Birth defects are an important cause of infant morbidity and mortality amongst developed countries (Dolk et al. 2010). Advances in prenatal and neonatal care have lead to a steady decline in infant mortality, leaving congenital birth defects responsible for a greater proportion of life-threatening conditions of infancy. Approximately, a fifth of all individuals with congenital malformations are stillborn or undergo therapeutic abortion (Dolk et al. 2010). The remaining survivors are faced with a significantly higher risk of death depending on the severity of their condition (Malcoe et al. 2011); nevertheless, such individuals will face numerous medical and surgical interventions with a lifetime of permanently compromised health.
Neural tube defects (NTDs) are common (1-4/2000 pregnancies worldwide) and often severe birth defects that occur due to complications in neural tube formation and closure during embryogenesis (Mitchell 2005; Copp & Greene 2010). NTDs present with a variety of complications and rank second to congenital heart disease as a leading cause of infant morbidity and mortality worldwide. NTDs that involve open lesions affecting the brain such as anencephaly and craniorachischisis are lethal and the affected individuals are stillborn or do not survive beyond birth. At the less severe end of the spectrum, spina bifida poses a lower risk of immediate death. Nonetheless, its open form is associated with various neurological impairments, involving incontinence, loss of sensation and inability to walk.
Closed spinal lesions are considered to be the least severe form of NTDs. However, even mild forms of this condition can become complicated, often due to abnormal development of the lumbosacral spinal cord, a condition that worsens with increasing age, affecting the lower limbs and the bladder, often necessitating surgical intervention (Copp & Greene 2013).
Extensive research on mouse models of NTDs has yieldeddan amalgam of informationnon the pathogenesis and causessof this condition. Emerging data from current literature and ongoing research in this field all hint at a genetic basis for most NTDs. Here we will focus on the pathogenesis of neural tube defects and attempt to delineate the signalling pathways and cellular mechanism through which the uncommon forms of NTDs such as craniorachischisis may occur.
Neural Tube Formation
Figure 1 | Neural tube closure in mouse and human embryos. Red labels indicate the NTDs associated with failure in specific closure events (red arrows). (Adapted from Copp & Greene 2010).
The neural tube serves as the embryos precursor to the central nervous system, and is therefore a requisite for the formation of the brain and spinal cord. The neural tube is formed during the process of neurulation in earlyeembryogenesis, beginning with neural plate (a thickening of dorsal surface ectoderm) formation and ending with neural tube closure. The neural plate contains a central indentation known as the neural groove, which gradually deepens leading to elevation of the neural folds, such that they’re ultimately juxtaposed towards one another at the midline; their subsequent fusion leads to transformation of the neural groove into a closed canal termed the neural tube. Neural tube development is separated into two phases: primary and secondary.
In mammals, the closure of the neural tube is a multi-site process, where closure events initiate at separate location along the body axis (figure 1). In mice, closure begins at the border between the cervical spine and the hindbrain (closure 1); hours later, closure continues at the border between future forebrain and midbrain (closure2); and shortly after it restarts at the rostral boundary of the upcoming forebrain (closure 3). The regions of the neural folds that remain open (neuropores) after the initial sequence of closure events, continue to fuse together rostrocaudally in a bidirectional fashion (zippering), the end ofwwhich marks theccompletion primary neurulation (Copp 2005).
The formation of the spinal cord at the lower sacral and coccygeal areas depends on the subsequent process of secondary neurulation. The tail bud, at the caudal end of the mice contains a group of multipotent stem cells thatggive rise toccells with a neuralffate (Cambray & Wilson 2002). The most posterior of these cells undergo a process knows as canalization, leading to a unique arrangement of cell around a central cavity which is then termed the secondary neural tube. The lumens of both primary and secondary neural tubes are continuous with one another. Ultimately, the same multipotential stem cells found in the tail bud form the lateral sclerotomal cells that transform into sacral and coccygeal vertebrae.
There is a high degree of conservation of neurulation events between mammalian species; nevertheless, there are some differences, notably the absence of closure 2 in human embryos. This absence of the closure point at the midbrain-forbrain boundary has been linked to the smaller midbrain in human embryo, which may have lead to the evolutionary elimination of closure 2 as an unnecessary process (Copp 2005).
Types of Neural Tube Defects
Figure 2 | Types of neural tube defects in relation to their corresponding closure event (Copp et al. 2013)
NTDs are categorised into two classes, open and closed. Open NTDs occur more commonly and involve conditions where vital structures such as the brain and/or spinal cord protrude through the skull and vertebrae, respectively. Phenotypically these manifest as exencephaly, open spina bifida and craniorachischisis. On the other hand, closed NTDs account for a lower proportion of neural tube defects; characteristically patient with closed NTDs have a layer of skin covering the spinal lesion, as seen in lipomyelomeningocele.
Disruptions in formation of the vertebrae covering the spinal cord increases the possibility of spinal cord protrusion through open defects in the vertebrae, which are due to abnormalfformation and fusion of vertebralssegments (figure 2D). These malformations mostly occur in the lumbar and sacral areas. In order to assist identification, spinal bifida is divided into three groups: spina bifida occulta (hidden), spina bifida cystica with meningocele and spina bifida cystica with myelomeningocele. The latter is the most severe of all three types, whereby the affected individuals will suffer from neurological impairments below the level of the lesion often resulting in walking disability, incontinence and general loss of sensation (Copp & Greene 2010).
Exencephaly & Anencephaly
The majority of embryos affected with NTDs undergo complete closure 1 and it’s only later in neurulation that specific failures occur, some presenting as open lesions of the cranial neural tube giving rise to exencephaly and the consequent anencephaly. In exencephaly the brain protrudes outside the head through deformities/openings in the skull (Copp & Greene 2010). Consequently, there is no skull formation over these deformities, leaving the neural tissue exposed; this leads to degeneration of the tissue and later the appearance of anencephaly in human pregnancy (figure 2A). In humans, anencephaly mainly affects either the rostral brain and skull (meroacrania) or the posterior brain and skull (holocrania) (Seller 1995).
At the most extreme end of the NTD spectrum, around 10% of affected individuals exhibit a significantly more extensive lesion affecting both the cranial and spinal neural tube, known as craniorachischisis. In this defect, following from failure in closure 1 (Ybot-Gonzalez, Savery, et al. 2007b), the neural tube remains exposed starting from the midbrain to lower spine (figure 2B). This specific anomaly has been linked to a failure in the process of convergent extension, which is a key morphogenic event for the development of a normal body axis. Mutations in genes involved in the signalling pathway (planar cell polarity) that regulates convergent extension have been specifically linked to craniorachischisis.
Signalling Pathways in Neurulation and NTDs
Figure 3 | Convergent-extension. Midline intercalation of mesodermal cells (from the node) cause narrowing and formation of the notochord. In the neural plate, cells converge at the midline, and lengthen (extend) in the rostro-caudal axis. Red arrows show midline intercalation in cells that emerge from the node and convergent extension in the neural plate (Ybot-Gonzalez, Savery, et al. 2007b).
Planar cell polarity pathway
The proximity of the neural folds to one another is a key determinant in initiation of neural tube closure. For the neural folds to be in close vicinity, the cells of the early disc shaped neural plate undergo a lengthening and narrowing process, known as convergent-extension (Ybot-Gonzalez, Savery, et al. 2007b). This process involves the movement of cells from the lateral aspects of the presumptive mesoderm and neural plate to the medial part. This gathering of cells in the midline results in convergence (medial-lateral narrowing) and extension (rostro-caudal lengthening) of the body axis (Keller et al. 2008).
Cellular polarization plays an important role in the development of the nervous system. Events such as neurulation andnneural tube closure depend on successful cellular migration and correct positioning of cells within the epithelial plane (planar cell polarity) (Montcouquiol et al. 2006). Amongst the many signalling pathways at work during neural tube closure, the non-canonical WNT signalling cascade acting through the planar cell polarity (PCP) pathway is a key regulator of cell movements in convergent-extension. At the molecular level, the Wnt-PCP pathway signals through membrane bound receptors (frizzled) and cytoplasmic proteins (dishevelled), bypassing the typical downstream stabilization of β-catenin usually observed in Wnt signalling (Montcouquiol et al. 2006).
Figure 4 | Non-canonical Wnt signalling in a mammalian cell. Signalling pathways essential for planar cell polarity pathway are indicated with black arrows. Blue arrows denote currently understood biochemical interactions and red arrows represent genetic interactions. * Components of PCP pathways that have altered genes associated in human NTDs. (Copp & Greene 2010)
On a cellular level, PCP signalling is thought to be involved in regulating the formation of stable and medio-laterally oriented Lamellipodia, which allow polarized cellular motility by providing cell-cell and cell-matrix traction (Keller et al. 2008).
The involvement of the PCP pathway in NTDs was first highlighted by studies carried out on mice, whereby those mice carrying mutations in various genes within the PCP pathway exhibited severe forms of NTDs. Mutations in transmembrane proteins encoded by core PCP pathway genes Vangl2 (loop-tail mutant), Celsr1 (Crash mice) and Fzd3/6 or the cytoplasmic proteins encoded by disheveled genes all result in suppression of convergent extension, and failure in closure of the neural tube from midbrain to low spine, giving rise to craniorachischisis (Greene et al. 1998).
Many studies have since focused on PCP genes and their variations between patients with NTDs and those without. There have been many reports of missense variants of PCP genes present in affected individuals, however promising, there needs to be further investigation into specific human mutations and obtaining functional evidence as to whether such mutations cause altered protein function in individuals with NTD, it is then that probable causal alleles can be identified.
Sonic Hedgehog & Bone morphogenetic proteins
Furthermore, discrepancies in core signal transduction machinery of Sonic hedgehog (SHH) and Bone morphogenetic proteins (BMP), which are key players in ventral and dorsal patterning of the neural tube, have been shown leading to NTDs in mice (Ybot-Gonzalez et al. 2002). SHH signalling is initiated by binding of the SHH ligand to its transmembrane receptor Patched1 (PTCH1), which then triggers downstream transcriptional activators (GLI proteins). When there is no ligand bound to PTCH1, its activity is inhibited by an associated membrane protein, Smoothened (SMO). Mutations in PTCH1 that render it unresponsive to inhibitory forces of SMO lead to NTDs; this mutation and other loss of function mutations of SHH inhibitory genes are all assumed to affect the dorsolateral bending of the neural plate, a process necessary for successful closure in both the midbrain and the lower spine (Murdoch & Copp 2010).
On the other hand, members of the TGFβ superfamily of proteins, BMPs, are involved in dorsal development of the early embryo. Experiments on mice have shown that BMP signalling inhibits the formation of dorsolateral hinge points (DLHP) during neurulation. DLHPs are important for proper neural tube closure; they anchor the ventral midline and elevate the neural folds bringing them close together (Eom et al. 2012). Failure in development of DLHPs leads to disruption of neural tube closure in the lower spinal regions, manifesting as severe spina bifida (Ybot-Gonzalez, Gaston-Massuet, et al. 2007a).
Environmental Factors & Folic acid
There are numerous teratogenic agents identified that cause NTDs in rodents. However, only a few non-genetic factors are believed to play a role in human NTDs. The anticonvulsant, valproic acid, is one such non-genetic factors that leads to an almost ten-times increase in risk of spinal NTDs, when consumed in early stages of pregnancy (Lammer et al. 1987). The mechanism behind this increased risk is thought to involve disruption of folate activity; valproic acid is shown to disturb the balance between protein acetylation and deacetylation, producing effects similar to trichostatin-A, a histone deacetylase inhibitor that causes NTDs in mice (Hernández-Díaz et al. 2001).
In addition to valproic acid, another environmental teratogen, fumonisin, a fungal product, has been associated with doubling the risk of NTDs; it is believed to negatively affect sphingolipid metabolism and disrupt expression of key embryonic genes (Gelineau van Waes et al. 2012). Furthermore, other non-teratogenic factors such as maternal obesity and diabetes are also believed to increase the risk of NTDs (Moretti et al. 2005).
Despite our ability to prevent exposure to predisposing environmental factors that are linked to congenital disorders, only a small proportion of birth defects have an identified environmental cause (Mitchell 2005). Even so, there seems to be a difference in susceptibility of individuals to different environmental factors, all hinting at complex genetic variation that determines levels of susceptibility (Copp & Greene 2013).
In 1980s, clinical trials focused on NTDs lead to the discovery of the preventive effects of folic acid supplementation in pregnant women against NTDs (Smithells et al. 1980) (Milunsky et al. 1989). To this day, folic acid supplements remain highly recommended to all women planning pregnancy. Folates are critical for production of pyramidines and purines, which are indispensable for DNA synthesis.
Contrary to popular belief, the majority of pregnancies complicated with NTDs are not folate deficient (KIRKE et al. 1993); meaning that the absence of folate does not necessarily cause NTDs, this has been replicated in mouse models, whereby severe folate deficiency does not lead to a defect, unless there was a genetic predisposition (Burren et al. 2008). Therefore, it is assumed that folic acid is able to trigger a specific cellular/molecular reaction that is able to counteract the detrimental effects of genetic/environmental factors that would otherwise cause NTDs. This may involve stimulation of cellular proliferation and survival pathways which depend on DNA synthesis, which itself is dependent on one-carbon metabolism for which folate is essential. Understanding the true underlying mechanism of folic acid supplementation and NTD prevention remains a top priority, results from which could lead to better understating of the different aetiologies seen in response to folic acid supplements (Copp & Greene 2010).
Despite significant breakthroughs in the filed of NTDs, including discovery of the preventive effects of folic acid against this abnormality, NTDs remains a complex and debilitating congenital disorder, capturing the interest and challenging the minds of clinicians and developmental biologists alike.
Continued research using genetic technologies holds great promise for identification of additional risk factors affecting neurulation in the embryo. Considering the multifactorial nature of NTDs in humans, genome wide sequencing of a large cohort of NTD patients will allows us to gain a better understanding of the genetic associations present in NTDs. Furthermore, In addition to animal models, stem cells have also proven extremely useful for the study of basic biological processes. It is suggested that stem cells can be used to model neural tube closure, this will allow direct and dynamic studies of neural morphogenesis; enabling comparison between normal and affected human tissue as they start the closure events.
Future studies will undoubtedly pave the way for the expansion of our currently limited knowledge concerning the complex molecular and cellular mechanisms involved in the pathogenesis of neural tube defects, where they will prove as a powerful tool for applications in the scientific and clinical disciplines.
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