How does folic acid prevent spina bifida in the embryo?

Hypothesis and Aims: 
Spina bifida and other neural tube defects (NTDs) can be prevented in some cases by folic acid supplementation early in pregnancy. But, it remains a mystery how folic acid acts on the developing embryo to prevent NTDs [1]. If we could understand this action, it would be an important step forward in improving the prevention of spina bifida. In this PhD project, the student will determine the embryonic effects of altered folate status in a ‘human-like’ mouse model of NTDs, in order to shed new light on the mode of action of folic acid. Specific aims are to:

1. Breed mutant mouse strains to create a new, ‘human-like’ model of spina bifida, in which both genetically altered and normal cells co-exist (‘mosaicism’) in the neural tube.
2. Determine the effects of a folate-deficient diet, and folic acid supplements, on this mouse model. These are expected to increase and decrease NTDs, respectively.
3. Use this model system to examine several aspects of neural tube closure in developing embryos, to determine which are faulty in folate-deficiency, leading to spina bifida, and which are corrected by folic acid.

First year plan:  
Months 1-6. The project will begin with breeding mice that lack Pax3 gene function in a proportion of neural tube cells. This will be achieved by breeding Nkx2.1CreERT2 x Pax3-flox mice (both strains already bred in our lab), and inducing Cre expression by tamoxifen. Complete loss of Pax3 causes spina bifida in all embryos [2], whereas we will adjust the level of mosaicism, by varying tamoxifen dosage, to generate NTDs in ~ 50% of embryos. Human NTDs are often mosaic, so this will produce a ‘human-like’ model. Alongside this breeding work, the student will learn skills of mouse embryo dissection, microscopy and other forms of analysis (e.g. gene expression studies).

Months 7-12. Once the genetic model is established, the student will determine the effects of a folate-deficient diet on the NTD rate, which is expected to increase, and to administer additional folic acid which is expected to reduce NTD rate. Embryos will be obtained from pregnancies raised under both types of folate status, and genotyped by PCR, to identify mutant and normal embryos for comparison. Analysis will begin of several key events in neurulation, including: (i) Analysis of cellular protrusions on neural folds tips, which we showed to be essential for closure [3]; (ii) Determination of closure rates (‘zippering’) as we have done previously [4]; (iii) Assessment of dorsal remodelling, which completes the closure process [5]. These experiments will continue through Years 2 and 3 of the PhD.

Ethics Approval:
Research is covered by a Home Office Project License.

References:
1.  Copp, A.J. et al. Neural tube defects: recent advances, unsolved questions, and controversies. Lancet Neurol 12, 799-810 (2013).
2.  Burren, K.A. et al. Gene-environment interactions in the causation of neural tube defects: folate deficiency increases susceptibility conferred by loss of Pax3 function. Hum. Mol. Genet 17, 3675-3685 (2008).
3.  Rolo, A. et al. Regulation of cell protrusions by small GTPases during fusion of the neural folds. Elife 5, e13273 (2016).
4.  Mole, M.A. et al. Integrin-Mediated Focal Anchorage Drives Epithelial Zippering during Mouse Neural Tube Closure. Dev Cell 52, 321-334 e326 (2020).
5.  Massa, V. et al. Apoptosis is not required for mammalian neural tube closure. Proc. Natl. Acad. Sci. USA 106, 8233-8238 (2009).