Neurulation and the Possible Etiologies of Neural Tube Defect

Abstract

Neural tube defects (NTDs) are variety of defects which result from abnormal closure of the neural tube during embryogenesis. Various factors are implicated in the genesis of neural tube defects, with contributions from both genetic and environmental factors. The clear understanding of the causes which leads to NTDs is lacking, but several non-genetic risk factors have been identified which can be prevented by maternal folic acid supplementation. Multiple genetic causes and several critical biochemical reactions have been identified whose regulation is essential for the closure of neural tube. Preventive therapies can be developed by identifying potential risk factors in the genesis of NTDs.

1. Introduction

Neural tube defects (NTDs) are birth defects which result from the abnormal closure of neural tube during embryogenesis [1]. Its etiology is multifactorial; nutritional, environmental, genetic, and exposure to various teratogenic drugs during pregnancy. The severity of NTDs varies from asymptomatic cutaneous manifestations to life threatening conditions where brain and spinal cord is completely exposed to the exterior.

Here authors describe the process of formation of central nervous system (brain and spinal cord) along with the possible etiologies of NTDs.

2. Neurulation

The initial process involved in the formation of nervous system (brain and spinal cord) is known as neurulation. It is further divided into:

1. Primary neurulation

2. Secondary neurulation

2.1 Primary neurulation

Primary neurulation is responsible for the future formation of brain and most part of the spinal cord. The thickened ectoderm, neural plate, elevates to form neural folds and subsequent fusion of neural folds give rise to neural tube (Figures 1 and 2). The fusion begins in cervical region and proceeds in cranial and caudal directions. Ends of the neural tube, neural pore, are closed first on the cranial side (21 days post fertilization) followed by the caudal side (28 days post fertilization) (Figure 3). Thus, neurulation is a process involved in the formation of neural tube and closure of neuropores by the end of the fourth week of embryo development [2, 3]. The defects resulting from the abnormalities in primary neurulation leads to open neural tube defects [4, 5].

Figure 1.

A—Section of blastocyst of 9 days embryo-epiblast and hypoblast in bilaminar germ disc. B—Transverse section through embryonic disc-trilaminar embryonic disc. C, D—Transverse section of the embryonic disc at approximately 18 days embryo-showing notochord.

Figure 2.

Illustrations of formation of neural groove, neural folds, neural tube and neural crest in transverse section.

Figure 3.

Dorsal view of embryonic disc.

2.2 Secondary neurulation

The formation of spinal cord distal to mid-sacral region is formed by the process of secondary neurulation. Some of the loosely packed cells of tail bud condense to form an epithelial rod, which later canalise and form a tubular lumen for the last part of sacral and coccygeal regions of spinal cord [6, 7, 8]. Studies of various pathways at molecular and cellular level in neurulation-stage embryos provide understanding of development of normal or abnormal neural tube [9]. The malformations resulting due to the abnormalities in secondary neurulation results in closed neural tube defects (Figure 4).

Figure 4.

Secondary neurulation.

3. Mechanisms of neurulation

Shaping of the neural plate with mediolateral narrowing and rostro caudal elongation is needed to initiate closure of neural tube [10]. This elongation depends on Wnt signalling pathway via Frizzled (Fzd) membrane receptors [11]. Convergent extension of neural plate takes place through planar cell polarity (PCP) mediators via PCP genes functioning including Vangl2, Celsr1, Dvl-1 and -2, Fzd-3 and 6, Scrb1, Ptk7, and Sec24b [12, 13].

Functional failure of PCP mediator genes results in broad neural plate and craniorachischisis due to disruption in the closure of neural plate [14]. Occurrence of closure in the forebrain and part of the midbrain in craniorachischisis implies that PCP-dependent mechanism is not necessary for whole of the brain. Exencephaly developing in mutants of the genes Fuz or Intu is more likely due to disturbed cilium-dependent hedgehog signaling, than altered regulation of convergent extension for neural tube closure initiation [15]. Thus, there are multiple mechanisms at cellular level on PCP signalling which potentially affects neural tube closure.

Tips of the neural folds approximate each other after bending of neuroepithelium to achieve closure. Median hinge points (MHP) and dorsolateral hinge points (DLHP) in a stereotypical manner bring out the bending. Regulating signals for the bending emanate from nonneural tissues around the neural folds. Notochord derived Shh causes induction of floor plate of the neural tube at the MHP. Factors enhancing Shh signals, for example, mutations in cilia-related genes such as Gli3, Rab23, Fkbp8, Tulp3, and Ift40 also results in NTDs [16]. DLHPs take a role for neural tube closure in low spinal region by Zic2, expression of BMP antagonist, noggin, sufficiently induces DLHPs in dorsal neural folds [17].

Complexity of cranial neurulation compared to the spinal neurulation appears due to more extensive and sensitive genetic mechanisms. As a result, exencephaly comprises three times of the cases as does spina bifida after induction by teratogens. Disruption of cranial neurulation is contributed by some specific factors; mesenchymal expansion under the neural folds, cytoskeleton disruption of actin filaments, and mutant genes (e.g., n-cofilin, vinculin) of various cytoskeletal components which are not essential for neurulation in spinal region [18].

Meeting of the neural folds in dorsal midline give rise to two different types of epithelial layers, fledgling neural tube and overlying intact surface ectoderm, eventually after process of adhesion, fusion, and remodelling. Ephrin receptors, protease-activated receptors and Grhl2 expressions, explain this process of adhesion at varying axial levels [19]. Cell proliferation, neuronal differentiation and programmed cell death can be regulated by the genes; neurofibromin 1, nucleoporin, Phactr4 for cell cycle progression, Notch pathway genes Hes1, Hes3, RBP-Jκ for neuronal differentiation, and caspase3 or Apaf1 genes for characteristic patterns of apoptotic cell death. NTDs are likely to occur in mutations of these genes, which hampers the regulated cell proliferation, differentiation, and cell death (Table 1) [20].

S. No.	Mechanism	Pathways/mediators/genes
1	Shaping and convergent extension of neural plate	Wnt signalling pathway, PCP mediator genes- Vangl2, Celsr1, Dvl-1 and -2, Fzd-3 and -6, Scrb1, Ptk7, and Sec24b
2	Adhesion, fusion, and remodelling	Ephrin receptors, protease-activated receptors and Grhl2 expressions
3	Cell proliferation	neurofibromin 1, nucleoporin, Phactr4
	Neuronal differentiation	Notch pathway genes Hes1, Hes3, RBP-Jκ
	Programmed cell death	caspase3 or Apaf1 genes for characteristic patterns of apoptotic cell death
4	Induction of floor plate	Notochord derived Shh, cilia-related genes- Gli3, Rab23, Fkbp8, Tulp3, and Ift40
5	Cranial Neurulation	mesenchymal expansion, genes for cytoskeleton components (e.g., n-cofilin, vinculin); cilium-dependent hedgehog signalling genes Fuz or Intu
6	Spinal Neural Tube Closure	by Zic2, Expression of BMP antagonist- noggin

Table 1.

Mechanism of neurulation.

3.1 Type of neural tube defects

NTDs are classically divided into two types:

1. Open neural tube defects

2. Closed neural tube defects

3.1.1 Open neural tube defects

Open NTDs or spina bifida cystica are craniorachischisis, exencephaly-anencephaly and myelomeningoceles (Figure 5A–F). Open defects are characterised by the exposure of neural tissue through the skin as well as through the bony defect and is obvious at birth. These defects present with neurological deficit and carries poor prognosis. They can be identified easily during pregnancy due to high levels of α fetoprotein and acetylcholinesterase in amniotic fluid.

Figure 5.

A–D cephalocele, A—meningocele, B—encephalo-meningocele, C—encephalo-meningo-cystocele, D—anterior, parietal and occipital cephalocele, E—myelomeningocele, F—myelocele.

Craniorachischisis is the most serious and rare type of open NTD, which involves the defect in both cranial and spinal region. Their reported prenatal terminations range from 0.51 to 10.7 per 10,000 births in different regions of the world. Neural tube gets open from brain stem to spinal cord resulting in anencephaly and spina bifida simultaneously with external exposure of tissue in hindbrain and spinal cord on its posterior aspect. Death of the new-born is certain in craniorachischisis making it a lethal condition [21].

Anencephaly due to exencephaly involves non closure of only cranial part of the neural tube. Absence of forebrain and the vault of skull with intact skull base can be seen. Forebrain and midbrain are absent, brain stem is less severely involved and pituitary gland is hypoplastic in most of these cases. It is a lethal condition causing death of new-born within few days after birth.

Myelomeningocele results from defect in posterior part of spine usually in the lumbosacral region. Meningeal sac hernia takes place posteriorly, containing cerebro-spinal fluid and nervous tissue, through a bony defect in the vertebral arch. Myelocele is a similar open NTD involving the spinal cord without protrusion of meningeal sac. Spinal cord is typically divided into two halves giving an appearance of “open book,” which exposes ependymal layer to the surface.

Survival of the babies with open spina bifida depends on the severity and level of the lesion. Some other associated conditions including hydrocephalus, Chiari malformation type II, and vertebral abnormalities make them more complicated [21].

3.1.2 Closed neural tube defects

Closed NTDs or spina bifida occulta are encephalocele, meningocele, lipomeningocoele, diastematomyelia, and tethered filum terminale. Here, the underlying neural defect is masked by the intact overlying skin. The defect lies in the lower lumbar and sacral regions, and represents closed defects with deficient vertebral arches, sacral agenesis, and other skeletal defects. Presence of nevi, depigmentation, haemangiomas, localized hypertrichosis, and lumps including subcutaneous lipomas are some cutaneous stigmata of the lower back, may be the only signs of spina bifida occulta. Symptoms may not develop until late childhood and they possess comparatively better prognosis than open neural tube defects [21].

Encephalocoele is a round, soft, compressible, and nodular sac like protrusion of brain and/or its meningeal covering through an opening in the skull. Majority of encephaloceles pass through the midline calvarial defects and are classified according to the site of herniation; anterior, parietal, and occipital. Among these locations, occipital comprises 75% of the total number of encephaloceles. Hypertrichosis, and bluish translucency could be seen over the lesions during increased intracranial pressure. A comparatively better prognosis is observed if site is more rostral [22].

Meningocoele consists of meningeal herniation through the defect in the vertebral column. The spinal cord in these cases lies within the spinal canal in normal position. Atrophic epidermis of the skin usually covers the pedunculated and compressible lesion of herniated mass. They generally present with normal neurological examination and without any deformity (Figure 5A–F).

Lipomyelomeningocele is a form of occult spinal dysraphism where fat herniates through the bony defect. Diastematomyelia refers to a split in the spinal cord by a bony or a fibrous septum. Majority of these patients have cutaneous manifestations (Table 2) [23].

Open NTDs- Spina bifida cystica
S.No	Type	Location of the defect	Findings of the defect	Prognosis
1	Craniorachischisis	Cranial and Spinal	Open neural tube from brain stem to spinal cord	Death of the new born, lethal condition
2	Exencephaly-Anencephaly	Cranial	Absent forebrain and skull, thick and flat skull base	Death of the new born, lethal condition
3	Myelomeningoceles	Posterior part of spine, lumbar region	Meningeal sac hernia containing CSF and nervous tissue; Associations with hydrocephalus, Chiari malformation type II, and vertebral abnormalities	Survival depends on severity and level of the lesion
Closed NTDs
1	Encephalocele	Cranial	Hernia through small opening in the skull	Depends on site, lesion more rostral with better prognosis
2	Meningocele	Spinal	Meningeal herniation covered by the skin without its appendages	normal neurological examination and functions of the body
3	Lipomeningocoele	Spinal	Fat along with meninges herniates through the bony defect	Usually have normal neurological function.
4	Diastematomyelia	Spinal	Spinal cord splitting by bony or fibrous septum	Neurological deficit with bowel and bladder involvement
5	Tethered filum terminale	Spinal	Conus medullaris is tethered by filum terminal	Usually become symptomatic in the late childhood

Table 2.

Types of neural tube defects.

4 Aetiology of neural tube defects

NTDs prevalence range from 0.5 to 10 per 1000 pregnancies, thereby poses significant public health problem [1]. Variations in the incidence are due to large variety of risk factors such as:

1. Environmental factors

2. Nutritional deficiencies

3. Genetic causes

4.1 Environment factors

Environmental exposure to air pollution, extremes of temperature, and exposure of toxins to the expectant mothers are some of the known risk factors contributing to the aetiology of NTDs [24]. Study on various animals suggests the effect of teratogens in development of NTDs. Anticonvulsant drug valproic acid and a fungal product fumonism are known teratogens to develop NTDs in humans.

Hyperglycemia in embryos of cultured rodents, maternal obesity and diabetes mellitus are recognised risk factors for NTDs. Increased oxidative stress, change in Pax3 gene functions, apoptosis of neuroepithelial cell, activation of apoptosis signal-regulating kinase 1(ASK1) enzyme are some effects brought about by the maternal and embryonic hyperglycemia resulting in NTDs (Table 3) [25].

Multifactorial (50%)
A	S.No.	Non-genetic causes	Examples
	1	Environmental	Air pollution; Extremes of temperature; Exposure of toxins to the expectant mothers; Teratogens; anticonvulsant-valproic acid, fungal product fumonism; Hyperglycemia in embryos; Maternal obesity and diabetes mellitus
	2	Nutritional	Poor nutritional status and folate deficiency in mothers
B	S.No.	Genetic causes	Examples
	1	Gene-gene interactions	Dvl1-Dvl2, Cdx1-Cdx2 double knockouts; Supplementary sequel of heterozygous mutations Dvl3 with Vangl2^Lp; Variable phenotypic expressions of Cecr1 mutation
	2	Effect of modifier genes	Variation in Lmnb1 in curly tail (Grhl3) embryos
	3	Implications through experimental models	PCP genes mutations- CELSR1, VANGL1, VANGL2, FZD6, SCRIB1, and DVL2
	4	Folate one-carbon metabolism in mitochondria	suboptimal levels of folate in maternal blood interact with mutated Pax3 genes
	5	Histone modifications	Mutations in histone demethylases Jarid2 and Fbxl10; Mutations in histone deacetylases Sirt1 or Hdac4; Teratogens- Valproic acid and trichostin A
	6	Syndromes	Trisomy 13, Trisomy 18 and Triploidy
Unknown factors (50%)

Table 3.

Aetiology of neural tube defects.

4.2 Nutritional deficiencies

Folate is a well recognized vitamin B supplement implicated in the causation of neural tube defects. Poor socioeconomic status with high risk of congenital anomalies focus the scientists to find out the nutritional deficiencies in such cases. In mothers of NTD fetuses, folate was found to be deficient. Mechanism of folic acid in prevention of NTDs was considered when it was seen that blood folic acid levels in some mothers of affected foetuses were normal. It was believed that some suboptimal levels of folate in maternal blood interact with mutated genes, such as Pax3 to cause NTDs in developing embryos. Neural tube closure requires complex reactions for various nucleotide biosynthesis and methylation. Deficient methylation and abnormal biosynthesis of purine base and thymidylate have been noticed in cases of NTDs [26].

4.3 Genetics causes

Genetic mutations in the aetiology of NTDs always depend on polygenic and multifactorial inheritance. The causations by gene variants are complicated by the multiple genes, modifier genes, epigenetic factors, and environmental effects. Some recognised mutations of different genes obtained by experiments on animals were found to be the causes in minimal number of NTD cases in humans. Increasing understanding of development of neural tube and NTDs on molecular and cellular level still needs more precision to identify genetic basis of occurrence in individual cases. More than 200 mutations in the genes, and association of the environmental risk affect folate metabolism to causes NTDs. Significance to focus more on individual genes by scientists comes from the fact of having very less percentage of NTD cases in syndromes of chromosomal aberrations as compared to the isolated cases of NTDs. Now a days, data analysis from large-scale genome sequencing of NTD patients is more promising and practicable to mark the contribution of various genes in the patients and mutational burden of associated risks.

4.3.1 Gene interactions and modifier genes

Three wide range mechanisms to explain gene interactions in development of NTDs are; 1—Functional incompetency of two non-comparable genes for example Dvl1-Dvl2 and Cdx1-Cdx2 double knockouts, 2—supplementary sequel of heterozygous mutations in Dvl3-Vangl2, 3—variable phenotypic expressions of inherent mouse strains, Cecr1 mutation [27]. A remarkable and rare example of modifier gene for growing the tendency of NTDs in curly tail (Grhl3) embryos is variation in Lmnb1.

4.3.2 Genetic implications through experimental models

Mutations in PCP genes—CELSR1, VANGL1, VANGL2, FZD6, SCRIB1, and DVL2 are well known mutations in mice to cause NTDs. Thus, known to be putative PCP gene mutations can cause multiple types of abnormal births including craniorachischisis, spina bifida, anencephaly, or closed forms of spina bifida in humans. A wide range of NTDs can be seen after combining PCP mutations with other genetic risk factors of NTDs- For example; VANGL2, a missense variant, was seen in a spina bifida patient having a putative mutation in DVL2.

4.3.3 Genetic factors relation with environmental risk factors

A known environmental risk factor when interacts with genetic alteration in the embryo, could eventually instigate the risk of NTDs. Folate one-carbon metabolism in mitochondria is highly studied category for finding cause of NTDs in such cases and genes related to folate metabolism enlighten the ambience of maternal folate levels.

Enhanced risk of developing spina bifida by the ‘risk’ genes GLUT1, SOD1, and SOD2 conglomerate in foetus of mothers having diabetes and obesity. Some genes related to maternal obesity- FTO, LEP, and TCF7L2 were identified as a risk factor for NTDs in embryos.

4.3.4 Gene-regulatory mechanisms and NTDs

Multigenic involvement of NTDs makes it more complicated and difficult to identify due to irregular expressions of genes. Such as, insufficient or excess expression of Grhl3 and Grhl2 cause NTDs in mice, positive and negative correlations to explain the relationships among folate status, DNA methylation, and risk of NTDs. Modifications of histone protein or chromatin remodelling are some probable causes of NTDs in mice and in few cases in humans. Histone modifications effectively mis regulate the genes responsible for neurulation. For example, mutations in histone demethylases Jarid2 and Fbxl10, mutations in histone deacetylases Sirt1 or Hdac4 and teratogenic inhibition of histone deacetylases by valproic acid and trichostin A.

5. Prevention of neural tube defects

Primary prevention is quite effective in reducing the birth defects related to NTDs. It has been suggested that folic acid supplementation in a dose of 0.4 mg per day prevents large number of NTDs. There is three fold reduction in NTDs recurrence with an intake of 4 mg folic acid per day by the expectant mothers. The exact mechanism of this prevention is yet to be elucidated, but folate plays an important role in numerous chemical reactions, including thymidine and purine production and S- adenosylmethionoine synthesis, which is the methyl donor for DNA, lipids and proteins.

6. Conclusion

The causes of NTDs are multifactorial in humans, including genetic and non-genetic factors. The combination of these factors leads to defective closure of neural tube and subsequent development of malformed fetal appearance. Folic acid supplementation during pregnancy and its awareness through various platforms are necessary to prevent further occurrence of NTDs in children.

Acknowledgments

I am thankful to my family for the support they have provided in writing this chapter.

Conflict of interest

“The authors declare no conflict of interest.”