprim Disturbance of folate-related metabolism

Gene expressiondysregulation

Fumonisin Disturbance of sphingolipid biosynthesis andmetabolism with effects on signaling pathways inneurulation

Valproic acid Histone deacetylase inhibition leading to disruption ofkey signaling pathways in neurulation

Glycemic dysregulation Hyperglycemia (in poorly controlled maternaldiabetes mellitus)

Increased cell death in neuroepithelium

Maternal obesity Unknown

Micronutrient deficiencies Folate Disturbance of folate-related metabolism

Inositol Disturbance of phosphorylation events downstream ofprotein kinase C

Vitamin B12 Disturbance of folate-related metabolism

Zinc Unknown

Thermal dysregulation Hyperthermia (e.g., maternal fever in weeks 34of pregnancy)

Unknown

For references to original literature, see Ref 27.



closure is initiated at the hindbrain/cervical boundary(termed closure 1) on embryonic day (E) 8.5. Closurethen progresses in a rostral direction to form theneural tube in the future brain, and simultaneouslyin a caudal direction along the future spinal cord.7

Closure initiates separately around 12 hours later atthe forebrain/midbrain boundary (closure 2) and alsoat the rostral end of the future forebrain (closure3).8 The open regions of neural folds between thesites of initial closure are termed neuropores, andthese close progressively as the neural tube zips upbidirectionally from the sites of closures 1 and 2,and in a caudal direction from the site of closure3. The anterior neuropore completes closure a fewhours after closures 2 and 3, and the hindbrainneuropore finishes closing around the 16 somitestage. From this stage onwards the cranial neuraltube is entirely closed, whereas spinal neurulationcontinues by zipping caudally along the spine untilthe posterior neuropore closes at the 30 somite stage,on embryonic day 10. This marks the end of primaryneurulation.

Variations in Cranial Neural Tube Closurein Humans and MiceIn human embryos, neural tube closure begins at1718 days post-fertilization and is discontinuousas in the mouse (Figure 2(b)). The site of closureinitiation resembles closure 1 in mice, and the onsetof closure from the extreme rostral end of theneural plate (adjacent to the chiasmatic plate) appearscomparable with mouse closure 3.30 However, theexistence of a closure 2-like event in human embryosis controversial: some authors describe a similar eventas in the mouse31 while others report the lack ofan initiation event between closures 1 and 3.30,32

If human embryos indeed lack a closure 2 event,then brain formation must be achieved by neurulationprogressing directly between closures 1 and 3, withcompletion of a single cranial (rostral) neuropore.Human populations could be polymorphic for closure2, an idea supported by the finding in mice of variablepositioning of closure 2 between inbred strains22

(see above). In the SELH/Bc strain, closure 2 isentirely absent33 leading to exencephaly in 17% ofembryos. Strikingly, however, the remaining 83%of SELH/Bc embryos manage to complete cranialneurulation between closures 1 and 3, demonstratingthat closure 2 is not needed for brain formation evenin the mouse. The human embryo, with its rathersmaller midbrain than in the mouse (Figure 2(c)), mayhave dispensed with closure 2 as an evolutionarilyunnecessary process.

BOX 1

SEX AND PREDISPOSITION TOEXENCEPHALY/ANENCEPHALY

Females are disproportionately representedamong fetuses with exencephaly/anencephalyboth in humans and mice, with a male : femalesex ratio often approaching 1:3.34,35 In contrast,open spina bifida exhibits a nearly equal sexratio, or even a male preponderance. Whileanencephaly could be a more severe condi-tion in males than females, leading to earlypregnancy loss of affected males, the analysisof mouse strains has provided strong evidenceagainst such a differential survival hypothe-sis. Moreover, male and female mouse embryosprogress through the stages of neurulation atidentical rates,36 even though females are at aslightly earlier mean developmental stage thanmale litter mates. This suggests that femalesbecome developmentally retarded earlier inpregnancy, an effect that appears to result fromthe possession of two X chromosomes: pres-ence or absence of a Y chromosome had noeffect on frequency of exencephaly in the Trp53(formerly known as p53) knockout mouse.37

The developmental mechanism of this interest-ing sex difference remains to be determined.

Secondary NeurulationFollowing completion of primary neurulation, theneural tube in the lower sacral and coccygeal regionsis formed by the process of secondary neurulation,a well-recognized feature of both mouse and humandevelopment.38,39 At the lowest spinal levels, the tailbud represents the remnant of the primitive streak andis the sole source of all non-epidermal tissues includingthe neural tube and the vertebrae. The tail budcontains a self-renewing stem cell population whosederivatives proliferate rapidly leading to longitudinalgrowth of the body axis. As cells are left in thewake of the retreating tail bud, they condense intocell masses that subsequently differentiate to formthe main structures of the post-lumbar region: theneural tube, notochord, somites, and hindgut. Theneural tube is formed when this cellular condensationundergoes canalization, converting the solid neuralprecursor into a hollow secondary neural tube. Celllineage analysis shows that tissues of the low bodyaxis arise from the same stem cell population,40 incontrast to the corresponding tissues at higher levelsof the body axis which arise from different germ



layers of the gastrulation stage embryo. It is probablyfor this reason that malformations and tumors (e.g.,teratomas) of the sacral and coccygeal regions areoften found to embrace several tissue types.

Assigning NTDs to Particular Eventsof NeurulationAnalysis of mouse mutants such as loop-tail(Vangl2) has demonstrated that failure of closure1 is the fundamental neurulation defect leading tocraniorachischisis, the most severe NTD41 (Figure 2).If closure 1 is completed successfully, then later arisingNTDs present as separate open lesions of the cranialand/or spinal regions: i.e., anencephaly and open spinabifida. In mice, incomplete closure of the cranialneural tube may result from failure of one of theinitiation events (closure 2 or 3) or from a defect inthe subsequent zippering and closure of the anterioror hindbrain neuropores.8 The commonest defect isfailure of zippering through the midbrain, betweenclosures 1 and 2, owing to the mechanical difficultyof achieving closure on the outer (convex) side ofthe cranial flexure which is located at midbrain level.Nevertheless, forebrain and hindbrain defects are alsoobserved in different mouse strains, as is failure ofclosure 3 which produces the specific phenotype ofsplit face with forebrain anencephaly. In the spinalregion, the wave of zippering down the body axis canbe arrested at any stage, yielding an open spina bifidaof varying length depending on the time of closurecessation. For example, spinal neurulation in theKumba (Zic2) mutant fails at the 1416 somite stage,when dorsolateral hinge point (DLHP) formation firstbecomes required,42 yielding a large spina bifida fromthoracic level downwards. In contrast, spinal closurein the curly tail (Grhl3) mutant fails after the 25somite stage, owing to enhanced axial curvature latein neurulation,9 producing a small sacral, or at mostlumbosacral, spina bifida.

Key Signaling Pathways in Neurulationand NTDsPCP SignalingAt the onset of neurulation, lengthening andnarrowing of the initially disc-shaped neural plate,termed convergent extension, is required to ensurethe neural folds are spaced sufficiently closely forclosure initiation.43 First described in amphibia,convergent extension comprises the lateral to medialdisplacement of cells in the presumptive mesodermand neural plate. Cell intercalation in the midlineleads to mediallateral narrowing (convergence) and

rostrocaudal lengthening (extension) of the bodyaxis.44 At the molecular level, convergent extensiondepends on a non-canonical WNT signaling cascade,the PCP pathway which signals via frizzled membranereceptors and cytoplasmic disheveled (DVL), but doesnot involve downstream stabilization of -catenin.45

Specific inhibition of PCP signaling in Xenopus laevis,by functional disruption of the key signaling moleculeDVL, resulted in inhibition of convergent extensionand gave rise to short, broad embryos whose neuralfolds failed to close.46 At the cellular level, PCPsignaling is thought to control polarized cellularmotility, in particular by regulating formation ofstable mediolaterally oriented actin-rich lamellipodia,which provide cellcell and cellmatrix traction.44

In mice, loss of function of the core PCPpathway genes Vangl2 (the homolog of Drosophilastrabismus/Van gogh) in loop-tail mutant, Celsr1(the homolog of Drosophila flamingo/starry night)in Crash mice, or double mutants for Fzd3 andFzd6, or two of the three disheveled genes (Dvl1and Dvl2) all suppress convergent extension cellmovements. Loss of function of other PCP-relatedgenes also cause craniorachischisis including Scrib(in the Circletail mouse) and the tyrosine kinasePtk7.27 A broad neural plate results in which closure1 fails, leading to craniorachischisis.47 Hence, thereis a specific relationship between PCP signaling,convergent extension, and initiation of neural tubeclosure.

SHH SignalingSonic hedgehog (SHH), one of three mammalianhomologs of Drosophila hedgehog, initiates an intra-cellular signaling pathway by binding to its transmem-brane receptor, Patched1 (PTCH1). In the absenceof SHH ligand, PTCH1 interacts with an associatedmembrane protein, Smoothened (SMO), to inhibitits activity. SHH binding to PTCH1 removes theinhibitory effect on SMO, allowing members of theGLI family of proteins to be processed as tran-scriptional activators. In addition to this core signaltransduction machinery, a variety of other proteinshave been identified that exert either positive or nega-tive influences on SHH signaling. Strikingly, however,it is primarily genetic changes in proteins with a nega-tive influence on the SHH pathway that lead to NTDsin mice.48 For example, mutations in PTCH1 thatrelieve the inhibition of SMO activity, and abolitionof inhibitory phosphorylation sites for protein kinaseA in GLI2, both lead to NTDs. Loss of function ofother SHH inhibitory genes, including Fkbp8, Gli3,Rab23, and Tulp3, also produce NTDs. In contrast,loss of function of proteins that activate signaling,



including SMO and SHH itself, does not produceNTDs,49,50 whereas overexpression of such proteinscan compromise neural tube closure. These findingsargue for an overall negative influence of SHH signal-ing on neural tube closure, an effect that appears to bemediated via the inhibition of dorsolateral neural platebending,51 which is essential for closure in both themidbrain and lower spinal region (see next section).

BMP SignalingBone morphogenetic proteins (BMPs) are members ofthe transforming growth factor (TGF)- superfamilyof proteins that signal via specific BMP receptors toregulate transcription via Smad proteins in a canonicalpathway and via several tyrosine kinases in non-canonical signaling. BMPs specify dorsal identity inthe early embryo, acting reciprocally to Shh whichis the main ventralizing factor of the early nervoussystem. Strikingly, however, both BMP and Shhsignaling have been found to inhibit DLHP formationduring mouse neurulation. Loss of activity in eitherpathway yields precocious DLHP formation, whereasgain of function inhibits bending.42,51 Factors thatinduce DLHP formation include the BMP antagonistsnoggin and neuralin. These proteins are secreted bythe dorsal neural plate and enable DHLP formationvia antagonism of the BMP inhibitory influence. Theirproduction is under negative regulatory control bySHH so that, at upper spinal levels where strongSHH expression occurs in the notochord, noggin, andneuralin production is inhibited and DLHP formationis blocked. Upper spinal neurulation therefore exhibitsonly midline bending, with a V-shaped neural groove.Lower in the spinal region, however, SHH expressionin the notochord declines and noggin/neuralinproduction is able to occur. This enables DLHPformation to break through, facilitating closureunder circumstances of convex body axis curvature,which characterizes the embryo after axial rotation iscomplete. Failure to develop DLHPs, as in the Kumba(Zic2) mutant, leads to failure of closure in the lowerspinal region, and severe spina bifida results.42

Grainyhead-Like GenesCurly tail (ct) is one of the best understood mousemodels of NTDs.9 Neural tube closure is delayed inthe low spinal region owing to a growth imbalancebetween the slowly proliferating hindgut and thenormally proliferating neural plate. This growthimbalance enhances curvature of the caudal region,which mechanically opposes neural fold elevationand fusion.52 The molecular basis of NTDs in curlytail was elucidated after mice null for Grhl3, agene within the ct critical region on chromosome 4,

were found to display spina bifida closely resemblingcurly tail.53 A Grhl3-containing bacterial artificialchromosome completely rescued spinal NTDs intransgenic curly tail embryos, demonstrating thatGrhl3 is the main defective gene in the ct mutant.54

Indeed, at the stage of low spinal closure, Grhl3transcripts localize specifically to the hindgut, thesite of the cell proliferation defect. Another familymember, Grhl2, also causes spina bifida in mice. Axialdefects (Axd) mutants, which exhibit spina bifidaand tail flexion defects, overexpress Grhl2, and theirphenotype can be ameliorated by introducing a nullGrhl2 allele.55 Moreover, mice homozygous for lossof Grhl2 function also exhibit NTDs, demonstratingthat both loss and gain of function of GRHL genes isa potent cause of neurulation disturbance.55

Retinoid SignalingRetinoic acid (RA) has long been studied as a potentteratogen in rodent systems, with NTDs among themalformations most often observed. It is now real-ized that endogenous synthesis of retinoids formsan integral part of early development, and that anydisturbance in the balance between production andturnover of retinoids can adversely affect develop-mental events including neural tube closure. Hence,NTDs are observed in mice with loss of function of thegenes encoding Aldh1a2 (formerly known as Raldh2),a principal enzyme of RA synthesis, Cyp26a1, a keyRA metabolizing enzyme and RA receptor genes Raraand Rarg through which RA signaling is mediated.56

While the existence of signaling pathways downstreamof the RA receptors is well known, it remains unclearprecisely how RA signaling participates in neural tubeclosure, at the cellular and tissue levels.

Key Cellular Functions in Neurulationand NTDsCytoskeletonA long standing question relates to the role of thecytoskeleton in neurulation. Actin microfilaments arelocalized circumferentially in the apices of all neu-roepithelial cells.57 Actomyosin contraction couldtherefore reduce the apical surface area of the neu-roepithelium and contribute to bending and closureof the neural folds. Consistent with this, experimentaldisruption of the cytoskeleton by actin-disassemblingdrugs such as cytochalasins causes exencephaly incultured rodent embryos indicating a role in cranialneural tube closure.58 Higher doses of cytochalasinD also inhibit closure 1, but spinal neurulation isresistant to cytochalasin D, with the stereotypicalmidline and dorsolateral bending points maintained



in the absence of apical microfilaments.59 These obser-vations using cytoskeletal inhibitors are mirrored instudies of mice with null mutations in genes encodingcytoskeletal proteins.27 Whereas, cranial NTDs areseen in several mice with compromised cytoskeletalfunction (e.g., Cfl1, Palld, Vcl mutants), spinal neuru-lation is completed successfully in such embryos. Onlymice null for the cytoskeleton-associated proteinsShroom3 and MARCKS-like 1 (formerly known asMARCKS-related protein) exhibit both exencephalyand spina bifida in a proportion of homozygotes.Hence, while regulation of the actomyosin cytoskele-ton appears essential for cranial neural tube closure,its role in spinal neurulation remains unclear.

ApoptosisExcessive cell death has been implicated in theNTDs resulting from many genetic and environmen-tal insults. It is crucial, however, to demonstratethat increased apoptosis precedes failed neural tubeclosure, as later-appearing abnormalities could be asecondary consequence of the failed morphogenesis.Genetic defects in which excessive cell death occursprior to or during neural tube closure include the anti-apoptoticBcl10 gene, and inhibitors of kappa B kinase(formerly known as IKK factors) that activate NFB inan antiapoptotic pathway, and the transcription fac-tors TFAP2A (formerly known as AP2) and BRCA1,which are required for cell survival. Conversely, NTDsalso occur in mice lacking gene functions includingCasp3 and Casp9 which are required for apoptosis.27

In these cases, diminished or abolished cell death isobserved and NTDs are localized to the midbrainand hindbrain, whereas neurulation in forebrain andspinal regions is completed normally. Similarly, in theApaf1 mutant, apoptosis is diminished and yet NTDsonly affect the cranial region. Chemical blockade ofapoptosis in mouse embryo culture, by inhibition ofeffector caspases with Z-vad-fmk or by inhibition ofTRP53 with pifithrin-, produced no defects of neuraltube closure,60 suggesting that Apaf-1 or Casp 3/9mutants are unlikely to develop exencephaly solelydue to diminution of apoptosis. It remains to be deter-mined what is the cause of the plentiful cell deathobserved in the dorsal midline of the closing neuraltube, although anoikis owing to transient remodelingof epithelial layers appears a possibility.

Cell ProliferationThe neurulation stage embryo is a rapidly prolifer-ating system, with neuroepithelial cell-cycle times asshort as 46 hours. On the other hand, neuroepithelialcells begin to exit the cell cycle and embark upon neu-ronal differentiation soon after neural tube closure,

suggesting that the balance between continued prolif-eration and onset of neuronal differentiation may becritical for closure. Indeed, several genes whose muta-tions lead to NTDs are essential for cell proliferationand/or prevention of premature neurogenesis.27 Theseinclude Jarid2 (formerly known as jumonji), whichhas homology to cell-cycle associated retinoblastoma(RB1)-binding proteins, NEUROFIBROMATOSIS 1and NUCLEOPORIN 50, which are negative reg-ulators of the cell-cycle inhibitors CDKN1A andCDKN1B (formerly known as p21 and p27), respec-tively, and PAX3 which promotes rapid dorsal neu-roepithelial cell proliferation. NTDs also result fromdefects in genes encoding negative regulators of theNotch signaling pathway including HES1, HES3, andRBPJ which are required to postpone neuronal differ-entiation until neural tube closure is complete. Con-versely, a number of genetically induced NTDs havebeen associated with excessive cell proliferation, thebest characterized example being loss of function ofPhactr4, which is required to maintain a high-dorsal,low-ventral gradient of cell proliferation in the neu-roepithelium. Phactr4 regulates a cascade of proteinphosphorylation, involving the phosphatase PPP1CCso that, in its absence, RB1 becomes hyperphosphory-lated and can no longer regulate E2F protens and theirtargets to limit cell-cycle progression. Excessive ven-tral cell proliferation ensues which causes exencephalyin Phactr4 mutants.61

CLINICAL DIAGNOSISAND MANAGEMENT OF NTDs

Historical Trends in ManagementPrior to the 1970s, management of NTDs consistedsolely of palliative surgical and medical support. Whilechildren with open spina bifida generally surviveif their lesion is closed surgically, thereby avoid-ing ascending infection, neurological outcome variesmarkedly with the vertebral level of lesion (i.e., higherdefects have greater neurological handicap). This ledto suggestions that surgery should be offered onlyin cases with a better prognosis.62 An ethical debateensued, around whether surgical treatment shouldbe withheld, but this was superseded in the 1970swhen methods for prenatal diagnosis of open NTDswere developed. Initially, diagnosis was based on mea-surement of alphafetoprotein (AFP) concentration inthe amniotic fluid and maternal blood, but latertechnological improvements enabled ultrasound toreplace AFP measurement as the mainstay of pre-natal diagnosis.63 Today, most fetuses with NTDsare diagnosed prenatally in developed countries, and



many are aborted therapeutically. In contrast, largenumbers of babies with NTDs continue to be bornin developing countries where prenatal diagnosis isnot routine, as well as in countries where therapeuticabortion is not available.

In Utero SurgeryIn both humans and mice, the open neural tube under-goes relatively normal neuronal differentiation, withdevelopment of spinal motor and sensory functioneven below the lesion level. As gestation progresses,however, neurons die within the exposed spinal cord,axonal connections are interrupted, and function islost.10 Hence, neurological disability in open spinabifida is a two-hit process: failed neural tube clo-sure followed by neurodegeneration in utero. This hasencouraged attempts to cover the persistently openneural tube during fetal development, in order to arrestor prevent the neurodegeneration. Surgical repair inutero for early open spina bifida is practised in severalcenters in the USA and the success of this procedurehas recently been evaluated in a randomized controlledtrial.64 This showed significant benefits for the childpostnatally, including a 50% reduction in shuntingfor hydrocephalus and a significant improvement inspinal neurological function. Against this was a sig-nificantly higher rate of premature birth and maternalcomplications such as uterine dehiscence at the oper-ation site. Clearly, surgery in utero for open spinabifida has both benefits and risks.

BOX 2

ECONOMIC EFFECT OF NTDs ANDCOSTBENEFIT ANALYSIS OF PRIMARYPREVENTION

The life-time medical and non-medical costs ofa person with spina bifida are estimated at$560,000,65 without taking into account the lostearnings of the individual. On the other hand,public health measures to prevent NTDs, whichinclude education programs to promote volun-tary FA supplementation and mandatory foodfortification, are themselves costly undertakings.A systematic review of costbenefit analyses hasconcluded that both voluntary FA supplemen-tation and food fortification with FA are costeffective in a range of countries.66 Benefitcostratios for food fortification were 4.3:1 in USA,11.8:1 in Chile and 30:1 in South Africa, empha-sizing the desirability of extending such primarypreventive measures for NTDs.

PRIMARY PREVENTION OF NTDs

FA and History of Prevention StrategiesAmong the most significant advances in preventionof birth defects has been the finding, through clini-cal trials, that use of maternal FA supplements cansubstantially reduce the risk of a pregnancy affectedby NTD.67,68 These results prompted the recommen-dation that all women planning pregnancy shouldconsume 0.4 mg FA per day, and that women athigh risk of NTD should receive 45 mg per day. Inorder to increase the population intake of FA, manda-tory food fortification was introduced in the USA in1999, and later in other countries. Subsequent stud-ies suggest this has contributed to a decline in NTDincidence.69 Nevertheless, it is apparent that a subsetof NTDs are not prevented by current therapeuticstrategies such that approximately 0.70.8 per 1000pregnancies persist despite FA usage.70

Folate One-Carbon Metabolism and NTDsIt is often assumed that NTDs are a vitamin-deficiencycondition, but in fact the great majority of humanNTD-affected pregnancies are not clinically folatedeficient.71 Moreover, severe folate deficiency inmouse models does not cause NTDs in the absence ofa genetic predisposition.26 It seems more likely thatexogenous FA is able to stimulate a cellular response,enabling the developing embryo to overcome theadverse effects of genetic and/or environmental distur-bances that would otherwise lead to NTDs. These dis-turbances could involve abnormalities in folate-relatedpathways, but might also affect systems unrelated tofolate metabolism. Folates are integral to intracellu-lar one-carbon metabolism which produces pyrim-idines and purines for DNA synthesis and s-adenosylmethionine, the universal methyl group donor for allmacromolecules. Hence, cell proliferation and/or cellsurvival, which depend on DNA synthesis, are likelyeffects of folate supplementation although, to date,no well-characterized example has been reported ofFA preventing NTDs in a model system via stimula-tion of cell proliferation, or suppression of apoptosis.Similarly, stimulation of DNA, protein, and/or lipidmethylation is a possible outcome of folate supple-mentation, although no specific examples of increasedmethylation linked to NTD prevention exist as yet.Interestingly, during mouse pregnancy, severe folatedeficiency was not found to decrease embryonic globalDNA methylation26 and conversely NTDs do not arisein models such as Mrhfr knockouts in which overallDNA methylation is significantly diminished.

Clearly, a priority for future research is toimprove our knowledge of the cellular mechanism(s)



underlying the preventive action of folate supplemen-tation. This could indicate why some NTDs are folateresponsive and others folate resistant, in turn focusingon the different etiologies in each case. This mightlead to better prediction of the likely importance offolate supplementation in pregnancy, or the need foradditional treatments such as inositol.

Folate-Resistant NTDs and InositolEstimates vary as to the effect of FA supplementa-tion on the prevalence of NTDs. Even after highdose (4 mg) therapy in the medical research council(MRC) trial, 1% of pregnancies still had recurrentNTDs,68 a 10-fold excess over the general populationfrequency. It appears, therefore, that not all cases ofNTDs are preventable by FA, a conclusion supportedby reports of NTD pregnancies recurring in familiesdespite high dose folate intake.72 Novel therapies areneeded, therefore, to improve NTD prevention partic-ularly for folate-resistant cases which currently cannotbe prevented.

Among mouse NTD models, some show pre-vention by FA, whereas others appear resistant. Forexample, NTDs in the splotch (Pax3) and Cited2mutant mice are reduced in frequency and severity byFA, whereas NTDs in the curly tail mutant mouse areresistant.73 Inositol is the only vitamin-like moleculeto be required for the normal rodent neural tube toclose,74 and both myo-inositol and d-chiro-inositolcan prevent NTDs in the FA-resistant curly tail NTDmodel.73 Direct treatment of curly tail embryos in cul-ture normalizes low spinal neural tube closure, demon-strating that inositols action is independent of thematernal environment. Moreover, inositol is also ableto prevent diabetes-induced NTDs in mice and rats,73

arguing for a general effect of inositol in enhancing

neural tube closure. Indeed, targeted mutations in theItpk1, Pip5kIc, and Inpp5e genes of mice produceNTDs via direct disturbance of inositol metabolism.27

The observations in mouse models together with thefinding that some human NTD pregnancies havelower maternal inositol concentrations than unaf-fected pregnancies75 has prompted an ongoing clinicaltrial to evaluate inositol as a preventive agent forNTDs, alongside FA (www.pontistudy.ich.ucl.ac.uk).

CONCLUSION

NTDs represent arguably the best understood cate-gory of human birth defects. Many decades of researchby developmental biologists, epidemiologists, andclinicians has led to significant advances in our knowl-edge of the embryonic process of neurulation and itsdisorders, the patterns of variation of NTDs in humanpopulations, and the role of FA in primary prevention.Clinical methods have been developed and refined forthe prenatal diagnosis and in utero surgical repair ofNTDs. Much less well understood is the area of NTDgenetics and, while there appears no doubt that geneticfactors play a key (perhaps predominant) role in etiol-ogy, we are yet to define the principal genes that deter-mine risk of human NTDs. Advances in NTD geneticsshould herald a new era in which the complex subtypesof these disorders will become discernible enabling, forexample, much more precise targeting of preventivetherapies. Hence, some genetic subtypes may requireonly low dose (e.g., 400 g) FA for prevention, othersmay require higher doses (e.g., 4 mg), and others maybe resistant to FA, necessitating the use of alterna-tive preventive agents. In few other areas of humanbiology is there a greater need for a multidisciplinaryapproach to such a complex developmental disorder.

ACKNOWLEDGMENTS

The authors research is funded by grants from the Wellcome Trust, Medical Research Council and Sparks.

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FURTHER READINGWyszynski DF, ed. Neural Tube Defects: From Origin to Treatment. Oxford: Oxford University Press; 2006, 1528.


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