Environmental effects on the epigenetics of neural crest cell development

expresses a different set of genes. The proper expression of the gene set for a given cell type is regulated and maintained through epigenetic mechanisms1–4. Two epigenetic mechanisms are responsible for the establishment and maintenance of cell properties in higher eukaryotes: DNA methylation and histone modification. Generally, the genetic information encoded within a DNA sequence tends to be static without variation. Even if variations occur as a result of mutations, the consequences are discrete: either the presence or absence of the DNA mutation accompanying certain phenotypes. In contrast, the epigenetic marks obtained through DNA methylation and histone modification are usually malleable in terms of their modification levels. This plastic nature of the epigenetic modifications can be further influenced by environmental factors such as diet, toxic chemicals and stress, which is a previously unrecognised but important issue for human health. Nevertheless, it is largely unknown to what extent the environmental exposure affects the epigenetic modification process. Owing to the recent development of new DNA sequencing technologies, we have gained an unprecedented global overview of epigenetic modifications at the genomic level. However, the biological roles of these epigenetic modifications are not well understood, possibly because the majority of epigenetic studies have focused primarily on cancer and stem cells, and not in a broader context of animal development5. In that regard, neural crest cells (NCCs) are an excellent choice for the following reasons. NCCs play important roles in the developmental process of vertebrates, providing a battery of different cell types. Some unique features of NCCs are their migration capability and maintenance of mulipotency during development, which probably makes them vulnerable to subtle environmental changes. In fact, a large number of human disorders are caused by defects in the development and migration of NCCs, collectively referred to as neurocristopathies. The neurocristopaties are well-known for their low levels of penetrance, suggesting the potential involvement of non-genetic components. These non-genetic components might be of epigenetic origin since epigenetic modifications are known to play significant roles in the establishment and maintenance of stem cells, including NCCs. In this short review, we will discuss possible roles of epigenetic mechanisms and environment in the development of NCCs.

expresses a different set of genes.The proper expression of the gene set for a given cell type is regulated and maintained through epigenetic mechanisms [1][2][3][4] .Two epigenetic mechanisms are responsible for the establishment and maintenance of cell properties in higher eukaryotes: DNA methylation and histone modification.Generally, the genetic information encoded within a DNA sequence tends to be static without variation.Even if variations occur as a result of mutations, the consequences are discrete: either the presence or absence of the DNA mutation accompanying certain phenotypes.In contrast, the epigenetic marks obtained through DNA methylation and histone modification are usually malleable in terms of their modification levels.This plastic nature of the epigenetic modifications can be further influenced by environmental factors such as diet, toxic chemicals and stress, which is a previously unrecognised but important issue for human health.Nevertheless, it is largely unknown to what extent the environmental exposure affects the epigenetic modification process.
Owing to the recent development of new DNA sequencing technologies, we have gained an unprecedented global overview of epigenetic modifications at the genomic level.However, the biological roles of these epigenetic modifications are not well understood, possibly because the majority of epigenetic studies have focused primarily on cancer and stem cells, and not in a broader context of animal development 5 .In that regard, neural crest cells (NCCs) are an excellent choice for the following reasons.NCCs play important roles in the developmental process of vertebrates, providing a battery of different cell types.Some unique features of NCCs are their migration capability and maintenance of mulipotency during development, which probably makes them vulnerable to subtle environmental changes.In fact, a large number of human disorders are caused by defects in the development and migration of NCCs, collectively referred to as neurocristopathies.The neurocristopaties are well-known for their low levels of penetrance, suggesting the potential involvement of non-genetic components.These non-genetic components might be of epigenetic origin since epigenetic modifications are known to play significant roles in the establishment and maintenance of stem cells, including NCCs.In this short review, we will discuss possible roles of epigenetic mechanisms and environment in the development of NCCs.

Discussion
The authors have referenced some of their own studies in this review.The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.cranial and trunk NCC.Each shows distinct patterns and restricted developmental potential.For this reason, NCCs have been regarded as a heterogeneous cell population with multipotency [6][7][8] .NCCs give rise to many different cell types for vertebrate organs, including enteric nervous tissues, endocrine tissues, facial cartilage and bone and melanocytes.A unique feature shared among these various NCCs is their migration capability from the neural crest to various locations within the developing vertebrate 9,10 .Several signalling pathways are involved in this migration process, including RET and endothelin receptor B (EDNRB) pathways.RET encodes a receptor tyrosine kinase that recognises glial cell line-derived neurotrophic factor.In contrast, EDNRB encodes a G protein-coupled receptor that recognises endothelin 3.Both signalling pathways are critical for the development and migration of NCCs during vertebrate development 9,10 .

Introduction
Besides the genes involved in these two signalling pathways, other genes play critical roles in the developmental and migratory process of NCCs.This includes Snail2, Pax3, Sox10 and Mitf.Snail2 is a major regulator for the epithelial mesenchymal transition process, by which early neuroectoderm-derived cells lose their epithelial properties and transform into mesenchymal cells, and finally into NCCs.Snail2 is also well-known as a cancer gene with a promi nent role in metastasis 11 .Both Pax3 and Sox10 are developmental genes, and play major roles in the lineage specification of NCCs.In particular, Sox10 plays a pivotal role in various cell types of NCCs, including melanocytes, nerve cells and ganglion cells of the enteric nervous system 12,13 .Mitf is a master gene regulating the development and differentiation of melanocytes.These NCC genes share one unusual feature.In embryonic stem cells, these NCC genes are epigenetically modified by a rare combination of two histone marks, active (H3K4me3) and repressive marks (H3K27me3) [14][15][16] .This rare combination, termed bivalent state, is also often found in the other developmental genes, and thus thought to indicate the neutral, uncommitted state of histone modification for the associated genes 17 .Since the NCC genes are marked by the two histone marks in the embryonic stem cells, the histone-modifying complexes responsible for these two marks, polycomb repressive complex 2 for H3K27me3 and trithorax group for H3K4me3, should also play critical roles in the development and migration of NCCs.However, it is largely unknown to what extent these complexes contribute to the development and migration of NCCs.

Neurocristopathy-NCC defects
A unique feature of NCCs is their migration and delayed cellular differentiation capability during development, which could cause them to be vulnerable to subtle changes in their environment.As a result, many human disorders are caused by defects in the migration process of NCCs.In particular, mutations in the two signalling pathways are often manifested as human genetic disorders, including Hirschsprung's disease (HSCR) and Waardenburg (WS) syndrome.The main disease phenotype of HSCR is obstruction of the gastrointestinal tract, resulting in severe constipation and an enlarged colon, or 'megacolon'.This is caused by the absence of the NCCderived ganglia and resulting inability of the colon to peristalse [18][19][20] .More than half of familial and sporadic cases have been shown to be linked to the proto-oncogene RET locus although some cases are also linked to the EDNRB pathway.HSCR is inherited as an autosomal dominance trait, and the identified disease alleles are usually loss-of-function mutations.Thus, haploinsufficiency is the primary mode for the dominance observed in the majority of the familial cases linked to the RET and EDNRB locus.This further suggests that the migration and development of the NCC-derived ganglia are very sensitive to the gene dosage of both RET and EDRNB pathways.In contrast, WS shows much greater genetic and phenotypic heterogeneity, and is divided into four subgroups: WS type 1-4 21,22 .All four share two core phenotypes: sensorineural hearing loss and pigmentary disturbance.Although WS2 is characterised by just the two core traits, each of the three remaining subtypes has additional disease traits.For example, WS4 (Waardenburg-Shah syndrome) exhibits a megacolon phenotype similar to HSCR.Several genes are linked to both WS2 and 4, including EDNRB, endothelin 3 and Sox10.Similar to HSCR, WS2 and 4 are also inherited as an autosomal dominant trait, and the main mode of this dominance is the haploinsufficiency driven by loss-offunction-type mutations on the above genes 21,22 .
Despite all the accumulated knowledge, the molecular basis of the migration process of NCCs as well as their associated neurocristopathies needs to be further investigated.In particular, all the disease alleles identified within the two signalling pathways usually show low penetrance (presence or absence) for the disease traits in both HSCR and WS cases 19,20 .In many sporadic cases of HSCR, the majority of segregation analyses tend to pinpoint the RET locus as a primary disease locus, yet relevant mutations that could explain the malfunction of the RET protein or pathway have not been clearly defined.On the other hand, a growing number of recent studies suggest involvement of epigenetics.Recent studies from the mouse mutant line of Aebp2, which is involved in the formation of the histone modification

Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)
Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Kim H, Kang TJ, Kim J. Environmental effects on the epigenetics of neural crest cell development.OA Molecular & Cell Biology 2013 May 01;1(1):4.
H3K27me3, revealed that aberrant levels of the histone modification H3K27me3 can cause similar neurocristopathies as seen in humans, highlighting the importance of epigenetics in the development and migration process of NCCs 23 .

Environmental connection to neurocristopathy
During vertebrate development, the migration process of NCCs continues throughout and after the formation of major organs.This, along with the migration itself, turns out to be another risk factor for neurocristopathies.NCCs separate from the other three germ layers at early stages, yet some NCCs do not develop and migrate until the formation of major organs from the three germ layers.During this long waiting period, NCCs have to maintain their multipotency, which may provide a window of time for environmental factors to affect their stem cell properties.Many studies suggest environmental intervention on the development of NCCs, as detailed below.
First, many congenital malformations are associated with defects in the development of NCCs 24 .Surprisingly, the severity of some of these disease phenotypes varies depending upon the environmental condition to which an individual is exposed.For example, cleft palates are more commonly seen in malnourished populations.In a recent case study in Nigeria, malnourished adults are shown to develop HSCR 25 .Intake of folic acid throughout pregnancy can prevent cases of spinal bifida, which is caused by the incomplete closure of the spine 26,27 .On the other hand, intake of folic acid antagonists can increase the incidence of birth defects 28 .
Second, overdose of alcohol or retinoic acid during pregnancy has been shown to impair the development of NCCs.In humans, alcohol consumption during pregnancy is the primary cause of many cases of foetal alcohol syndrome, another well-known neural crest defect.Similarly, in mouse models, embryos that are exposed to alcohol have severe problems in the migration process of NCCs.The migrating NCCs often differentiate prematurely or undergo apoptosis.In one experiment, the ethanol-induced apoptosis in mouse embryos were rescued by injection of sonic hedgehog 29 .The sonic hedgehog pathway is known to be important in the development of craniofacial NCCs 30 .Retinoic acid is another important molecule for the proper development of NCCs 31 .In particular, the dosage of retinoic acid is critical: overdose or deprivation of retinoic acid can affect the proper NCC development 31 .For instance, overdose of retinoic acid has been shown to upregulate the expression levels of Hox genes, thus impacting animal development 31,32 .
Third, a number of nutritional supplements involved in methyl donor pathways, such as the folate and homocysteine (Hcy) pathways, are shown to affect the development and migration of NCCs.Hcy is thought to inhibit retinoic acid synthesis and subsequently affects signals for cell fate determination 33 .Overdose of Hcy levels in the blood stream during pregnancy have also been associated with congenital defects 33 .In general, any subtle environmental change, in the methyl donor pathway will affect the global levels of histone and DNA methylation since these two epigenetic modifications require a methyl group for their methylation.In this situation, the NCCs are thought to be vulnerable due to their long waiting period as a population of transient and uncommitted stem cells during development.

Conclusion
Among all the cell lineages constituting the body of vertebrates, NCCs are unique in many ways.NCCs are cells with multipotency, giving rise to various cell types that are unique to vertebrates.Yet NCCs have a challenging task during the development of animals, namely migration from the neural crest to various destinations within developing embryos.Thus, NCCs are susceptible to changes in environment because the transient and uncommitted status of NCCs can be easily affected during the migration process.According to recent studies, epigenetic mechanisms play many important roles in the development and migration processes of NCCs, such as the establishment and maintenance of stem cell properties and the lineage specification and differentiation of NCC-derived various cell types.It has also been well-known that many neurocristopathies display low levels of penetrance.It is therefore likely that environmental factors are accountable for the observed low levels of penetrance through various epigenetic mechanisms.In fact, human neurocristopathies appear to be driven by both genetic and epigenetic factors.Many epigenetic changes triggered by environmental exposures can be reversed or at least prevented with proper education.Thus, future studies on this epigenetic aspect of NCCs will provide useful information for human health including which environmental exposures have the most effect and to what extent these exposures are responsible for the disease phenotypes of the neurocristopathies.