PATTERNS

Folic Acid and Homocysteine Affect Neural Crestand Neuroepithelial Cell Outgrowth andDifferentiation In VitroMarit J. Boot,1 Regine P.M. Steegers-Theunissen,2,3 Robert E. Poelmann,1 Liesbeth Van Iperen,1

Jan Lindemans,4 and Adriana C. Gittenberger-de Groot1*

The beneficial effect of additional folic acid in the periconceptional period to prevent neural tube defects, orofacialclefts, and conotruncal heart defects in the offspring has been shown. Folate shortage results in homocysteineaccumulation. Elevated levels of homocysteine have been related to neural tube defects. We studied the behavior ofneuroepithelial cells and cranial and cardiac neural crest cells in vitro. Neural tube explants were cultured for 24 and48 hr in medium after addition of folic acid and/or homocysteine. Folic acid addition increased neuroepithelial celloutgrowth and increased neural crest cell differentiation into nerve and smooth muscle cells. Addition ofhomocysteine increased neural crest cell outgrowth and migration from the neural tube and inhibited neural crest celldifferentiation. Our findings suggest that neural tube defects caused by folate deficiency and hyperhomocysteinemiadevelop due to increased neuroepithelial to neural crest cell transformation. This increased transformation leads toa shortage of neuroepithelial cells in the neural tube. Defects in orofacial and conotruncal development are explainedby abnormal differentiation of neural crest cells in the presence of high homocysteine concentrations. Our findingssupports a critical role for folic acid and homocysteine in the development of neural tube defects and neural crestrelated heart malformations. Developmental Dynamics 227:301–308, 2003. © 2003 Wiley-Liss, Inc.

Key words: congenital heart defects; neural tube defects; chick embryo; smooth muscle; actin

Received 4 November 2002; Accepted 17 February 2003

INTRODUCTION

Periconceptional use of multivita-mins reduces the risk of neural tubedefects (Mulinare et al., 1988; Czei-zel and Dudas, 1992), orofacial clefts(Tolarova, 1982; Shaw et al., 1995a),and limb and conotruncal heart de-fects (Shaw et al., 1995b; Botto et al.,1996) in the offspring. To evaluatewhether the B vitamin folate is re-sponsible for the risk reduction, sev-eral studies have been performedsupplementing women with the syn-

thetic form of folate, i.e., folic acid(Laurence et al., 1981; MRC VitaminStudy Research Group, 1991). Theclinical trial performed by the MRCVitamin Study Research Groupshowed a 72% protective effect ofmaternal folic acid intake againstneural tube defects in their offspring(MRC Vitamin Study ResearchGroup, 1991), whereas supplemen-tation with other vitamins showed nosignificant protective effect. Thisfinding is supported by an experi-

mental study in rats showing that fo-late deficiency in early pregnancyresulted in impaired embryonic de-velopment, displaying a range ofconotruncal and pharyngeal archartery malformations (Baird et al.,1954). More evidence that folate is akey player in facial and cardiovas-cular development was given by astudy in which an increased risk ofcardiovascular defects and oralclefts was shown after exposure tofolate antagonists during the sec-

1Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands2Department of Epidemiology/Biostatistics University Medical Center, Nijmegen, The Netherlands3Department of Obstetrics/Gynaecology, Erasmus Medical Center, Rotterdam, The Netherlands4Department of Clinical Chemistry, Erasmus Medical Center, Rotterdam, The Netherlands*Correspondence to: Dr. A.C. Gittenberger-de Groot, Department of Anatomy and Embryology, Leiden University Medical Center, PO Box9602, 2300 RC Leiden, The Netherlands. E-mail: a.c.gittenberger-de_groot@lumc.nl

DOI 10.1002/dvdy.10303

DEVELOPMENTAL DYNAMICS 227:301–308, 2003

© 2003 Wiley-Liss, Inc.

ond or third month after the lastmenstrual period. The use of multivi-tamin supplements containing folicacid diminished these risks (Hernan-dez-Diaz et al., 2000).

Although the beneficial effect ofadditional folate or folic acid in thepericonceptional period has beendemonstrated, little is known aboutthe underlying mechanisms. Severalstudies point toward an importantrole of homocysteine as well. Folateis involved in nucleic acid synthesisand serves as a methyl group donorto homocysteine, resulting in the for-mation of methionine. S-Adenosyl-methionine is the activated form ofmethionine and is an essential sub-strate for the methylation reactionsinvolving proteins, tRNA, DNA, andlipids. A lack of folate results in anaccumulation of homocysteine(Kang et al., 1987). Furthermore,mildly elevated levels of homocys-teine in blood and amniotic fluidhave been related to neural tubedefects (Steegers-Theunissen et al.,1991, 1994, 1995; Mills et al., 1995).Mothers who gave birth to childrenwith a neural tube defect showedmore frequently abnormal homo-cysteine metabolism than controlmothers (Steegers-Theunissen et al.,1991, 1994). Experiments using highconcentrations of homocysteine inchick embryos show neural tube,orofacial, and conotruncal defectsin a time- and concentration-de-pendent manner. Folic acid supple-mentation was effective in the pre-vention of these defects (Rosenquistet al., 1996).

Cell types involved in neural tubedefects, orofacial clefts, andconotruncal heart defects are neu-roepithelial cells, cranial neural crestcells, and cardiac neural crest cells,respectively (Kirby et al., 1985;Poswillo, 1988; Poelmann et al., 1998;Botto et al., 1999). The beneficial ef-fect of supplemented folic acid issuggested to be specific for neuro-epithelial and neural crest cells(Rosenquist and Finnell, 2001). An ex-planation could be provided by thehigh expression of folate receptors inthe cells of the neurectoderm, whichare the precursor cells of both neuralcrest cells and the neuroepithelialcells of the neural tube (Rosenquistand Finnell, 2001). The direct effects

of folate and homocysteine on pro-liferation, differentiation and celldeath of cranial, and cardiac neuralcrest as well as neuroepithelial cellsare largely unknown. The aim of thisstudy is to analyze the in vitro behav-ior of neuroepithelial cells and neu-ral crest cells after folic acid and/orhomocysteine addition to the me-dium.

RESULTS

Neural Tube ExplantAttachment and InitialOutgrowth of Cells

After 3 hr in culture, differences inexplant attachment to the fibronec-tin-coated glass and outgrowth pat-terns were noted and appeared tobe dependent on the compositionof the culture media. In control me-dium, the neural tube explants wereonly loosely attached to the glassand no outgrowth of cells was ob-served (Fig. 1A,B). Migration of thefirst cells started after 4 to 5 hr inculture (data not shown). In the pres-ence of both 9 and 90 �M folic acid,migration of cells from the neuraltube explants was detected in 80%of the cultures within 2 to 3 hr. Thefirst cells that left the initial explantswere compactly organized neuro-epithelial cells (Fig. 1C,D), and theexplants were firmly attached to thefibronectin layer. Addition of 30 �Mhomocysteine to the medium re-sulted after 2 to 3 hr in outgrowth ofneural crest cells, which were recog-nized by their protrusions giving thecells their typical web-shaped mor-phology. The cultures exposed to300 �M homocysteine (Fig. 1E) dis-played both neuroepithelial andneural crest cells (Fig. 1F) leaving theexplants after 2 to 3 hr. In the pres-ence of 90 �M folic acid and 300 �Mhomocysteine, a low number ofneuroepithelial and neural crest cellswas observed outside the explants in40% of the cultures, whereas no out-growth of cells was observed in 60%of the cultures (Fig. 1G,H).

Neuroepithelial and NeuralCrest Cell Differentiation After24 Hours in Culture

Neural tube explants were culturedfor 24 hr before HNK-1 staining (Fig.

2A–H). In control cultures (M199),compactly organized neuroepithe-lial cells were surrounding the neuraltube explant as well as distinct areasof neural crest cells (Fig. 2A). A fewHNK-1–positive cells showed protrud-ing axons (arrow, Fig. 2E). Cultureswith 9 or 90 �M folic acid additiondisplayed large areas of neuroepi-thelial cells surrounding the explantsand a low number of neural crestcells (Fig. 2B). Some of the neuralcrest cells were smaller and showeda more round shape compared withthe neural crest cells in the controlcultures. Nerve cells with protrudingaxons were more abundant in the 90�M folic acid cultures (Fig. 2F). Addi-tion of 30 or 300 �M homocysteineresulted in a high number of neuralcrest cells (Fig. 2C). The cells were ofsimilar sizes as the neural crest cells inthe control cultures. No differentia-tion into nerve tissue was observedafter 24 hr in the presence of homo-cysteine (Fig. 2G). The combinationof folic acid (90 �M) and homocys-teine (300 �M) resulted in areas ofneural crest cells (Fig. 2D), withoutaxon formation (Fig. 2H).

Neural Crest Cell DifferentiationAfter 48 Hours in Culture

After 48 hr (Fig. 2I–P), the differentia-tion patterns of the cultures with folicacid addition were more pro-nounced. Whereas the control cul-tures had a low number of nerve fi-bers (arrows, Fig. 2I), the folic acidcultures displayed a high number ofaxons (arrows, Fig. 2J). The cells cul-tured in medium with homocysteinedid not have nerve fibers, althoughsome cells showed small protrusionsand had lost their web-shaped ap-pearance (arrowhead, Fig 2K). Thecombination of folic acid and ho-mocysteine resulted in a low numberof nerve fibers (arrows) in the culture(Fig. 2L).

The cultures were also studied forsmooth muscle actin expression withthe marker HHF35. The control cul-tures displayed actin expression onlyin a few cells (arrowheads, Fig. 2M)in contrast to the folic acid culturesthat showed intense actin staining inlarge round cells with blunt protru-sions (arrowheads, Fig. 2N). The ho-mocysteine cultures only faintly ex-

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pressed actin (Fig. 2O). Thecombination of folic acid and ho-mocysteine resulted in several small,elongated cells with actin expres-sion (Fig. 2P).

Necrosis and Apoptosis in thePresence of Folic Acid and/orHomocysteine

In all cultures, several dark browncells were observed after immuno-histochemistry (arrows, Fig. 2H).These cells were attached to the fi-bronectin layer with only a smallfraction of their cell surface and theirplane of focus was different from thesurrounding cells. The cells did notshow the typical chromosome con-figuration of dividing cells and ap-peared to be dying cells. The cellswere analyzed for necrosis and apo-ptosis by TUNEL staining. After 24 hr,none of the cultures showed TUNELactivity; however, all cultures dis-played areas of necrotic neuroepi-

thelial and neural crest cells. After 48hr in culture, approximately 5–10cells per culture were TUNEL positiveand no differences between the fo-lic acid and homocysteine cultureswere observed. The necrosis intensi-fied after 48 hr, especially at the bor-ders of the initial explant and theneural crest cells that are not in con-tact with neuroepithelial cells. Thenumbers of necrotic cells were simi-lar in all cultures (not shown).

Morphometric Analysis ofNeural Tube and Neural CrestOutgrowth

The size of the explant, the cell sur-face area of neuroepithelial cellsoutside the explant, and the cell sur-face area occupied by neural crestcells were analyzed in the 24 hr HNK-1–stained cultures. The area of neu-roepithelial cells was normalizedagainst the explant size (Fig. 3A). Inthe control cultures, the area of neu-

roepithelial cells outside the explantwas on average 2.7 times the size ofthe explant. Interestingly, in the cul-tures with folic acid, the area of neu-roepithelial cells was 4.5 times theexplant size. This finding means thata significant, 40% larger area of neu-roepithelial cells was observed in thepresence of folic acid comparedwith the control. The addition of ho-mocysteine and the combination offolic acid and homocysteine did notsignificantly change the neuroepi-thelial cell outgrowth comparedwith the controls.

The area occupied by neural crestcells was determined and normal-ized against the explant size (Fig. 3B).The area of neural crest cells in thecontrol cultures was on average 0.64times the size of the explants. In thepresence of folic acid, the area ofneural crest cells was marginally re-duced, although not significantly.The addition of homocysteine re-sulted in a significantly larger area of

Fig. 1. Brightfield microscopy images of neural tube explants after 3 hr in culture. A,B: In control medium, no cells were observed outsidethe neural tube explants. C,D: Folic acid at 90 �M resulted in an outgrowth of neuroepithelial cells. Note that the neural tube was alreadypartly closed at the time of explantation. This finding was not an effect of folic acid supplementation. The initial borders of the explant areindicated with dotted lines. Explants cultured with 300 �M homocysteine showed high amounts of neuroepithelial (E) and neural crest cells(F) outside the explant. G,H: Neural tube explants with both 90 �M folic acid and 300 �M homocysteine displayed no outgrowth. Originalmagnification: �70 in A,C,E,G, �210 in B,D,F,H, representing an enlargement of the boxed areas in A,C,E,G, respectively.

FOLIC ACID AND HOMOCYSTEINE AFFECT CELL BEHAVIOR 303

Fig. 2. Neural tube explants cultured for 24 or 48 hr. A–H: Neural tube explants cultured for 24 hr that were HNK-1 stained to mark neuralcrest cells. Neural crest cells were observed in distinct areas flanking the otic placode region that did not produce neural crest cells (A).The neural crest cell area was smaller in folic acid (B) and larger in cultures with homocysteine addition (C). Control cultures showed lownumbers of axons (arrow, E), while numerous axons were observed in the folic acid cultures (arrows, F). Nerve tissue differentiation wasabsent in homocysteine (G) and combined folic acid and homocysteine cultures (H). All cultures showed dark brown necrotic cells(arrows, H). I–L: Explants cultured for 48 hr and stained for neural crest and nerve tissue with HNK-1. Control cultures showed some axondevelopment (arrows, I). Cultures with folic acid showed intensive differentiation into nerve tissue (axons indicated with arrows, J),whereas in cultures with homocysteine, hardly any nerve differentiation was observed, although some cells showed axon-like protrusions(arrowhead, K). M–P: HHF35 muscle actin-stained cultures. Small cells with actin expression were observed in control cultures (arrowhead,M). Folic acid resulted in intense muscle actin expression in large differentiated neural crest cells (arrowheads, N). Addition of homo-cysteine, or both folic acid and homocysteine resulted in less actin staining. Note also the difference in morphology (O, P). Originalmagnification: �16 in A–D, �80 in E–P.

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neural crest cells, namely 1.05 timesthe size of the explant; this area was30% more than observed in the con-trol cultures. If both folic acid andhomocysteine were added to theculture medium, the relative out-growth of neural crest cells wascomparable to the control cultures.

DISCUSSION

The formation of neural folds, theclosure of the neural tube, and themigration of neural crest cells requirecritical changes in neuroepithelialcell number, shape, size, adhesion,and position (Colas and Schoen-wolf, 2001). So far, the morphoge-netic influences and the way nutri-ents are involved in these processesare largely unknown.

In this study, we cultured the cra-nial and cardiac neural tube regionat the time of neural tube closureand observed a direct effect of ahigh concentration of folic acid onthe neuroepithelial cells. The area ofneuroepithelial cell outgrowth is 40%larger compared with control cul-tures, whereas cell death numbersand cell sizes were comparable tocontrols, suggesting that folic acidmight stimulate the proliferation ofneuroepithelial cells. These findingsare in line with the hypothesis thatfolate deficiency decreases the pro-liferative capacity of neural tube orneural crest cells, and the embryomay react by developing neural

tube defects (Antony and Hansen,2000). The beneficial effect of folicacid supplementation in pregnantwomen to prevent neural tube de-fects (Mulinare et al., 1988; Czeizeland Dudas, 1992) might be ex-plained by a direct effect of folicacid on the outgrowth of neuroepi-thelial cells. A direct effect of folicacid on the neurepithelium is alsolikely when analyzing the folate re-ceptor (Folbp1) -deficient mousemodel. These embryos displayedabnormalities specifically at the siteof the neural tube and the branchialarches (Piedrahita et al., 1999;Rosenquist and Finnell, 2001). Fur-thermore, a protective effect of folicacid against neural tube defectshas been observed in Pax3-deficientSplotch mice (Fleming and Copp,1998; Epstein et al., 2000).

Of interest is that folic acid directlyaffected neural crest cell differenti-ation. We noticed a high degree ofdifferentiation reflected by nerve tis-sue markers and muscle actin ex-pression, which suggests an impor-tant function of folate in thedifferentiation of neural crest cells.

High concentrations of homocys-teine have been reported to induceneural crest– and neural tube–re-lated defects in chick embryos(Rosenquist et al., 1996). This couldbe due to the ability of homocys-teine to inhibit the NMDA receptor,which is involved in intracellular and

intercellular processes that are cen-tral to neural tube closure and neu-ral crest migration (Rosenquist et al.,1999; Rosenquist and Finnell, 2001).

In one of our culture groups, wemimicked the human situationpresent in a subset of mothers of off-spring with a neural tube defecthaving low folate and elevated ho-mocysteine concentrations in theamniotic fluid (Steegers-Theunissenet al., 1991, 1994, 1995). We addedhigh concentrations of homocys-teine to medium that already con-tained low concentrations of folicacid and observed a profound ef-fect on the cranial and cardiac neu-ral crest cells. The neural crest cellarea was 30% larger in the presenceof homocysteine, whereas no differ-ences in neural crest cell death orcell shape were observed, suggest-ing that proliferation or migration ofneural crest cells from the neuraltube explant is increased. Further-more, the neural crest cells hardlyshowed any differentiation. The con-centration of homocysteine deter-mined how strong the inhibition ofdifferentiation was. The increase inneural crest cell outgrowth inducedby homocysteine that we describe issupported by recent findings thathomocysteine changes neural crestcell motility, migration distance, cellsurface area, and cell perimeter(Brauer and Rosenquist, 2002). Incontrast to the Brauer and Rosen-

Fig. 3. Relative outgrowth of neuroepithelial (A) and neural crest cells (B) in relation to the addition of folic acid (FA) and/orhomocysteine (Hcys) to the culture medium. The cultures with folic acid and/or homocysteine were compared with the control cultures.The Mann-Whitney U test was used to compare independent groups. The relative outgrowth is represented in a box plot as the medianwith the 95% confidence interval, also showing the minimum and maximum value. *P � 0.005; #P � 0.05.

FOLIC ACID AND HOMOCYSTEINE AFFECT CELL BEHAVIOR 305

quist study, we investigated the ef-fects of folic acid on neuroepithelialcell outgrowth and showed the ef-fects of both folic acid and homo-cysteine on the differentiation ofneural crest cells.

By extrapolating the in vitro effectsof hyperhomocysteinemia to the invivo situation, we hypothesize thatdefects in neural tube closure couldbe explained by an increased neu-roepithelial to neural crest cell trans-formation leading to a lower num-ber of neuroepithelial cells in theneural tube. Defects in orofacialand conotruncal developmentmight be explained by aberrant dif-ferentiation of neural crest cells.However, if high concentrations ofhomocysteine can cause neuraltube, orofacial, and conotruncalheart defects, it is surprising to findonly 1.1% offspring with the combi-nation of multiple midline defects(Coddington and Hisnanick, 1996).This might be explained by the highembryonic lethality rate in earlypregnancy, the period in which neu-ral tube closure and the most drasticchanges in heart morphology oc-cur.

Folic acid supplementation hasbeen reported to counteract the ef-fects of mildly increased to very highhomocysteine concentrations (Stee-gers-Theunissen et al., 1994; Rosen-quist et al., 1996; Carmody et al.,1999; Buemi et al., 2001). We ob-served neuroepithelial and neuralcrest cell outgrowth after combined

addition of folic acid and homocys-teine to be similar to the controls,implicating that folic acid counter-acts the effects of homocysteine oncell outgrowth. On the other hand,the morphology of the neural crestcells was different compared withcontrols. This finding might be ex-plained by a toxic effect of the highconcentrations of folic acid and ho-mocysteine or a disturbance in thebalance between folate and homo-cysteine.

These findings indicate that folatehas a variety of functions importantfor embryogenesis. Folate partici-pates in DNA biosynthesis during thecomplete pregnancy, but the ma-ternal requirement for folate is espe-cially high during the second trimes-ter, which led to the conclusion thatfolic acid supplementation in preg-nant women should be strongly in-creased especially during the sec-ond trimester (McPartlin et al., 1993).We focused on the effect of folicacid on neural crest cell differentia-tion during early embryonic devel-opmental stages, and our findingsindicate that we have to be careful,because high folic acid concentra-tions disturb normal differentiationpatterns. Genes encoding folate re-ceptors are not expressed uniformlyduring pregnancy and are affectedby the folate concentration (Antonyand Hansen, 2000). This finding sug-gests that correct timing of folic acidsupplementation is as essential asthe concentration. Furthermore, the

supplementation might have to beadjusted to the special requirementsduring each phase of the preg-nancy.

We demonstrated that folic acidcounteracted the effects of homo-cysteine, which partly explains thebeneficial effect of folic acid supple-mentation in hyperhomocysteine-mic women. These new findings aresteps in the process to unravel thedevelopmental mechanisms under-lying neural tube defects, orofacialclefts, and conotruncal heart de-fects.

EXPERIMENTAL PROCEDURES

In Vitro Culture System

Chicken embryos staged HH9 andHH10 (Hamburger and Hamilton,1951) were used for in vitro culture ofthe neural tube. HH9 and HH10 em-bryos were equally distributed amongthe experimental groups. The em-bryos were excised from the yolk sacand washed in Locke’s solution. Theneural tube region containing themesencephalon, rhombencephalon,and the neural tube up to the level ofthe fourth somite pair (Fig. 4A) wasexcised by using a tungsten needle.The somites and mesenchymal tissueattached to the neural tube regionwere removed by placing the explant(Fig. 4B) in a collagenase solution(0.5% in Locke’s solution) for 10 min.The neural tube explant (Fig. 4C) wasrinsed with medium and subsequentlytransferred onto a fibronectin (20 �g/

Fig. 4. Techniques used to culture neural tube explants. The mesencephalon, rhombencephalon, and neural tube posterior to the levelof the fourth somite pair was excised (A) and placed in a collagenase solution (B) to remove mesenchyme and somites. C: The neuraltube region was placed on a fibronectin-coated glass and cultured for 24 or 48 hr. D: Subsequently, the culture was fixed andimmunohistochemistry was performed.

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ml) -coated coverslip in a 24-wellplate and cultured in 1 ml of medium.The neural tube cultures were moni-tored for 24 to 48 hr with recordingsevery hour for the first 5 hr by using aLeica DM IRBE microscope withQFluoro software and subsequentlyfixed and immunohistochemicallyprocessed (Fig. 4D).

Culture Medium and FolicAcid and HomocysteineAdditions

The neural tube explants were cul-tured in minimal medium M199 (Me-dium 199, Life Technologies; 10% fe-tal calf serum; 0.5% gentamicin; 1%penicillin/streptomycin; 1% ITS). Con-trols consisted of neural tube ex-plants cultured in M199 with the ad-dition of a small volume of distilledwater. We compared the controlcultures with cultures supplementedwith chicken embryo extract (CEE,Life Technologies). The control cul-tures and cultures with chicken em-bryo extract showed similar cell out-growth and differentiation patterns,suggesting the control may be usedas a representation of the normalembryonic situation.

We studied the effects of a stableform of folic acid (pteroylglutamicacid, Sigma) and/or homocysteinethiolactone (L-homocysteine thio-lactone hydrochloride, Sigma). Weused supraphysiological concentra-tions of folic acid (9 �M and 90 �M),which are comparable to concen-trations used in other in vitro studies(Buemi et al., 2001; Dogan et al.,2001). The folate concentrations inthe culture medium were measured48 hr after administration by usingthe Roche Elecsys 2010 assay(Roche diagnostics, Modular analyt-ics E170).

Neural tube explants were ex-posed to 30 �M or 300 �M L-homo-cysteine thiolactone hydrochloride.These concentrations are toxic in hu-man and comparable to concen-trations used in other in vitro studies(Carmody et al., 1999). Homocys-teine concentrations were mea-sured after 48 hr culture by usinghigh performance liquid chroma-tography as previously described(Pfeiffer et al., 1999). Neural tube ex-plants were also cultured in the pres-

ence of both 90 �M folic acid and300 �M homocysteine.

Folate and homocysteine con-centrations were stable in the cul-ture medium (data not shown). After48 hr in culture, the variation be-tween the medium samples and thestock solution was less than 5%, bothfor separate and combined addi-tion of folic acid and homocysteine.

The number of cultures studied foreach condition were control cul-tures (n � 42), chicken embryo ex-tract (n � 6), folic acid 9 �M (n � 6),folic acid 90 �M (n � 31), homocys-teine 30 �M (n � 10), homocysteine300 �M (n � 16), folic acid 90 �M,and 300 �M homocysteine (n � 24).

Immunohistochemistry

After 24 or 48 hr culture, the neuraltube explants were fixed for 30 min in4% paraformaldehyde in 0.1 M phos-phate buffer and stored in 70% eth-anol at 4°C. Before the antibody in-cubation, the cultures were washedin phosphate-buffered saline (PBS)and treated with 0.3% H2O2 in PBS.Routine immunohistochemical stain-ing was performed by using over-night incubations with the primaryantibodies HNK-1 (Hybridomabank,Iowa City; Abo and Balch, 1981) andHHF35 (DAKO Denmark) (Tsukada etal., 1987) to mark neural crest anddifferentiated nerves (Poelmann etal., 1998) or muscle-specific actin,respectively (Bergwerff et al., 1996).All antibodies were diluted in PBSwith 0.05% Tween and 1% chickenegg albumin (HNK-1 1:50, HHF351:500). After thorough washing withPBS, the cultures were incubated for2 hr with horseradish peroxidase–conjugated rabbit anti-mouse anti-bodies (1:200, DAKO). Rinsing withPBS was followed by treatment with0.04% diaminobenzidine tetrahydro-chloride (DAB)/0.006% H2O2 in 0.05M Tris-maleic acid (pH 7.6) for 10 min.The cultures were counter-stainedwith Mayer’s hematoxylin, dehy-drated, and mounted in Entellan.

Cell Death Analysis

To determine whether dying cells inthe cultures were necrotic or apopto-tic, we performed a TUNEL procedure(Boehringer, Mannheim) to mark ap-

optosis and propidium iodine coun-terstaining to visualize necrosis. Thecultures were treated with proteinaseK 0.02 �g/ml in PBS for 15 min, rinsedwith PBS followed by the TUNEL reac-tion at 37°C for 2 hr. After washing withPBS and water, the cultures weremounted in anti-fade reagent (2% 1,4-diazabicyclo[2.2.2]octane in glycerin)with 0.1% propidium iodide.

Morphometric Analyses

By using Adobe Photoshop, digitalimages of the 24 hr HNK-1–stainedneural tube cultures were automati-cally analyzed and cell areas wheredivided into three different groupsbased on the color the cells ob-tained during immunohistochemis-try. Group one contained the darkpurple neural tube explant. Grouptwo consisted of hematoxylin blue,densely organized neuroepithelialcells that have grown out of the ex-plant. Group three contained theDAB brown, HNK-1–positive neuralcrest cells. The surface occupied byeach group as detected by thenumber of pixels was measured. Therelative outgrowth of neuroepithelial(group 2) and neural crest cells(group 3) were both normalizedagainst the explant area (group 1).The HNK-1–stained 24 hr cultureswere analyzed by one observerwithout prior knowledge of the com-position of the culture medium.Number of cultures: control (n � 10),90 �M folic acid (n � 10), 300 �Mhomocysteine (n � 10), 90 �M folicacid, and 300 �M homocysteine(n � 10). Statistical analysis was per-formed by using the Mann-WhitneyU test to compare independentgroups. The results are presented asthe median with the 95% confi-dence interval and the minimumand maximum values.

ACKNOWLEDGMENTSWe thank Montsy Brouns (Depart-ment of Clinical Chemistry, ErasmusMedical Center Rotterdam) for bio-chemical analyses. We also thankBas Blankevoort for artwork, Jan Lensfor photographic contributions andsupport with the morphometricalanalyses (Department of Anatomyand Embryology, Leiden University

FOLIC ACID AND HOMOCYSTEINE AFFECT CELL BEHAVIOR 307

Medical Center), and NicoletteUrsem (Department of Obstetrics/Gynaecology, Erasmus MedicalCenter Rotterdam) for assistancewith statistical programs.

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