Lamin B1 and Neural Tube Defects Figure 6. Cell cycle progression is impaired in curly tail cells and embryos expressing the Lmnb18E variant. In cultured embryonic fibroblasts (A–C), analysis of growth curves (A) shows that ct8E cells proliferate significantly slower than ct9E cells (*p,0.05; multiple linear regression for days 0–4) and then undergo a ‘growth crisis’ after 4 days culture. (B) Cell cycle analysis was performed at day 0 (5 hours of culture) using EdU to label cells as they progress through S phase (Bi) and anti-phospho Histone H3 (pH 3) to label cells in G2/M (Bii–vi). Mitotis was scored visually as cells that were in prophase, metaphase (Biv), anaphase (Bv) or telophase (Bvi). Data represents the mean of three experiments, each using an independent cell line, plated in triplicate. ct8E cells show significantly reduced EdU labelling (* p,0.05; t-test). There is a trend towards reduced pH 3 labelling and mitotic index in ct8E cells, but this difference is not statistically significant. The proportion of cells double-labelled with EdU and pH 3 (Bii) does not differ between ct8E and ct9E cells. (C) Expression of cell cycle regulators determined by qRT-PCR. For each gene, significant differences in the comparison of ct8E and ct9E cells cultured for the same period are indicated (* p,0.05, ** p,0.01; ANOVA with Holm-Sidak Pairwise Comparison). Expression differences between 0 and 5 days in culture for cells of the same genotype are indicated (# p,0.05). (D) Analysis of proliferation in embryos at E10.5 showed that EdU labeling index was significantly diminished in the hindgut of ct8E compared with ct9E embryos (* p,0.02; t-test). doi:10.1371/journal.pgen.1003059.g006 Discussion The multifactorial, partially penetrant genetics of the curly tail mouse provided an opportunity to investigate the Lmnb1 polymorphism as a potential modifier of susceptibility to NTDs. In the context of the genetic background of the curly tail mouse, we observed a major effect of lamin B1 on development of the neural tube, the embryonic precursor of the brain and spinal cord. Curly tail sub-strains expressing the Lmnb18E variant demonstrate failure of neural tube closure with significantly higher frequency than those that express wild-type protein. Thus, although both the curly tail sub-strains (ct9E and ct8E) are homozygous for the Grhl3ct mutation, which results in diminished Grhl3 expression [9], there is a three-fold difference in the frequency of NTDs depending on the co-existing Lmnb1 genotype. Strikingly, although exencephaly occurs at much lower frequency than spina bifida, Lmnb1 also affected the penetrance of these defects to a similar extent as spinal NTDs, with approximately 65% reduction in frequency among ct9E compared with ct8E embryos. Interestingly, it appears that the Lmnb18E variant may confer susceptibility to NTDs even in the absence of a Grhl3 mutation, at least in the context of the ct genetic background. Thus, +ct;8E embryos that are wild-type for Grhl3 but which carry the Lmnb18E variant developed occasional tail flexion defects and/or exencephaly. In contrast, spinal NTDs can be prevented by transgenic over-expression of Grhl3 expression (ctTgGrhl3) [9], despite the presence of the Lmnb18E variant. The possible functional effect of polymorphic variants has been explored in very few proteins, to date. We found that the loss of a single Glu, in Lmnb18E, compromises the stability of lamin B1’s interaction within the nuclear lamina. This effect is predicted to result from disturbance of the conformation of the C-terminal region of the protein, owing to the location of the variant Glu residue in a predicted alpha-helix. The effect of the Glu repeat polymorphism on lamina stability in FLIP analysis correlates with the observation of a higher proportion of dysmorphic nuclei in ct MEFs that express the PLOS Genetics | www.plosgenetics.org 9 November 2012 | Volume 8 | Issue 11 | e1003059