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Review Article Spina Bifida: Pathogenesis, Mechanisms, and Genes in Mice and Humans Siti W. Mohd-Zin, 1 Ahmed I. Marwan, 2 Mohamad K. Abou Chaar, 3 Azlina Ahmad-Annuar, 4 and Noraishah M. Abdul-Aziz 1 Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Laboratory for Fetal and Regenerative Biology, Colorado Fetal Care Center, Division of Pediatric Surgery, Children’s Hospital Colorado, University of Colorado, Anschutz Medical Campus, E th Ave, Aurora, CO , USA Training and Technical Division, Islamic Hospital, Abdali, Amman , Jordan Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Correspondence should be addressed to Noraishah M. Abdul-Aziz; [email protected] Received June ; Revised November ; Accepted December ; Published February Academic Editor: Heinz Hoer Copyright © Siti W. Mohd-Zin et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Spina bida is among the phenotypes of the larger condition known as neural tube defects (NTDs). It is the most common central nervous system malformation compatible with life and the second leading cause of birth defects aer congenital heart defects. In this review paper, we dene spina bida and discuss the phenotypes seen in humans as described by both surgeons and embryologists in order to compare and ultimately contrast it to the leading animal model, the mouse. Our understanding of spina bida is currently limited to the observations we make in mouse models, which reect complete or targeted knockouts of genes, which perturb the whole gene(s) without taking into account the issue of haploinsuciency, which is most prominent in the human spina bida condition. We thus conclude that the need to study spina bida in all its forms, both aperta and occulta, is more indicative of the spina bida in surviving humans and that the measure of deterioration arising from caudal neural tube defects, more commonly known as spina bida, must be determined by the level of the lesion both in mouse and in man. 1. Introduction Spina bida is the most common and complex central nervous system malformation in humans. Management of these patients involves various disciplines to ensure the best possible outcome achieved and provide a good quality of life for its patients [, ]. e study of this condition is extremely relevant in that even in the years since the discovery of the benets of folic acid this condition is highly prevalent around the world and its occurrence does not seem to decrease []. Interestingly, the debate is very much ongoing upon the evidence that the United States of America has seen a decline in cases of spina bida (https://www.cdc.gov/ncbddd/spinabifidadata.html). is review paper intends to compare and contrast spina bida in humans and spina bida in the mouse, which is the leading animal model of this devastating condition in light of the information studies on animal models have shed on the human counterpart []. 2. Spina Bifida in Humans Development of the central nervous system including the brain and spinal cord is a complex process beginning with a at sheet of cells which undergoes sequential thickening, elevation, mediolateral convergence accompanied by rostro- caudal extension, and nally adhesion to form the neural tube (NT) which is the precursor of the brain and the spinal cord. Perturbations of these interconnected processes result in neural tube defects (NTDs), which are the most common congenital malformation aecting this system and are associated with signicant complications. NTDs can occur in two major forms: spina bida (SB) aperta, which Hindawi Publishing Corporation Scientifica Volume 2017, Article ID 5364827, 29 pages https://doi.org/10.1155/2017/5364827

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Review ArticleSpina Bifida: Pathogenesis, Mechanisms, and Genes inMice and Humans

Siti W. Mohd-Zin,1 Ahmed I. Marwan,2 Mohamad K. Abou Chaar,3

Azlina Ahmad-Annuar,4 and Noraishah M. Abdul-Aziz1

!Department of Parasitology, Faculty of Medicine, University of Malaya, "#$#% Kuala Lumpur, Malaysia&Laboratory for Fetal andRegenerative Biology, Colorado Fetal CareCenter, Division of Pediatric Surgery, Children’sHospital Colorado,University of Colorado, Anschutz Medical Campus, !&'## E !'th Ave, Aurora, CO (##)", USA%Training and Technical Division, Islamic Hospital, Abdali, Amman &)!), Jordan)Department of Biomedical Science, Faculty of Medicine, University of Malaya, "#$#% Kuala Lumpur, Malaysia

Correspondence should be addressed to Noraishah M. Abdul-Aziz; [email protected]

Received !" June !"#$; Revised #% November !"#$; Accepted # December !"#$; Published #! February !"#&

Academic Editor: Heinz Ho'er

Copyright © !"#& Siti W. Mohd-Zin et al. (is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Spina bi)da is among the phenotypes of the larger condition known as neural tube defects (NTDs). It is the most common centralnervous systemmalformation compatiblewith life and the second leading cause of birth defects a*er congenital heart defects. In thisreview paper, we de)ne spina bi)da and discuss the phenotypes seen in humans as described by both surgeons and embryologists inorder to compare and ultimately contrast it to the leading animal model, the mouse. Our understanding of spina bi)da is currentlylimited to the observations we make in mouse models, which re'ect complete or targeted knockouts of genes, which perturb thewhole gene(s) without taking into account the issue of haploinsu+ciency, which is most prominent in the human spina bi)dacondition. We thus conclude that the need to study spina bi)da in all its forms, both aperta and occulta, is more indicative of thespina bi)da in surviving humans and that the measure of deterioration arising from caudal neural tube defects, more commonlyknown as spina bi)da, must be determined by the level of the lesion both in mouse and in man.

1. Introduction

Spina bi)da is the most common and complex centralnervous system malformation in humans. Management ofthese patients involves various disciplines to ensure the bestpossible outcome achieved and provide a good quality oflife for its patients [#, !]. (e study of this condition isextremely relevant in that even in the !" years since thediscovery of the bene)ts of folic acid this condition ishighly prevalent around the world and its occurrence doesnot seem to decrease [,]. Interestingly, the debate is verymuch ongoing upon the evidence that the United Statesof America has seen a decline in cases of spina bi)da(https://www.cdc.gov/ncbddd/spinabifidadata.html). (isreview paper intends to compare and contrast spina bi)da inhumans and spina bi)da in the mouse, which is the leadinganimal model of this devastating condition in light of the

information studies on animal models have shed on thehuman counterpart [%–$].

2. Spina Bifida in Humans

Development of the central nervous system including thebrain and spinal cord is a complex process beginning witha 'at sheet of cells which undergoes sequential thickening,elevation, mediolateral convergence accompanied by rostro-caudal extension, and )nally adhesion to form the neuraltube (NT) which is the precursor of the brain and thespinal cord. Perturbations of these interconnected processesresult in neural tube defects (NTDs), which are the mostcommon congenital malformation a-ecting this system andare associated with signi)cant complications. NTDs canoccur in two major forms: spina bi)da (SB) aperta, which

Hindawi Publishing Corporation

Scientifica

Volume 2017, Article ID 5364827, 29 pages

https://doi.org/10.1155/2017/5364827

! Scienti)ca

is the open-lesion NTD, and the closed-lesion NTD, morecommonly known as SB occulta.

3. Epidemiology

Spina bi)da is the most common nonlethal malformationin the spectrum of NTDs and has an incidence generallyaround ".. per #,""" births, although higher frequencies havebeen reported [&–##]. In the United Kingdom, the populationprevalence of spina bi)da is &./–/.% per #",""" for males and0."–0.% per #",""" for females [#!]. While the prevalence inthe United States of America is more than , in every #","""births [/, #,], studies in parts of Asia, such as Malaysia, havealso shown a lower occurrence of spina bi)da than that of theUK [#%]. More recent e-orts by our group (“Spina Bi)da: A#"-Year Retrospective Study at University of Malaya MedicalCentre, Malaysia,” manuscript in submission), however, havefound that the lower rate of NTDs may not be completelyrepresentative as in our hospital alone from the years !"",to !"#! we have had over #" cases of neural tube defects peryear (spina bi)da and anencephaly). Furthermore, certainregions of China have shown much higher preponderanceof this condition than in other parts of the world [#.–#/]. InAfrica, for example, spina bi)da has been recorded as beinglow in occurrence in comparison to other birth defects butquestions have arisen with regard to record-taking and datamanagement [#0]. Gender preponderance di-ers accordingto country; in the USA, spina bi)da is thought to be moreprevalent in girls than in boys [!", !#].

4. Pathogenesis

Spina bi*da aperta (SBA), sometimes referred to as spinabi)da cystica, is usually visible at birth as an exposed neuraltissue with or without a protruding sac at the site of the lesion.SBAmay be referred to as either myeloschisis (Figure #(a)) ormyelomeningocele (Figure #(b)). Myelomeningocele is whenthe spinal cord protrudes from the spinal canal into a 'uid-)lled sac resulting from incomplete closure of the primaryneural tube. Myeloschisis is when the incomplete closure ofthe primary neural plate results in a cle* spinal cord withthe edges 'ush with the defect. (e extent and severity ofthe neurological de)cits depend on the location of the lesionalong the neuraxis [!!].

Meningocele (Figure #(c)) is o*en described as a lesssevere variant of myelomeningocele in which the spinal cordis not found in the sac and is described by embryologiststo be absent of neural matter in its herniated sac; and itsdescription is o*en coupled with that of myelomeningocelewhich clearly has neural matter herniating at the site of theopen lesion. (erefore, the status of meningocele being anopen (aperta) or closed (occulta) defect is still debatablein terms of embryogenesis. However, imaging evidence byradiologists has )rmly placed meningocele as spina bi)daocculta [,, &, #!#–#!,].

Myelomeningocele (MMC) is usually associated with atype II Chiari hindbrain malformation, ventriculomegaly,and hydrocephalus [#!%, #!.]. Chiari type II malformationis the downward displacement of the cerebellar vermis into

the cervical vertebral canal [!!, #!.]. It is o*en symptomaticand is diagnosed prenatally with ultrafast fetal magneticresonance imaging (MRI) [#!$, #!&]. (is malformationcauses elongation of the brain stem and obliteration ofthe fourth ventricle, leading to obstruction of cerebrospinal'uid circulation and development of hydrocephalus in 0"%of patients [!!]. Treatment of such accompanying hydro-cephalus is needed in about /!% of cases and involvesdraining of cerebrospinal 'uid into either the peritoneal orother body cavity via a subcutaneous shunt [#!/].

Spina bi*da occulta (SBO) is the second major form ofNTDs, where the site of the lesion is not le* exposed [#!0,#,"]. Spina bi)da occulta encompasses lipomyelomeningo-cele (Figure #(d)), lipomeningocele (Figure #(e)), and spinaldorsal dermal sinus tract (Figure #(f)) ranging phenotypicallyfrom (i) dysplastic skin, (ii) tu* of hair, and (iii) vestigialtail as well as other forms of spinal dysraphism, whichlack a pathogenic representation when the vertebrae developabnormally leading to absence of the neural arches [#,#, #,!].In symptomatic cases, tethering of the spinal cord within thevertebral canal can result in pain, weakness, and incontinencein otherwise normal, healthy children or adults [#,,].

5. Treatment and Management

Management of patients with myelomeningocele hasimproved drastically from the mid-#0&"s when patientswere sometimes denied treatment based on the severityof their condition [#,%] to the current state-of-the-artprenatal in utero repairs performed at highly specializedcenters [#!&, #!/]. Neonatal surgical closure of the lesionis considered the standard of care against which all novelmanagement options are compared [!!, #,., #,$].

NTDs have a profound impact on society.(e morbidityand mortality rates of spina bi)da patients decrease withimproving medical care. Taking the United Kingdom as anexample, Bowman et al. [#,&] in their !.-year follow-up of&# spina bi)da aperta patients found that at least &.% of thesechildren can be expected to reach their early adult years [#,&].Moreover, as many as /.% are attending or have graduatedfrom high school and/or college. More than /"% of youngadults with spina bi)da have social bladder continence. In thesame study, %0% had scoliosis, with %,% eventually requiringa spinal fusion. Approximately one-third of patients wereallergic to latex, with six patients having experienced a life-threatening reaction. Renal failure was $./–0." times morecommon for males and 0.!–##.. times more common forfemale patients comparedwith the general population in eachof the years #00%–#00& in the UK [#,/].(erefore, longer lifeequates with the need for progressively better quality of life.

(e sequelae of NTDs are staggering and appear tohave not only anatomical e-ects secondary to the primarydefect but also functional, emotional, and psychologicalmorbidities including bladder and bowel incontinence, paral-ysis, musculoskeletal deformity, and shunt malfunctions andinfections, among others. Moreover, the costs involved inmaintenance of spina bi)da patients include mobility aids(orthoses, wheelchairs, and crutches), medications, and thecost associated with shunt revisions, in addition to the cost

Scienti)ca ,

Myeloschisis

Spina bi!da aperta

(a)

Myelomeningocele

Spina bi!da aperta

(b)

Spina bi!da aperta/spina bi!da occulta

Meningocele

(c)

Spina bi!da occulta

Lipomyelomeningocele

(d)

Spina bi!da occulta

Lipomeningocele

(e)

Spina bi!da occulta

Spinal dorsal Dermal sinus tract

(f)

F12345 #: Schematic representation of the open (aperta) and close (occulta) types of spina bi)da. (a) Myeloschisis which represents the mostsevere form of open spina bi)da. (b) Myelomeningocele which represents another typical severe form of open spina bi)da (spina bi)daaperta/spina bi)da cystica).(e typical representation is that of the spinal cord lying outside the spinal canal. (c) Meningocele that representsopen or close spina bi)da (the skinmay ormay not be present) but spinal cord does not lie outside the spinal canal. (d) Lipomyelomeningocelethat represents closed spina bi)da (spina bi)da occulta) (covered with skin) but spinal cord is intermeshed with lipid globules (in yellow). (e)Lipomeningocele that exhibits closed spina bi)da but spinal cord does not lie outside spinal canal even though lipid globules are present. (f)Spinal dorsal dermal sinus tract; spina bi)da occulta with vertebral arches missing (o*en asymptomatic and is thought to be a mesodermaldefect and a defect of secondary neurulation).

of modi)cations to public utilities that are required to enabledisabled access. Ultimately, its compound nature results in animmense )nancial burden amounting to 6#,%"",""" per childa-ected by NTD over a !"-year life span [#,0–#%!].

".!. Syndromic and Nonsyndromic (Isolated) Spina Bi*da. Asmall proportion of NTDs in live born infants are associated

with speci)c syndromes that are associated with chromo-somal or single-gene disorders [#%,]. NTDs are currentlyconsidered as “complex” disorders with genetic and envi-ronmental factors playing roles in causation [#%%], whichhave been summarized in Table #. Craniorachischisis andencephalocoele have the highest rate of syndromic associa-tion, anencephaly and high spina bi)da have intermediate

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Scienti)ca .

rates, and caudal spina bi)da has the lowest rate [#%.]. (erole of folic acid in preventing syndromic NTDs turnedout to be not as gratifying as for nonsyndromic (isolated),multifactorial NTDs [#%$]. It should be noted that whilesyndromic NTDs may have identi)able genetic causes, manyof the nonsyndromic (isolated) NTDs have unidenti)edgenetic etiology. Most of human neural tube defects arenonsyndromic with NTD being the only defect. (e focusof this review paper is on nonsyndromic (isolated) spinabi)da apart from the clearly stated syndromic spina bi)damentioned speci)cally in Table #.

".&. Causative Factors, Detection, and Prevention of SpinaBi*da. (e etiology of spina bi)da is heterogeneous [#%&–#."]. Most nonsyndromic spina bi)da is thought to be ofmultifactorial origin [#.#] with in'uence of both genetic andenvironmental factors [#%%, #.!]. Among the environmentalfactors associated with increased risk of spina bi)da areincreased pregnancy weight [#.,–#./], maternal smoking[#.0–#$#], drug intake speci)cally of antiepileptic drugs [#$!–#$%], and maternal illnesses such as diabetes [#$., #$$] andhyperthermia [#$&]. Dietary factors includingwater chlorina-tion [#$/–#&"], inositol intake [#&#], simple sugar intake [#&!],and the intake of trace elements and other micronutrients[#&,–#&$] have been proposed to act as either contributoryor preventive factors for spina bi)da. Isolated spina bi)da iscaused by cytogenetic abnormalities in !–#$% of cases [#&&–#&0].

Elevated levels of maternal serum alpha-fetoprotein areusually indicative of spina bi)da aperta [#/", #/#] but canbe associated with other conditions (e.g., twin gestationand abnormalities of placentation including placental lakesand placenta previa) and ultrasound is needed to con)rmthe diagnosis. Screening obstetrical ultrasonography is theinitial routine method for the detection of NTDs duringpregnancy in many countries. However, it sometimes failsto detect closed spina bi)da [#/!, #/,]. In highly specializedfetal centers, use of ultrafast fetal MRI has enabled detailedanatomical evaluation of the defect and accurate assessmentof its accompanying e-ects [#!$].

It has been over #. years since the Medical ResearchCouncil Vitamin Trial involving ,, centers around the worldconclusively showed that &!% of recurrent NTD cases couldbe prevented by folic acid supplements in the periconcep-tional period [#/%]. A further study [#/.] showed that the)rst occurrence of spina bi)da could also be prevented byfolic acid. However, not all NTDs are responsive to folicacid and inositol has been shown as a possible additionaltherapy, based on prevention of spina bi)da in folate-resistantNTDs in mice as well as the PONTI human trial [#/$, #/&].Calcium formate too has been shown to have preventivee-ects on NTD in mice but evidence is not yet forthcomingin prevention of human NTDs [#//–#0"].(ere still remainsroom to study whether there are other supplements out therethat can prevent spina bi)da.

6. Surgical Management of Spina Bifida

Surgical management of spina bi)da here is discussed as a!-point discussion: )rst is surgical management prior to the

advent of in utero repair of open spina bi)da and second is inutero repair leading to the Management of Myelomeningo-cele Study (MOMS) trial [#!/]. Postnatal repair of open spinabi)da repair is a requirement in order to prevent furthermechanical damage and infection. (e lesion either may beclosed primarily with the aid of skin and muscle 'aps or mayrequire a synthetic patch such as AlloDerm (LifeCell Corp.,Branchburg, NJ) [#0#], gelatin, or collagen hybrid sponges[#0!]. In utero MMC repair in humans was )rst reportedin the landmark paper published in #00/ [#!&]. However,since then, a handful of centers have been o-ering in uterorepair. Furthermore, its popularity has increased in Europe[#0,]. (e principle of in utero repair is to prevent the !-hit hypothesis much described in previous literature that thechild is exposed to neurological deterioration contributed)rst by failure of the neural tube to form and secondly byphysical and chemical perturbation in'icted on the exposedneurological tissue of the open lesion [#!/, #0%]. In an elegantexperimental study, Meuli et al. [#0.] concluded that surgicalexposure of the normal spinal cord to the amniotic space in a&.-day sheep fetus results in a MMC-type pathology at birthwith clinical, histological, andmorphological attributes com-parable to humanMMC. He-ez et al. [#0$] has demonstratedthat spinal cord injury caused by exposure to the intrauterinemilieu can be prevented by primary closure of the fetal skinincision as late as hours a*er creating the defect. It alsodemonstrated that ongoing exposure beyond !% hours leadsto spinal cord damage and permanent neurological de)cit.Moreover, animal studies have previously shown that prenatalcoverage of a spina bi)da-like lesion preserves neurologicfunction and improves hindbrain herniation [#0., #0&, #0/].

(e )rst human prenatal repair of MMC was reportedin Tulipan et al. [#00]. Cumulative data suggested not onlya dramatic improvement in hindbrain herniation but alsoincreased maternal and neonatal risks including pretermlabor, uterine dehiscence, and increased risk of fetal andneonatal death among others. Adzick et al. [#!/] investigatedthe e-ects of prenatal repair of MMC via a randomizedprospective study. It reported that prenatal surgery for MMCperformed before !$ weeks of gestation decreased the riskof death or need for shunting by the age of #! months andalso improved scores on a composite measure of mentaland motor function, with adjustment for lesion level, at ,"months of age. Prenatal surgery also improves the degree ofhindbrain herniation associated with Chiari II malformation,motor function, and the likelihood of being able to walkindependently, as compared with postnatal surgery [#!/].Open prenatal repair comes with an increased maternal andneonatal risk including preterm labor, uterine dehiscence,premature rupture of membranes, and increased risk of fetaland neonatal death. (e main goal for prenatal repair ofMMC is to achieve skin closure to prevent further damageof the placode and arrest the CSF leak.

7. Human Spina Bifida Genes

Despite the !."mouse mutants with NTDs to date, there hasyet to be a signi)cant breakthrough for human NTD gene(s)both causal and/or associated with NTDs that can be used for

$ Scienti)ca

genetic screeningworldwide [%, &].(e importance of )ndingcandidate gene(s) as a genetic screening tool for potentialparents cannot be undervalued as it has been estimated thatthe total lifetime costs for patients with spina bi)da (spinalNTDs) amount to about 6#.% million in the US and morethan :.""k in Europe, with ,&.#% of the total cost attributedto direct medical costs and the remainder in indirect costs,including the needs of the caregiver [!""].

Despite observation of multiplex nonsyndromic NTDcases in multigenerational NTD families as seen in #&US and#% Dutch families with more than # NTD-a-ected person,there are other NTD cases that are simplex and sporadic asseen in identical twins with lumbosacral lipomyelomeningo-cele with no known familiar history of NTDs [!"#, !"!].(issuggests that NTDs have a multifactorial genetic etiology.

To date, the strongest candidate thus far for a poten-tial NTD screening gene is the methylenetetrahydrofolatereductase (MTHFR) C$&&T (rs#/"##,,) polymorphism inpopulations of non-Latin origin (meta-analysis study) [!",].In recent meta-analysis study, Zhang et al. support thesigni)cant association between C$&&T and NTDs in case-control studies (!! studies, !,$"! cases, and %,"&" controls)[!"%]. (e second most studied MTHFR variant is A#!0/C,which did not report any signi)cant increase in risk of NTDs[!"%]. Another meta-analysis study by Blom et al. (!""$)reported increased risk in mothers and associated with NTDinfants who are homozygous for C$&&T variant [!".]. Inspina bi)da case studies, MTHFR C$&&T variant was clearlyreported as associated gene or risk factors in Irish ()"! spinabi*da patients), mixed USA, mixed UK, and Italian cohortbut not in other #/" Dutch patients (Table ,), while A#!0/Cvariant was reported with no association to spina bi)dacases in Italian, Mexican (Yucatan), and Dutch population(Table ,). MTHFR is the most studied human spina bi)dagene, as its role in folate one-carbon metabolism )ts into aclearmechanism of NTD.However, the studies have not beenwell replicated in many other populations across the world,indicating that it is not likely to be either a major contributoror a common factor in NTD globally.

Other genes such as the planar cell polarity (PCP) genes,which have been studied in spina bi)da cohorts amongItalians, Americans, and the French, areVANGL! andCESLR![%%, /!–/%]. (e noncore PCP gene SCRIB has also beenimplicated as a spina bi)da gene among the American cohort[/.]. However, noncore PCP gene association needs to beexplored further in larger NTD cohorts. To date, over #""human spina bi)da genes have been used to screen for spinabi)da with %/ genes reported as a potential risk factor aslisted in Table , which was reviewed in Greene et al. [0,];further candidates since then are NKX&-(, PTCH!, Glypican-", PARD%, Paraoxonase !, COMT, AMT, andGLDC genes [#$,#&, !"$–!##]. All of these do not represent a potential globalspina bi)da gene. (erefore, a strong candidate spina bi)dagene(s) for the world population has yet to be discovered.

8. Spina Bifida in Mouse

(ere exist more than !." mouse models with neural tubedefects, of which &% are of spina bi)da (Table !) [%], yet there

does not exist a single mouse gene which can be used toscreen the orthologous human gene of neural tube defect norspina bi)da to date [!#!]. (at said, it does not mean thatthe studies on the structural changes a-orded by the mousemodel cannot be used as a tool to understand human spinabi)da. We discuss the various studies on mouse neurulationbelow and why it is still an invaluable tool for understandinghuman neurulation.

(.!. Mechanisms of Neural Tube Closure. In vertebrates, thedevelopment of the CNS starts with the formation of theneural plate on the dorsal surface of the embryo during lategastrulation [!#,, !#%]. A complex morphogenetic processtransforms the neural plate into the hollow neural tube in aprocess known as “neurulation” [!#,]. Primary neurulation isresponsible for formation of the neural tube throughout thebrain and the spinal cord rostral to the mid-sacral level [!#.].At more caudal levels, an alternative mechanism (secondaryneurulation) operates whereby the neural tube is formed bycanalization of a condensed rod of mesenchymal cells in thetail bud [!#$].

(e process of neurulation in mammals and some othervertebrates is considered discontinuous because it occurssimultaneously at multiple sites along the neuraxis [!#.–!#0]. (ere are three points of de novo neural tube fusionin the mouse, which is the most studied mammalian model([!!"]; see Figure !(a)). Closure ! occurs adjacent to somite, in embryos with $-& somites and progresses rostrally andcaudally, closure & occurs at themidbrain-forebrain boundaryat around the #"-somite stage and progresses caudally, andclosure % occurs at the rostral end of the forebrain, soon a*erclosure !.

Considering this discontinuous process of neurulation,it can be understood why NTDs are such a complex groupof heterogeneous birth defects, with various phenotypicpresentations. Failure of closure ! leads to craniorachischisis(Figure !(b)); failure of closures ! and/or , causes exen-cephaly and/or anencephaly, respectively (Figure !(c)), whilefailure of neurulation to progress from the site of closure# caudally along the spinal axis leads to spina bi)da aperta(Figure !(d)).

During neurulation, the neuroepithelium must undergovarious structural changes in order to achieve closure. (eadvent of molecular biology has allowed scientists to identifythe genes that are required for these structural changes tooccur.(e next section gives a brief overview of the researchto date on how gene expression a-ects structural changesin neural tube development, with an emphasis on generegulation in the spinal region.

(.&. +e Structural Changes of the Mouse Neural Tube duringthe Process of Closure. Morphologically, the mouse neuraltube undergoes distinct structural changes prior to its closure[&, !#., !!#–!!%]. A summary of the spatiotemporal expres-sion of genes in the mouse neural tube during neurulationis as shown in Table .. (e neuroepithelium narrows andlengthens, a process referred to as convergent extension(Figure ,(a)), in which the polarized cells which form theneuroepithelial plate converge towards the midline, elongateanteroposteriorly, and then intercalate [!#., !!.].

Scienti)ca &

T7895 !: List of mouse models that exhibits spina bi)da (reviewedin [%, .%]).

Mouse modelsGene or mutants (., genes)

(#) Aln(!) Ambra!(,) Apapb(%) Axdmutant(.) Axin!($) Cyp&$a!(&) Dsmutant(/) Dvl&(0) Fgfr! chimera(#") Fkbp( hypomorph(##) Fpn!(#!) F&r, F&rl! (Par!, Par&) (digenic)(#,) Grhl%(#%) Gnaz!/!; Grhl%Cre/+ (digenic)(#.) gGpr!$! (vlmutant)(#$) ct mutant (Grhl% hypomorph)(#&) Itgb!(#/) ltpk!(#0) Lrp$(!") Lrp$ (rs mutant, hypomorph)(!#)Map%k)(!!)Mapk) partial function(!,)Marcksl! (Mlp)(!%)Med!&(!.)Msgn!(!$)Msx!, Msx& (digenic)(!&) Ndst!(!/) p&(IP(!0) Pax% (Spmutant)(,") Pax% (Sp&H)(,#) Pax% (Spd)(,!) Ptpn, (Meg&)(,,) Rab&% (opbmutant)(,%) Rab&% (opb&)(,.) Rac!!/!; !"ℎ$3"#$/+ (digenic)(,$) Sfrp!, Sfrp& (digenic)(,&) Shroom%(,/) Sp((,0) Spint& (HA#!)(%") T (Tc/tw" mutant)(%#) Terc(%!) Traf)(%,) Trpm$(%%) Tulp%(%.) Tulp% (hhkr mutant)(%$) vlmutant(%&)Wnt%a (vt mutant hypomorph)(%/) Z-x!a(%0) Zic& hypomorph(.") Zic&Ku(.#) g&e(.!) !B(.,) ,'c&

T7895 !: Continued.Mouse modelsMouse strain (# gene)(.%) NOD

PCP genes (% genes)(..) Lp, Crcmutants (digenic, heterozygous)(.$) Lp, Ptk' (digenic, heterozygous)(.&) VanglLp/+, Sec&)b+/! (digenic)(./) VanglLp/+, Sfrp!!/!, Sfrp&+/!, Sfrp"!/! (% genes)

Lethal before gestation day #! (, genes)(.0) Brca!($") Fgfr! (alpha isoform)($#) !!C

Spina bi)da occulta (#, genes)($!) Foxc! (chmutant)($,) Foxc&($%) Lrp$($.) Nog($$) Pdgfra($&) Pdfgc($/) Pkd!($0) Prrx!(&") snomutant(&#) Tg.&(&!) T (Tc/+ mutant)(&,) Traf)(&%) Zic!

Mouse models studied in nutrient supplement rescue (. genes)(#) Sp&Hmutant at Pax% gene(!) Spmutant at Pax% gene(,) Axdmutant(%) ct mutant (hypomorph at Grhl%)(.) Grhl% null

Convergent extension leads to narrowing and lengthen-ing of the neuroepithelium, a process that has been suggestedalso to assist neural fold elevation via axial elongation [#".,!!$–!!/]. However, the lengthening of the body axis isdisrupted by manipulation of gene function required forconvergent extension; whilst the neural folds are still ableto elevate, convergent extension still fails [!!&, !!0, !,"].Hence, convergent extension and neural fold elevation areseparable processes. Elevation of the neural folds at highlevels of the spinal neuraxis results from the formation of amedian hinge point (MHP) (Figure ,(b)) in a process termedMode # neurulation [!#., !,#, !,!]. (e neural folds remainstraight along both apical and basal surfaces, resulting in aneural tube with a slit-shaped lumen. Mode # neurulationoccurs during formation of the spinal neural tube in $–#"-somite stage embryos, as shown in Figures %(a) and %(b).

A second set of hinge points are formed dorsolaterallyat more caudal levels of the spinal neuraxis, the dorsolateralhinge points (DLHPs), a process that appears to enhance theability of the apposing tips of the neural folds to come close toeach other (Figure ,(c)). Mode ! occurs during formulationof the spinal neural tube in #!–#.-somite stage embryos andgenerates a diamond-shaped lumen, as depicted in Figures%(c) and %(d). InMode !, amedian hinge point is also present,

/ Scienti)ca

T7895,:Co

mprehensiv

elist

ofhu

man

spinab

i)da

genes.

Genes

(%"genes)

Popu

lationandsamples

ize(in

brackets)

ofspinab

i)da

cases

References

Stud

iesshow

edassocia

tionwith

genesrela

tedNTD

sor

riskfactor

forspina

bi)d

aStud

iesshow

edno

associa

tionwith

genesrela

tedNTD

s

One

carbon

metabolism

(inclu

ding

homocysteiner

emethylation)

(/genes)

ALDH

!L!

Dutch

(#/"patie

nts)

[..]

BHMT

Mixed

USA

(!.0

cases)

Mixed

USA

(!.!

cases),D

utch

(#/"patie

nts)

[..–.&]

CHKA

Mixed

USA

(#",c

ases)

[./]

MTR

RMixed

USA

(!.0

cases),m

ixed

UK(!!0

patie

nts),

Dutch

(#"0cases)

Irish

(.&.

mixed

families),Dutch

(#"0cases;#/"

patie

nts)

[..,.$,.0–

$#]

NOS%

Mixed

USA

(,"#

families),Dutch

(#"0cases)

Mixed

USA

(!.0

cases),D

utch

(#/"patie

nts)

[..,.$,$!,

$,]

PYCT

!AMixed

USA

(#",c

ases)

[./]

SARD

HDutch

(#/"patie

nts)

[..]

TRDM

T!Dutch

(#/"cases)

[..]

NTD

sinmou

semutant(&g

enes)

BRCA

!Mixed

USA

(!$/

patie

ntsa

ndparents)

[$%]

CFL!

Mixed

USA

(!%$

cases)

[$.]

PAX%

USA

(&%cases)

[$$]

PDGF

RADutch

(//c

ases

and.$

mothers)

Mixed

USA

(%"&

triads)

[$&,$/]

TXN&

Mixed

USA

(%/c

ases)

[$0]

ZIC&

Dutch

(##&m

ixed

patie

nts)

[&"]

ZIC%

Dutch

(##&m

ixed

patie

nts)

[&"]

Folatem

etabolism

(.genes)

CBS

Mixed

USA

(!.0

cases)

Dutch

(#/"patie

nts)

[..,.$]

DHFR

Irish

(!/,

cases),m

ixed

USA

($#cases,m

ultia

-ected

families)

Mixed

USA

(!.0

cases),m

ixed

UK(!!0

patie

nts),

Dutch

(#/"patients;#"0patients)

[..,.$,&#–

&,]

MTH

FD!

Irish

(."0

mixed

cases),m

ixed

USA

(!.0

mixed

cases),

Italian(#%

!cases),Irish

(#&$mixed

cases)

Mixed

UK(!!0

patie

nts),D

utch

(#",c

ases),Dutch

(#/"

patie

nts)

[..,.$,.0,

$!,&#,&%–&$]

MTH

FRIrish

(%.#

cases),m

ixed

USA

(!.0

cases),m

ixed

UK

(!!0

patie

nts),Italian(#.

cases)

Dutch

(#/"patie

nts),M

exica

n(Yucatan)(0&

cases),

Italian(#.

cases)

[..,.$,.0,

$",&&,&/]

TYMS

Non

-Hisp

anicwh

iteUSA

(!$%

cases),m

ixed

USA

(!.0

cases)

Dutch

(#/"patie

nts)

[..,.$,&0]

Glucose

metabolism

(%genes)

GLUT

!Mixed

USA

(."&

cases)

[/"]

HK!

Mixed

USA

(."&

cases)

[/"]

LEP

Mixed

USA

(."&

cases)

[/"]

LEPR

Mixed

USA

(."&

cases)

[/"]

Scienti)ca 0

T7895,:Co

ntinued.

Genes

(%"genes)

Popu

lationandsamples

ize(in

brackets)

ofspinab

i)da

cases

References

Stud

iesshow

edassocia

tionwith

genesrela

tedNTD

sor

riskfactor

forspina

bi)d

aStud

iesshow

edno

associa

tionwith

genesrela

tedNTD

s

DNA

repairandDNA

methylation(,

genes)

APE!

Mixed

USA

(,/"

patie

nts)

[/#]

XPD

Mixed

USA

(,/"

patie

nts)

[/#]

SOX!(

Belgium

(/,p

atien

ts)(Rochtus

etal.,!"#$)

Folatetranspo

rt(!

genes)

CUBN

Dutch

(#&0patie

nts)

[..]

SLCA

!,A!

,RFC

-!Dutch

(#/"patie

nts)

Mixed

USA

(!.0

cases),m

ixed

UK(!!0

patie

nts)

[..,.$,.0]

PCPgenes(%gene)

VANG

L!Ita

lianandmixed

USA

($./

patie

nts),Italianand

French

(#"!p

atien

ts)Mixed

UKandUSA

(!%patie

nts)

[%%,/!,/,]

CELSR!

California(

#0!p

atien

ts)[/%]

SCRIB

California(

#0!p

atien

ts)[/.]

DVL!

Han

Chinesec

ohort(!"

cases)

[/$]

Retin

olmetabolism

(#gene)

ALDH

!A&

Mixed

USA

(,#/

families)

[/&]

Axiald

evelo

pmentinmou

se(#gene)

T(brachyury)

Mixed

USA

(,#$

cases)

[//]

Methylationreactio

ns(#gene)

PCMT!

Mixed

USA

(#.!c

ases)

Dutch

(#/"patie

nts)

[..,/0]

Oxidativ

estre

ss(!

genes)

SOD!

Mixed

USA

($#"

trios

ordu

os)

[0"]

SOD&

Mixed

USA

($#"

trios

ordu

os)

[0"]

Interm

ediate)

lamentp

rotein

(#gene)

LMNB

!Mixed

UK,

USA

,and

Swedish

(!,,

patie

nts)

[0#]

Celladhesio

nmolecules

(#gene)

NCAM

!USA

(!"%

patie

nts)

[0!]

Atotalof%

"genesreportedshow

inga

ssociation/ris

kfactor

forspina

bi)d

aasreview

edin

Greenee

tal.[

0,].

#" Scienti)ca

ANP

HNP

(1)

(2)

(3)(B)

(C)

(D) (E)

(F)

(A)

(a) (b)

(c) (d)

F12345 !: Points of closure in the mouse embryo and phenotypes of failure of closure of the various points along the neuraxis. (a) Schematic)gure illustrating the multiple points of closure of the neural tube, directions of closure, and the di-erent locations of neuropores in thedeveloping embryo. (#), site of closure (#) which occurs at the level of somite , in the $-&-somite embryo. Closure (#) is the initiation eventof neurulation. Closure then progresses caudally and is completed by closure of the posterior neuropore (PNP) at the !0-,"-somite stage ofdevelopment; (!), second closure site at around the #"-somite stage; (,), closure (,) site which begins soon a*er closure (!). Arrows depictspreading of neural tube closure to neighbouring regions with completion of anterior neuropore closure soon a*er initiation of closure (,)and closure of the hindbrain neuropore at the #/–!"-somite stage. (b) Phenotype resulting from failure of closure (#): craniorachischisis;(c) phenotype resulting from failure of closure (!): exencephaly; (d) phenotype of failure of the caudal wave of spinal closure, leading to anenlarged PNP and later development of spina bi)da. (A), posterior neuropore; (B), branchial arches; (C), developing heart; (D), hindbrain;(E), midbrain; (F), forebrain; ANP: anterior neuropore; HNP: hindbrain neuropore.

whereas the remaining portions of the neuroepithelium donot bend. At the #&–!&-somite stage, the neural tube closeswithout a median hinge point, whereas dorsolateral hingepoints are retained. (is is known as Mode , neurulationand generates an almost circular shaped lumen, as shown inFigures %(e) and %(f).

Adhesion of the tips of the apposing neural folds is the)nal step in primary neurulation, enabling the neural tubeto complete its closure [!#.]. (e tips of the apposing neuralfolds and the eventual point of adhesion are reported tocontain cell to cell recognition molecules (as demonstratedin red in Figure ,(c)) which may be required for the speci)cadhesion process to occur [!,,–!%,]. (is is supported byprevious evidence that the cell surface of the neuroep-ithelium is lined by carbohydrate-rich material that is notobserved in the rest of the neuroepithelium [!,/]. Removal of

GPI-anchored proteins from the cell surface during neuru-lation results in delayed spinal neural tube closure [!%%].Interestingly, work performed by Abdul-Aziz et al. andPyrgaki et al. demonstrated protrusions emanating from theneural fold tips that interdigitate leading to eventual adhesion[!%%, !%.] (Figure ,(d)). Ultimately, the newly formed neuraltube undergoes remodelling via apoptosis to enable theneural tube to separate from its surface ectoderm [!!/, !%$](Figure ,(e)).

(.%. Primary Neurulation Versus Secondary Neurulation. Pri-mary neurulation and secondary neurulation are importantdevelopmental processes and have been described in manymodels. In the chick, there does not exist a clear distinctionas to when primary neurulation ends and secondary neu-rulation begins; the lower spinal cord has been described

Scienti)ca ##

Se

Ne

Neuroepithelium

(a)

Se

MHP

Me

Ne

Neuroepithelium

(b)

Se

MHP

Me

DLHPDLHP

Neuroepithelium

Lamellipodial protrusions

(c)

POAF

Nt

Neuroepithelium

(d)

NeuroepitheliumPossible site of apoptosis to allowseparation of neuroepithelium andsurface ectoderm

(e)

F12345 ,: Schematic representation of the formation of the mouse spinal neural tube. Process of closure of the PNP of embryos undergoingMode # (a, b, d) or Mode ! (a, c, d) neurulation. (a) Neuroepithelium thickens and converges; (b) formation of bilateral neural folds whichare elevated (Mode #); (c) apposing tips of neural folds aided by bending at the dorsolateral hinge points (DLHP) of the bilateral neural folds(Mode !); (d) adhesion and fusion at the tips of the neural folds; (e) remodeling of the neural tube. Ne, neuroepithelium; Se, surface ectoderm;Me, mesoderm; MHP, median hinge point; DLHP, dorsolateral hinge points; POAF, point of adhesion and fusion; Nt, notochord.

as junctional neurulation, whereby ingression and accretionaccompany the process of de)ning the area which straddlesprimary and secondary neurulation and is therefore thoughtto somehow represent human thoracolumbar spina bi)da[!%&].

In mouse and humans, spina bi)da occulta has largelybeen described as a result of failure of secondary neurulation[,, !#.]. However, much has been described of the severity of

lipomyelomeningocele [#,#, !%/] in comparison to the some-what neurologically unperturbed tethered cord phenomenonwhich is brought on by trapped nerves due to missingvertebral arches [#,,]. What is evident is that, irrespective ofwhether or not there is skin covering the neural tube defectlesion, the severity of the condition depends on the levelwhere the site of the lesion is located. Secondary neurulationin the mouse is described as occurring at sacral level ! [!!%].

#! Scienti)ca

Level of spinal axis sectioned

6–10-somite stage (E8.5)

(a)

Mode 1

(b)12–15-somite stage (E9.5)

Level of spinal axis sectioned

(c)

Mode 2

(d)

17–27-somite stage (E10.5)

Level of spinal axis sectioned

(e)

Mode 3

(f)

F12345 %: Schematic )gure showing progressive developmental stages of the mouse embryo and sections through the PNP at these stages.(a) Schematic of embryo at $–#"-somite stage, which has already undergone closure (#); (b) section through PNP of (a), depicting Mode #neurulation; (c) schematic of embryo at #!–#.-somite stage; (d) section through PNP of (c) exhibiting Mode ! neurulation; (e) schematic ofembryo which has undergone closures (#), (!), and (,) with PNP being the only remaining unfused section of the neural tube; (f) sectionthrough PNP of (e) depicting Mode , neurulation.

(erefore, to describe lipomyelomeningocele as resultingfrom failure of secondary neurulation would be arti)cial.

9. The Genetics behind the StructuralChanges in Spinal Neural Tube Closure

(is section summarizes the various genes that are switchedon during neurulation and whose functions have been impli-cated in the various structural changes that the spinal neuraltube undergoes in order for closure to be achieved.

,.!. PlanarCell Polarity andConvergent Extension. Planar cellpolarity (PCP) is a process in which cells develop with uni-form orientation within the plane of an epithelium [!%0].(ePCP pathway is a noncanonicalWnt pathway [!!., !."–!.!].

Various Wnt molecules are known to play roles in the PCPpathway such as Wnt## and Wnt.a [!.", !.,].

PCP signaling has been suggested to be primarilyrequired for cytoskeletal activity, for example, cellular pro-trusion, cell-cell adhesion, and cell-matrix adhesion [!.%].Skin development, body hair orientation, polarization of thesensory epithelium in the inner ear, and the directed move-ment of mesenchymal cell populations during gastrulationare among the processes requiring proper PCP signaling invertebrates [!!&, !.%–!.$]. In vertebrates, function of thePCP pathway appears to be required for convergent extension(CE). Lamellipodia have been the type of cell shown to driveCE. (ese broad sheet-like protrusions exert traction onadjacent mesodermal cells causing mediolateral intercalation[!.&–!.0]. PCP signaling causes the regulation of cytoskeletal

Scienti)ca #,

organization that redistributes subcellular PCP componentsasymmetrically causing polarization of these cells [!$"].Moreover, components of the signaling cascade converge orare expressed asymmetrically in the lamellipodia [!.", !.,].

Among the genes implicated in this net movement ofcells, known as convergent extension, are ! asymmetricmolecular systems that control PCP behaviour, the “core”genes and the “Fat-Dachsous” PCP system [!$#, !$!]. (e“core” genes give rise to multipass transmembrane proteins:Frizzled (Fzd-,, -$, and -&), Van Gogh (Vangl-# and -!),Flamingo (Celsr-#, -!, and -,), and cytosolic components,Dishevelled (Dvl-#, -!, and -,), Diego (Inversin), and Prickle(Pk-# and -!) [!$,]. (e Fat-Dachsous (Ft-Ds) pathwayincludes the large protocadherins Ft and Ds, acting as itsligand, and Four-jointed (Fj) as a Golgi resident transmem-brane kinase [!$%]. Downstream of the PCP system are PPE(Planar Polarity E-ector) genes: Inturned (In), Fritz (Frtz),and Fuzzy (Fy) [!$., !$$].+eMultipleWing Hairs (mwh) actdownstream of both PCP and PPE [!$&] with Wnt%, Wnt.a,Wnt&a, and Wnt## as regulators [!$,].

Vangl-& (formerly known as Ltap and Lpp!) has beenidenti)ed as the causative gene in the loop-tail mouse[#"., !$/, !$0]. Mutations in Celsr-! cause craniorachis-chisis in the Crash mouse [!&"]. (e Dvl-#/Dvl-!, Dvl-!/Dvl-,, Dvl-!/Vangl-!, and Fzd-,/Fzd-$ double knockoutmice also have severe NTD forms, mainly craniorachischisisand exencephaly [!$0, !&#–!&,]. (e Vangl-# and Vangl-! compound heterozygote exhibits craniorachischisis [!&%].(e noncore PCP genes also exhibit severe NTD in theirmouse mutants including Protein Tyrosine Kinase & (PTK&),Scribbled PCP protein, the gene responsible for the circle tailmouse phenotype, Scrib, andDishevelled BindingAntagonistof Beta-Catenin # (Dact-#) [!.!, !&", !&%–!&&]. All of thesegenes have been implicated in the PCP pathway. Failure ofconvergent extension results in an open neuraxis (the entireneural tube from midbrain to low spine remains exposed)and a shortened embryo, more commonly described ascraniorachischisis.

,.&. Neural Fold Elevation and Bending. Dorsoventral pat-terning in the neural development of vertebrates is controlledby the induction and polarizing properties of the 'oor plate[!&/]. Expression of various genes such as sonic hedgehog(Shh), bone morphogenetic protein (BMP) ', HNF%%, andVangl-! emanating from the notochord and 'oor plate isthought to cause cell speci)cation which in'uences themorphogenesis of the neural tube [#"$, #"&, ##!, ##,, !.!].(e 'oor plate and notochord appear to control the patternof cell types that appear along the dorsoventral axis of theneural tube [!!$, !&/]. Morphogenesis of the spinal neuraltube, in particular, the formation of the median hinge point(MHP), is most likely a nonneuroepithelial cell autonomousaction as it is dependent on the di-erentiation of ventral celltypes by signals transmitted from axial mesodermal cells ofthe notochord to overlying neuroepithelial cells [!&/–!/%].

Implantation and ablation experiments which manip-ulated the notochord in both chick and mouse embryos[!!#, !/%–!/&] veri)ed that the notochord is required forformation of the MHP. It was proposed that the notochord

releases a morphogen that may regulate MHP formation.Shh protein is expressed in the notochord at this stage[##,, !//] and application of either Shh-expressing cells orpuri)ed protein to intermediate neural plate explants leadsto induction of the 'oor plate [##,], suggesting that Shh isthe MHP-inducing morphogen. However, MHP formation isnot totally abolished in Shh-null mouse embryos, suggestingthat other factors from the notochord may also have MHP-inducing properties [!/&].

(e second site of neural fold bending as described inSection /.! and Figure ,(c) is the dorsolateral hinge point(DLHP). Bending of the neuroepithelium at the DLHP isregulated by mutually antagonistic signals external to theneural fold, as reviewed by Greene and Copp [!!%, !/0]. Incontrast to midline bending, Shh has been shown to inhibitdorsolateral bending in the mouse [!/&] consistent with anabsence of NTDs in Shh-null embryos. Signal(s) arising fromthe surface ectoderm (SE) comprise(s) a second antagonisticsignal involved in the regulation and formation of the DLHPs[!0"].(is has been suggested as further evidence that bend-ing of the neural folds involves signaling from the SE. Bonemorphogenetic proteins (BMPs) are candidates to mediatethis signaling. (ree BMPs (BMP&, BMP), and BMP') areexpressed in the spinal neural tube. BMP& and BMP' areexpressed in the surface ectoderm adjacent to the open spinalneural tube, while BMP) is expressed in the surface ectodermoverlying the closed spinal neural tube [!0#].

Recent studies suggest that Noggin may also play a rolein regulating DLHP formation [!0!, !0,]. Noggin is aninhibitor of BMP signaling and is expressed at the tips ofthe apposing neural folds [!0,, !0%]. Homozygous mouseembryos null for Noggin exhibit both exencephaly and spinabi)da (#""%) [!0!, !0.]. However, spina bi)da does not arisein homozygous Noggin mutants until embryonic day ##-#!when the neural tube ruptures. (e spinal neural tube ofhomozygous null Noggin embryos during neurulation takeson the appearance of a wavy neural tube before the neuraltube reopens [!0,], possibly suggesting an unstable initialclosure mechanism. Shh works in an antagonistic mannertowardsNoggin, as doesNoggin towardsBMP signaling [!0$].(is suggests that Nogginmay facilitate bending of the spinalneural tube [!0,] by overcoming the inhibitory in'uence ofBMPs.

Stottmann et al. [!0,] suggest that the spinal defect inNoggin null embryos results from a failure to maintain aclosed neural tube due to a defective paraxial mesoderm[!0,]. Yip et al. [!0&] also had shown that the mesodermalextracellular matrix plays an important role in maintainingneuroepithelial rigidity of the spinal neural tube duringneurulation [!0&]. Embryos were cultured in the presenceof chlorate, which functions to inhibit sulfation of heparansulphate proteoglycans (HSPGs) in the extracellular matrixof the mesoderm. (is treatment not only resulted in anexpedited bending of the DLHPs but also elicited an unnat-ural shape of neural tube due to a convex shaped mesoderm.However, removal of the paraxialmesodermdoes not preventclosure of the spinal neural tube [!/&].

Interestingly, there are , genes which, when mutated, notonly a-ect paraxial mesoderm production in the mouse [#"0,

#% Scienti)ca

!0!, !0/] but also result in an NTD phenotype in the mouse.(ese are Cyp&$, Noggin, and Fgfr! [0%, #"0, !0,].(e Wnt,a[!00], Lef#/Tcf# double null [#"/] and Raldh! [,""] mutantsalso have defective paraxial mesoderm production, with anabnormal neural tube during neurulation. Whether or notthe paraxial mesoderm plays a primary role in successfulneurulation in these mutants remains unknown.

Neural tube closure does not depend exclusively on theMHP or DLHPs, since closure can occur in the absenceof either, as in Mode , and Mode # spinal neurulation,respectively. However, cell shape changes of some type,a-ecting morphogenesis of the spinal neural tube, are clearlyrequired for closure to occur in all species studied, includingthe mouse [!.%]. Table . demonstrates the lack of speci)cexpression of genes at the DLHPs. However, overlappinggene expression throughout the neuroepithelium and tips ofneural foldsmay facilitate the bendingmechanism seen in theDLHPs.

,.%. Adhesion of the Neural Folds. In all animal speciesstudied, a zone of altered cell morphology with numerousrounded cell blebs has been observed along the tips ofthe spinal neural folds, immediately prior to adhesion. (eobserved surface alterations may re'ect a change in theproperties of the cells at the adhesion site which correlatewith initial adhesion between the folds [!,%, !,$, ,"#, ,"!].Structural observations of the point of adhesion in humanembryos have yet to be reported, possibly due to insu+cientor poor preservation of material so that surface structurescannot be observed.

Adhesion is the )nal process in the sequence of primaryneurulation events. Suchphysical zippering state of the neuraltube has been suggested, in previous studies, as evidence thatneural tube closure is a continuous process [,",]. However, adebate exists as towhether the physical process of neurulationactually equates to continuous zippering or, more accurately,to a button-like process in which neural tube adhesioninitially occurs at various slightly separated points along theaxis. According to the latter idea, neural tube adhesion isactually a discontinuous process of closure [!!!].

PCP regulation may play a role in adhesion and fusionas suggested in both zebra)sh and Xenopus studies. Firstly,cell division regulated by PCP signaling leads to rescueof neural tube morphogenesis in the trilobite zebra)shmutant [,"%]. Secondly, the Xenopus adhesion molecules,NF-protocadherin, and its cytosolic partner TAF#/Set havebeen suggested to participate in CE a*er the neural folds areformed. Disruptions in NF-protocadherin and TAF# can leadto a shortenedAP axis that was not evident until stages !!–!.,some time a*er neural tube closure [,".].

Ultrastructures that emanate from the neural folds at thesite of closure have been regarded as a secondary process inthe frog. (is is because wound healing which acts via actinpurse-string contraction is thought to be the primary cause ofclosure in the frog neural tube [,"$]. Adhesion of the neuraltube and epidermis have been suggested to be separate eventsbased upon the observation that the epidermal ectoderm isstill able tomigrate and cover the open neural tube in both thechick and the frog [,"!, ,".]. However, the issue of whether

or not the neural folds could adhere even in the absence ofepidermal fusion in both the chick and the frog has yet to beanswered.

Adhesion in the neural tube of rodents has been describedpreviously but the mechanism of this highly specializedprocess is poorly understood [#",, !%", !%,, ,"#, ,"&, ,"/]. Ina recent study, a direct requirementwas shown for the bindingof a speci)c ligand (ephrinA.) to a speci)c type of receptor(EphA&) in order to enable adhesion to occur in the neuraltube [!%,].

Cell to cell adhesion provides impetus for positional cellmigration [,"0].(is may suggest that PCP driven events inthe surface ectodermmay play a role in neural tube closure, assuggested in the chick embryo [,#"]. Epidermal constrictionhas also been shown to be crucial for spinal neural tubeclosure in the frog, while the surface ectoderm was shownto be necessary for spinal neural tube closure in the mouse[!/&, ,##].

10. Mouse Mutant Models with a SpinalDefect, Not a Neural Tube Defect

Table % summarizes the ten mouse mutant models thatexhibit a spinal defect alone. Spinal defects encompassmouse mutants with spina bi)da (without any other NTDphenotype, e.g., exencephaly and/or craniorachischisis) andabnormal spinal neural tubes with no spina bi)da.

(e mutants which display only spina bi)da are theFGFR#& chimeric mutant [0%], Traf% mutant [0.], the Shp!chimeric mutant [0$], the axial defects mutant [0&], glial cellmissing-# [0/], and vacuolated lens [00].

All of these mutants have spina bi)da, which denotesincomplete closure of the spinal neural tube. A large majority(% out $ of these mutants which have only spina bi)da) havea second phenotype that is a second neural tube. Vacuolatedlens mutant embryos develop spina bi)da and, in addition,an ectopic neural tube is observed, ventral to the open neuraltube [00]. In Shp!, FGFR#&, and vacuolated lens mutants,an ectopic neural tube is observed during the period ofneurulation between E/.. and E0.. [0%, 0$]. In contrast, anectopic neural tube has only been observed at E#!.. and laterstages in Gcm#mutant embryos [0/].

(e prevalence of an ectopic neural tube in ! out of $mutants at E0..–E#".. seems to suggest that a second neuraltube may be a common occurrence and that this predispo-sition may be the result of an underlying fault in primaryneurulation instead of failure of secondary neurulation.

(ere are many di-erent examples of mouse mutants inwhich the caudal neural tube is abnormal but the phenotypedi-ers from spina bi)da. In many cases, these are describedas spinal neural tube defects [#""–#",]. Apart from the ,mutants with only spina bi)da (Fgfr#, Shp!, andGcm#) whichhave ! neural tubes with one notochord, ! othermutants withspinal defect but no spina bi)da share the same predicament.(ese are the EphA! null mouse [#"#] and PAK% null mouse[#""]. Another abnormal spinal neural tube phenotype is awavy spinal neural tube that occurs in the WASP null mouseand the Vinculin null mouse [#"!, #",]. Vinculin is a large

Scienti)ca #.

T7895 %

Mutant name Genemutated

Function ofprotein

Possiblemechanismof NTD

Schematicrepresentations of ectopic

spinal neural tube

Rate ofoccurrenceof spinabi)da

Phenotypeand reference

Fibroblastgrowth factorreceptor #(knockoutproducingchimeras)

Fgfr! Growth factorreceptor

Unknown(NTDs occur

only inchimaeras)

or

E#".., !0..%have spinabi)da and#.% haveectopic

neural tube

Spina bi)da,second NT;NT in NT &kinky tail

[0%]

Tumournecrosis factorreceptorassociated factor%(knockout)

Traf) Intracellularsignaling adaptor Unknown No ectopic neural tube

%"%homozygousnulls havespina bi)da

Spina bi)da[0.]

Shp!(knockoutproducingchimeras)

Shp&Tyrosine

phosphatase(dephosphorylates

proteins)

Unknown(NTDs occur

only inchimaeras)

E#".., ,$% ofhigh contentchimeras

have secondneural tubeand .0% havespina bi)da

Spina bi)da,second NT

[0$]

Axial defects(spontaneousmutant; genenot identi)ed)

ND ND ND No ectopic neural tube#"%

penetrance inCD#

Spina bi)da[0&]

Glial cellsmissing-#(knockout)

Gcm!Transcription

factor

Ectopicexpressioncauses NTDsby unknownmechanism

and

!../%transgenicshave spinabi)da; #""%transgenicshave ectopicneural tube

Spina bi)da;multiple NT

[0/]

Vacuolated lens(spontaneousmutant)

ND NDSuggestedfailure inappositionand fusion?

."% ofhomozygousnulls show

spina bi)da at#! dpc

Spina bi)da[00]

#$ Scienti)ca

T7895 %: Continued.

Mutant name Genemutated

Function ofprotein

Possiblemechanismof NTD

Schematicrepresentations of ectopic

spinal neural tube

Rate ofoccurrenceof spinabi)da

Phenotypeand reference

PAK%(knockout) PAK) Cytoskeletal

organization

Target forRho GTPase

Cdc%! None

Doubleneural tubewith onenotochordNo spinabi)da[#""]

EphA!(knockout) EphA& Adhesion and

fusion?

Receptortyrosinekinase None

Kinky tailwith doubleneural tubeNo spinabi)da[#"#]

WASP(knockout) WASP Cytoskeletal

organization

Formation ofcell-surfaceprojections()lopodia)required for

cellmovement

andactin-basedmotility

None

Wavy neuraltube

No spinabi)da[#"!]

Vinculin(knockout)(E#" Lethal)

Vinculin Cytoskeletalorganization

Majorconstituent ofcell junctions(cell matrix &

cell-cell)

NoneWavy neural

tubeNo spinabi)da[#",]

Scienti)ca #&

T7895 .

Neural tube structure Genes expressed at

NeuroepitheliumZic& [#"%]Vangl& [#".]

Floor plate and notochordHNF% [#"$]Vangl! [#"&]Wnt%a [#"/]Cyp!$a# [/, #"0]Shh [##]Map,k% [##"]Marcksl# (Mlp) [#/, ###]Traf% [!#, 0.]

NotochordBMP' [##!]Brachyury [$]Shh [#", ##, ##,, ##%]

Surface ectodermFgf( [##.]Grainyhead-like & [##$]BMP' [#"%]Wnt$ [#"%]Notch! [#"/]

Dorsolateral hinge pointsNone

Tips of neural folds at E0..Axin& [##&]Pax% [##/]

Dorsal roof of closed neural tube bridgeZic& [#"%]Msx! [#"%]Wnt! [0, ##0]BMP$ [##0]

Tips of neural folds (surface ectoderm)Grainyhead-like % (in neural ectoderm at E/..) [##$]Par! and Par& [#!"]

#/ Scienti)ca

protein that binds multiple cytoskeletal proteins, actin, &-actinin, talin, paxillin, VASP, ponsin, vinexin, and proteinkinase C (PKC)which have been suggested to be the adhesionsca-old that connects early adhesion sites to actin-drivenprotrusive machinery in enabling motility [,#!].

Abnormal and ectopic spinal neural tubes may beregarded as variant forms of NTDs as it may be possible thatthe neural tube reopens a*er closure due to various reasons.Ectopic neural tube may take on many di-erent variationsapart from the expected second or multiple neural tubes.Among them are a neural tube positioned above anotherneural tube as well as a wavy neural tube phenotype thatis observed in many knockout mice with NTDs. (e wavyregion in these knockout mice has not had its spinal neuraltube sectioned; thus it remains unknown whether the neuraltube remains adhered. Spina bi)da occulta in humans isusually accompanied by various physical abnormalities suchas lipoma, rachischisis, hair tu*s, ectodermal sinuses, skinpigmentation, or diastematomyelia. (ese associated defectsoccur in either syndromic or nonsyndromicNTDs. However,they may be missed and not categorized properly in casesof transgenic mice with possible NTDs. (ere is only oneexample of a null mouse in which these abnormalities havebeen well described which is the Gcm# mouse mutant thatexhibits both open (meningomyelocele) and close (lipomaand diastematomyelia) spina bi)da in its litters [0/].

11. Haploinsufficiency in Mouse and Man

Haploinsu+ciency is poorly studied in both man andmouse.Furthermore, the study of the occurrence of spina bi)dain genes acting in an additive or subtractive manner isalmost unknown. Currently, there are . studies in the mouse,which have demonstrated spina bi)da and the interaction ofthe involved genes mechanistically. (ese include Lrp$ andWnt"a [,#,], Zac! and Suz!& [,#%], Hira and Pax% [,#.], Rybpencompassing Ring! and YYP! [,#$], and haploinsu+ciencyof the components in the primary cilium of the hedgehogpathway [,#&].

(e scenario in humans is somewhat similar in thatthere are % studies to date demonstrating the involvement ofhaploinsu+ciency in the causation of spina bi)da. (e Pax%gene and the EphA) gene act in concert with each other incausing spina bi)da due to interstitial deletion at position!q,$ [,#/]. Furthermore, in the same paper, Goumy et al.[,#/] suggested that a similar phenomenon occurs in themouse when taking into account the spina bi)da phenotypeseen on the Splotch mouse that is a-ected by both Pax% [0,]and EphA) [,#0], albeit the link between the two in themousehas yet to be ascertained. (e hedgehog pathway has alsobeen implicated in humans, where spina bi)da occurs whenPatched is perturbed when implicated with Gorlin syndrome[,!"].(e third and fourth studies implicating human spinabi)da involve haploinsu+ciency in the region of #,q [,!#] and&q [,!!].

12. Conclusion

(is review paper aims to probe spina bi)da, the survivingform of neural tube defects, closely and to analyze the

relationship of what can be learnt from the mouse model ofspina bi)da and to use that knowledge in order to shine abrighter understanding with regard to the human form.

What is very obvious is that there have been amultitude ofgenes (&% according to this review) which regulate speci)callyspina bi)da in themouse.(is is a very high number of genes;therefore the take homemessagewould be in our opinion thatthere are a multitude of genes that can, if perturbed, causespina bi)da. Whether or not these genes cause the conditionor are in fact a player in a pool of numerous genes, which cando the job of closing the spinal neural tube, is a tantalisingidea. (erefore, we put forth the idea that perhaps these &%may be working with other genes in their family or othergenes which share a common pathway in order to close theneural tube. Furthermore, the idea of gene-gene interactionwhich promotes heterogeneity among genes is incompletewithout also considering the idea of haploinsu+ciency ofgenes, where many mutations in mankind are somehowprotected from having a deleterious phenotype by havingother genes compensate the job of the gene or genes beingperturbed. A very good example of this would be the Vangl-# and Vangl-! compound heterozygote mouse mutant whichlacks a single allele of both Vangl-# and Vangl-!; therefore theprobability that the ! genes compensate each other is highand both genes are required in a certain amount of dose,lack of which translates into a neural tube defect phenotype.(erefore, the mouse model which examines the delineationof genes has not completed its true worth until scientistsunderstand the biology of the disease or condition betterby also taking into account (i) the amount (the functioningallele) of the said gene and (ii) the interactionwith other genesin its family which may be able to compensate its functionas well as (iii) the interaction with other genes which share acommonpathway.(emouse is a powerful tool to study spinabi)da because it is amammal like humans and its embryologyis similar to humans and therefore it is an indispensabletool to mechanistically study the structural changes involvedin spinal neural tube closure. (e genes involved in spinalneural tube defects may di-er in man and mouse; however,parallels may be drawn between the principles of how thegenes interact in in'uencing spinal neural tube closure inboth man and mouse.

Competing Interests

(e authors declare that they have no competing interests.

Acknowledgments

(is paper is supported by High Impact Research Grant fromtheUniversity ofMalaya toNoraishahM.Abdul-Aziz (UM.C/$!./#/HIR/"$!–J-!""##-&,.0. and UM.C/$!./#/HIR/#%//!–J-!""##-&,/%,), High Impact ResearchGrant from theMinistryof Higher Education Malaysia to Noraishah M. Abdul-Aziz (UM.C/$!./#/HIR/MOHE/MED/"//"%–E-"""",!), andpostgraduate grant from University of Malaya to Siti W.Mohd-Zin and Noraishah M. Abdul-Aziz (PPP PG#.,-!"#.A). (e authors would like to acknowledge Professor

Scienti)ca #0

Andrew J. Copp and Professor Nicholas D. E. Greene (Uni-versity College London) for helpful discussions.

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