The human neural tube defects (NTD), anencephaly, spina bifida and craniorachischisis, originate from a failure of the embryonic neural tube to close. Human NTD are relatively common and both complex and heterogeneous in genetic origin, but the genetic variants and developmental mechanisms are largely unknown. Here we review the numerous studies, mainly in mice, of normal neural tube closure, the mechanisms of failure caused by specific gene mutations, and the evolution of the vertebrate cranial neural tube and its genetic processes, seeking insights into the etiology of human NTD. We find evidence of many regions along the anterior–posterior axis each differing in some aspect of neural tube closure—morphology, cell behavior, specific genes required—and conclude that the etiology of NTD is likely to be partly specific to the anterior–posterior location of the defect and also genetically heterogeneous. We revisit the hypotheses explaining the excess of females among cranial NTD cases in mice and humans and new developments in understanding the role of the folate pathway in NTD. Finally, we demonstrate that evidence from mouse mutants strongly supports the search for digenic or oligogenic etiology in human NTD of all types. The mechanisms of closure of the neural tube and the development of neural tube closure defects (NTD) are genetically complex and diverse. With an incidence of one to ten per thousand births, NTD are among the most common severe birth defects . DefectsThe formation of a neural tube by the mechanism of rolling up a neural plate into a tube evolved before the evolutionary emergence of vertebrates, and some mammalian molecular mechanisms likely reflect this ancient origin. We briefly consider the process of neural tube formation in the animal usually used as a proxy for the ancestral chordate, Amphioxus, and in Ascidian embryos, representative of another ancient pre-vertebrate lineage, the Tunicates. There are extensive differences in the physical process of neural tube closure among modern species and even among mammals; we shall briefly also consider these to point to the need for assessment of these differences before using observations from model animals to predict the etiologies of human NTD. We give considerable emphasis to the anterior–posterior regional differences in the mechanisms of neural tube closure at the morphological and molecular levels, underlining the concept that defects of closure of the neural tube are mechanistically heterogeneous with distinct or partially overlapping etiologies. This heterogeneity predicts genetic differences in the etiologies of human NTD located at different sites along the anterior–posterior axis, as well as genetic heterogeneity at any given site. We focus on cranial neural tube development, its genetic mechanisms of failure to close, and the evolutionary New Head hypothesis that predicts consequential differences between the cranial and spinal regions in the mechanisms of neural tube closure. These differences should inform genetic studies of human NTD. We consider recent advances in understanding of the epigenetic mechanism by which the sex chromosome complement of mammalian embryos influences risk of cranial NTD, seeking new insight into the neural tube closure process. Similarly, we focus on the phenomenon of the preventative effect of maternal folic acid supplementation on occurrence of NTD in humans and in mice and recent developments in understanding of folate mechanisms, seeking possible insights into the etiologies of NTD. That there is a genetic etiology for human NTD is clear, with 30× elevated risk in siblings of NTD cases, but it is complex. Typical recent studies are based on exon sequencing of candidate genes in cohorts of NTD cases, including a variety of locations and types of lesion. This approach for several planar cell polarity (PCP) genes has detected heterozygous rare putatively deleterious variants in a small number of cases .Despite many genetic studies, the etiology is not well understood , and likFailure to close the neural tube leads to three principal open neural tube defects: craniorachischisis, in which the entire spinal region and the hindbrain fail to close; spina bifida aperta, in which a limited region of the spinal neural tube fails to close, and anencephaly or exencephaly, in which the midbrain and sometimes also the hindbrain fail to close. There are other defects that arise subsequently from defects in the tissue of the closed neural tube or surrounding mesenchyme, such as spina bifida occulta  and encephalocele . Semantically or clinically, they are often included in NTD, but they are not within the scope of the etiology of failure to form a closed neural tube ,13.The major physical components of the complex that will comprise the neural tube  include:Prickle [Wnt5a and Wnt11). The “core” proteins include the transmembrane receptors Vangl1/2, Celsr1/2/3, and Fzd (Frizzled) and the cytosolic proteins Dvl, Prickle, Inversin (Invs) and Scrib [Fat and Dchs, which bind to each other and are regulated by Fjx [Ptk7, Sec24b, Intu and Fuz [Immediately preceding the initiation of neural tube formation, the vertebrate embryo undergoes convergent extension, which involves the lateral-to-medial convergence and anterior–posterior extension of axial tissues, including the neural plate. The non-canonical Wnt/PCP (planar cell polarity) pathway is crucial to this process. Cells change position in the tissue, intercalating between surrounding cells to shift toward the midline. A prerequisite is the directional orientation of cells relative to a morphogenic signal. The cells become asymmetrical in the positioning of various subcellular structures, including the core proteins of the PCP pathway ,15. MutaPrickle . The “cond Scrib . The Fat and Fuz . In addition to its essential role in planar cell polarity required for convergent extension, the PCP pathway affects ciliogenesis, the arrangement of cytoskeletal actin and microtubules, the basal body and the centriole within cells ,17,18. STwo areas of the neural plate undergo pronounced bending that strongly contributes to forming a tube; these are the medial hinge point (MHP) located at the midline base of the neural tube throughout most of its length, and the dorsolateral hinge point (DLHP) located in the dorsal third of each fold in some zones along the length of the neural tube  Figure . In addiShh [Shh signaling molecules from the ventral neural plate, BMP signaling from the surface ectoderm, and Noggin from the neuroepithelium [The mouse spinal DLHPs form at the dorsoventral location where the neuroepithelium ceases to have basal contact with the paraxial mesoderm and begins basal contact with the surface ectoderm ; howeverShh , does noShh . There iithelium . The dorSonic hedgehog (Shh) signaling. Other pathways, such as signaling through Wnt, Calcium, Pdgfr, Notch, and Tgfb, may also function via cilia [Most cells have a primary cilium. In the neural tube, primary cilia can be seen extending from the neuroepithelium into the incipient lumen . Vital ia cilia ,26,27. Sia cilia , and somia cilia ,29. TherSonic hedgehog (Shh) signaling pathway, including positive and negative regulators, are present in the cilia [Shh signaling pathway. The Shh signaling pathway affects the transcription of downstream target genes via the Gli transcription factors; there are many transcriptional targets and complex feedback loops [Shh signaling pathway inhibits the processing of a full-length activator form of Gli3 (Gli3FL) into a repressor form (Gli3R) and the ratio of Gli3FL:Gli3R is a measure of functionality of the Shh signaling pathway [Several components of the he cilia . Shh is ck loops . An acti pathway ,30.Shh is produced by the notochord and prechordal plate extending from the future forebrain area to the caudal area [Shh production from the midline of the neural plate with a ventral to dorsal gradient in the developing neural tube [Shh signaling is required for development of the floor plate, it is not clear that Shh is required for formation of the MHP; the MHP is not always required for neural tube closure [Shh signaling [Shh signaling in the dorsal neural folds permits a neuroepithelial response to Noggin, which suppresses Bmp signaling from the adjacent surface ectoderm, enabling formation of dorsolateral hinge points (DLHP) [Shh is expressed in a rostral to caudal and temporal gradient in the developing neural tube from the upper spinal region to the caudal spinal region, being expressed in the caudal notochord and floor plate after neural tube closure [Shh signaling in the ventral midline seems contradictory to the Shh concentration-dependent inhibition of DLHP in the upper spine. However, in many species, the cranial neural folds are clearly much larger than the spinal neural folds; this is particularly notable in mouse embryos. Therefore, the physical distance for diffusion of signaling molecules from the midline to the dorsal tips is greater than in the spine. We suggest that the large size of the cranial neural folds creates the necessary lack of signaling concentration in the cranial dorsal folds, enabling cranial DLHP formation. During neural tube development, dal area , and indral tube . While t closure ,22. The s (DLHP) . Shh is  closure . In the  closure  and FiguShh gene, or loss of function of components of the Shh signaling pathway such as Smo, do not cause NTD; the neural tube forms a midline bend and DLHP and closes. Typically, loss of function of the Shh signaling pathway causes a lack of development of the cranial midline tissues that results in holoprosencephaly and related facial defects [Shh signaling pathway downstream of Shh due to the loss of negative regulators such as Ptch1, Rab23, Tulp3 or Sufu [Tulp3 [Loss of function of the  defects ,33,34. I or Sufu  leads tou [Tulp3 ) and cauShh signaling [Ttc21b (intraflagellar transport), Kif7 (kinesin motor protein) and Arl13b , seem to increase Shh signaling pathway activity based on expansion of ventral markers, and cause exencephaly, parallel to the other mutants with increased Shh signaling. Others, such as mutants for several intraflagellar transport (Ift) genes, dynein components , PCP effector genes , and C2cd3 (which recruits Ift proteins for ciliogenesis) seem to reduce Shh signaling pathway activity, based on reduced ventral markers in the neural tube, but contrary to the mutations with loss of Shh signaling, they also cause exencephaly. Notably, it seems that cilia mutants that cause exencephaly have an increased Gli3FL:Gli3R ratio [Mutations in genes that affect the structure or function of cilia fall into two major types with respect to ignaling . Some mu3R ratio . The proEph receptor tyrosine kinases and their membrane bound ligands, the Ephrins, which are generally involved in cell–cell attraction and repulsion interactions, are found in the neural folds, including the dorsal tips, just prior to fusion and are possibly involved in initiation of adhesions between apposed folds [To complete neural tube closure, the neuroepithelium and surface ectoderm of the neural fold must make contact with their counterparts in the apposed fold and form de novo adhesions to create a continuous surface ectoderm covering a continuous neuroepithelium. The neuroepithelia and surface ectoderms do not necessarily make contact simultaneously, and the order varies along the anterior–posterior axis. Some details have emerged from recent studies. Filopodia and ruffles project from apposed surface ectoderms into the gap between the apposed folds and are required for successful closure, although their role is unknown ,38,39. Ved folds . Shh signaling along the anterior to posterior spine [Grhl gene family [Ephrin/Eph gene families [Studies of mouse embryos have demonstrated that different regions along the anterior–posterior axis have different major mechanisms for bending the neuroepithelium to meet in the midline and also differ in fusion mechanisms . These ror spine , presence family  and the families ,42,43, tThe upper spine region depends on planar cell polarity for cell movements and convergent extension that narrows the midline gap between the future neural folds, and on cell cycle dynamics in the neural plate midline that cause cell wedging to create a medial hinge point (MHP). The mid-spinal region also depends on the effects of convergent extension, and has the MHP, but additionally has DLHPs. The most caudal area of primary neurulation, the posterior neuropore (PNP), likely also depends on initial convergent extension to narrow the neural plate, but unlike the rest of the neural tube, the notochord is not present before closure . The MHPCaudal to the PNP, neural folds do not form and a tube is hollowed out from the caudal mesenchyme in a process termed “secondary neurulation”. Defects in secondary neurulation per se would not cause closure defects, but there is evidence that mis-regulation of the development of the junction between the PNP and secondary neurulation may cause some lumbosacral open neural tube defects ,46. Overall, given the various overlapping regional differences in molecular genetic mechanisms and closure mechanisms from head to tail, it is likely that NTD in different anterior–posterior regions have region-specific causes that can be predicted. For example, in families with recurrence of NTDs, in about 70% of families, the second cases have the same defect as the first case, but in about 30% of families, one has spina bifida and one has anencephaly . Given tAt several specific locations along the anterior–posterior axis the neural folds achieve close apposition and initiate fusion. Closure then is thought to “zip” along the neural folds until it meets a closed region “zipped” from a different initiation site—an intermittent pattern rather than a single “zipper”. The mechanisms that determine the locations of closure initiation sites are not known. The locations of cranial closure initiation sites differ somewhat between normal mouse strains ; the posThe relevance to NTD of species differences in locations of closure initiation sites is debatable. Generally, the locations of open cranial NTD and open spinal NTD in mice and humans appear to be the same, i.e., the same parts of the neural tube have failed to close. As we have said previously “… if the defect in both species is a failure of elevation itself, a species difference in the location of first contact of already elevated folds would be subsequent to, and unrelated to, the cause of NTDs” . FurtherThe tips of the neural folds expose the border between the neuroepithelium and surface ectoderm. The cell type involved in initial contact between apposed folds varies regionally. In the mouse forebrain region, first contact across the gap is between the apposed neuroepithelia, whereas in the midbrain and hindbrain, the initial contact and apparent adhesion is between cells of the surface ectoderm, which in the midbrain wraps around the neuroectoderm as the folds meet so that surface ectoderm faces across the closing gap ,67. In tThe fusion of the neuroepithelium and the surface ectoderm may be independent; a closed cerebellar defect arising in an unfused neuroepithelium beneath a fused surface ectoderm has been described in mice .Fusions between epithelia in various tissues and species are typically preceded by various cellular projections such as ruffles, filopodia and lamellipodia ). DynamiRac and Cdc42 [Rac1 induces the formation of branched actin networks needed for lamellipodia and ruffles, whereas Cdc42 leads to assembly of unbranched actin bundles that form filopodia [The regional differences in the abundance of filopodia and ruffles reflect, at least in part, differences in regulators of the actin cytoskeleton, nd Cdc42 . These Gilopodia .Ephs) and their membrane-bound ligands, the Ephrins, are involved in cell–cell interactions throughout development. Interaction between Ephrins and their receptors may cause cell adhesion or repulsion. They are involved in the adhesion of apposed neural folds for fusion and may have a role in the etiology of NTD. This area has not been deeply studied and roles of more members of these gene families are likely to be identified in future studies. Expression of some combinations of Ephrins and Ephs may differ between cranial regions and also differ from the caudal region. EphA4 is expressed in the midbrain region in early neural fold stages [EphA2 and EphA4 are expressed in the hindbrain neural folds before closure [EphrinA5 and its receptor EphA7 splice forms are expressed in the dorsal cranial neural folds (midbrain and hindbrain) before and during closure, but not in the spinal neural folds [EphA1 is expressed in the neural plate and other tissues, EphA2 in the neural plate and tips of the apposed neural folds, EphA4 in the mesoderm and tips of the apposed neural folds, and EphA5 in the neural plate [EphA2 in the tips of the neural folds located its expression to surface ectoderm cells and their lamellipodia-like protrusions that extend towards the apposed neural fold [The gene families of membrane-bound Ephrin receptor tyrosine kinases  in all homozygotes and concomitant exencephaly in a few [Grhl2 causes split face and open cranial neural tube extending through the hindbrain to the cervical region and, depending on the mutant allele and genetic background, spina bifida [Grhl2+/−;Grhl3+/−) have lumbo-sacral spina bifida with curled tail or exencephaly and/or curled tail. Grhl2 null and Grhl3 null embryos fail to form DLHP in the regions that will have failed neural tube closure [The regionality of the requirement for expression of different members of the in a few . Loss ofa bifida ,41. HeteGrhl2 is essential to closure of the forebrain and cooperates with Grhl3, whereas Grhl3 is essential to closure posterior to the mid-thoracic region, and cooperates with Grlh2 for closure of the posterior neuropore (PNP). Neither Grhl gene is required for closure of the cervical region to the thoracic region, the rostral part of the Closure 1 zone; this is the only region of the neural tube that does not form a concave shape of neural folds before closure, and may therefore be the only region that does not have DLHPs. DLHPs are lacking in the PNP of both Grhl2 and Grhl3 mutants; other regions were not studied. In summary, the Grhl2 and Grhl3 genes may be respectively essential for DLHP formation in separate zones of the developing neural tube, and may also function cooperatively for DLHP formation in other zones. The Grhl mutants did not alter the expression of some genes known to be important in DLHP formation, such as Bmp2, Noggin, and Zic2 [Grhl2, which is specifically expressed in the surface ectoderm of the cranial neural folds, is required to suppress transformation of ectoderm to mesenchyme [Grhl3. In the caudal region Grhl3, is expressed in surface ectoderm during neural tube closure, and lack of Grhl3 seems to cause delay of specification of surface ectoderm [Grhl2 [Grhl3 is expressed in the gut endoderm that underlies the neural tube, and lack of Grhl3 expression there is associated with defective hindgut cell proliferation that causes excessive axial bending that mechanically interferes with closure of the posterior neuropore [Further study  indicateand Zic2 . The faisenchyme . The acqectoderm , a situam [Grhl2 . Later, europore .Vangl2) on their differentiation as well as on neuroepithelial actomyosin contractility [Actomyosin “machinery”  and RhoGTPases) is located circumferentially in the apices and apical junctions of all neuroepithelial cells. Its contraction is thought to be responsible for apical constriction, contributing to neural fold bending, and is required for cranial neural tube closure but not spinal neural tube closure. Mice with mutations affecting actin-associated proteins develop cranial, but not spinal, NTDs. Conversely, the caudal spinal region as well as the cranial region requires actin turnover for neural tube closure and abnormal accumulation of apical actomyosin does cause failure of neural fold elevation in both regions . RhoA anactility .Hox genes, which convey anterior–posterior positional information, are expressed in many different combinations along the midline axis before neural tube formation, with the earliest and fewest expressed in the hindbrain region and the latest and most numerous expressed in the caudal spinal region; in contrast, none are expressed in the cranial region rostral to the hindbrain [Other regional differences in the neural tube also have an unknown or uncertain role in closure mechanisms. For example, numerous indbrain ,75. Anotindbrain ,76. Therindbrain ,77. Most of our understanding of neural tube development is based on a small number of species: mouse, chick, frog (Xenopus), rat, and zebrafish. Among these, there are very important differences in the basic mechanisms by which the neural tube is formed. All likely share the basic planar cell polarity/convergent extension mechanism . DetailsNoggin/BMP signaling and parts of the Wnt/PCP signaling pathway are present [The Cephalochordate, Amphioxus (lancelet), whose lineage diverged from vertebrates 550–600 million years ago, is used as a proxy for the ancestral chordate ,82. Like present  but a deThe lineage of Tunicate chordates, the Ascidians (sea squirts), diverged from the vertebrate lineage around the same time as Cephalochordates . The larThe similarities of neural tube development in comparisons of mammals and Amphioxus and Ascidian embryos suggests that the rolling up of the neural plate into a tube is the ancient mechanism, and from Ascidian embryos that the formation of neural folds is also the ancient mechanism. The divergences from this pattern in species such as zebrafish are therefore new evolutionary products. Some of the ancient processes, such as migration of the neural plate border cells to form the surface ectoderm over the tube in Amphioxus suggest the possibility of atavistic capabilities lurking in the mammalian genome, potentially available in some circumstances.An important context for understanding cranial neural tube closure is that the vertebrate head and its neural tube are considered by the “New Head” hypothesis to be a new structure that was added by evolution, stepwise, onto the basic chordate ancestor which already had the spinal neural tube ,89. The The ancestral pre-vertebrate chordate representative, Amphioxus, is a filter-feeder with tentacle-like structures around a small mouth, pharyngeal gill slits, a notochord that extends all the way to the front of the head, a neural tube, and a post-anal tail; it does not have jaws, skull, neural crest or ectodermal placodes . It has a very simple tiny brain with rudiments of hindbrain, diencephalon, and perhaps some midbrain, but no telencephalon . AmphioxSoxE gene that causes its expression in cranial neural crest cells [Evolution of a neural crest set the stage for the development of the New Head . Most ofst cells . A consiTcf7l1 expressed in the anterior forebrain neural folds acts to repress the Wnt/B-catenin pathway that otherwise would induce neuroectoderm cells to become neural crest cells [Tcf7l1 leads to conversion of the neuroectoderm of the most rostral neural folds to neural crest cells and a subsequent reduction of the rostral forebrain, which if severe is accompanied by exencephaly [Unlike the rest of the vertebrate neural tube, the anterior forebrain neural folds do not generate neural crest cells, and the neural crest cells of the forebrain mesenchyme migrate in from the caudal forebrain and the midbrain ,94,95,96st cells . Loss ofncephaly . InteresHox genes, some of which are present in Amphioxus, are expressed in the neuroectoderm and paraxial mesoderm of vertebrates and impart anterior–posterior positional identity. Under the regulation of Fgf, Wnt and other genes, various combinations of Hox genes are expressed in the newly formed axial tissues from the primitive streak stage onward [Hox expression extends from the embryonic hindbrain through the tailbud; there is no cranial Hox gene expression rostral of the border between rhombomere 1 and 2 of the hindbrain [Hox gene expression affects the subsequent genetic program determining cranial neural tube closure.e onward ,98. In vShh, but it also differs from the notochord, by producing factors such as Goosecoid (Gsc), an inhibitor of convergent extension [cNot1 [The prechordal plate ,99 underxtension  and by nn [cNot1 ,101,102.Although the prechordal plate is initially located in line with the notochord, it is bent to lie at right angles to the notochord just before and during the time the mesencephalic folds are elevating ,99,103. A further complexity in the mechanical context of closure of the cranial neural tube is the development of the optic sulci in the forebrain. These begin as indentations in the neuroepithelium and balloon outward toward the surface ectoderm as the forebrain neural folds elevate toward each other, so that the apposed forebrain folds resemble a pair of castanets  , but thpalladin, vinculin, cofilin 1 (Cfl1) and Marcks, or in genes for actin regulatory proteins such as Mena, Vasp and Evl, or in genes for protein kinases with cytoskeletal influence such as Abl1, Abl2, Mapk8 and Mapk9 all cause exencephaly in mice [Mammalian cranial neural fold bending for closure requires the actomyosin cytoskeleton, and mutations in its components or chemical disruption of apical actin microfilaments cause exencephaly (summarized in ). Thus,  in mice . In contCasp3 and Apaf1 null mutants, the midbrain and hindbrain fail to close but the rest of the neural tube closes normally. Live imaging of the midbrain–hindbrain neural folds undergoing bending and closure in normal embryos show conventional and unconventional apoptotic cells [Casp3 and Apaf1 null mutants, neither type of apoptotic cell is found and the apical bending of the folds towards the midline is severely reduced. The mechanism of the effect of apoptosis on bending is not known.Programmed cell death, apoptosis, normally occurs in the neuroepithelium during development of the neural tube , with a ic cells . ConventTcfap2a and the cell adhesion factor Cdh2 (N-cadherin). In mice on E8.5, Tcfap2a is expressed in the cranial neuroepithelium and cranial neural crest; Cdh2 is expressed in the cranial neural plate. Conditional mutants in which either Tcfap2a or Cdh2 is deleted in the neural crest cells in which Wnt1 is normally expressed, suggest that some aspect of cranial neural crest abnormality causes midbrain exencephaly . Althoue border . A complal crest . The potTcfap2a null embryos have split face and forebrain/midbrain/hindbrain exencephaly [Cdh2 null mutants rescued from early lethality [Wnt1-Cre conditional null Tcfap2a and Cdh2 mutants, the mechanisms are unclear. We have found no definitive evidence from mouse mutants for delayed or abnormal neural crest cell emigration as a cause for exencephaly.It is interesting that whereas 100% of ncephaly ,112, theethality ,114, whiThe vertebrate cranial mesoderm has an important role in cranial neural fold elevation. The vertebrate cranial mesoderm, created by gastrulation , may havThe role of cranial mesenchyme  in the cranial neural tube has been studied in mouse, rat and chick embryos . During twist causes severe craniofacial defects, including midbrain exencephaly; the neural folds fail to become concave and remain everted [twist is highly expressed in the cranial mesenchyme just before and during the time of neural tube closure [twist in the precursors of the cranial mesoderm causes reduced proliferation and abnormal clustering of mesoderm cells in the lateral regions of the head folds and subsequent exencephaly [Cart1 (Alx1) and Alx3 in twist null mutant embryos suggests that they are in the same regulatory framework [Several mouse mutants demonstrate the importance of the cranial mesenchyme to cranial neural fold elevation. Loss of function of the transcription factor  everted . twist i closure ,122. At  closure . The exe closure  and on cncephaly . No evidncephaly . The neuncephaly  and the ncephaly . Based oramework , and it Cart1 (Alx1) transcription factor is expressed in mesenchyme cells of the forebrain but not the midbrain, and not in neuroepithelium, during neural tube development [Cart1 (Alx1) have increased apoptosis and transient deficiency of mesenchyme in the forebrain, with delayed closure of the forebrain folds; subsequently, the midbrain neural folds fail to elevate, leading to exencephaly [Cart1 (Alx1) early expression may be limited to the forebrain mesenchyme, a homolog, Alx3, appears to be expressed in paraxial mesoderm of the body and the mesenchyme of the head, but also not in the neuroectoderm, at the time the cranial folds are elevating [Cart1 (Alx1) and Alx3 expression in the neural fold mesenchyme is in the neural crest cell component, this seems unproven. About 10% of mutants lacking Alx3 develop midbrain exencephaly and/or split face ; the neural folds of these embryos fail to undergo the shift from convex to concave or development of a dorsolateral hinge point and therefore fail to meet in the midline [Alx3 expression is lost during folic acid deficiency at the time of cranial neural tube closure, whereas expression of other neural tube genes such as Cart1 (Alx1), twist, and Cited2 is not affected [The elopment . Null muncephaly . Whereaslevating . Althoug midline . Increas midline , but is affected .Inka1 is first expressed in the cranial mesenchyme prior to and during neural tube formation and then in the cranial neural crest during its migration in the mesenchyme [Inka1 protein inhibits the Pak4 kinase that may function in cell–cell contacts and adherent junction formation [Inka1 is regulated by Tcfap2a in non-mammalian vertebrates, but not in mice [Inka1 causes a predisposition to midbrain exencephaly (5–7%); otherwise, individuals are normal and fertile. The mechanism by which the neural tube fails to close has not been demonstrated, and the mechanistic role of the mesoderm or neural crest or both is unknown.senchyme . It is normation . Inka1 i in mice . Loss ofSki, [Loss of function of the transcriptional regulator, Ski,  which caSki, , is someSki,  but cranSki, ,131, andSmarca4 (Brg1 or SW1/SNF), involved in chromatin remodeling, die before implantation. Heterozygotes often have midbrain exencephaly, and no other defects [Smarca4 is expressed strongly in the cranial mesenchyme before elevation [Null mutant embryos for  defects . Smarca4levation . The mecHectd1 (opm) lead to midbrain exencephaly in all homozygotes and some heterozygotes [Hectd1 is ubiquitously expressed in embryos, including the head mesenchyme, and the cause of exencephaly is attributable to the effects of Hectd1 deficiency on the cranial mesoderm cells, which are abnormally shaped and packed more densely than normal near the neural tube at the normal time of elevation, and which fail to orient parallel to the neuroepithelium [twist has an expanded area of expression in the midbrain folds in the Hectd1 mutants, and after failure of full elevation, Snail and Pdgfr are abnormally expressed in the dorsal area of the midbrain folds [Hectd1 substrates, heat shock protein 90 (Hsp90), is known to enhance migration in various cell type, is secreted at elevated rates from Hectd1 mutant cranial mesenchyme cells which are abnormally migratory in vitro [Hectd1 in the midbrain neural folds lead to excess Hsp90 secretion, which in turn alters the behavior of the mesoderm cells, leading to their failure to contribute to elevation of the folds.Mutations causing loss of function of the E3 ubiquitin ligase ozygotes . Hectd1 ithelium ,134. Thein vitro . It seemCecr2 chromatin-remodeling factor develop exencephaly [Cecr2 is expressed in neuroepithelium and mesenchyme during cranial neural tube development [Cart1/Alx1 transcription factor [Cecr2 mutants may be attributable to defective cranial mesenchyme. The observation that in Cecr2 mutants, the cranial neural folds do not elevate sufficiently for contact between the apposed folds, despite forming DLHP [Loss-of-function mutants for the ncephaly . Cecr2 ielopment . Studiesn factor . As thisn factor , it appeing DLHP , is consExencephaly in mouse mutants is usually caused by a failure of midbrain neural fold elevation and it appears that defects in cranial mesoderm are a common etiology for this defect. Exencephaly becomes anencephaly, owing to degradation of exposed neural tissue during later gestation, and therefore is seen as anencephaly at birth in humans . InteresThe actomyosin-dependent shaping and bending of the cranial neural folds is based in the neuroepithelium, and it seems predictable that the many actomyosin-related genes that cause NTD would show neuroepithelium-specific expression, but data to demonstrate this is lacking. Many studies report whole mount in situ hybridization in the cranial neural folds without demonstration of the cell type involved . A few genes that cause cranial neural tube defects when dysfunctional have been reported to be expressed in the neuroepithelium.Cfl1  is expressed in the pre-closure cranial and caudal neuroepithelium and neural crest [Cfl1 enables the dynamic reorganization of the actin cytoskeleton by its function in F-actin, severing and depolymerization and by promoting the recycling of monomeric actin [Cfl1 mutants is owing to actomyosin accumulation, lack of F-actin turnover and actomyosin disassembly [al crest ,142. Cflic actin . Mutantsic actin ,141. Oriassembly .Fat1 cadherin, a large cell adhesion molecule, is expressed in the early anterior neural plate in the forebrain and midbrain neural folds during neural fold elevation and in the caudal neural folds [Fat1 is thought to regulate actin cytoskeletal organization at cell peripheries. In cell cultures, it is found in filopodia and cell–cell boundaries, overlapping with dynamic actin structures; it regulates actin polymerization [Fat1 null mutants have exencephaly, but not spina bifida. Although mutations of the family member Fat4 do not cause NTD, and Fat4 binds different actin-regulating and junctional proteins than Fat1, they form heterodimers, and on some strain backgrounds, the combination of homozygous null mutations at both Fat1 and Fat4 cause increased frequencies of exencephaly compared with Fat1 alone [al folds . It seemrization . Therefot1 alone ; this isCited2 (Mrg1) is a transcription regulator that serves as a co-activator for several transcription factors such as Tfcap2, Smad4, Lhx2, Nanog and Tbx3 [and Tbx3 ,147,148.and Tbx3 ,149. Nuland Tbx3 .Sall gene family Sall1, Sall2, and Sall4 are transcription repressors that interact with chromatin remodeling complexes, can form heterodimers [Sall genes cause exencephaly, depending on strain background [Sall1 with Sall4 or Sall2 with Sall4 also cause exencephaly. Based on embryos with a combination of null Sall1 and hypomorphic Sall4, it appears that the cause of exencephaly is a deficiency of elevation of the cranial neural folds, which do elevate part way but do not reach approximation in order to fuse [Members of the rodimers  and are rodimers ,152. Losckground ,152,153,Nuak gene family, Nuak1 and Nuak2, are serine-threonine kinases that have many functions [Nuak1 is expressed in many embryonic tissues including the neuroepithelium, but null mutants do not have NTD; Nuak2 is specifically expressed in the entire neuroepithelium during neural tube closure, and some of the null mutants have midbrain and hindbrain exencephaly; digenic null mutants for both genes all have exencephaly of the forebrain, midbrain and hindbrain [Members of the unctions . Nuak1 iindbrain . The craindbrain , which wShroom3 (Shrm), encodes a cytoskeletal protein that binds F-actin and regulates its distribution within the cell. During neural fold elevation, Shroom3 is strongly expressed in the cranial neuroepithelium, rostral to the otic vesicle, but not in the adjacent cranial mesenchyme [Shroom3 mutant homozygotes have midbrain and hindbrain exencephaly, most also have split face , and some also have spina bifida aperta. The morphology of the neuroepithelium of the closed spinal neural tube is also abnormal [Shroom3-mutant embryonic neuroepithelial cells, F-actin is shifted away from the apical surface, whereas in wild-type cells, it is predominantly localized at the apical surface of the cells. This pattern suggests that the mechanism of the neural tube closure defects in Shroom3 mutants is the lack of actomyosin-dependent apical constriction in the neuroepithelium. There is evidence, based on various digenic heterozygous mutants involving Vangl2 (Lp) and Shroom3, that the actomyosin system is linked to the PCP-convergent extension system by Shroom3 [Shroom3/+ nor Lp/+ mutants have spina bifida aperta, the digenic mutants  have almost 40% spina bifida aperta. At the molecular level, there is evidence that that a PCP factor (Dvl2) determines the location of Shroom3 at mediolateral cell junctions along with F-actin, Rock1 and Myosin IIb, components of the actinomyosin pathway [Shroom3 and Dvl2 may interact to link PCP signaling to actomyosin contractility. This is an example of how simultaneous reduction in a genetic component of each of two pathways can cause NTD.senchyme . Later, senchyme ). All Shabnormal . In Shro Shroom3 . For exa pathway , suggestDlx5 and Msx2 with each other and with the downstream membrane-bound cellular adhesion/repulsion factors EphrinA5 and EphA7. Dlx5 is expressed in the dorsal tips of the neural fold before, during, and after neural tube closure, extending from the forebrain to the posterior neuropore [Msx2 is expressed before closure in the dorsal edges of the neural tube, except in the forebrain region [Msx2 and Dlx5 have a high frequency (73%) of exencephaly that seems to be caused by their joint regulatory effect on the splicing of EphA7 transcripts [EphrinA5 and its receptor EphA7 splice forms are expressed in the dorsal cranial neural folds (midbrain and hindbrain) before and during closure [EphrinA5 with full length EphA7 causes cellular repulsion, but a co-expressed alternative splice form of truncated EphA7 converts the effect of the interaction to adhesion [EphrinA5 null mutants sometimes develop exencephaly (17%), a few of which have failed forebrain closure as well as midbrain and hindbrain, having failed to fuse despite elevating to become juxtaposed at the midline; some (25%) EphA7 null mutants also have exencephaly [Msx2 or Dlx5 have fairly normal expression of EphrinA5, full-length EphA7 and truncated EphA7 in the cranial neural tube. In contrast, in Dlx5/Msx2 digenic null mutants during the stage of neural tube closure, the expression of EphrinA5 at the apex of the neural folds is decreased, full length EphA7 expression is unchanged but the truncated form of EphA7 is greatly reduced, a situation that is expected to cause repulsion, rather than adhesion of the apposed midbrain and hindbrain neural folds. It appears, therefore, that the mechanism leading to exencephaly is likely lack of adhesion of neural folds in each of the EphrinA5, EphA7 and digenic null Msx/Dlx5 mutants, and the limitation of the NTD to the midbrain and hindbrain reflects the limited expression domain of EphrinA5 and EphA7.An example of the potential molecular complexity underlying neural tube fusion and leading to a cranial-specific etiology of an NTD is an apparent regulatory relationship between the transcription factors europore ,159, andeuropore . Similarn region ,162, butn region ,163. In nscripts . The lig closure . The intadhesion . EphrinAncephaly . Null muEphrinB1, express exencephaly at a higher rate in heterozygous females than in homozygotes and null males [EphrinB1-positive and -negative cells in the neuroepithelium, affecting its structural integrity [Demonstrating another kind of complexity, null mutation carriers for the X-linked gene, ll males . Embryosntegrity .In general, it seems likely that genetic defects whose mechanisms cause cranial neural tube defects through failure of fusion of apposed neural folds, would demonstrate defects in the surface ectoderm projections normally present at the fusion sites. However, it appears that data is lacking in this area.Myo10), an unconventional myosin known for its roles in formation of filopodia and localization to the tips of filopodia, causes exencephaly, along with other non-neural-tube defects [Loss of function of Myosin-X  with 72% females among exencephalics [Gldc [Cited2 [A striking predominance of females in cranial neural tube closure defects has been recognized for many years; about two-thirds of human anencephalics and mouse exencephalics are female (reviewed in ). Human ephalics , and Scaephalics . The 14 cs [Gldc  and Cite [Cited2  requiresThe causes of the female excess among cranial neural tube closure defects in humans are difficult to identify, but in mice, several of the possible explanations have been ruled out by the data. For example, the neural tube develops long before gonadal hormones can be present, the developmental rate of the cranial neural tube during closure does not differ between males and females, and there is no evidence of preferential death of affected males during gestation . In miceDnmt3b (DNA methyltransferase 3 beta) transcript and protein levels are reduced in XX embryos relative to embryos with one X [There is an intriguing phenomenon of methylation differences correlated to number of X chromosomes in mouse and bovine embryos, during the early developmental period when both X’s are normally active, such as in blastocysts or embryonic stem cells. The global DNA is hypomethylated and the th one X ,172. WheSry testis-determining gene; females have two copies of the large X, of which one copy has been mostly silenced beginning from about the time of implantation [In mammals such as humans and mice, males have one large X chromosome with over a thousand genes and one small Y chromosome, which contains, amongst a few genes, the antation  by a comantation . ChromatA mechanism that has been suggested to explain a variety of disorders that occur at higher frequencies in females than in males is that some genes on the inactive X variably escape from silencing and cause a gene expression imbalance; there is evidence in support of this mechanism for the autoimmune disease lupus erythematosus .Smchd1, a gene involved in the methylation of DNA (formerly Momme D1) causes reduced methylation and silencing of a reporter transgene and autosomal retrotransposons, but only in females. The female-specific effects led Blewitt et al. [Sry has been moved to an autosome, comparing XX females, XX males, XY males, and XY females. It shows that the expression level of hundreds of genes in mouse thymus cells is determined by the number of X chromosomes present and not by gender [Another hypothesis to explain the female excess is that the use of cellular resources to inactivate a large X chromosome in females after every mitosis would competitively reduce the availability of those resources for gene regulation on the autosomes, weakening the process of neural tube closure ; updatedt et al.  to suggey gender . Genes dy gender . The seny gender . If someThere is an interesting interaction between embryo gender and the preventative effects of folate on human NTD. In at least three populations from Chile, Argentina and Canada, the female excess among anencephalic births disappeared soon after the start of folic acid fortification of wheat flour ,178. PriMuch of the understanding of basic mechanisms of neural tube development is based on studies of the spinal region, and spinal neural tube closure has been well reviewed by others ,6. We haGoosecoid (Gsc), present in the prechordal plate and not in the notochord, appears to repress convergent extension; i.e., because of Goosecoid, normally there is no convergent extension in the forebrain. The role of a disruption in the planar cell polarity (PCP) pathway and the consequent lack of convergent extension in causing craniorachischisis have been well reviewed recently [The failure of convergent extension  results recently . Vangl2, Celsr1, Dvl1/2/3 and Scrib; and the less central factors, Ptk7, Cdx1/2, Sec24b, Sfrp1/2/5 and Smurf1/2 [In mice, among the numerous mouse gene mutations that cause NTDs, those that produce craniorachischisis are all components of the planar cell polarity pathway, including the PCP core factors, Smurf1/2 .Sonic hedgehog expression domain; if the neural tube in these embryos appeared closed caudal to the notochord duplication, two parallel neural tubes side by side were found housed in a single dural tube [Lp mouse mutant at the Vangl2 gene (Vangl2-Lp/Lp), the notochord at the time of initiation of neural tube closure is loosely organized and comprised of two or three parallel longitudinal structures [In humans, craniorachischisis is a relatively rare NTD for which the only known genetic cause is mutations in PCP genes . The devral tube . In the ructures . The proructures .Vangl2-Lp mutant that has 2% spina bifida [Inturned (Intu) with 30% exencephaly [Shroom3, has been found to interact with PCP pathway proteins during neural tube morphogenesis in mice [It is becoming clear that PCP genes can have roles other than in convergent extension that can influence neural tube closure. One possibility (for spina bifida) is that caudal neural tube closure is delayed in some PCP-mutant heterozygotes, as has been observed in the mouse heterozygous a bifida . A secona bifida , includincephaly . A thirdncephaly . Convers in mice . PCP gen in mice . Vangl2 mutant, Lp, and one of several other PCP genes. In addition to the PCP–PCP gene interactions, there are digenic mutant examples of PCP genes interacting with genes in actomyosin dynamics, as in Vangl2;Cfl1 [Vangl2;Shroom3 [In mice, a large and growing number of digenic mutants, heterozygous for mutations at two gene loci, have been observed to have NTD. The molecular mechanisms by which these digenic or oligogenic combinations lead to NTD are largely unknown, but likely include both cumulative effects along the same pathway or deficiencies in interacting pathways. The known digenics involve at least twenty different kinds of genes ,10,12, bgl2;Cfl1  and Vang;Shroom3 ,157.Vangl2, Celsr1, Scrib or Sec24b) and all embryos have craniorachischisis; heterozygotes do not. Although virtually all mouse mutants with craniorachischisis have mutations in genes of the PCP pathway, often mutations at two or more PCP genes are involved. In one type of digenic example, mutants are homozygous at both of two homologous PCP genes with functional redundancy  and nearly all embryos have craniorachischisis; homozygotes for any one of these genes do not. In yet another type of digenic cause, high frequencies of craniorachischisis are found in mutants that are double heterozygotes for mutations at two PCP genes of different gene families  [Mouse PCP-mutant studies focused on the NTD craniorachischisis demonstrate the genetic complexity of the role of PCP genes in craniorachischisis . In some1;Scrib) ,185. It Fzd3, Intu or Fat1, or digenic mutants at Vangl2;Fzd1 (both heterozygous) or Vangl2;Cthrc1 . Only spina bifida is found in digenic heterozygotes at Vangl2;Ptk7, Vangl2;Grhl3, Vangl2;Sec24b, and Celsr1;Ptk7 [Vangl2;Scrib or Celsr1;Scrib [Craniorachischisis is not the only NTD caused by PCP mutations in mice. Mutations of various single PCP genes or digenic combinations of PCP genes do not cause craniorachischisis, but do cause the more common types of NTD ; severasr1;Ptk7 . Finallyr1;Scrib .Wnt-PCP pathway on the caudal neural tube that cause spina bifida include effects on cell differentiation, cell shape, and actomyosin contractility [The mechanisms by which these non-craniorachischisis defects of neural tube closure are caused by PCP pathway mutations are largely unknown. However, the clear differences known for neural tube formation between anterior–posterior regions (discussed above) raise the possibility that the causal mechanisms involve the ciliogenesis, actomyosin, and microtubule functions of PCP genes, in addition to their convergent extension role . Effectsactility .VANGL2, CELSR1, SCRIB or DACT1) [CELSR1;SCRIB; one anencephaly digenic at CELSR1;DVL3 and one spina bifida digenic at PTK7;SCRIB [Mouse mutants support the digenic (or oligogenic) hypothesis for human craniorachischisis and, further, the hypothesis that digenic heterozygotes for PCP genes can also cause risk for human exencephaly and spina bifida. Human NTD cases of various types have been screened for rare deleterious variants in several PCP genes. Human craniorachischisis is relatively rare and few studies are of craniorachischisis only. Most variant screening studies include exencephaly, spina bifida and other neural tube abnormalities . Among cr DACT1) ,186,187.K7;SCRIB .Overall, the mouse digenic PCP mutants support the strategy of screening cases with anencephaly, spina bifida or craniorachischisis for PCP gene mutations and, given the usual observation of human heterozygosity for the PCP mutations found, strongly encourage the search for second gene locus mutations in these NTD cases.In addition to genetic susceptibility, environmental factors contribute to risk of developing NTD, including various aspects of diet and maternal health ,189. OneCart1 and Cited2, the histone actetyltransferase Gcn5, and the “Crooked” gain-of-function mutation of the transmembrane co-receptor for Wnt, the Lrp6 gene [Lrp6 also causes exencephaly and spina bifida but responds to maternal folate supplementation with increased frequency of these NTDs [In parallel to humans, in mice, mutations in most folate metabolic pathway genes that have been tested do not cause NTD . A smallrp6 gene . Interesese NTDs . These mese NTDs .Pax3 transcription factor mutations [Splotch” mutants have spina bifida, exencephaly, or both. These mutants have a defect in the folate metabolic pathway that leads to deficiency of de novo pyrimidine biosynthesis; this defect seems to be ameliorated by folate supplementation, which reduces the frequency of NTD by about 40% [The mutant genes in folate-responsive mouse NTD models may have unexpected connections to folate pathways. An example is the utations ,204, whibout 40% ,204. Slc19a1 each causes reduced cilia formation, indicating that the folate and methylation pathways are required for cilium formation. The lack of cilia is attributable to the demonstrated deficiency of methylation of Septin2, a cytoskeletal protein whose methylation state affects whether it contributes to cilia formation [Hedgehog signaling pathway [Hedgehog pathway, the truncated form of Gli3 (Gli3R), is decreased in mouse embryonic fibroblast cells lacking folate or methylation, indicating that the lack of cilia in these cells leads to dysregulation of the Hedgehog pathway [Sonic hedgehog signaling pathway in the developing neural tube, which in turn interferes with dorsal bending of the neural folds and causes exencephaly or spina bifida [Hedgehog signaling. A recent study has suggested that folate deficiency or lack of the folate-dependent methylation process may exert adverse effects on neural tube development through an effect on formation of primary cilia . In Xenoormation . Cilia a pathway . An indi pathway . Cilial a bifida  (SectionFolr1 (Fbp1) is a membrane protein that takes folate into cells. Normally, Folr1 in mouse embryos is expressed in the neural folds of the upper spine just before and during closure and expression expands bidirectionally along the anterior–posterior axis with advancing closure; it is also expressed in the cranial neural folds just before and during closure, similarly expanding with the advancing closure; it is not detected in the forebrain region and posterior neuropore [Folr1 null mutant is lethal before neural tube closure, but with maternal folate supplementation, null Folr1 mutant embryos survive to express NTD, particularly exencephaly [FOLR1 gene variants do not seem to be important in human anencephaly [FOLR2 and FOLR3 may have a small role [Lrp2, a multi-ligand membrane receptor that seems to mediate uptake of folic acid into cells by Folr1 at the apical surface of the developing neuroepithelium [Lrp2 mutants by half; dietary supplementation with folic acid is not effective [europore ,207. Thencephaly . FOLR1 gncephaly  nor in sall role . A functithelium . Maternaffective . Folr1 and Lrp2 cause exencephaly. There are other mechanisms of uptake of folate into cells that may be more efficient than Folr1, such as the reduced folate carriers [Folr1 can also function as a transcription factor in mammalian cells, with a large number of developmentally important genes as its target [Xenopus have pointed to a novel mechanism for the role of Folr1 and folate in neural tube closure [Folr1 during neurulation in Xenopus causes failure to develop a neural tube. In Xenopus, Folr1 is expressed at the apical surface of neural plate cells, and interaction between Folr1 and folate in the medial neural plate is required for these cells’ apical constriction and the bending of the neural plate. Folr1 co-localizes and interacts with C-cadherin and beta-catenin in the medial neural plate cells. Apical constriction requires internalization of apically localized C-cadherin by endocytosis; in Folr1 knockdown neural plates, there is a decrease in C-cadherin–containing endosomes. Overall, the observations indicate that the primary function of Folr1 interacting with folate in Xenopus neural tube development may not be related to its functions in metabolism, but rather as a regulator of the cytoskeleton and cell adhesion remodeling [Folr1-folate interaction to cytoskeletal remodeling can be found in mammals, then a fresh interpretation of the folate-responsiveness of human and mouse NTD occurrences should be undertaken. Perhaps supplemental availability of folate to the Folr1 receptor can maximize its activity, and can also compensate for weaknesses in other unrelated modules that also regulate the cytoskeleton.Cellular folate deficiency may not be the mechanism by which mutants for carriers . In thiss target . Further closure . Knockdomodeling ,214. If Hedgehog signaling, and cytoskeletal-driven bending of the neural plate. Given the genetic heterogeneity of NTD, individual cases may have different types of weakness in the closure process and interact with the aspect of supplemental folate that matches the functional deficit.Overall, the mouse NTD mutants that interact with folate present a complex story that suggests that some folate effects on NTD risk may function through connections to the known metabolic roles of folate; other folate effects on NTD may be through previously unknown roles in ciliogenesis and regulation of the cytoskeleton. Given the great variety of cellular functions essential for successful neural tube closure, abundant levels of folic acid may be important via different aspects of its function in multiple aspects of the closure process, such as cell proliferation, regulation of a complex repertoire of developmental gene signaling via DNA and histone methylation, Amt, Gldc, Mthfd1l and Slc25a32 [Folr1), almost all of the mitochondrial folate-pathway mutants reported to date have exencephaly ; none have spina bifida. The exception among the mitochondrial folate-pathway mutants that does not cause NTD is Mthfd2, which has redundancy with Mthfd2l [A new set of NTD mutants in folate-pathway genes is emerging. The genes are all involved in folate one-carbon metabolism in the mitochondria, the process that supplies one-carbon formate molecules to the cytosol for nucleotide biosynthesis and DNA methylation . The repSlc25a32 ,216,217.Slc25a32  do not h Mthfd2l .Gldc, Mthfd1l, Slc25a32), the frequency of NTDs is reduced by between 60% and 100% by sodium formate supplementation (in drinking water) to the pregnant dams [Amt mutant with formate has not been reported. In none of these mutants has folate/folic acid treatment reduced the NTD frequency.Interestingly, in three of the mitochondrial mutants , treatment of curly tail strain dams with prenatal sodium formate was found to reduce the spina bifida frequency by 75% [A further example is the curly tail mouse strain, in which 10–20% of embryos have spina bifida. Folic acid has no preventative effect in curly tail strain embryos, but nucleotide precursors reduce spina bifida by 85% . Recently by 75% .Because the main source of one-carbon units for cytoplasmic processes comes from the mitochondria, any failure in a step of mitochondrial folate one-carbon metabolism results in a deficiency of one-carbon components (formate) for essential cellular processes, such as DNA synthesis and methylation. Momb et al.  suggest Understanding of the genetic architecture of human NTD is largely derived from two approaches: family-based data and population-based data. The family-based approach led to the multifactorial model that explains the pattern of recurrence of NTD in families. Rates of recurrence of NTD in various types of relatives of NTD cases demonstrate that NTD have a strong genetic etiology, with a 30- to 50-fold increased rate of occurrence in siblings of NTD cases, compared to a population rate of about one per 1000 births ,47. The Population-based studies aimed at finding statistical associations with single nucleotide polymorphisms (SNPs) in over 200 candidate genes have found a few weak associations often lacking statistical significance ,223,224.Studies based on sequencing of coding regions of candidate genes usually are done as a population-based approach, often using pools of various types of NTD cases . This apA current hypothesis about the genetic architecture of various multifactorial, post-reproductive diseases (“complex traits”)  suggestsThe common forms of NTD that are explained by the multifactorial model are non-syndromic, meaning the only primary defect is the defect in neural tube closure. There are some rare human syndromes of multiple unrelated defects that include anencephaly or spina bifida, and they are caused by chromosomal defects or Mendelian mutations . They arFor a multifactorial trait, it is expected that environmental factors contribute to the trait to some degree. Theoretically, the effects of environmental factors add to the genetic risk factors in individuals. Individual embryos with a load of genetic risk factors but marginal ability to close the neural tube may be hampered enough by the additional effects of environmental factors to fail to close, whereas an exposed embryo with fewer genetic risk factors would still be able to successfully close. Folate supplementation  is an enThe almost 300 mouse NTD mutants cannot model the human genetic architecture of NTD. The mouse mutants are maintained on inbred strain backgrounds, within which there is essentially no genetic variation between individuals. Very few strain backgrounds are used, and the strains themselves are a very limited sample of the variation in the mouse species’ genome. Some mutations show different penetrance or expressivity of NTD on different strain backgrounds, demonstrating their potential for multifactorial genetics. In the studies of digenic effects, to test the combined effects of two different genes on NTD risk, the specific digenic combination of interest is created by crosses between mutant stocks. Unexpected genetic interactions are unlikely to be discovered in this hypothesis-driven system.Wnt9b versus the non-syndromic cleft lip phenotype from a hypomorphic mutation at Wnt9b [The mouse mutations are usually highly penetrant recessive “knockouts” that cause loss of function of the gene, and for many of the genes, these cause syndromes of multiple defects. In general, gene loci that cause syndromic phenotypes from null (knockout) mutations may present non-syndromic phenotypes from hypomorphic (reduced function) mutations. An example in mice is the kidney defect and cleft lip syndrome caused by a null mutation at at Wnt9b . TherefoThe use of candidate genes from mice should be tempered by awareness of the differences in the morphological process between human and mouse neural tube development , and ideMany of the candidate genes that have been tested in human NTD cases are based on various hypotheses, such as roles related to folate metabolism or folate transport, and the lack of finding gene variants associated with human NTD concurs with the mouse mutants; i.e., they do not cause NTD in mouse mutants. The pools of human cases studied usually focus on spina bifida. The majority of the NTD genes in mouse mutants cause cranial NTD, and based on the regional heterogeneity of etiology discussed in Two successful sequencing studies are discussed in Sonic hedgehog cilia system on which it depends; the actomyosin and cytoskeletal system responsible for some bending of the neuroepithelium; the various types of cell projections and the Ephrin system that facilitate contact and fusion. Each of these systems involves many genes. Despite the understanding of many details of the genetic determination of neural tube development, the genetic causes of human NTD are mostly undiscovered. Various insights emerge from an overview of the many studies of neural tube closure. The mammalian way of forming a neural tube by elevating and bending the lateral edges of a neural plate to meet in the midline is an ancient mechanism that evolved before vertebrates. Several systems contribute to forming the neural tube. Among them are the planar cell polarity/convergent extension system; the medial hinge point and related cell cycle phenomena; the dorsolateral hinge point and the The evidence that the planar cell polarity system is involved in various aspects of neural tube closure in addition to convergent extension, such as the cytoskeleton, ciliogenesis, and the actomyosin system, offers an explanation for the observed role of planar cell polarity genes in exencephaly/anencephaly and spina bifida as well as in craniorachischisis.Although the mouse has been used extensively as the mammalian model for human NTD, the development of the neural tube in other mammals, such as pig and rabbit, is more similar to human in morphology. Gene expression arrays of regions of neural folds from these species during elevation and closure might point to the subset of genes and mechanisms previously identified in mice that are likely to be involved in human neural tube closure. The vertebrate head evolved as an “add-on” to the pre-vertebrate body plan and its neural tube. The formation of the cranial neural tube consequently differs in several ways from that of the spinal region. For example, the mouse cranial mesoderm has a much greater role in neural tube closure and etiology of NTD. The neuroepithelial actomyosin system is also more important in the cranial region than in the spinal region. The large number of genes involved in these two systems may account for the predominance of genes causing exencephaly among mouse NTD mutants. The genes identified by these mutants may be good candidates for examination in the etiology of human anencephaly. Cranial NTD occur more frequently in females than in males. This phenomenon is seen in humans and in nearly all mouse NTD mutants examined. Recent advances in the understanding of epigenetics suggest the hypothesis that the second, inactive X chromosome of female embryos is a “heterochromatin sink” that sequesters essential epigenetic factors, altering expression of genes in the rest of the genome and thereby increasing the risk of NTD. The limitation of the effect to cranial NTD suggests that specific genes are involved. Folr1 membrane protein that takes folate into cells seems to act also as a regulator of the cytoskeleton and of cell adhesion remodeling in the neural plate. Secondly, independent evidence in Xenopus demonstrates a requirement for folate in ciliogenesis, which is known to be involved in NTD. These observations may shift the focus of candidate gene research in folate-responsive human NTD away from the metabolic functions of folate.It is well established that peri-conceptional dietary folic acid supplementation reduces the risk of occurrence of NTD in humans and in some mouse mutants, but the genetic risk of folate-responsive NTD does not seem to involve genes in the folate metabolic pathway in either species. There is recent evidence for a function of folate-pathway genes that is separate from the established metabolic functions in DNA methylation and nucleotide synthesis. In Xenopus, the Msx2/Dlx5 double mutant, where the combined effect of the two mutant genes alters the balance of splice forms of a third gene, EphA7. The extensive study of the planar cell polarity system has generated several examples of digenic combinations of different planar cell polarity genes that cause a variety of types of NTD. The observations in mice suggest that many of the cases of human NTD in which a putative mutation has been detected at one planar cell polarity gene locus may well harbor an undetected mutation at a second planar cell polarity gene locus or other type of gene.There are many examples in mouse mutants of NTD caused by combinations of mutant genes at two or more loci from the same or different genetic pathways. Usually, the molecular mechanism of interaction between the mutant genes is unknown. The mechanism of the interaction effect can be surprising, as in the case of the Overall, the cumulative data show that whereas the neural tube appears to be a single anterior–posterior structure, in contradiction, the gene expression, cell behavior, and closure systems occupy a variety of different regions. The neural tube along its length is neither homogeneous nor segmented. Each gene and mechanism is expressed in areas along the length that are different from the expression patterns for many of the other genes or mechanisms so that each linear area is the result of a unique combination. NTD may be a series of genetically distinct defects occurring along a contiguous physical structure. Setting aside the planar cell polarity system mutations, the remainder of human NTD may have genetic etiology that varies by anterior–posterior location of the lesion, a concept that could inform human genetic studies in search of causative variants. For example, families within which there are both anencephalic and spina bifida cases may tend to have mutations in the DLHP system. Perhaps the most important conclusion from this review is recognition of the degree to which the neural tube is regionalized along the anterior–posterior axis.


Herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) have co-evolved with humans for thousands of years and are present at a high prevalence in the population worldwide. HSV infections are responsible for several illnesses including skin and mucosal lesions, blindness and even life-threatening encephalitis in both, immunocompetent and immunocompromised individuals of all ages. Therefore, diseases caused by HSVs represent significant public health burdens. Similar to other herpesviruses, HSV-1 and HSV-2 produce lifelong infections in the host by establishing latency in neurons and sporadically reactivating from these cells, eliciting recurrences that are accompanied by viral shedding in both, symptomatic and asymptomatic individuals. The ability of HSVs to persist and recur in otherwise healthy individuals is likely given by the numerous virulence factors that these viruses have evolved to evade host antiviral responses. Here, we review and discuss molecular mechanisms used by HSVs to evade early innate antiviral responses, which are the first lines of defense against these viruses. A comprehensive understanding of how HSVs evade host early antiviral responses could contribute to the development of novel therapies and vaccines to counteract these viruses. Herpesviridae family and Alphaherpesvirinae subfamily, similar to varicella zoster virus (VZV)  type 1  and type 2 , are members of the Davison, . HSVs arDavison, , with twDavison, , and ~11Davison, . HSV-1 aDavison, . AdditioDavison, . AlthougHSV-1 is mainly associated with orofacial lesions, yet it is also the leading cause of infectious blindness in developed countries and the number one cause of viral encephalitis in adults , due to synergistic aspects related to the co-infection with both viruses , and glycoprotein L (gL) were present in the virion  for attaching to target cells  genes, with many of their functions being related to limiting host immediate antiviral mechanisms. This set of genes also encodes proteins that act as transcription factors that promote the transcription of the second set of viral genes  viral genes are expressed, which are involved among others in promoting the replication of the viral genome  viral genes, which are occasionally separated into late early (or gamma-1) and late (or gamma-2) genes, and are involved among others in providing the structural components that are present in the virion  and de-envelope from the outer nuclear membrane (ONM)  and D (gD) have been described to produce, at least partially the inhibition of Fas-mediated apoptosis in a neuroblastoma cell line and Jurkat cells , was shown to be non-lethal for DCs, yet it is unknown if the mutated or deleted viral gene is directly involved in cell death or if its deletion interferes with viral processes that relate to cell death  that may be produced during viral infections or abnormal cellular processes, has been reported to promote effective antiviral responses  and restrict viral replication  has been reported to induce a response that reduces HSV infection and disease in a genital mouse infection model when engaged with the synthetic agonist Imiquimod . Interestingly, intranasal application of CpG ODNs that are TLR9 agonists in BALB/c mice previous to HSV-1 infection was reported to reduce viral load and the production of pro-inflammatory cytokines IL-6, CCL2, and CCL5 by neurons in the CNS, which increased the survival rate of the infected mice -like receptors and a broad class of putative DNA sensors  is a cytosolic DNA sensor that triggers cytosolic GMP-AMP (cGAMP) production upon binding to an activating DNA  has been described to target cGAS for degradation even at 20 h post-infection, significantly reducing the chances that this receptor signal for IFN-β production upon HSV infection of epithelial cells and fibroblasts  Su and .Although some level of interference has been described by HSVs over the nucleic acid sensors described above, the DNA sensor termed DNA-dependent activator of interferon (DAI), which is expressed in primary vaginal tissue has been reported to readily detect HSV-2 and lead to IL-6 and IFN-β release upon infection , are two host sensors specialized in recognizing dsRNA , a host factor that once activated can help hamper the replication of viruses by inducing NF-κB activation and the expression of cytokines that control virus replication and infection (IFNs) , can lead to the activation of immune and non-immune cells and trigger antiviral responses that restrict and interfere with virus replication. A significant antiviral response elicited by the sensing of viruses is the IFN response. IFNs are cytokines that once bound to their receptor can potentiate antiviral activities both, in the cell that secretes these molecules and neighbor cells  prior to HSV-1 infection promoted the expression of numerous antiviral proteins in primary human keratinocyte cultures. One of them, IFN-β helped prevent their infection. This effect was dependent on TLR3 engagement and JAK-STAT signaling events   cells are innate immune cells capable of sensing and destroying virus-infected cells that either lack the expression of major histocompatibility complex I (MHC-I) molecules, or express NK-activating molecules on the surface because of abnormal cellular processes betray infection . NKTs recognize antigens in the form of glycolipids presented on CD1d molecules that share structural similarities with MHC-I  are key immune cells that promote and regulate immune responses by modulating the activity of innate and adaptive immune cells  and monocyte-derived DCs. Importantly, the effects of HSV infection over the capacity of DCs to activate T cells seems to more pronounced in vitro than in vivo , thereby likely reducing the efficacy of DCs at activating CD4+ plasmacytoid DC subset increased the expression of markers associated with the migration of these cells to lymph nodes, and that these DCs were able to promote the activity and functions of T cells, B cells and NK cells, which were recruited to the infection site  (Iijima et al., RM CD8+ are recruited to the genital tissue by chemokines such as CXCL-9 and CXCL-10, which are expressed by the infected epithelium (Nakanishi et al., + cells and stimulated them to establish TRM CD8+ cells (Smith et al., + subset were found to be present at the site of infection after applying “prime and pull” strategy mentioned above and were held responsible for re-stimulating HSV antigen-specific memory CD8+ T cells (Shin et al., Despite numerous studies describing approaches that elicit protective immunity against HSVs, identifying a correlate of protection for HSV infection has remained elusive. Interestingly, recent studies suggest that the outcome of the DC-HSV interaction may relate to the establishment of protective immunity, as specific HSV mutants that are attenuated in DCs confer particularly protective and robust immunity against HSV infection HSVs have also been reported to induce the synthesis and release of pro-inflammatory cytokines by DCs that promote their infection with HIV and the replication of this virus from previously-infected cells, likely increasing the dissemination of the latter virus during co-infections (Stefanidou et al., +CD8α+ DC subset reduced the amounts of latent HSV-1 in neurons after ocular infection (Mott et al., + DCs had increased amounts of latent HSV-1 and more recurrences (Mott et al., Recent studies support the notion that DCs may promote neuron infection with HSV, thus contributing to virus latency within the host. In addition, it has been observed that animals depleted of DCs display up to fivefold less latently infected neurons in the trigeminal ganglia, as compared to wild-type mice suggesting that DCs participate in processes related to neuron infection (Mott and Ghiasi, Overall, HSVs have evolved multiple mechanisms to negatively modulate the function of DCs, which likely results in a reduced capacity of the host to control HSV infection and mount and effective antiviral response. Identifying strategies that improve the interaction between HSVs and DCs should likely ameliorate the overall host response to these viruses either, immediately upon infection or during the establishment of long-term protection.Herpes simplex viruses elicit a diverse array of diseases in humans, both in individuals that have immune-related complications, as well as otherwise healthy persons. The capacity of HSVs to elicit disease during primary infections, as well as recurrences after establishing lifelong infection relates, among others to their ability to evade and neutralize host antiviral mechanisms that act in immune and non-immune cells. Importantly, HSVs interfere with early antiviral steps, such as the capacity of the host to sense viral determinants, signaling pathways that lead to cellular antiviral effects and the function of innate immune cells that act early after infection against these viruses. Evasion of these processes gives HSVs the chance to infect host cells and reach neurons favoring viral latency and lifelong infections, altogether dampening antiviral activities that could help immune cells establish effective and protective immunity against these viruses. The fact that the host somewhat fails at initiating an effective early antiviral response may provide grounds for the establishment of ineffective adaptive immunity, mainly through the interference of DC function, which is crucial for linking innate and adaptive immunity. Thus, improving the outcome of the early host antiviral responses against HSVs could help both, the generation of better anti-HSV therapies, as well as the design of prophylactic strategies intended at preventing infection with these viruses.All authors listed have made substantial, direct and intellectual contributions to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


IFITMs not only inhibit viral entry into host cells by interrupting the membrane fusion between viral envelope and cellular membranes, but also reduce the production of infectious virions or infectivity of progeny virions. Not surprisingly, some viruses can evade the restriction of IFITMs and even hijack the antiviral proteins to facilitate their infectious entry into host cells or promote the assembly of virions, presumably by modulating membrane fusion. Similar to many other host defense genes that evolve under the selective pressure of microorganism infection, IFITM genes evolved in an accelerated speed in vertebrates and many single-nucleotide polymorphisms (SNPs) have been identified in the human population, some of which have been associated with severity and prognosis of viral infection . Here, we review the function and potential impact of genetic variation for IFITM restriction of viral infections. Continuing research efforts are required to decipher the molecular mechanism underlying the complicated interaction among IFITMs and viruses in an effort to determine their pathobiological roles in the context of viral infections  IFITM3 gene that have been identified in human populations, several are associated with disease severity and prognosis of influenza A virus (IAV) and other viral infections (Interferon-induced transmembrane proteins (IFITMs) are a family of small proteins that can be found in single cell organisms and are evolutionally conserved across vertebrates . The humfections . Mechanifections . In thisIn vivo studies in IFITM3 knockout mice demonstrate the critical role of IFITM3 in restricting infection and reducing disease severity of infection by IAV , which enters cells via direct fusion with the plasma membrane , which abrogated endolysosomal fusion with the viral envelop  to IFITMs, HPV infection of keratinocytes inhibits the expression of IFITM1 and RIPK3 to escape from IFN-γ and TNF-α-mediated antiproliferation and necroptosis, which is essential for establishing a persistent infection  in its promoter region besides the additional gamma-activated sequence (GAS) in the promoter region of IFITM1 gene  result in an accumulation of IFITM3 in the plasma membrane and reduced inhibition of viruses that enter the cytoplasm at endocytic vesicles/lysosomes, such as IAV, human coronaviruses NL63 and -229E , which results in its plasma membrane accumulation and decreased antiviral activity against influenza viruses  in IFITM1 reduced its distribution in LAMP1-positive lysosomes but enriched its localization in CD63-positive multivesicular bodies  glycoprotein  . While CHuman IFITM proteins possess four conserved lysine residues Figure  that canIFITM proteins function as homo- or hetero-oligomers. Two phenylalanine residues (F75 and F78) are essential for IFITM homo- and hetero-oligomerization . IFITM3 + T cells  . Zhang e+ cells . In addi+ cells . A highe+ cells . Thus, rIFITMs are a group of small fusogenic proteins that restrict the infection of broad spectrum of viruses by inhibiting the fusion of viral and cellular membranes. However, whether IFITMs inhibit hemifusion, the process whereby the outer, but not the inner, leaflet of the viral and cellular membranes merge, or the transition from hemifusion to pore formation remains to be rigorously determined. It is also important to note that IFITMs do not always inhibit virus entry, but may promote membrane fusion under selected conditions, such as in the case of HCoV-OC43 infection . MoreoveAll the authors participated in the draft and revision of this review article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Micronutrient deficiency, also known as “hidden hunger,” is an increasingly serious global challenge to humankind. Among the mineral elements, Fe (Iron) and Zn (Zinc) have earned recognition as micronutrients of outstanding and diverse biological relevance, as well as of clinical importance to global public health. The inherently low Fe and Zn content and poor bioavailability in cereal grains seems to be at the root of these mineral nutrient deficiencies, especially in the developing world where cereal-based diets are the most important sources of calories. The emerging physiological and molecular understanding of the uptake of Fe and Zn and their translocation in cereal grains regrettably also indicates accumulation of other toxic metals, with chemically similar properties, together with these mineral elements. This review article emphasizes breeding to develop bioavailable Fe- and Zn-efficient cereal cultivars to overcome malnutrition while minimizing the risks of toxic metals. We attempt to critically examine the genetic diversity regarding these nutritionally important traits as well as the progress in terms of quantitative genetics. We sought to integrate findings from the rhizosphere with Fe and Zn accumulation in grain, and to discuss the promoters as well as the anti-nutritional factors affecting Fe and Zn bioavailability in humans while restricting the content of toxic metals. Overall, the Green Revolution has paid rich dividends in food grain production, particularly cereals, that have led to a significant reduction in the proportion of undernourished people worldwide; however, the problem of malnutrition or lack of quality food still persists, leading to an economic burden for society  deficiency and affecting 32.9% people worldwide; meanwhile, zinc (Zn) deficiency affects 17% of the world's population, with the highest risk occurring in sub-Saharan Africa and South Asia ], tubers [sweet potato (Ipomoea batatas) and yam (Dioscorea spp.)], and plantain (Musa spp.) that play a very important role in the daily diets of resource-poor people lack high enough amounts of micronutrients . Agronomic biofortification is a fertilizer-based approach that relies on soil and/or foliar application of micronutrients either alone or in combination with other fertilizers. It is well-established that a Zn fertilizer strategy is an effective way to biofortify cereal crops with Zn, but recurrent cost is involved . Biofortifying staple crops through plant breeding is therefore a key option to improve micronutrient deficiency in human diets , subsequent long-distance transport from roots to shoots, and further redistribution toward the developing seeds  and Strategy II utilizing ferric Fe3+ (Poaceae), referred to as reducing and chelating strategies, respectively, or a combination of strategies I and II , maize (Zea mays L.), and wheat (Triticum aestivum L.) follow Strategy II, and their root epidermis secretes phytosiderophores (PSs) that form stable Fe(III) chelates in the rhizosphere /ZIFL4 belongs to the major facilitator superfamily (MFS) that exports Fe3+-PS chelates in rice and barley  that allows direct uptake of Fe2+ from the rhizosphere, thus clearly showing the different uptake strategies for Fe2+ and Fe3+ in these cereal crops.Plants have developed different strategies for Fe uptake from the rhizosphere: Strategy I involving ferrous Fe2+) or as a Zn-PS complex formed with PSs known as Fe3+-chelators, which are secreted by roots of the plant  HarvestPlus in collaboration with international and national research institutes emphasized biofortification of staple food crops as a cost-effective, easily applicable, and sustainable approach to benefit low-income households. This may complement other efforts aimed at reaching rural populations in developing countries (www.harvestplus.org). Considering the average content of Fe and Zn in cereal grains and their retention after processing, as well as addressing the issues related to the bioavailability of these mineral nutrients, HarvestPlus established target levels of these nutritionally important traits in cereal grains , sorghum (Sorghum bicolor L.), and pearl millet (Pennisetum glaucum L.), which are the six most important crops and represent 89% of all cereal production worldwide, was reviewed, and the existence of significant genetic differences for these minerals was reported , ICP-mass spectrometry, near-infrared reflectance spectrophotometry, X-ray fluorescence spectrometry, elemental distribution maps secondary ion mass spectrometry, synchrotron X-ray, fluorescence spectroscopy, micro-X-ray fluorescence spectroscopy, and Laser-induced breakdown spectroscopy], which can differ substantially with respect to the many attributes describing method performance Ihnat, . TherefoModern cereal cultivars have a lower concentration of Fe and Zn in grains than landraces. This is because breeding has been mainly aimed at increasing grain yield or improving host plant resistance, among other target traits, instead of also improving the micronutrient concentration in grain. Utilization of landraces or crop wild relatives for genetic gain is not a new concept. The genetic variability for content of micronutrients is becoming acknowledged as a desired trait of crop wild relatives, particularly for rice and wheat.Triticum and Aegilops species have very high grain Fe and Zn contents when compared with both bread and durum wheat , Aegilops tauschii, and Aegilops speltoides were found to have substantially higher levels of these minerals (two- to three-fold) in their grain than modern wheat cultivars  led to dissection of complex multigenic traits that were difficult to improve through crossbreeding before the progress made in DNA-aided analysis. QTL mapping for mineral nutrients in cereal grains has allowed the identification of many QTLs for both Fe and Zn Table . Most ofUnfortunately, there is no literature indicating so far a success story for marker-aided selection (MAS) for improving Fe and Zn in cereal grains, but some progress has been made that has laid the foundation stone toward breeding for Fe and Zn biofortification in cereals using MAS. For instance, some of the QTLs identified for Fe and Zn are co-localized, thereby suggesting common mechanisms for their transport. Furthermore, some QTLs for these mineral nutrients are also co-localized with those for other mineral elements such as phosphorus (P) and calcium (Ca), or other agronomically important traits including grain protein content and grain weight Table . Fine maOsNAC5—a transcription factor that appears to be related with the remobilization of Zn from green tissues to the seed  for grain protein content to chromosome 6BS in a population of recombinant inbred lines (RILs) that derived after crossing “Langdon” (LDN)—a durum wheat cultivar—and DIC6B—a chromosome substitution LDN line including wild emmer wheat. Subsequently, the Gpc-B1 locus was also found to be related to high concentrations of both Fe and Zn, as well as with fast leaf senescence. The dissection of the Gpc-B1 locus by positional cloning revealed that the gene underlying the Gpc-B1 locus encodes NAM1, which is a NAC transcription factor that belongs to a protein group that includes “No Apical Meristem” (NAM) in Arabidopsis thaliana  or promoters such as prebiotics enhancing their absorption in the gut—to ensure their effective availability from cereal-based diets , potassium (K), and Zn, which after human or animal consumption binds to these minerals in the intestines forming mixed salts that are further excreted, thus resulting in mineral deficiency in human populations  mutants have been found in barley, maize, rice, and wheat exhibiting 50 to 95% reduced phytic acid P , and/or metalloids , which have no beneficial role in plants, animals, or humans  (Hanikenne et al., HMA2 was determined to contribute to Cd and Zn translocation in rice (Clemens et al., Soil is a natural source of heavy metals, and their elevated concentration in soil can occur either naturally or through anthropogenic activities such as urban and industrial activities as well as from agricultural practices. These toxic metals contamination is a non-reversible accumulation process due to their long estimated half-life in soil. Thus, accumulation of toxic metals in cereal grains impacts significantly on nutritional quality and crop safety. Generally, metals commonly enter plants as divalent cations. It has been reported that increasing accumulation of Fe and Zn in seeds leads to a higher accumulation of Cd, which chemically resembles Fe and Zn. Thus, uptake of Cd in roots and then translocation to seeds appears to occur inside plants along nutrient translocation pathways Krämer, . The firCdu1 locus on 5BL in durum wheat (Knox et al., OsNRAMP5 (Ishikawa et al., The concentrations of essential mineral nutrients and non-essential metals in grains appear to be independently regulated because some independent grain Cd accumulation loci have been reported in cereals, such as the Globally, the committed efforts by CGIAR HarvestPlus have led to the integration of essential micronutrients as a core activity in the breeding programs of almost all major cereal crops. Considering the complex genetic mechanism of Fe and Zn accumulation in cereal grains, eradication of these mineral nutrient deficiencies by increasing their levels in cereal grains through conventional breeding is simply too difficult. In the post-genomic and computational systems biology era, the combination of high-throughput genomics and robust statistical analysis, particularly QTL mapping studies, has helped to dissect the molecular basis of natural diversity for complex quantitative traits in a better way. Recent molecular mapping studies clearly indicate the co-localization of QTLs for Fe and Zn with those for other potentially toxic metals such as Cd, Pb, and As. Available knowledge can be used to design targeted crosses for MAB targeting cereal cultivars with high levels of Fe and Zn.Undoubtedly, QTLs detected only for Fe or Zn have also revealed that plants may be able to differentiate between nutrients and chemically similar toxin ions. Although no information is available so far about the enhancement of toxic metals in cereal grains through cross breeding, there is fear of inadvertent breeding for these non-essential metals that are toxic to both plants and animals even in low concentrations. Moreover, modifications in the accumulation of these toxic elements that are of concern for food safety are rarely determined during research on mineral nutrients. Thus, utilization of natural genetic variation for these mineral nutrients through a molecular breeding approach seems to be more attractive in the future. Furthermore, existence of substantial genetic variability for Fe and Zn bioavailability inhibitors and promoters also offers good opportunities to increase the bioavailable forms of these mineral nutrients in cereal grains. Genes accounting for this variability have rarely, however, been found and, therefore, are not yet being used in breeding; however, this also seems to be a promising approach for the near future. Hence, Fe and Zn bioavailability from cereal grains may be improved through breeding by accumulating either anti-nutrient agents or prebiotics. Furthermore, both functional and genetic evidence along with genome sequencing will provide means for gaining more insights regarding the emerging biofortification genomics.AG-O conducted the literature survey and together with SC wrote the first draft. RO edited and together with AG-O, SC, MG, and AM improved the manuscript writing. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.