Kaitlyn Greenwood

BIOL334: Dr. Ealy

Literature Review
May 6, 2020
Literature Review of Spina Bifida Development
Spina bifida affects nearly one out of every one thousand births around the world each
year. This developmental disorder is caused by the incomplete closure of the neuroepithelial cells
in the caudal region of the neural tube1. There are four different types of spina bifida (occulta,
closed neural tube defects, meningocele, and myelomeningocele) that are classified by where in
the spine the defect occurs. Patients with the occulta form rarely suffer from symptoms caused
by the defect, but patients with myelomeningocele typically suffer severely from symptoms2.
Although there is a 79-96% one-year survival rate, mortality increases as the patient ages and
grows. Growth and development of the spine typically exacerbate the issues caused by the
malformations2. Symptoms range from birthmarks and muscle weakness to total paralysis. It is
common for patients to have bowel and bladder issues throughout their lives such as leaking of
urine and/or stool and constipation or even bowel obstructions. Without proper regulatory
treatment, constipation and bowel obstructions could be deadly. A portion of patients are
paralyzed from the spot of malformation in their spine down. Paraplegic patients are much more
susceptible to low blood pressure, bed sores, pneumonia, and cardiovascular issues due to lack of
mobility. Without proper maintenance care, any of these conditions can become deadly. As of
now, there is no set cure for spina bifida. There are surgical options that have proven success in
reduction of symptom complication, but most other treatments focus on treating the symptoms3.
Due to the immense risk posed by surgical procedures, efforts are becoming more concentrated
on nonsurgical options.
A genetic cause for spina bifida is not currently known but there have been many studies
attempting to uncover this mystery. Common ground to start on is the development process of
the neural tube, called neurulation. The neural tube develops from the ectoderm in an embryo but
communicates closely with the neighboring mesoderm. Neurulation is broken down into two
stages, primary and secondary. In the primary stage, the folding in of the neural plate creates the
neural tube. At this point the neural crest cells that were most lateral on the neural plate have
almost converged on the dorsal side of now the neural tube. Complete closure of the neural tube
begins where the brain and spinal cord meet at the bottom of the hindbrain and continues towards
the primitive knot then ends at the posterior neuropore. Spina bifida results in the failure to close
the posterior neuropore4. Because we know that this specific step is the physical cause for spina
bifida, scientist can focus on gene expression from fertilization to this point.
A large number of genes have become associated with the correct develop of the neural
tube. All of the implicated genes work together to form a complex network of expression and
inhibition to cause the end result of the posterior neuropore closing. The gene that seemingly
plays the biggest role is bone morphogenic protein (BMP). The BMP proteins play roles in
neurogenesis regulation, axon development guidance, cell development, cell transition from
epithelial to mesenchymal, and many more. BMP has been shown associated with Wnt genes to
regulate mesoderm formation5. Near the beginning of neurulation, Wnt needs to be expressed to
induce neural crest cell formation. A Wnt and planar cell polarity pathway (PCP) acts together to
elongate the neural tube until embryo day 9.5. At this point of development, Wnt is inhibiting
Axin1-APC-Gsk3 which allows -catenin levels to rise. The increase in -catenin levels during
gastrulation causes activation of Cdx2 and Pax3 genes, which are both crucial to posterior
neuropore closure6. Expression of those genes at the correct time promote closure, but there are
other genes that specifically need to be inhibited for it to correctly occur. During elongation,
BMP needs to be mostly inhibited. For BMP to be effectively inhibited, noggin and Fgf3 need to
be expressed. When it is time for closure of the neuropore, noggin and Fgf3 become inhibited so
that BMP becomes expressed and promotes the closure along with crest cell differentiation5,7,8.
Working together in different combinations for expression and inhibition, these genes result in
correct closure of the posterior neuropore.
Unfortunately, errors in the expression and inhibition of genes result in neural tube
defects, one of which is spina bifida. Patients with spina bifida have been shown to have reduced
bone mass. Due to the role of Wnt and PCP in neurulation and bone mass formation, one group
of scientists looked into the potential connection of this pathway and development of bone mass
disorders correlating with spina bifida. It was shown that Wnt and PCP act as antagonists for
each other to induce correct bone mass formation. Wnt promotes high levels of PCP so that
higher levels of bone mass are produced in the developing embryo. When Wnt is not present in
an embryo, spina bifida develops, along with lower bone mass1. This can be understood by
seeing that Wnt signaling is required to the posterior neuropore closure and promotion of bone
mass production1,7. Wnt signaling defects is only one of the many ways spina bifida can develop.
Due to the crucial roles BMP plays in neural tube development, there are many points
where perturbed BMP signaling could cause defects. During the early part of neurulation when
the neural plate is beginning to fold, BMP needs repressed. Without BMP repression, the dorsal
lateral hinge point will not form. This point is where the neural plate folds back towards the
medial plane where it will eventually connect to close7. Without this point, the plate will not be
able to fold back, and the neural tube will never be completely formed. Noggin expression in the
ventral portion of the neural tube allows for inhibition of BMP in this region as well7. If there is
over expression of BMP in the embryo, the neural tube does not close and there is excessive
neural crest cell development. If there is too little expression of BMP throughout neurulation,
there is premature closure of the neural tube without elongation4. Clearly BMP regulation is key
to correct development and closure of the neural tube.
The Pax3 gene has been closely associated to the correct function of Wnt and -catenin.
Without expression of -catenin, the Pax3 gene is not expressed and closure of the posterior
neuropore does not occur, resulting in a form of spina bifida6. One study has shown the in vivo
replacement of the Pax3 gene rescues its function so that the neural tube correctly develops9.
Although the specific mode of action is unknown, supplementation of folic acid in carrying
mothers can rescue Pax3 function and prevent neural tube defects. This study found that there
was altered methylation to the Pax3 gene in spina bifida mouse infants. They presume that the
folic acid supplement somehow corrects the altered methylation of Pax3 so that the gene
becomes functional again9. Pax3 is closely associated with the homeobox gene Cdx2. It is shown
that in mice mutants lacking -catenin and Pax3 result in a loss of function of the Cdx2 gene as
well. When experiments were produced on the mutant mice in attempt to rescue Cdx2 function,
they found that replacing -catenin and Pax3 separately did not rescue Cdx2 function. This
shows that -catenin and Pax3 mush be present together to have function of the Cdx2 gene
which is a crucial element in posterior neuropore closure6. Due to studies showing
supplementation of folic acid in a mother’s diet can rescue Pax3 function that had altered
methylation, there could potentially be a connection between maternal methylation and fetal
gene methylation.
 A study published in 2015, looked into the placental DNA methylation in patients that
were born with spina bifida. Ultimately, they found that 914 regions in placental DNA were
hypermethylated. These regions make up about 163 genes. The hypermethylation of these genes
means there are genes being repressed that should not be so that loss of their expression
contributes to development of spina bifida. They also found that 2925 regions were
hypomethylated. These regions make up about 810 genes. The hypomethylation of these genes
means that there is an immense excess of genes being expressed in the developing embryo. It can
be concluded that this convoluted number of excess genes contribute heavily to the development
of spina bifida5. These pattern findings are significant because they show how important
maternal factors are for a developing embryo. An error in maternal DNA methylation could lead
to life-long conditions in their children.
As of now the closest treatment to a cure for spina bifida is open fetal surgery. There can
be surgical procedures done on infants to correct the malformation in the spine, but these have
shown many complications such as hydrocephalus, a buildup of cerebrospinal fluid around the
brain and spinal cord. The hydrocephalus complication would then require another surgery to
place a shunt in the fourth ventricle between the brain and spinal cord so that the excess fluid
could drain. Shunt placement could mean that the patient will have to have repeat surgeries
throughout life if the shunt becomes blocked or infected3. These complications could lead to
death. As with almost all surgeries in The United States, fetal surgery comes with a hefty price
tag and immense risk. Due to the fact that fetal surgery is done with the fetus still in the mother’s
uterus, there becomes the chance of the mother and the baby developing life-threatening
complications. Due to these risks and potential complications of surgery, it is important that we
work of finding cures and treatment options that are nonsurgical. This is a difficult task since the
exact genetic mechanism of spina bifida is unknown. There are an innumerous number of genes
that must cooperate in the correct way for correct neural tube development and posterior
neuropore closure. Because there are so many different genes working together, there are
numerous places throughout the process for something to be thrown off and effect the final
development. This leads to a variety of ways to genetically treat spina bifida before birth, but
countless hours would need to be spent to completely understand the full picture of spina bifida
development and how to prevent it. Due to spina bifida being one of the most prevalent
developmental disorders, it is important we work towards finding better ways to treat patients
suffering the consequences of their disease.
 References
1. Orriss, I, et al. (2018) “Spina Bifida-Predisposing Heterozygous Mutations in Planar Cell
Polarity Genes and Zic2 Reduce Bone Mass in Mice”. Scientific Reports. 8: 1-14.

2. “Spina Bifida Fact Sheet.” National Institute of Neurological Disorders and Stroke, U.S.
Department of Health and Human Services, 17 Mar. 2020,
www.ninds.nih.gov/Disorders/Patient-Caregiver-Education/Fact-Sheets/Spina-Bifida-
Fact-Sheet.

3. Texas Children's Fetal Center. “Spina Bifida (Myelomeningocele).” Pavilion for Women,

2020, women.texaschildrens.org/program/texas-childrens-fetal-center/conditions-we-
treat/spina-bifida-myelomeningocele?
_vsrefdom=p.3024&npclid=Cj0KCQjwncT1BRDhARIsAOQF9LnfHj1_MCXOGxqpXt
TYHB9vaHwiPjI8sD9AGyYAphjrEKb0B5o4gZAaAo1uEALw_wcB&utm_source=goo
gle&utm_medium=cpc&utm_term=spina%2Bbifida
%2Btreatment&utm_campaign=Texas%2BChildrens%2BHospital%2B%7C%2BFetal
%2B%7C%2BGeneral%2Bl%2BTCH048CP.
4. Anderson, M, et al. (2016) “An FGF3-BMP Signaling Axis Regulates Caudal Neural
Tube Closure, Neural Crest Specification and Anterior-Posterior Axis Extension”. PLOS.
12(5): 1-30.
5. Zhang, X, et al. (2015) “Spina Bifida in Fetus is Associated with an Altered Pattern of
DNA Methylation in Placenta”. Journal of Human Genetics. 60: 605-611.
6. Zhao, T, et al. (2014). “-catenin Regulates Pax3 and Cdx2 for Caudal Neural Tube
Closure and Elongation”. The Company of Biologists. 141: 148-157.
7. Nikolopoulou, E, et al. (2017). “Neural Tube Closure: Cellular, Molecular, and
Biomechanical Mechanisms”. The Company of Biologists. 144: 552-566.

8. Steventon, B, et al. “Differential Requirements of BMP and Wnt Signalling during

Gastrulation and Neurulation Define Two Steps in Neural Crest Induction.” Development
(Cambridge, England), Company of Biologists, Mar. 2009,
www.ncbi.nlm.nih.gov/pmc/articles/PMC2685944/.
9. Sudiwala, S, et al. (2019). “Cellular Mechanisms Underlying Pax3-related Neural Tube
Defects and Their Prevention by Folic Acid”. Disease Models and Mechanisms. 12: 1-18.