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. 2016 May 28;37(2):371–376. doi: 10.1007/s10571-016-0383-y

Effects of Folic Acid and Homocysteine on the Morphogenesis of Mouse Cephalic Neural Crest Cells In Vitro

Fernanda Rosene Melo 1,#, Raul Bardini Bressan 2,#, Bruno Costa-Silva 3, Andrea Gonçalves Trentin 1,✉
PMCID: PMC11482145  PMID: 27236697

Abstract

Folate deficiency and hyperhomocysteinemia have long been associated with developmental anomalies, particularly neural tube defects and neurocristopathies—a group of diverse disorders that result from defective growth, differentiation, and migration of neural crest (NC) cells. However, the exact mechanisms by which homocysteine (Hcys) and/or folate deficiencies disrupt NC development are still poorly understood in mammals. In this work, we employed a well-defined culture system to investigate the effects of Hcys and folic acid (FA) supplementation on the morphogenetic processes of murine NC cells in vitro. We demonstrated that Hcys increases outgrowth and proliferation of cephalic NC cells and impairs their differentiation into smooth muscle cells. In addition, we showed that FA alone does not directly affect the developmental dynamics of the cephalic NC cells but is able to prevent the Hcys-induced effects. Our results, therefore, suggest that elevated Hcys levels per se cause dysmorphogenesis of the cephalic NC and might contribute to neurocristopathies in mammalian embryos.

Keywords: Neural crest, Neurocristopathy, Hyperhomocysteinemia, Folate deficiency, Smooth muscle

Introduction

The neural crest (NC) is a migratory, multipotent cell population that originates a vast array of vertebrate structures including the craniofacial skeleton and vasculature, cardiac structures, and the peripheral nervous system (Le Douarin and Kalcheim 1999). Defective growth, differentiation, and migration of NC cells result in a broad spectrum of congenital malformations, collectively called neurocristopathies (Bolande 1997).

Folate deficiency has long been associated with congenital abnormalities, particularly neural tube defects (NTDs) and neurocristopathies. In humans, epidemiological studies have shown that folic acid (FA) supplementation during preconception and early gestation prevents the development of NTDs, craniofacial, and heart malformations (Shaw et al. 1995; Kalter 2003; Obican et al. 2010). In addition, experimental studies in chick embryos have shown that imbalanced FA levels increase the frequency of NTDs and congenital heart, limb, and craniofacial malformations in a dose- and time-dependent manner (Rosenquist et al. 1996; Epeldegui et al. 2002; Boot et al. 2004; Tierney et al. 2004; Kobus et al. 2009).

Folate is a cofactor in one-carbon metabolism and is a crucial regulator of nucleotide synthesis and methylation reactions (Crider et al. 2012). Dietary folate deficiency results in elevated serum levels of homocysteine (Hcys), which is cytotoxic and can induce DNA breakage, oxidative stress, and apoptosis (Mattson and Shea 2003; Stover 2009). A substantial body of literature suggests that elevated Hcys during pregnancy acts as a teratogen and that the protective effect of folate is due to its ability to reduce serum Hcys levels by shunting excess Hcys into the methionine metabolic pathway (reviewed in Beaudin and Stover 2007; van Mil et al. 2010; Rosenquist 2013).

Despite the epidemiological and experimental evidence, little is known about the effects of hyperhomocysteinemia and folate deficiency on mammalian NC morphogenesis. In this work, we investigated the effects of FA and Hcys on murine NC cell outgrowth, survival, and differentiation in vitro. We demonstrated that elevated Hcys increased outgrowth and proliferation of cephalic NC cells and impaired their differentiation into smooth muscle cells. Such effects were prevented by concomitant administration of FA, which alone had no effect on cephalic NC cell behavior. Therefore, our results suggest that elevated Hcys levels per se cause dysmorphogenesis of the cephalic NC, which may contribute to neurocristopathies in mammalian embryos.

Experimental Procedures

Mouse NC Cell Cultures

Primary cultures of cephalic NC cells were prepared as previously described (Ito and Morita 1995; Costa-Silva et al. 2009) using C57BL/6 mouse embryos at 8.5 dpc. Briefly, neural folds at the mesencephalic level were microdissected and plated onto fibronectin-coated culture dishes (Gibco, 20 μg ml−1). After 48 h, the primary explants were mechanically removed and the emigrated cells harvested and replated onto fresh dishes. Cells were maintained in culture for additional 6 or 12 days in a complex culture medium consisting of α-minimum essential medium (α-MEM; Gibco) enriched with 10 % fetal bovine serum (FBS; Cultlab), 2 % chicken embryonic extract, hydrocortisone (0.1 µg ml−1), transferrin (10 µg ml−1), insulin (1 ng ml−1), 3-3′-5 triiodothyronine (0.4 ng ml−1), glucagon (0.01 ng ml−1), EGF (0.1 ng ml−1), FGF2 (0.2 ng ml−1), penicillin (200 U ml−1), and streptomycin (10 μg ml−1) (all purchased from Sigma). Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 and 10 % O2. The media was changed every 3 days.

Homocysteine and Folic Acid Treatment

Cultures were maintained in the complex medium alone (control) or supplemented with Hcys (150 and 300 µM) or FA (45 and 90 µM). Concomitant treatment consisted of 300 µM Hcys and 90 µM FA. These supraphysiological concentrations are comparable to other in vitro studies (Brauer and Rosenquist 2002; Boot et al. 2003).

Analysis of NC Cell Outgrowth

Outgrowth of NC cells to the culture dishes was analyzed 48 h after initial plating of neural fold explants in the primary cultures as previously described (Costa-Silva et al. 2009).

Immunofluorescent Staining

Cells were fixed with 4 % paraformaldehyde (30 min), permeabilized with 0.1 % Triton X-100 (Sigma, 30 min), and blocked with 5 % FBS (1 h) at room temperature. Samples were then incubated with primary antibodies against lineage-specific markers (overnight, 4 °C) and followed by incubation with appropriate secondary antibodies (1 h, room temperature). The following primary antibodies were used: anti-GFAP (Dako), anti-βIII-tubulin (Promega), anti-α-SMA (Sigma), anti-nestin (Abcam), and anti-p75NTR (Chemicon). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma) for 5 min at room temperature. Immunoreactive cells were quantified on a minimum of 5000 cells in at least 20 random fields. Total cell number was determined by DAPI nuclear staining.

Cell Proliferation and Cell Death Assessment

Cell proliferation was analyzed by 5-bromo-2′-deoxyuridine (BrdU) incorporation as previously described (Bressan et al. 2014). Cultures were incubated with 10 µM BrdU (Invitrogen, 24 h, 37 °C), fixed, and immunostained with anti-BrdU antibody (Calbiochem) according to manufacturer’s instructions. The ratio of proliferation was determined as the proportion of BrdU-positive cells in relation to total cell number. Cell death was assessed by quantification of pyknotic nuclei after DAPI staining as described elsewhere (Costa-Silva et al. 2009). A total of 20 random fields (×200 magnification) were counted for each condition.

Statistical Analysis

Statistical differences were evaluated by one-way ANOVA with Bonferroni’s post-test using GraphPad Prism 6.0 (Graphpad Software, Inc.). The level of significance was set at P < 0.05 in all cases. Experiments were performed in triplicate, and results represent the mean of at least three independent experiments.

Results

In order to investigate whether exposure to Hcys and FA affect the outgrowth of cephalic NC cells, we measured the area occupied by the emigrated NC cells from NT explants on the culture dishes after 48 h of primary culture (Fig. 1a–c). Cultures treated with 300 μM Hcys showed increased area of NC cell occupation compared to the control condition (1.75-fold increase), while FA alone (45 and 90 μM) had no significant effects (Fig. 1c). The co-administration of 90 μM FA, however, was able to partially prevent the effect of 300uM Hcys on cell outgrowth (Fig. 1c). No substantial differences in NC cell morphology upon the treatments were observed (Fig.1a,b).

Fig. 1.

Fig. 1

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Hcys increases outgrowth and proliferation of mouse cephalic NC cells. a Phase photomicrograph of a primary culture (control condition) 48 h after initial plating of the neural tube explants (NT). b Morphology of emigrated NC cells in primary cultures. c Quantification of the area occupied by emigrated NC cells after 48 h of primary culture in the presence of different concentrations of Hcys and/or FA. Values were obtained from the analysis of at least six explants in each condition of three independent experiments and expressed as the mean ± SEM. Representative images of the control condition are displayed. d Quantification of BrdU-positive cells in relation to the total cell number after 6 days of secondary culture. Results were obtained from three independent experiments. Values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni post-test (Color figure online)

Importantly, treatment with 300 μM Hcys also significantly increased the overall proliferation of NC cells (2.6-fold increase in relation to the control), as determined by BrdU incorporation at day 6 of secondary culture (i.e., after replating the cells that had initially grown away from the NT explant; Fig. 1d). FA alone did not affect the proliferation rate of the cells but, when co-added with Hcys, was able to completely prevent the Hcys-induced effects (Fig. 1d). Quantification of pyknotic nuclei by DAPI staining 12 days after replating was used to assess the effects of Hcys and FA on cell death (Fig. 2). We observed a small percentage of apoptotic cells (ranging from 1.45 to 1.82 %) with no statistically significant differences among the treatment conditions.

Fig. 2.

Fig. 2

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Effects of Hcys and FA on NC cell death. Cell death was assessed by quantification of pyknotic nuclei after DAPI staining at day 12 of secondary cultures. Image displays a representative field of the control condition. Values represent the percentage of pyknotic nuclei in relation to total cell number and are expressed as the mean ± SEM of seven independent experiments

We next examined the effects of Hcys and FA on the differentiation of cephalic NC cells at day 12 of secondary culture (Fig. 3). Immunofluorescence staining revealed an increased proportion of cells expressing the NC progenitor/stem markers p75NTR and nestin in Hcys-treated cultures (Fig. 3a). In this condition, undifferentiated p75NTR-positive and nestin-positive cells contributed to, respectively, 23 and 40 % of total cells versus 13 and 26 % in the control condition (1.8- and 1.5-fold increase, respectively). Hcys effects were prevented by FA supplementation, while FA alone did not induce any phenotype change (Fig. 3b). Double staining for BrdU and nestin was subsequently performed in order to specifically analyze the proliferation rate of NC progenitors. This analysis revealed a significant increase in the proportion of proliferating nestin-positive progenitor cells in the presence of Hcys (2.4-fold increase compared to the control) (Fig. 3a, b). The treatment with FA alone did not affect the proportion of the nestin/BrdU double-stained cells. However, FA was able to completely prevent the Hcys-induced effects on NC progenitor proliferation.

Fig. 3.

Fig. 3

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Hcys retains cephalic NC cells in an undifferentiated and proliferative state and impairs their differentiation into smooth muscle cells. a The proportion of undifferentiated NC cells was assessed by immunofluorescence for the NC progenitor markers p75NTR and nestin. Proliferating progenitors were identified by double staining for nestin (green) and BrdU (red). Representative images of positive cells in the control condition are displayed. b Percentage of p75NTR-positive, nestin-positive, and proliferating nestin-positive progenitors in relation to the total cell number in the presence of Hcys and/or FA. c NC-derived phenotypes were identified by the expression of the lineage-specific markers βIII-tubulin for neurons GFAP for glia and αSMA for smooth muscle cells. Representative images of positive cells in the control condition are displayed. d Quantification of the respective cell types in relation to total cell number. Values are expressed as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA with Bonferroni post-test (Color figure online)

The above results indicated that Hcys could maintain the NC cells in a more proliferative, undifferentiated state. Therefore, to address the possibility that Hcys affects the differentiation of NC cells into one or more specific phenotypes, we evaluated the proportion of cells expressing differentiated phenotypic markers at day 12 of secondary culture. Figure 3c shows representative images of NC-derived cell types according to the expression of the lineage-specific markers βIII-tubulin (neurons), GFAP (glial cells, including Schwann and/or olfactory enshealting cells) and αSMA (smooth muscle cells). In control cultures, glial cells, and neurons corresponded to approximately 3 and 2 % of the total cells, respectively. No statistically significant differences in the proportion of these phenotypes were observed upon the different treatments. However, the proportion of smooth muscle cells—the most frequent differentiated cell type in the cultures—was reduced by five-fold in the presence of Hcys (Fig. 3c, d). Treatment with FA prevented the Hcys effects on the smooth muscle differentiation, while FA alone had no significant effect on this cell phenotype.

Discussion

Maternal folate deficiency and hyperhomocysteinemia have long been associated with NTDs and neurocristopathies. However, little is known about the teratogenic effects of elevated Hcys and folate deficiencies on the morphogenetic processes of mammalian NC. Here we demonstrated that elevated Hcys in vitro increases outgrowth and proliferation of murine cephalic NC cells and impairs their differentiation into smooth muscle, most likely by maintaining cells in a more undifferentiated and proliferative state. Such effects were prevented by the concomitant administration of FA, which alone had no effect on cephalic NC cell behavior.

We first showed that Hcys increases the outgrowth of cephalic NC cells, as demonstrated by the greater area occupied by NC cells migrating away from the NT explants. Such effect on cephalic NC outgrowth is supported by observations in the chick embryo that Hcys alters cardiac NC cell motility, migration distance, cell surface area, and cell perimeter (Brauer and Rosenquist 2002; Boot et al. 2003). The Hcys-induced increase in motility and migration of avian NC cells is thought to occur via inhibition of N-methyl-d-aspartate (NMDA) receptor (Brauer and Rosenquist 2002; Boot et al. 2003). However, as the expression of NMDA receptor in murine embryos is not detected until later stages of gestation—at a time when the NT is closed and the NC cells have already migrated (Bennett et al. 2006)—we speculate that a NMDA receptor-independent mechanism mediates Hcys-induced effects on murine cephalic NC cell migration and further investigation is needed to clarify the question.

In addition, we observed that exposure to Hcys stimulates mouse cephalic NC cell proliferation as shown by increased BrdU incorporation rate. This result, however, differs from previous observations in the chick embryo, in which the Hcys treatment decreased the proliferation of newly formed cardiac NC cells via inhibition of cell cycle progression or S-phase entry (Tierney et al. 2004). Biochemical/molecular differences between avian and murine embryos and/or along the NC axial level (cardiac versus cephalic NC), together with potential Hcys secondary effects in adjacent cells present in the described in vivo settings, might explain the differences between our results and the previous study. Noteworthy, the differences observed are supported by the idea that Hcys-induced teratogenic effects are time- and cell type-dependent (reviewed in van Mil et al. 2010).

As the increased proliferation rate suggests a possible effect of Hcys on the differentiation of cephalic NC cells, we next analyzed the proportion of cells expressing the NC progenitor/stem cell markers p75NTR and nestin. This revealed a significant increase of undifferentiated cells in Hcys-treated cultures, with no alterations in the other conditions. Although p75NTR expression has been reported in differentiating peripheral glial cells, we did not observe its co-expression with GFAP (not shown), as it would be expected for immature Schwann cells and olfactory enshealthing glia (Jessen and Mirsky 2005; Katoh et al. 2011). This, therefore, suggests that p75NTR-positive cells observed in our cultures indeed correspond to undifferentiated NC progenitors (including those endowed with glial potential), as extensively described in the literature (reviewed in Dupin and Sommer 2012, Dupin and Coelho-Aguiar 2013). Furthermore, we verified a remarkable increase in the proportion of nestin/BrdU double positive cells, which indicates that Hcys maintains cephalic NC cells in a more undifferentiated and proliferative state.

Confirming the ability of Hcys to impair cephalic NC cell differentiation, we observed a dramatic reduction in the proportion of smooth muscle cells upon exposure to Hcys, with no significant effects on the other NC-derived phenotypes analyzed. Similarly, Boot et al.(2003) have reported that Hcys disturbs the differentiation of avian cardiac NC and inhibits αSMA expression in vitro. Moreover, several reports have demonstrated the mitogenic effect of Hcys on vascular smooth muscle cells and its involvement in atherosclerosis progression in adults (Tsai et al. 1994; Kartal Ozer et al. 2005; Liu et al.2009; Zhang et al. 2012; Chiang et al. 2011). However, our results are the first to indicate that elevated Hcys promotes proliferation of progenitor cells endowed with smooth muscle potential, rather than fully differentiated cells, during mammalian development. Considering the cephalic NC-origin of smooth muscle cells of all blood vessels of the face and forebrain (Etchevers et al. 2001), our study indicates that Hcys-induced teratogenesis in mammalian embryos may occur through impairment of cephalic vascular system formation.

Additionally, we showed here that Hcys-induced effects can be partially or completely prevented by the administration of FA, which alone had little or no effect on cephalic NC cell behavior. The ability of FA to consistently counteract Hcys-induced alterations in the developmental dynamics of cephalic NC cells may partly explain the well-known beneficial effects of periconceptional folate supplementation in preventing neurocristopathies and NTDs (reviewed in Antony and Hansen 2000; Brauer and Tierney 2004; Imbard et al. 2013).

In summary, our study shows that elevated Hcys directly disrupts normal morphogenesis of mammalian NC in vitro and supports the idea that the protective effect of folate is due to its ability to prevent Hcys-induced teratogenesis. The results suggest that Hcys may particularly impair the development of the cephalic vascular system by increasing outgrowth and delaying the differentiation of cephalic NC cells into smooth muscle cells. These new findings are steps forward in the process of unraveling the cellular mechanisms underlying NTDs, craniofacial malformations, and conotruncal heart defects.

Acknowledgments

This work was supported by Ministério da Ciência, Tecnologia e Inovação/Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCTI/CNPq/Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Instituto Nacional de Neurociência Translacional (MCTI/INNT), and Fundação de Amparo à Pesquisa do Estado de Santa Catarina (FAPESC, Brazil). RBB is supported by a fellowship from the Science Without Borders Program (CAPES, Brazil).

Compliance with Ethical Standards

Conflict of Interest

The authors declare no conflict of interest.

Footnotes

Fernanda Rosene Melo and Raul Bardini Bressan have contributed equally to this work and are both first authors.

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