Mini-review: toward understanding mechanisms of genetic neural tube defects in mice

Mini-Review: Toward UnderstandingMechanisms of Genetic Neural TubeDefects in MiceM.J. HARRIS* AND D.M. JURILOFFDepartment of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

ABSTRACT We review the data from studiesof mouse mutants that lend insight to the mechanismsthat lead to neural tube defects (NTDs). Most of the 50single-gene mutations that cause neural tube defects(NTDs) in mice also cause severe embryonic-lethalsyndromes, in which exencephaly is a nonspecificfeature. In a few mutants (e.g., Trp53, Macs, Mlp or Sp),other defects may be present, but affected fetuses cansurvive to birth. Multifactorial genetic causes, as arepresent in the curly tail stock (15–20% spina bifida), orthe SELH/Bc strain (15–20% exencephaly), lead tononsyndromic NTDs. The mutations indicate that ‘‘spinabifida occulta,’’ a dorsal gap in the vertebral archesover an intact neural tube, is usually genetically anddevelopmentally unrelated to exencephaly or ‘‘spinabifida’’ (aperta). Almost all exencephaly or spina bifidaaperta of genetic origin is caused by failure of neuralfold elevation. The developmental mechanisms in ge-netic NTDs are considered in terms of distinct rostro-caudal zones along the neural folds that likely differ inmechanism of elevation. Failure of elevation leads to:split face (zone A), exencephaly (zone B), rachischisis(all of zone D), or spina bifida (caudal zone D). Thedevelopmental mechanisms leading to these geneticNTDs are heterogeneous, even within one zone. At thetissue level, the mutants show that the mechanism offailure of elevation can involve, e.g., (1) slow growth ofadjacent tethered tissue (curly tail), (2) defective fore-brain mesenchyme (Cart1 or twist), (3) defective basallamina in surface ectoderm (Lama5), (4) excessivebreadth of floorplate and notochord (Lp), (5) abnormalneuroepithelium (Apob, Sp, Tcfap2a), (6) morphologicaldeformation of neural folds (jmj), (7) abnormal neuroepi-thelial and neural crest cell gap-junction communication(Gja1), or (8) incomplete compensation for a defectivestep in the elevation sequence (SELH/Bc). At thebiochemical level, mutants suggest involvement of: (1)faulty regulation of apoptosis (Trp53 or p300), (2)premature differentiation (Hes1), (3) disruption of actinfunction (Macs or Mlp), (4) abnormal telomerase com-plex (Terc), or (5) faulty pyrimidine synthesis (Sp). TheNTD preventative effect of maternal dietary supplemen-tation is also heterogeneous, as demonstrated by: (1)methionine (Axd), (2) folic acid or thymidine (Sp), or (3)inositol (curly tail). The heterogeneity of mechanism ofmouse NTDs suggests that human NTDs, including the

common nonsyndromic anencephaly or spina bifida,may also reflect a variety of genetically caused defectsin developmental mechanisms normally responsiblefor elevation of the neural folds. Teratology 60:292–305, 1999. r 1999 Wiley-Liss, Inc.

In mice, more than 50 genes whose mutations lead toneural tube defects (NTDs) are now known. There arealso several inbred strains with multifactorial geneticliabilities to ‘‘spontaneous’’ NTDs. The variety of typesof NTD caused by these genotypes parallels the varietyof NTD types in humans. Several of the mutants andstrains have been studied during the period of embry-onic neural tube formation, and abnormalities contrib-uting to NTDs have been identified. The purpose of thisbrief review is to outline the current understanding ofmechanisms that lead to NTDs in mouse mutants andstrains.

NEURAL TUBE CLOSURE IN MICE

In mouse embryos, the neural tube forms during days8–10 of gestation. The neural folds, comprised of neuro-epithelium (neuroectoderm) and underlying mesen-chyme within a surface ectoderm, elevate on either sideof the midline, bend toward each other, meet at theirtips, and fuse to form a tube (see Figs. 2 and 3). Trunkneural fold elevation occurs first, at the level of the 3rdand 4th somites, and proceeds as a wave both rostrally(a short distance) and caudally to the base of the tail (along distance), requiring about a day and a half forcompletion. At the time when about one-third thelength of the trunk folds have elevated, regions encom-passing the prospective forebrain to the rostral hind-brain begin to elevate. The formation of the optic sulcileads to complete elevation of the forebrain region whilethe midbrain is only half elevated. Midbrain elevation

Grant sponsor: Medical Research Council of Canada; Grant numbers:MT-6766, MT-13675; Grant sponsor: British Columbia Health Re-search Foundation; Grant number: 5(94-1).

*Correspondence to: Dr. M.J. Harris, Department of Medical Genetics,University of British Columbia, 6174 University Boulevard, Vancou-ver, B.C., Canada V6T 1Z3. E-mail: [email protected]

Received 1 January 1999; Accepted 7 July 1999

TERATOLOGY 60:292–305 (1999)

r 1999 WILEY-LISS, INC.

is completed before that of the most caudal region of thetrunk.

Apposed neural folds fuse in the midline and theregional differences in timing of completion of elevationlead to a series of initiation sites of closure, which alsotravels as waves from the points of first contact. Thusthe elevation and subsequent fusion of the neural foldsare intermittent along the length of the neural folds.The sequence of elevation and initiation sites of contactand fusion has been described previously (Macdonald etal., ’89; Sakai, ’89; Copp et al., ’90; Golden and Chernoff,’93). The last regions of the tube to complete elevationand fuse close on day 9 at the head and during early day10 at the base of the tail (Theiler, ’89; Kaufman, ’92).Failure of these closures leads to the common neuraltube defects, exencephaly and spina bifida, respectively(Fig. 1).

LOCATIONS OF NTDS IN MICE REFLECTINGA ZONAL PATTERN OF NEURAL

FOLD ELEVATION

Almost all NTDs in mice involve a failure of neuralfold elevation along some portion of the neural tubeaxis. We suggest that the distinct sites of NTDs inmutant mice, along the rostro-caudal axis — exen-cephaly, spina bifida, split face, or rachischisis — reflectregional differences in mechanisms of normal neuralfold elevation. It can be deduced, both from the morpho-logical sequence of normal neural fold elevation andfrom the specificity of rostro-caudal sites of NTDs in themutants and strains, that in the mouse embryo thereare at least four independent zones of elevation of theneural folds (Fig. 4b) and that the zones depend, at leastin part, on different mechanisms of elevation. Withineach zone there are distinct sites where the tips of the

Fig. 1. Day 13–14 mouse embryos with typical exencephaly (a) or spina bifida (b).

Fig. 2. Conceptual summary of the normal process of neural foldelevation in the mouse. Panels a–c are progressive steps through time.Details and proportions vary across rostro-caudal zones.

GENETIC NEURAL TUBE DEFECTS 293

elevated neural folds initiate contact and fuse, previ-ously described as Closures 1–4 (Figs. 3a,4a) (Goldenand Chernoff, ’83, ’93; Macdonald et al., ’89). To under-stand the mechanisms underlying each of the varioustypes of NTD, and to explain the similarity of NTDsamongst species (e.g., mouse and human), we suggestthat the zonal character of the process of elevation,rather than the fusion initiation sites along the ele-vated folds, should be emphasized (Fig. 4b,c). In mostinstances, failure of neural tube closure results fromfailure of neural fold elevation, not failure of fusion. In

this review, we name the rostro-caudal zones of neuralfold elevation ‘‘zone A’’ through ‘‘zone D’’ (Fig. 4b).

In zone A, the development of the optic sulci (Fig. 3a)appears likely to contribute to the apposition of theneural folds, and failure of contact and fusion of theseapposed neural folds, from initiation site 3 (Fig. 4a),results in a midline cleft of the face from upper lip toforehead. In zone B, the region between the caudalborder of the forebrain and the rostral border of thehindbrain, failure of elevation of the neural folds re-sults in exencephaly. In zone C, the roof of the rhomben-

Fig. 3. Normal cranial neural fold elevation in early Day 9 mouse embryos. (a) Rostral SEM view of14-somite-stage normal embryo, showing neural folds elevating in zone A (optic sulci) and zone B, withneuroepithelia having made contact at Closure 2 initiation site. (b) Transverse histological sectionthrough zone B in 8-somite-stage normal embryo, showing loose mesenchyme, M, and compactneuroepithelium (neuroectoderm), N.

Fig. 4. Zonal pattern of normal neural fold elevation and fusion initiation sites in mouse embryos.Diagrammatic sideviews. (a) Locations of sequential initiation sites of neural fold fusion (1–4). Lines witharrows show directions of closure from initiation sites (triangles). PNP 5 posterior neuropore. (b).Locations of independent zones of neural fold elevation labelled rostral to caudal (A–D). (c) Relation ofzones of neural fold elevation in Day 8–9 embryo to morphology in Day 14 embryo. ex and sb indicatelocations of open neural folds in exencephaly and spina bifida aperta, respectively.

294 HARRIS AND JURILOFF

cephalon, closure is not well understood; isolated lack ofclosure of this region has not been recognized in mousemutants. In zone D, failure of elevation of the neuralfolds for all of the zone results in rachischisis. Elevationwith conversion of the neural folds to concave shapeappears to travel as a rostral to caudal wave throughzone D, caudal to initiation site 1 (see Fig.6a,b in Coppet al., ’90). Failure of the wave of elevation to extend tothe caudal limit of zone D, i.e., failure to close theposterior neuropore, results in spina bifida aperta(SBA), commonly called spina bifida. Although thedefects encephalocele (protrusion of brain and menin-ges through an abnormal opening in the skull, mostcommonly in the occipital or frontal regions) (O’Rahillyand Muller, ’92) and spina bifida occulta (SBO, a dorsalgap in the vertebral arches) arise after neural tubeelevation and fusion, in mouse mutants their locationsalso appear to be related to specific zones.

Clear regional differences are also evident in thecellular events of initial contact and fusion of neuralfolds during neural tube closure. For example, in zoneA, midline contact and fusion of apposed neuroepithe-lium precedes that of surface ectoderm; in zone B,initial midline contact and fusion involves both surfaceectoderm and neuroepithelium; whereas in zone C,fusion of surface ectoderm precedes that of neuroepithe-lium (Geelen and Langman, ’77; Harris et al., ’94).Another example is the change in relative prominenceof median and dorsolateral hinge points (Fig.2b) as thewave of elevation moves through zone D (Shum andCopp, ’96).

HETEROGENEITY OF MECHANISMSLEADING TO NTD

For most of the 50-plus mouse ‘‘NTD mutants,’’ theNTD is a part of a syndrome that includes primarydefects in other developmental systems. Many of themutations cause embryonic death soon after the normaltime of neural tube closure. For these early lethals, thefailure to complete neural tube closure may be areflection of the moribund state and not of a geneticdefect specific to the mechanism of neural tube closureitself. For some mutants that survive longer, the NTD ispart of a syndrome involving other tissues, such asneural crest-derived tissues and limb muscles in thesplotch (Sp) mutant (Franz, ’93). For a few mutants,either the open neural tube is the only major develop-mental defect (nonsyndromic), or the other developmen-tal defects are present in only a subset of the mutantembryos. Individuals with these types of NTD generallysurvive to birth (reviewed in Harris and Juriloff, ’97;Juriloff and Harris, ’98). Most NTD mutations in micecause either exencephaly or spina bifida or both, equiva-lent to the common NTDs in humans (exencephaly inmice is the equivalent of anencephaly in humans).

In inbred strains with multifactorial genetic liability,NTDs are expressed in some individuals in each genera-tion (e.g., 2–20%). The fetuses with NTDs survive tobirth, with no other major primary developmental

defects (reviewed in Harris and Juriloff, ’97; Juriloffand Harris, ’98). In parallel to the NTDs of mousestrains, the common forms of neural tube defects inhumans also are nonsyndromic in this sense, appear tohave multifactorial inheritance, and to survive to lategestation or birth (Seller, ’94).

Taken together, the mouse mutants show that thereare probably different mechanisms specific to each typeof NTD, as well as a few mechanisms held in commonbetween particular types of NTD (Table 1). Many of themutants and strains have exencephaly as their onlyNTD, indicating that there are mechanisms essential toelevation only in zone B. A few mutants have spinabifida aperta only, reflecting a caudal zone D-specificessential mechanism. Some mutations can cause bothexencephaly and spina bifida aperta, demonstratingmechanisms shared between zone B and caudal zone D.Only one mutant (looptail, Lp) has cranio-rachischisis(all of zone D and zone C), and interestingly it com-pletes neural tube closure in zones A and B (Gerrelliand Copp, ’97). A few mutations cause both exencephalyand split face, indicating that some mechanistic aspectsof cranial neural tube closure are shared by zone A andzone B. These appear to be of two types. Embryoshomozygous for the Ap2 (Tcfap2a) gene knockout lackneural fold elevation in both zone A and zone B,demonstrating that some elevating mechanisms areshared between these zones (Schorle et al., ’96; Zhanget al., ’96). In contrast, the Hes1 (Ishibashi et al., ’95)and Ski (Berk et al., ’97) knockouts, and the Rara, Rargdouble knockout (Lohnes et al., ’94) lack neural foldelevation in zone B and have a split face that weinterpret to be due to a lack of fusion of the elevatedfolds of zone A.

There is one exceptional mutant in which a closedcaudal neural tube re-opens. In the Tc/tw5 compoundmutant, the original caudal zone D folds elevate, fuse,and subsequently rupture to form a ‘‘blister’’ under anintact epidermis, a spina bifida aperta, due to a defec-tive dorsal neuroepithelial basal lamina (Park et al.,’89).

The mouse mutations suggest that another form ofNTD, encephalocele, is not a microform of exencephaly.One mutant, ‘‘brain hernia’’ (bh), often has cerebralencephalocele, but it has no exencephaly (Bennett, ’59).None of the strains or mutants that has exencephalyalso has encephalocele.

The mutations also indicate that most spina bifidaocculta (SBO) in mice is caused by a different geneticliability than spina bifida aperta (SBA), involves adifferent set of developmental mechanisms than SBA,and therefore is probably not a microform of SBA. Ofthe mutations examined for both traits, six cause SBObut not SBA and two cause SBA but not SBO. For SBOonly, these are: congenital hydrocephalus (ch, Mf1;Gruneberg, 1963; Kume et al., ’98), patch (Ph, Pdgfra;Payne et al., ’97), snubnose (sno; Hollander, ’76), andengineered mutations of paired-related homeobox gene1 (Prx1, formerly MHox; Martin et al., ’95), transform-


TABLE 1. Selected examples of genetic NTD in mice for which there are glimpses of mechanism

Mutant* or strainand details

Morphological, cellular, or biochemicalprecursor of failure of elevation

of neural foldsPrevention agentand mechanism References

Exencephaly (EX) only: zone B

Apob truncatedprotein30% EX

E9: excessive cell death in neuroepithe-lium of caudal zone B and zone C.

Apolipoprotein B is a lipoprotein thattransports cholesterol, lipids, andvitamin E.

— Homanics et al., ’95

Cart1 knockout65–100% EX

E8.75: excessive cell death in mesen-chyme of zone A.

Cart1 is a transcription factor; targetsunknown.

folic acid; mechanismunknown

Zhao et al., ’96

Gja1 transgene25% EX (homo)10% EX (hetero)knockout‘‘some EX’’

E8.5: Gja1 is expressed in dorsal neuralfolds, neural tube, and neural crest inzones A, B, and D in normal embryos.

Gja1 codes for connexin 43, a compo-nent of gap junctions. Altered expres-sion leads to altered communicationbetween cells.

— Ewart et al., ’97Lo et al., ’97

jmj jumonjigene trap0–50% EX

E8.5: transverse groove across zone Bneural folds.

— Takeuchi et al., ’95

Lama5 knockout60% EX

E8.7: Laminin alpha 5, a component ofbasal laminae, is present in surfaceectoderm and neuroectoderm of neuralfolds. In Lama52/2 embryos, the basallamina of surface ectoderm in zone Bneural folds is thin and patchy.

— Miner et al., ’98

Macs knockout25% EX

E8.5–9: Macs is expressed specificallyin zone B and caudal zone D neuralfolds (neuroepithelium, mesenchyme,surface ectoderm) in normal embryos.

MARCKS is a cell membrane-associatedprotein, phosphorylated by activatedprotein kinase C (PKC), that cross-links actin when unphosphorylated.

— Blackshear et al., ’96Stumpo et al., ’95

nt no turningspontaneous‘‘some’’ EX

E8.5: neural fold elevation is delayed(zones A–D).

E9: some neural tubes remain open incaudal zone B.

Embryos do not complete ‘‘turning’’;heart loop is to right or left (lateralityrandom). All die by E12.

— Melloy et al., ’98

p300 knockout100% EX ( p3002/2)5–20% EX ( p3001/2)

Both zone B and zone C remain open inhomozygotes; only zone B is open inheterozygotes.

p300 is a transcription coactivator thatinteracts with multiple transcriptionfactors, including retinoic acid recep-tors (RARs). RAR activity is reducedin knockout.

— Yao et al., ’98

SELH/Bc spontaneousmultigenic15–20% EX

E8.5: zone B neural folds are abnormallyflat instead of convex and fail to elevate.

E9.5: failure of zone B elevation in allE8 embryos is ‘‘rescued’’ in 80–85% ofembryos by a later elevation, pro-ceeding caudally from zone A.

alternative normaldiet; mechanismunknown

Macdonald et al., ’89Juriloff et al., ’89Gunn et al., ’93Harris and Juriloff,

’98Juriloff and Harris, ’98

Trp53 ‘‘p53,’’ knockout20–30% EX in females;0–1% EX in males

p53 is a transcription factor involved inapoptosis.

p53-induced transcription is modulated bya complex of p300 and p53.

— Sah et al., ’95Armstrong et al., ’95Lill et al., ’97

twist knockout100% EX

E8.5: zone A mesenchyme cells are abnor-mally round.

E9: zone A mesenchyme cells are deficientand zone B mesenchyme cells areabnormally round.

Twist is a transcription factor expressedin cranial mesenchyme during neuraltube closure in normal embryos.

— Chen and Behringer,’95

TABLE 1. Selected examples of genetic NTD in mice for which there are glimpses of mechanism (continued)




xn exencephalyspontaneous35–85% EX

E8.5: zone B folds fail to elevate. — Wallace et al., ’78Anderson, ’81

Exencephaly (EX) and split face (SF): zones A and B

Hes1 knockout70% EX & SF

E8.5: zone B folds fail to elevate; zone Afolds elevate but fail to fuse.**

Hes1 is a transcription factor expressedin cranial neuroepithelium and mes-enchyme in normal embryos.

E8.5: Mash1, a neural differentiationfactor antagonized by Hes1, isexpressed in only midbrain neuralfolds in normal embryos, but in bothmidbrain and forebrain (zones A & B)neural folds in Hes12/2 embryos.

— Ishibashi et al., ’95

Rara/Rargdouble knockout45% EX, 100% SF

Based on E18 phenotype, zone A foldsappear to elevate but not fuse,whereas zone B folds fail to elevate.**

— Lohnes et al., ’94

Ski knockout85% EX, 15% SF

Based on E18 phenotype, zone A foldsappear to elevate but not fuse,whereas zone B folds fail to elevate.**

— Berk et al., ’97

Tcfap2a ‘‘Ap2,’’ knockout100% EX & SF

E8.5–E9: zones A, B, and C folds fail toelevate. Ap2 is a transcription factorstrongly expressed in cranial neuralfolds and emigrating neural crestcells in normal embryos.

E9: ectopic expression of Ncamthroughout cranial neuroepithelium,instead of at only the tips of the folds,and excessive cell death in zone Bneuroepithelium.

— Zhang et al., ’96Schorle et al., ’96

Exencephaly (EX) and spina bifida aperta (SBA): zone B and caudal zone D

Axin Fu, fused(Axin gene)spontaneous10% EX25% SBA

E9: irregular folding of neural tube.Secondary neural tubes form parallelto the primary caudal neural tube. Axinis a negative regulator of the Wnt sig-nalling pathway in normal embryos.

— Theiler and Glueck-sohn-Waelsch, ’56

Zeng et al., ’97

ct curly tailspontaneous1–5% EX15–20% SBA

E9: Slow growth of ventral tail bud(hindgut, notochord), tethered todorsal tail bud (neuroepithelium)which is growing normally, causesventral bend in body that interfereswith elevation of caudal zone Dneural folds and posterior neuropore(PNP) closure.

Rarb is underexpressed in hindgut ofembryos that have delayed PNP clo-sure.

growth retardants(e.g., fasting, reti-noic acid, DNAinhibitors) realigngrowth schedules.

insertion of eyelashstraightens caudalembryo.

inositol upregulatesRarb in hindgutand reduces delayin PNP closure.

Copp et al., ’88Seller, ’94Peeters et al., ’96Peeters et al., ’98Brook et al., ’91Chen et al., ’95Greene and Copp, ’97

Mlp knockout55–100% EX0–15% SBA

E8–8.5: Mlp is expressed over wholelength of neural tube in lateral edgesof neuroepithelium; enriched in cranialmedian hingepoint; detectable in cra-nial mesenchyme in normal embryos.

Mlp (MARCKS-like protein) has func-tion similar to MARCKS (see Macsabove), with role in actin organization.

— Wu et al., ’96Chen et al., ’96

Nog knockout‘‘some’’—100% EX‘‘some’’ SBA

E8.5: Nog is expressed in dorsal neuralfolds in zone D in normal embryos. ZoneB neural folds are flat at the 8–9-somitestage in Nog2/2 embryos. Noggin is asignalling molecule that antagonizesBMPs.

— McMahon et al., ’98

opb open brainspontaneous80–100% EX2% SBA

E9.5: zone B neural folds fail to completeelevation.

— Sporle et al., ’96Gunther et al., ’97

TABLE 1. Selected examples of genetic NTD in mice for which there are glimpses of mechanism (continued)




Pax3 Sp, splotch(Pax3 gene)spontaneous50–100% EX25–100% SBA

E8–E9: Pax3 is expressed in dorsal neu-roepithelium of cranial and trunkneural folds and in subsets ofmigrating neural crest cells innormal embryos.Pax3 is a transcription factor, whosetargets are thought to include Ncamand Ncad.

E8.5–E9.5: pyrimidine synthesis is defi-cient in splotch homozygotes.

folic acid or thymi-dine; mechanismunknown

Goulding et al., ’91

Fleming and Copp,’98

Fleming and Copp,’98

E9: splotch homozygotes have completeloss of emigration of neural crest cellsfrom caudal neural folds. The defectappears to be extrinsic to the neuralcrest cells, but in the neural tube.

Serbedzija andMcMahon, ’97

E9: excess chondroitin sulfate proteo-glycans in basement membrane ofneuroepithelium in splotch homozy-gotes.

Trasler and Morriss-Kay, ’91

E9: versican, a chondroitin sulfate pro-teoglycan that interfers with neuralcrest cell migration, is overexpressedin splotch postotic paraxial meso-derm.

Henderson et al., ’97

E9: NCAM, a cell adhesion moleculenormally expressed in neural plateprior to neural tube formation, isabnormally glycosylated in splotchneural folds.

Moase and Trasler,’91

Neale and Trasler,’94

E9: Ncad, a cell adhesion molecule withnormal peak expression in cranialneural folds at time of zone B eleva-tion, is overexpressed in splotchneural folds.

Bennett et al., ’98

E9: Msx2, a transcription factor that isnormally expressed in neural tubeand migrating neural crest cells, isoverexpressed in splotch neural tubeand premigratory and migratory (car-diac) neural crest.

R. Maxson, personalcommunication

E9: neuroepithelium is disorganized insplotch neural folds, with excessintercellular space.

Yang and Trasler, ’91

Spina bifida aperta (SBA) only: caudal zone D

Axd axial defectsspontaneous50–100% SBA

unknown methionine; mecha-nism unknown

Essien, ’92Essien and Wann-

berg, ’93

vl vacuolated lensspontaneous,40% SBA

E9–E10: caudal zone D neural foldshave an excessively wide angle at theventral midline.

Apical actin microfilaments in caudalneuroepithelium are less prominentand more delicate in vl/vl than innormal.

— Wilson and Wyatt,’92a

Cranio-rachischisis: zones D and C

Lp looptailspontaneous100% cranio-rachischisis

E8.5: floorplate and notochord areexcessively broad under entire lengthof zone D neural folds; zone D neuralfolds fail to evaluate.

Shh and Netrin are overexpressed infloorplate.

Brachyury is overexpressed in noto-chord.

Zone A and zone B neural folds elevateand fuse; zone C fails.

— Wilson and Wyatt,’92b

Gerrelli and Copp,’97

Greene et al., ’98

*NTD phenotype in homozygote unless indicated otherwise.**Our interpretation of the published phenotype.

ing growth factor beta 2 (Tgfb2; Sanford et al., ’97), ortype II collagen (Col2a1; Savontaus et al., ’97). For SBAonly, these are: curly tail (ct; Embury et al., ’79) andsplotch (Sp, Pax3; Auerbach, ’54; Kapron-Bras andTrasler, ’85). Possible exceptions to this pattern are themutant fused (Fu), in which some homozygotes haveSBA and some may have SBO (Theiler and Gluecksohn-Waelsch, ’56), and the curtailed (Tc) mutation which,like Fu/Fu, causes ventral duplication of the caudalneural tube, with SBA in Tc/tw5 and SBO in Tc/1 (Parket al., ’89). The mechanism leading to SBO has beenstudied in detail in only one mutant, homozygous patch(Ph, at the Pdgfra locus). In Ph/Ph embryos, the neuralfolds elevate and fuse normally on day 9–10 of gesta-tion. A later defect of cell density in the somitic meso-derm on days 11–14 causes lateral displacement of theforming vertebral arches. On day 17 the vertebralarches are splayed laterally along the entire spinalcolumn (SBO) and the neural tube is normal (Payne etal., ’97). A second mechanism underlying SBO may bedemonstrated by homozygotes for a truncated type IIcollagen gene Col2a1, which have normal neural tubeclosure in embryos and subsequent vertebral defectswith frequent lumbar SBO in fetuses, due to retardedgrowth of cartilage in the vertebral arches (Savontauset al., ’97).

MECHANISMS LEADING TO FAILUREOF NEURAL FOLD ELEVATION

The morphological and histological processes of neu-ral tube maldevelopment (days 8–10 of gestation) havenot yet been examined closely in most of the mutantsand affected strains and, although the gene ‘‘knockout’’mutations that cause NTDs inactivate genes for pro-teins whose cellular biochemical functions are at leastpartly known, the role of these biochemical functions inneural tube closure is not yet known. However, for somemutants and strains part of the sequence of the morpho-logical or biochemical events has been observed andeach illuminates one or more aspects of the mecha-nisms that lead to NTDs (Table 1). The fragmentaryinsight into mechanisms gleaned from mutants is basedon patterns of gene expression, biochemical pathways,cellular anomalies, or morphology of tissues; i.e., thetype of knowledge of mechanism in the various mutantsis not parallel.

All the mouse mutants and strains with exencephalythat have been examined developmentally (Apob, Cart1,Gja1, Hes1, jmj, Lama5, Macs, Mlp, Nog, nt, opb, Sp,Tcfap2a, twist, xn, SELH/Bc; references in Table 1)have demonstrated the same mechanism, a failure ofelevation of the midbrain (zone B) neural folds. The onemutant with cranio-rachischisis (Lp; Table 1) has afailure of elevation of trunk neural folds, from hind-brain to tail (all of zone D). Similarly, in three mutantswith spina bifida aperta (ct, Sp, vl; Table 1) the neuralfolds in caudal zone D fail to complete elevation and,therefore, do not fuse.

Although the ‘‘mechanism’’ of NTD has been observedin almost all cases to be a failure of neural foldelevation, the complete sequence of steps involved, fromabnormal genes to failure of elevation of neural folds, isnot known for any mutant or strain. The assortment ofglimpses of mechanisms leading to failure of neural foldelevation is summarized below, with mutants groupedtogether that appear to share mechanisms.

At the tissue level

1. Primary defect in adjacent tethered tissue (caudalzone D). The mechanism of cause of nonsyndromicspina bifida aperta is best understood for the curly tail(ct) model, in which spina bifida occurs in 15–20% ofembryos (Embury et al., ’79). The primary developmen-tal defect in curly tail appears to be in the adjacenttissue ventral to the posterior neuropore (PNP) and notin the neural folds themselves. Slow growth of theventral tail bud (which becomes the hindgut and noto-chord), tethered to the overlying dorsal tail bud (neuro-epithelium), which is growing normally, causes a tran-sient abnormal ventral bend that interferes withapposition of the caudal neural folds (Peeters et al., ’96,’98). Treatments that slow overall growth at this spe-cific time (day 9) and allow synchronization of growth ofthe neuroepithelium with the underlying tissues (Coppet al., ’88; Seller,’94; Peeters et al., ’96), or that physi-cally straighten the caudal region (Brook et al., ’91)result in more successful PNP closure and lower fre-quency of spina bifida.

The genetic cause of spina bifida in the curly tailmodel involves the joint effects of several genes, ofwhich two are mapped (Neumann et al., ’94; Letts et al.,’95). None of the genes has been identified. However, astep in the path between the causal mutations and thedeficiency in hindgut growth appears to involve theretinoic acid receptor beta (Rarb). Rarb is implicatedfrom a variety of approaches. First, a study of genesthat are normally expressed in the PNP region at thetime of PNP closure showed that the curly tail embryoswith delayed PNP closure have a deficiency of Rarbtranscripts in hindgut endoderm, the slow-growingtissue (Chen et al., ’95). Second, retinoic acid treatmenton day 9 of gestation upregulates Rarb and prevents50% of the tail defects in curly tail mice (Chen et al., ’94;Chen et al., ’95). Third, the delay of PNP closure isreduced and spina bifida in curly tail mice is prevented(70%) by supplementation with the vitamin inositol,which also leads to upregulated Rarb expression in thehindgut (Greene and Copp, ’97). The signalling path-way to Rarb is hypothesized to be mediated by upregu-lated protein kinase C (PKC), and this hypothesis issupported by the observation that a phorbol ester thatupregulates PKC also leads to upregulated Rarb andless delayed PNP closure (Greene and Copp, ’97).Whether the upregulation of Rarb by these treatmentsis a cause or a reflection of increased hindgut growth isnot known. Target genes regulated by Rarb in the curlytail hindgut are yet to be identified.


2. Defective mesenchyme in zone A causing failure ofelevation in zone B. The forebrain mesenchyme ofCart1 homozygous knockout embryos has abnormalextensive cell death during the early stages of cranialneural fold elevation. It appears that the midbrainmesenchyme (Fig. 3b) may be displaced in part, to fillthe void, and that the failure of zone B neural foldelevation may be attributable to a lack of supportingmesenchyme. It was reported that maternal treatmentwith folic acid reduced the frequency of exencephaly(Zhao et al., ’96), but the effects on cell death and themetabolic link between Cart1, a transcription factor,and folate metabolism are unclear.

Another knockout mutant, twist, also has abnormalfunction and deficiency of forebrain mesenchyme dur-ing cranial neural tube closure and failure of zone Belevation and exencephaly (Chen and Behringer, ’95).

3. Defective basal lamina in surface ectoderm (zoneB). In normal day 8.7 embryos, the basal laminaunderlying the surface ectoderm of the neural folds iscontinuous. In the cranial neural folds of embryoslacking laminin alpha 5 (Lama5), this basal lamina isthin and patchy. The folds (zone B) often fail to elevate,and 60% of Lama5-/- embryos have exencephaly (Mineret al., ’98). Schoenwolf and colleagues (Hackett et al.,’97) have suggested, from experiments in the trunkregion of chick, that the strip of surface ectoderm thatborders the neural folds provides a necessary compo-nent of the normal force required to elevate the neuralfolds. Miner et al. (’98) suggest that a lack of thiselevating force, caused by weakness in the basal laminaof the surface ectoderm, may be the mechanism offailure in Lama5-/- mouse embryos.

4. Mechanical interference by abnormal floorplateand notochord (zone D). In looptail (Lp) homozygotes,the neural folds fail to elevate along the entire length ofthe neural tube caudal to the midbrain (zone C and allof zone D), leading to cranio-rachischisis in all embryos.In looptail embryos on day 8.5, just prior to the start ofnormal zone D elevation, the notochord and ventralmidline of the neural plate are excessively broad in theregion at and caudal to the Closure 1 initiation site(Wilson and Wyatt, ’92b; Gerrelli and Copp, ’97; Greeneet al., ’98). Shh and Netrin are overexpressed in themidline floorplate and Brachyury is overexpressed inthe notochord (Greene et al., ’98). It is thought that thebroad floorplate and enlarged notochord interfere withneural fold apposition along the length of the zone Dneural tube (Greene et al.,’98). Zone C closure also failsin Lp/Lp, suggesting that rostral zone D closure maybe necessary as an initiation point for zone C closure.

5. Abnormality of neuroepithelium (zones A, B, andC). Two mutants, the Tcfap2a (Ap2) knockout and theApob truncation mutation, indicate that excessive celldeath in the cranial neuroepithelium (Fig. 3b) duringthe normal time of neural fold elevation creates risk offailure of zone B neural fold elevation and exencephaly.In the Tcfap2a mutant, cell death occurs in zone Bneuroepithelium (Schorle et al., ’96), and elevation fails

in zone A as well as in zone B. Ncam expression in theunelevated neural folds is subsequently abnormallywidespread instead of being normally restricted to thetips of the neural folds (Zhang et al., ’96). In the Apobmutant, cell death occurs in the neuroepithelium ofcaudal zone B and rostral zone C and elevation oftenfails in zone B (Homanics et al., ’95). Excessive celldeath in the cranial neuroepithelium has also beenreported in a third mutant, the Ski knockout, but theembryos were examined for cell death subsequent tofailure of zone B elevation (Berk et al., ’97), andwhether cell death has a causal role is unclear.

Failure of zone B neural fold elevation arising fromeither lack of expression or overexpression of the gap-junction protein gene, Gja1 (a connexin, Cx43), through-out all zones in the dorsal neural tube and neural crestsuggests that gap-junction intercellular communica-tion is particularly important to zone B neural foldelevation (Ewart et al., ’97; Lo et al., ’97).

The mutant jumonji (jmj) demonstrates a novel typeof mechanical interference with neural fold elevation inzone B. At the normal time of cranial neural foldelevation, a deep transverse groove or fold affecting allcell layers (including neuroepithelium) at caudal zoneB has been reported (Takeuchi et al., ’95). The cellularevents leading to the abnormal fold have not beendescribed.

6. Loss of semiredundant zone B elevation mecha-nism. In the SELH/Bc strain, there is failure of zone Bcranial fold elevation in all embryos at the usual6–8-somite (pair) stage, followed by ‘‘rescue’’ by lateelevation at the 15–20 somite stage in 80–90% ofindividuals (Macdonald et al., ’89). This leads to anunusual pattern of initiation sites of fusion, with clo-sure extending from the rostral Closure 3 initiation sitethrough zone B, and with absence of the normal Closure2 initiation site. Notably, about 10–20% of embryosapparently fail to meet the threshold of ‘‘elevatingforce’’ required for rescue, and their neural folds remainsplayed open, leading ultimately to exencephaly (Juri-loff et al., ’89; Macdonald et al., ’89; Gunn et al., ’95).The difference between those embryos that succeed inelevating their neural folds and those that do not isthought to derive from the stochastic nature of biochemi-cal processes within cells.

The defect in SELH/Bc mice appears to be a failure ofa normal elevation mechanism in zone B neural folds.The shape of the normally convex open cranial neuralfolds is abnormally ‘‘flat’’ in SELH/Bc embryos at the3–5 somite stage, just prior to the time of normalelevation (Gunn et al., ’93), possibly signifying anabnormality of cell behavior related to the elevationprocess, although neuroepithelial cellular shape andorganization appear normal (Macdonald et al., ’89;Gunn et al., ’93). An unknown component differingbetween two commercial Purina diets alters the fre-quency of exencephaly by threefold (10% vs. 30%;Juriloff and Harris, ’98).


Like the SELH/Bc strain, other strains and somemutants (e.g., NZW-xid strain, Trp53-/-, Macs-/-) haveexencephaly with ‘‘low penetrance’’ (Harris and Juriloff,’97). It is not known whether, as in SELH/Bc, thenonexencephalic mutant embryos have been rescued bya secondary late elevation that compensates for aprimary defect in the zone B elevation mechanism. Thelarge number of mutations that, in contrast, causehighly penetrant exencephaly in mice (at least 30)suggests that there are also mechanistic pathways thatare absolutely required for zone B neural fold elevationand that cannot be replaced by a compensatory mecha-nism.

At the biochemical level

1. Fragments of molecular genetic regulation path-ways involved in neural fold elevation (zones A and B).Knockout of the gene for p53 (Trp53), a transcriptionfactor and ‘‘tumor suppressor’’ that regulates apoptosis,causes exencephaly in 20–30% of female, but not male,homozygotes (Armstrong et al., ’95; Sah et al., ’95).Neither the developmental mechanism nor the reasonfor the strong gender difference is known. The molecu-lar pathway could involve p300, a transcription coacti-vator known to complex with p53 that may modulatep53-induced transcription-dependent apoptosis (Lill etal., ’97). Interestingly, the p300 gene knockout itself hasexencephaly (Yao et al., ’98). p300 also interacts withother transcription factors, including the retinoic acidreceptors (RARs). RAR activity was observed to bereduced in the p300 knockout (Yao et al., ’98), andhomozygotes for a double knockout of RARs, Rara andRarg, also often have exencephaly (Lohnes et al., ’94).

Hes1 is a transcription factor whose function antago-nizes the neural differentiation factor, Mash1, andwhose knockout has exencephaly. In Hes1 knockouts,Mash1 is ectopically expressed in zone A neuroepithe-lium, leading to premature differentiation of zone Aneuroepithelial cells (Ishibashi et al., ’95). The mecha-nism leading to failure of elevation of zone B andexencephaly is not known.

Noggin is a signaling molecule known to be anantagonist of bone morphogenetic proteins (BMPs).Nog knockout mutants have failure of elevation of zoneB neural folds (McMahon et al., ’98). It has beensuggested that the role of noggin is to temporarilyshield the dorsal aspect of the neural folds from theinfluence of BMP4, because the BMP-responsive gene,Msx1, has been observed to be precociously expressed inthe dorsal neural tube of Nog-/- embryos (caudal, day10.5; McMahon et al., ’98; Smith, ’99).

mTR or Terc, the gene for the RNA component of thetelomerase complex,which extends telomeres by addi-tion of telomeric repeats, is abundantly expressed inneural folds of day 8.5 mouse embryos (Herrera et al.,’99); 10–30% of embryos lacking Terc have NTDs on day10.5, with no other apparent defect. The zones affectedappear to be an unusual mix of zone A, B, C, or caudal D(Herrera et al., ’99).

2. Role for actin in neural fold elevation (zone B andcaudal zone D). MARCKS (Macs) and MARCKS-likeprotein (Mlp) are associated with the inside of the cellmembrane, are phosphorylated by activated proteinkinase C (PKC) and cross-link actin when unphosphory-lated, thus regulating the arrangement of actin mol-ecules at the cell membrane (Blackshear et al., ’96). Anactin-based change in neuroepithelial cell shape, wedg-ing, is hypothesized to provide a major force for eleva-tion of the neural folds (Sadler et al., ’82). The signal toredistribute actin to produce the wedge shape likelyinvolves PKC (Chen et al., ’96). Knockout of Macscauses failure of neural fold elevation in zone B (Stumpoet al., ’95; Blackshear et al., ’96); knockout of Mlpcauses failure in both zone B and caudal zone D (Chenet al., ’96; Wu et al., ’96).

At the time of normal elevation of the caudal neuralfolds, those of vacuolated lens (vl) mutant homozygoteshave an excessively wide angle at the midline. Theactin microfilaments in the apices of the neuroepithe-lial cells in the caudal neural folds are less prominentand more delicate in vl/vl than in normal and approxi-mately 40% of these homozygotes later have spinabifida (SBA; Wilson and Wyatt, ’92a). The molecularproduct of the vl gene is not known.

3. Requirement for methionine in neural fold eleva-tion (zone B and caudal zone D). ‘‘Axial defects’’ (Axd) isone of the few mutants with spina bifida aperta and noexencephaly (Essien et al., ’90). It is also the onlymutant for which methionine supplementation has hada preventative effect on the NTD (Essien, ’92; Essienand Wannberg, ’93). The preventative effect of methio-nine in Axd/Axd is specific; neither folinic acid (themetabolically active form of folic acid) nor vitamin B12(a cofactor in the synthesis of methionine and folic acidfrom homocysteine and 5-methyl-tetrahydrofolate) iseffective (Essien and Wannberg, ’93). Methionine isessential for elevation and apposition of cranial neuralfolds in normal embryos grown in vitro (Coelho andKlein, ’90) and for normal methylation and localizationof actin and ab-tubulin in neuroepithelial cells; withoutmethionine the cells become round rather than colum-nar (Moephuli et al., ’97). By contrast to Axd, in splotch(Sp) mutants, methionine supplementation increasesrather than decreases the frequency of spina bifida(Fleming and Copp, ’98). The Axd gene locus is not yetmapped, a necessary step before Axd/Axd embryos canbe identified so that neural fold elevation and theembryonic mechanisms of failure and rescue can beobserved.

4. Compensation for faulty pyrimidine biosynthesis byfolic acid or thymidine (zone B or caudal zone D).Splotch (Sp) homozygotes have a high frequency of

both exencephaly (zone B) and spina bifida (caudal zoneD) (Auerbach, ’54; Kapron-Bras and Trasler, ’85; Moaseand Trasler, ’87). At the time of neural tube closure,splotch embryos have a metabolic deficiency in thesupply of folate for the biosynthesis of pyrimidine, adeficiency that can be compensated for by supplemental


thymidine or folic acid, either of which reduces the riskof subsequent NTD. This provides the first evidencethat faulty folate metabolism has a direct role in thecause of NTDs (Fleming and Copp, ’98). How thesplotch mutation causes lack of folate, and how the lackof folate causes NTD risk are unknown.

Previous studies on neural fold elevation in splotchhave pointed toward several different potential mecha-nisms for NTDs in this mutant. One potential mecha-nism involves the observed failure of emigration ofneural crest cells from the neuroepithelium of theelevating neural folds of the caudal trunk (Serbedzijaand McMahon, ’97). Chondroitin sulfate proteoglycansare present in excess in the neuroepithelial basementmembrane in splotch neural folds (Trasler and Morriss-Kay, ’91). The gene for versican, a chondroitin sulfateproteoglycan that is nonpermissive for neural crest cellmigration, is overexpressed in splotch postotic, par-axial mesoderm (Henderson et al., ’97), at least afterthe normal time of cranial neural tube closure. The celladhesion molecule, NCAM, is abnormally glycosylatedin cranial tissue of day 9 splotch embryos (Moase andTrasler, ’91; Neale and Trasler, ’94), and Ncad, the genefor another cell adhesion molecule, is overexpressed insplotch neural folds (Bennett et al., ’98). Any or all ofthese might interfere with neural crest cell emigration,as has been demonstrated for versican (Henderson etal., ’97). Other studies have shown abnormal spacesbetween neuroepithelial cells in splotch embryos (Yangand Trasler, ’91).

Splotch mutations are defects in a gene for a transcrip-tion factor, Pax3, and some of the genes regulated byPax3 are known. The mechanism causing exencephalyin splotch may involve the regulation of Msx2. Msx2 is atranscription factor normally expressed in the dorsalneural tube on day 9 (Wang et al., ’96) and it causesexencephaly when overexpressed (Winograd et al., ’97).In splotch, with Pax3 disrupted, Msx2 is overexpressedin the neural tube (R. Maxson, personal communica-tion).

The splotch mutant is an example of the complexityof deducing the molecular mechanism leading to theNTD even when the function of the disrupted gene isknown. There is no predictable connection betweenPax3 gene function and pyrimidine biosynthesis, and itis not yet known how the faulty pyrimidine biosynthe-sis relates to the multiple potential mechanisms forNTD in splotch.

PERSPECTIVES

The fragments of data that bear on mechanisms ofNTDs in mutant mice show that there are numerousways in which the elevation of the neural folds can becompromised. So far, most of the engineered NTDmutations in mice are null (total loss of function)mutations, most cause NTDs as part of severe embry-onic-lethal syndromes and would seem to give littleinsight into specific mechanisms of NTDs. It is not yetknown whether other types of alteration of these same

genes, changes that retain partial gene function, areinvolved in the mouse strains with ‘‘low penetrance’’(15–20% risk) nonsyndromic NTD, or whether anothertype of gene is involved in multigenic NTD. That someof the genes could be the same is suggested by muta-tions at the Apob locus. All homozygotes for the Apobnull mutation have exencephaly and other lethal de-fects and die by embryonic day 11 (Farese et al., ’95),whereas homozygotes for an Apob truncated mutationhave 30% nonsyndromic exencephaly and survive tobirth (Homanics et al., ’95).

Any human homologs of the many mouse gene knock-out mutations in which homozygotes have NTDs butdie as embryos are expected to be very rare (as is usualfor recessive lethal mutations), and if the humancounterpart is also an embryonic lethal, the NTD wouldlikely go unrecognized among early spontaneous abor-tuses. Notably, PAX3 mutation homozygotes are rareamong human NTD cases (e.g., Chatkupt et al., ’95).However, the nonsyndromic ‘‘low penetrance’’ NTDmouse mutants and strains, like p53 mutation homozy-gotes (Trp53-/-), curly tail, and SELH/Bc, that surviveto birth could have clinically recognizable human coun-terparts.

The several mouse NTD mutants that respond tovarious maternal nutritional supplements (Table 1;Axd, Cart1, Sp, curly tail, SELH/Bc) provide a resourcefor study of mechanisms of modification of risk of NTDs.The findings of reduction of risk of human NTDs bymaternal periconceptional folate supplementation(Wald, ’94), the high homocysteine level in mothers ofNTD infants (Mills et al., ’96), the association of aparticular allele at the MTHFR locus with NTD risk(Christensen et al., ’99) are striking biological corre-lates, but it is not possible in humans to observe theunderlying developmental mechanisms. That each ofthe responsive mouse mutants responds to a differentsupplement again indicates heterogeneity of NTD mech-anism.

CONCLUSIONS

Studies of the mouse mutations that cause NTDs inhomozygous embryos that are viable to fetal life andthat are therefore potential homologs of human NTDs(e.g., Apob, Cart1, ct, Gja1, Lama5, Lp, Macs, Mlp, Sp,Tcfap2a, Terc, Trp53), suggest that the primary me-chanical defect leading to the NTD can be, e.g., in theneuroepithelium itself, in the adjacent surface ecto-derm, or in the adjacent ventral tissues. Heterogeneityof NTD mechanism is also apparent in the zonalpattern of NTD susceptibility in mouse mutants (Table1). For example, SELH/Bc embryos are at risk for onlyexencephaly (zone B), curly tail embryos are at riskmostly for spina bifida (caudal zone D), and splotchembryos are at high risk for both exencephaly andspina bifida. It is likely that the mechanisms that canlead to NTDs in humans, as in mice, are heterogeneous.As in mice, human NTDs have a zonal location, witheither anencephaly or spina bifida being the common


forms. Whether there is a mixture of human genotypesthat differ in risk for one or other of these two types ofNTD is not clear (e.g., Hall et al., ’88).

We anticipate that, as a benefit of the genome sequenc-ing projects, there will soon be conceptual bridgesbetween the current fragments of understanding ofbiochemical pathways leading to NTDs in mice, as moreof the steps in essential pathways will be identified byNTDs in homozygotes with complete and partial knock-outs of newly discovered genes. It seems likely thatmany of the mouse NTD mutants will shuffle intogroups that represent sequential steps in shared bio-chemical pathways. One challenge will be to under-stand how molecules translate into a variety of cell-based mechanical forces causing a major morphogenetictissue reorganization that creates the neural tube. Forteratologists, a key challenge will be to identify themechanisms that compensate for defective molecularpathways and to use them in further approaches toprevention of NTDs.

ACKNOWLEDGMENT

The authors thank Eric Leinberger for technicalassistance with Figures 2 and 4.

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Mini-review: toward understanding mechanisms of genetic neural tube defects in mice

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