DEVELOPMENTAL DYNAMICS 227:301–308, 2003
PATTERNS
Folic Acid and Homocysteine Affect Neural Crest and Neuroepithelial Cell Outgrowth and Differentiation In Vitro Marit J. Boot,1 Re ´ gine P.M. Steegers-Theunissen,2,3 Robert E. Poelmann,1 Liesbeth Van Iperen,1 4 Jan Lindemans, and Adriana C. Gittenberger-de Groot1*
The beneficial effect of additional folic acid in the periconceptional period to prevent neural tube defects, orofacial clefts, and conotruncal heart defects in the offspring has been shown. Folate shortage results in homocysteine accumulation. Elevated levels of homocysteine have been related to neural tube defects. We studied the behavior of neuroepithelial cells and cranial and cardiac neural crest cells in vitro. Neural tube explants were cultured for 24 and 48 hr in medium after addition of folic acid and/or homocysteine. Folic acid addition increased neuroepithelial cell outgrowth and increased neural crest cell differentiation into nerve and smooth muscle cells. Addition of homocysteine increased neural crest cell outgrowth and migration from the neural tube and inhibited neural crest cell differentiation. Our findings suggest that neural tube defects caused by folate deficiency and hyperhomocysteinemia develop due to increased neuroepithelial to neural crest cell transformation. This increased transformation leads to a shortage of neuroepithelial cells in the neural tube. Defects in orofacial and conotruncal development are explained by abnormal differentiation of neural crest cells in the presence of high homocysteine concentrations. Our findings supports a critical role for folic acid and homocysteine in the development of neural tube defects and neural crest related 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 multivitamins reduces the risk of neural tube defects (Mulinare et al., 1988; Czeizel and Duda ´ s, 1992), orofacial clefts (Tolarova, 1982; Shaw et al., 1995a), and limb and conotruncal heart defects (Shaw et al., 1995b; Botto et al., 1996) in the offspring. To evaluate whether the B vitamin folate is responsible for the risk reduction, several studies have been performed supplementing women with the syn-
1
thetic form of folate, i.e., folic acid (Laurence et al., 1981; MRC Vitamin Study Research Group, 1991). The clinical trial performed by the MRC Vitamin Study Research Group showed a 72% protective effect of maternal folic acid intake against neural tube defects in their offspring (MRC Vitamin Study Research Group, 1991), whereas supplementation with other vitamins showed no significant protective effect. This finding is supported by an experi-
mental study in rats showing that folate deficiency in early pregnancy resulted in impaired embryonic development, displaying a range of conotruncal and pharyngeal arch artery malformations (Baird et al., 1954). More evidence that folate is a key player in facial and cardiovascular development was given by a study in which an increased risk of cardiovascular defects and oral clefts was shown after exposure to folate antagonists during the sec-
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands Department of Epidemiology/Biostatistics University Medical Center, Nijmegen, The Netherlands Department of Obstetrics/Gynaecology, Erasmus Medical Center, Rotterdam, The Netherlands 4 Department 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 Box 9602, 2300 RC Leiden, The Netherlands. E-mail:
[email protected] 2 3
DOI 10.1002/dvdy.10303
© 2003 Wiley-Liss, Inc.
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ond or third month after the last menstrual period. The use of multivitamin supplements containing folic acid diminished these risks (Hernandez-Diaz et al., 2000). Although the beneficial effect of additional folate or folic acid in the periconceptional period has been demonstrated, little is known about the underlying mechanisms. Several studies point toward an important role of homocysteine as well. Folate is involved in nucleic acid synthesis and serves as a methyl group donor to homocysteine, resulting in the formation of methionine. S-Adenosylmethionine is the activated form of methionine and is an essential substrate for the methylation reactions involving proteins, tRNA, DNA, and lipids. A lack of folate results in an accumulation of homocysteine (Kang et al., 1987). Furthermore, mildly elevated levels of homocysteine in blood and amniotic fluid have been related to neural tube defects (Steegers-Theunissen et al., 1991, 1994, 1995; Mills et al., 1995). Mothers who gave birth to children with a neural tube defect showed more frequently abnormal homocysteine metabolism than control mothers (Steegers-Theunissen et al., 1991, 1994). Experiments using high concentrations of homocysteine in chick embryos show neural tube, orofacial, and conotruncal defects in a time- and concentration-dependent manner. Folic acid supplementation was effective in the prevention of these defects (Rosenquist et al., 1996). Cell types involved in neural tube defects, orofacial clefts, and conotruncal heart defects are neuroepithelial cells, cranial neural crest cells, and cardiac neural crest cells, respectively (Kirby et al., 1985; Poswillo, 1988; Poelmann et al., 1998; Botto et al., 1999). The beneficial effect of supplemented folic acid is suggested to be specific for neuroepithelial and neural crest cells (Rosenquist and Finnell, 2001). An explanation could be provided by the high expression of folate receptors in the cells of the neurectoderm, which are the precursor cells of both neural crest cells and the neuroepithelial cells of the neural tube (Rosenquist and Finnell, 2001). The direct effects
of folate and homocysteine on proliferation, differentiation and cell death of cranial, and cardiac neural crest as well as neuroepithelial cells are largely unknown. The aim of this study is to analyze the in vitro behavior of neuroepithelial cells and neural crest cells after folic acid and/or homocysteine addition to the medium.
RESULTS Neural Tube Explant Attachment and Initial Outgrowth of Cells After 3 hr in culture, differences in explant attachment to the fibronectin-coated glass and outgrowth patterns were noted and appeared to be dependent on the composition of the culture media. In control medium, the neural tube explants were only loosely attached to the glass and no outgrowth of cells was observed (Fig. 1A,B). Migration of the first cells started after 4 to 5 hr in culture (data not shown). In the presence of both 9 and 90 M folic acid, migration of cells from the neural tube explants was detected in 80% of the cultures within 2 to 3 hr. The first cells that left the initial explants were compactly organized neuroepithelial cells (Fig. 1C,D), and the explants were firmly attached to the fibronectin layer. Addition of 30 M homocysteine to the medium resulted after 2 to 3 hr in outgrowth of neural crest cells, which were recognized by their protrusions giving the cells their typical web-shaped morphology. The cultures exposed to 300 M homocysteine (Fig. 1E) displayed both neuroepithelial and neural crest cells (Fig. 1F) leaving the explants after 2 to 3 hr. In the presence of 90 M folic acid and 300 M homocysteine, a low number of neuroepithelial and neural crest cells was observed outside the explants in 40% of the cultures, whereas no outgrowth of cells was observed in 60% of the cultures (Fig. 1G,H).
Neuroepithelial and Neural Crest Cell Differentiation After 24 Hours in Culture Neural tube explants were cultured for 24 hr before HNK-1 staining (Fig.
2A–H). In control cultures (M199), compactly organized neuroepithelial cells were surrounding the neural tube explant as well as distinct areas of neural crest cells (Fig. 2A). A few HNK-1–positive cells showed protruding axons (arrow, Fig. 2E). Cultures with 9 or 90 M folic acid addition displayed large areas of neuroepithelial cells surrounding the explants and a low number of neural crest cells (Fig. 2B). Some of the neural crest cells were smaller and showed a more round shape compared with the neural crest cells in the control cultures. Nerve cells with protruding axons were more abundant in the 90 M folic acid cultures (Fig. 2F). Addition of 30 or 300 M homocysteine resulted in a high number of neural crest cells (Fig. 2C). The cells were of similar sizes as the neural crest cells in the control cultures. No differentiation into nerve tissue was observed after 24 hr in the presence of homocysteine (Fig. 2G). The combination of folic acid (90 M) and homocysteine (300 M) resulted in areas of neural crest cells (Fig. 2D), without axon formation (Fig. 2H).
Neural Crest Cell Differentiation After 48 Hours in Culture After 48 hr (Fig. 2I–P), the differentiation patterns of the cultures with folic acid addition were more pronounced. Whereas the control cultures had a low number of nerve fibers (arrows, Fig. 2I), the folic acid cultures displayed a high number of axons (arrows, Fig. 2J). The cells cultured in medium with homocysteine did not have nerve fibers, although some cells showed small protrusions and had lost their web-shaped appearance (arrowhead, Fig 2K). The combination of folic acid and homocysteine resulted in a low number of nerve fibers (arrows) in the culture (Fig. 2L). The cultures were also studied for smooth muscle actin expression with the marker HHF35. The control cultures displayed actin expression only in a few cells (arrowheads, Fig. 2M) in contrast to the folic acid cultures that showed intense actin staining in large round cells with blunt protrusions (arrowheads, Fig. 2N). The homocysteine cultures only faintly ex-
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Fig. 1. Brightfield microscopy images of neural tube explants after 3 hr in culture. A,B: In control medium, no cells were observed outside the neural tube explants. C,D: Folic acid at 90 M resulted in an outgrowth of neuroepithelial cells. Note that the neural tube was already partly closed at the time of explantation. This finding was not an effect of folic acid supplementation. The initial borders of the explant are indicated 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. Original magnification: ⫻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.
pressed actin (Fig. 2O). The combination of folic acid and homocysteine resulted in several small, elongated cells with actin expression (Fig. 2P).
Necrosis and Apoptosis in the Presence of Folic Acid and/or Homocysteine In all cultures, several dark brown cells were observed after immunohistochemistry (arrows, Fig. 2H). These cells were attached to the fibronectin layer with only a small fraction of their cell surface and their plane of focus was different from the surrounding cells. The cells did not show the typical chromosome configuration of dividing cells and appeared to be dying cells. The cells were analyzed for necrosis and apoptosis by TUNEL staining. After 24 hr, none of the cultures showed TUNEL activity; however, all cultures displayed areas of necrotic neuroepi-
thelial and neural crest cells. After 48 hr in culture, approximately 5–10 cells per culture were TUNEL positive and no differences between the folic acid and homocysteine cultures were observed. The necrosis intensified after 48 hr, especially at the borders of the initial explant and the neural crest cells that are not in contact with neuroepithelial cells. The numbers of necrotic cells were similar in all cultures (not shown).
Morphometric Analysis of Neural Tube and Neural Crest Outgrowth The size of the explant, the cell surface area of neuroepithelial cells outside the explant, and the cell surface area occupied by neural crest cells were analyzed in the 24 hr HNK1–stained cultures. The area of neuroepithelial cells was normalized against the explant size (Fig. 3A). In the control cultures, the area of neu-
roepithelial cells outside the explant was on average 2.7 times the size of the explant. Interestingly, in the cultures with folic acid, the area of neuroepithelial cells was 4.5 times the explant size. This finding means that a significant, 40% larger area of neuroepithelial cells was observed in the presence of folic acid compared with the control. The addition of homocysteine and the combination of folic acid and homocysteine did not significantly change the neuroepithelial cell outgrowth compared with the controls. The area occupied by neural crest cells was determined and normalized against the explant size (Fig. 3B). The area of neural crest cells in the control cultures was on average 0.64 times the size of the explants. In the presence of folic acid, the area of neural crest cells was marginally reduced, although not significantly. The addition of homocysteine resulted in a significantly larger area of
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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 neural crest 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 low numbers of axons (arrow, E), while numerous axons were observed in the folic acid cultures (arrows, F). Nerve tissue differentiation was absent 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 axon development (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 homocysteine, or both folic acid and homocysteine resulted in less actin staining. Note also the difference in morphology (O, P). Original magnification: ⫻16 in A–D, ⫻80 in E–P.
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Fig. 3. Relative outgrowth of neuroepithelial (A) and neural crest cells (B) in relation to the addition of folic acid (FA) and/or homocysteine (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 median with the 95% confidence interval, also showing the minimum and maximum value. *P ⬍ 0.005; #P ⬍ 0.05.
neural crest cells, namely 1.05 times the size of the explant; this area was 30% more than observed in the control cultures. If both folic acid and homocysteine were added to the culture medium, the relative outgrowth of neural crest cells was comparable to the control cultures.
DISCUSSION The formation of neural folds, the closure of the neural tube, and the migration of neural crest cells require critical changes in neuroepithelial cell number, shape, size, adhesion, and position (Colas and Schoenwolf, 2001). So far, the morphogenetic influences and the way nutrients are involved in these processes are largely unknown. In this study, we cultured the cranial and cardiac neural tube region at the time of neural tube closure and observed a direct effect of a high concentration of folic acid on the neuroepithelial cells. The area of neuroepithelial cell outgrowth is 40% larger compared with control cultures, whereas cell death numbers and cell sizes were comparable to controls, suggesting that folic acid might stimulate the proliferation of neuroepithelial cells. These findings are in line with the hypothesis that folate deficiency decreases the proliferative capacity of neural tube or neural crest cells, and the embryo may react by developing neural
tube defects (Antony and Hansen, 2000). The beneficial effect of folic acid supplementation in pregnant women to prevent neural tube defects (Mulinare et al., 1988; Czeizel and Duda ´ s, 1992) might be explained by a direct effect of folic acid on the outgrowth of neuroepithelial cells. A direct effect of folic acid on the neurepithelium is also likely when analyzing the folate receptor (Folbp1) -deficient mouse model. These embryos displayed abnormalities specifically at the site of the neural tube and the branchial arches (Piedrahita et al., 1999; Rosenquist and Finnell, 2001). Furthermore, a protective effect of folic acid against neural tube defects has been observed in Pax3-deficient Splotch mice (Fleming and Copp, 1998; Epstein et al., 2000). Of interest is that folic acid directly affected neural crest cell differentiation. We noticed a high degree of differentiation reflected by nerve tissue markers and muscle actin expression, which suggests an important function of folate in the differentiation of neural crest cells. High concentrations of homocysteine have been reported to induce neural crest– and neural tube–related defects in chick embryos (Rosenquist et al., 1996). This could be due to the ability of homocysteine to inhibit the NMDA receptor, which is involved in intracellular and
intercellular processes that are central to neural tube closure and neural crest migration (Rosenquist et al., 1999; Rosenquist and Finnell, 2001). In one of our culture groups, we mimicked the human situation present in a subset of mothers of offspring with a neural tube defect having low folate and elevated homocysteine concentrations in the amniotic fluid (Steegers-Theunissen et al., 1991, 1994, 1995). We added high concentrations of homocysteine to medium that already contained low concentrations of folic acid and observed a profound effect on the cranial and cardiac neural crest cells. The neural crest cell area was 30% larger in the presence of homocysteine, whereas no differences in neural crest cell death or cell shape were observed, suggesting that proliferation or migration of neural crest cells from the neural tube explant is increased. Furthermore, the neural crest cells hardly showed any differentiation. The concentration of homocysteine determined how strong the inhibition of differentiation was. The increase in neural crest cell outgrowth induced by homocysteine that we describe is supported by recent findings that homocysteine changes neural crest cell motility, migration distance, cell surface area, and cell perimeter (Brauer and Rosenquist, 2002). In contrast to the Brauer and Rosen-
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Fig. 4. Techniques used to culture neural tube explants. The mesencephalon, rhombencephalon, and neural tube posterior to the level of the fourth somite pair was excised (A) and placed in a collagenase solution (B) to remove mesenchyme and somites. C: The neural tube region was placed on a fibronectin-coated glass and cultured for 24 or 48 hr. D: Subsequently, the culture was fixed and immunohistochemistry was performed.
quist study, we investigated the effects of folic acid on neuroepithelial cell outgrowth and showed the effects of both folic acid and homocysteine on the differentiation of neural crest cells. By extrapolating the in vitro effects of hyperhomocysteinemia to the in vivo situation, we hypothesize that defects in neural tube closure could be explained by an increased neuroepithelial to neural crest cell transformation leading to a lower number of neuroepithelial cells in the neural tube. Defects in orofacial and conotruncal development might be explained by aberrant differentiation of neural crest cells. However, if high concentrations of homocysteine can cause neural tube, orofacial, and conotruncal heart defects, it is surprising to find only 1.1% offspring with the combination of multiple midline defects (Coddington and Hisnanick, 1996). This might be explained by the high embryonic lethality rate in early pregnancy, the period in which neural tube closure and the most drastic changes in heart morphology occur. Folic acid supplementation has been reported to counteract the effects of mildly increased to very high homocysteine concentrations (Steegers-Theunissen et al., 1994; Rosenquist et al., 1996; Carmody et al., 1999; Buemi et al., 2001). We observed neuroepithelial and neural crest cell outgrowth after combined
addition of folic acid and homocysteine to be similar to the controls, implicating that folic acid counteracts the effects of homocysteine on cell outgrowth. On the other hand, the morphology of the neural crest cells was different compared with controls. This finding might be explained by a toxic effect of the high concentrations of folic acid and homocysteine or a disturbance in the balance between folate and homocysteine. These findings indicate that folate has a variety of functions important for embryogenesis. Folate participates in DNA biosynthesis during the complete pregnancy, but the maternal requirement for folate is especially high during the second trimester, which led to the conclusion that folic acid supplementation in pregnant women should be strongly increased especially during the second trimester (McPartlin et al., 1993). We focused on the effect of folic acid on neural crest cell differentiation during early embryonic developmental stages, and our findings indicate that we have to be careful, because high folic acid concentrations disturb normal differentiation patterns. Genes encoding folate receptors are not expressed uniformly during pregnancy and are affected by the folate concentration (Antony and Hansen, 2000). This finding suggests that correct timing of folic acid supplementation is as essential as the concentration. Furthermore, the
supplementation might have to be adjusted to the special requirements during each phase of the pregnancy. We demonstrated that folic acid counteracted the effects of homocysteine, which partly explains the beneficial effect of folic acid supplementation in hyperhomocysteinemic women. These new findings are steps in the process to unravel the developmental mechanisms underlying neural tube defects, orofacial clefts, and conotruncal heart defects.
EXPERIMENTAL PROCEDURES In Vitro Culture System Chicken embryos staged HH9 and HH10 (Hamburger and Hamilton, 1951) were used for in vitro culture of the neural tube. HH9 and HH10 embryos were equally distributed among the experimental groups. The embryos were excised from the yolk sac and washed in Locke’s solution. The neural tube region containing the mesencephalon, rhombencephalon, and the neural tube up to the level of the fourth somite pair (Fig. 4A) was excised by using a tungsten needle. The somites and mesenchymal tissue attached to the neural tube region were 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) was rinsed with medium and subsequently transferred onto a fibronectin (20 g/
FOLIC ACID AND HOMOCYSTEINE AFFECT CELL BEHAVIOR 307
ml) -coated coverslip in a 24-well plate and cultured in 1 ml of medium. The neural tube cultures were monitored for 24 to 48 hr with recordings every hour for the first 5 hr by using a Leica DM IRBE microscope with QFluoro software and subsequently fixed and immunohistochemically processed (Fig. 4D).
Culture Medium and Folic Acid and Homocysteine Additions The neural tube explants were cultured in minimal medium M199 (Medium 199, Life Technologies; 10% fetal calf serum; 0.5% gentamicin; 1% penicillin/streptomycin; 1% ITS). Controls consisted of neural tube explants cultured in M199 with the addition of a small volume of distilled water. We compared the control cultures with cultures supplemented with chicken embryo extract (CEE, Life Technologies). The control cultures and cultures with chicken embryo extract showed similar cell outgrowth and differentiation patterns, suggesting the control may be used as a representation of the normal embryonic situation. We studied the effects of a stable form of folic acid (pteroylglutamic acid, Sigma) and/or homocysteine thiolactone (L-homocysteine thiolactone hydrochloride, Sigma). We used supraphysiological concentrations of folic acid (9 M and 90 M), which are comparable to concentrations used in other in vitro studies (Buemi et al., 2001; Dogan et al., 2001). The folate concentrations in the culture medium were measured 48 hr after administration by using the Roche Elecsys 2010 assay (Roche diagnostics, Modular analytics E170). Neural tube explants were exposed to 30 M or 300 M L-homocysteine thiolactone hydrochloride. These concentrations are toxic in human and comparable to concentrations used in other in vitro studies (Carmody et al., 1999). Homocysteine concentrations were measured after 48 hr culture by using high performance liquid chromatography as previously described (Pfeiffer et al., 1999). Neural tube explants were also cultured in the pres-
ence of both 90 M folic acid and 300 M homocysteine. Folate and homocysteine concentrations were stable in the culture medium (data not shown). After 48 hr in culture, the variation between the medium samples and the stock solution was less than 5%, both for separate and combined addition of folic acid and homocysteine. The number of cultures studied for each condition were control cultures (n ⫽ 42), chicken embryo extract (n ⫽ 6), folic acid 9 M (n ⫽ 6), folic acid 90 M (n ⫽ 31), homocysteine 30 M (n ⫽ 10), homocysteine 300 M (n ⫽ 16), folic acid 90 M, and 300 M homocysteine (n ⫽ 24).
Immunohistochemistry After 24 or 48 hr culture, the neural tube explants were fixed for 30 min in 4% paraformaldehyde in 0.1 M phosphate buffer and stored in 70% ethanol at 4°C. Before the antibody incubation, the cultures were washed in phosphate-buffered saline (PBS) and treated with 0.3% H2O2 in PBS. Routine immunohistochemical staining was performed by using overnight incubations with the primary antibodies HNK-1 (Hybridomabank, Iowa City; Abo and Balch, 1981) and HHF35 (DAKO Denmark) (Tsukada et al., 1987) to mark neural crest and differentiated nerves (Poelmann et al., 1998) or muscle-specific actin, respectively (Bergwerff et al., 1996). All antibodies were diluted in PBS with 0.05% Tween and 1% chicken egg albumin (HNK-1 1:50, HHF35 1:500). After thorough washing with PBS, the cultures were incubated for 2 hr with horseradish peroxidase– conjugated rabbit anti-mouse antibodies (1:200, DAKO). Rinsing with PBS was followed by treatment with 0.04% diaminobenzidine tetrahydrochloride (DAB)/0.006% H2O2 in 0.05 M Tris-maleic acid (pH 7.6) for 10 min. The cultures were counter-stained with Mayer’s hematoxylin, dehydrated, and mounted in Entellan.
Cell Death Analysis To determine whether dying cells in the cultures were necrotic or apoptotic, we performed a TUNEL procedure (Boehringer, Mannheim) to mark ap-
optosis and propidium iodine counterstaining to visualize necrosis. The cultures were treated with proteinase K 0.02 g/ml in PBS for 15 min, rinsed with PBS followed by the TUNEL reaction at 37°C for 2 hr. After washing with PBS and water, the cultures were mounted in anti-fade reagent (2% 1,4diazabicyclo[2.2.2]octane in glycerin) with 0.1% propidium iodide.
Morphometric Analyses By using Adobe Photoshop, digital images of the 24 hr HNK-1–stained neural tube cultures were automatically analyzed and cell areas where divided into three different groups based on the color the cells obtained during immunohistochemistry. Group one contained the dark purple neural tube explant. Group two consisted of hematoxylin blue, densely organized neuroepithelial cells that have grown out of the explant. Group three contained the DAB brown, HNK-1–positive neural crest cells. The surface occupied by each group as detected by the number of pixels was measured. The relative outgrowth of neuroepithelial (group 2) and neural crest cells (group 3) were both normalized against the explant area (group 1). The HNK-1–stained 24 hr cultures were analyzed by one observer without prior knowledge of the composition of the culture medium. Number of cultures: control (n ⫽ 10), 90 M folic acid (n ⫽ 10), 300 M homocysteine (n ⫽ 10), 90 M folic acid, and 300 M homocysteine (n ⫽ 10). Statistical analysis was performed by using the Mann-Whitney U test to compare independent groups. The results are presented as the median with the 95% confidence interval and the minimum and maximum values.
ACKNOWLEDGMENTS We thank Montsy Brouns (Department of Clinical Chemistry, Erasmus Medical Center Rotterdam) for biochemical analyses. We also thank Bas Blankevoort for artwork, Jan Lens for photographic contributions and support with the morphometrical analyses (Department of Anatomy and Embryology, Leiden University
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Medical Center), and Nicolette Ursem (Department of Obstetrics/ Gynaecology, Erasmus Medical Center Rotterdam) for assistance with statistical programs.
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