Figure 4. Variation in mean PNP length of embryos of the curly tail sub-strains. (A–C) Curly tail embryos at the 28–29 somite stage (E10.5), showing whole embryo (A) and enlarged views of two different caudal regions (B, C) to illustrate variable PNP lengths (between the arrows and indicated by dotted lines). An enlarged PNP (e.g. 800 mm length in B) is indicative of delay or failure of closure compared with a smaller PNP (e.g. 375 mm in C). Scale bars represent 1 mm in A and 0.5 mm in B, C. (D) PNP length measurements (mean in mm 6 SEM) at somite stages around the time of PNP closure. At 28–29 and 30–31 somites, the mean PNP lengths of embryos of the ct, ct8E and ct9E strains (i.e. Grhl3 mutants; grey bars) are significantly larger than those of stage-matched embryos wild-type at the Grhl3 locus (i.e. +ct and +ct;8E; white bars), regardless of Lmnb1 genotype (* significantly different from Grhl3 wild-type strains, p,0.001; ANOVA and Holm-Sidak pairwise comparisons). This difference is also observed for the ct strain at the 26– 27 somite stage. At each stage, the mean PNP length of +ct8E embryos is larger than for +ct embryos, but this difference does not reach statistical significance. Among ct sub-strains, embryos carrying the Lmnb19E allele had the smallest PNP lengths at each stage analysed (# significantly different from ct strain; p,0.001). Number of embryos corresponding to each bar: n = 5–16 at the 26–27 somite stage; n = 14–66 at the 28–29 and 30–31 somite stages. doi:10.1371/journal.pgen.1003059.g004 replication [24], prompted us to investigate the effect of the Glu variant on proliferative capacity in ct cells. MEFs were plated and counted after 4 hours (t = 0) and after successive 24 hour periods up to 5 days. Growth curves showed that ct8E MEFs proliferate significantly more slowly than their ct9E counterparts over the first four days in culture (p,0.05; Multiple linear regression, Lamin B1 and Neural Tube Defects R2 = 0.948) and then undergo a ‘proliferative crisis’ where cell numbers cease to increase (Figure 6A). The experiment was performed on three separate occasions using independent cell lines, with the same result each time. Therefore, in addition to nuclear dysmorphology, the Lmnb18E variant is associated with an apparent reduction in proliferative capacity in ct cells. In contrast, ct9E cells continued to proliferate at a similar rate to wild-type +8E cells at day 5 (Figure 6A). In accordance with the growth curve data, we also noted that when MEFs were repeatedly passaged, ct8E fibroblasts show a dramatic loss of proliferative capacity from passage 5 onwards, whereas ct9E continue to exhibit similar doubling times up to at least passage 8. To further investigate cell cycle properties of ct8E and ct9E cells, labelling with 5-ethynyl-29-deoxyuridine (EdU; to monitor S-phase progression) and immunostaining for phospho-histone H3 (pH 3; a marker of mitosis) were performed on day 0. This is well before the profound loss of proliferative capacity that occurs in ct8E cells after extended culture and it was therefore predicted that differences, if present, may be subtle. However, corresponding with growth curve data, we observed significantly fewer EdU-labelled ct8E cells than ct9E cells (Figure 6B), together with a non-significant reduction in pH 3 labelling (cells in G2/ M phase) and mitotic index. Consistent with the reduced EdU labelling in ct8E cells, indicating that fewer cells had passed through S-phase, we observed a slightly lower proportion of EdU/pH 3 double-labelled nuclei. However, there was no difference between strains in the number of double-labelled cells as a proportion of the total number of EdU-labelled cells (Figure 6B), suggesting that progression from S-phase to G2 is not defective in ct8E cells. We next examined the expression of key regulators of cell cycle expression by qRT-PCR, 4 hours after plating (t = 0, as for cell cycle analysis) and after 5 days of culture (t = 5). The reduced proliferative capacity of ct8E cells during the initial growth period was associated with significantly lower expression of Ccnd1, encoding cyclin D1 (Figure 6C). After 5 days, the expression of Ccna2 and Ccnb1 (encoding cyclin A2 and cyclin B1, respectively) was also significantly reduced in ct8E compared with ct9E cells, consistent with diminished cell cycle progression [43,44]. Con- versely, there was a dramatic increase in expression of p16Ink4a (Figure 6C), which suppresses cell cycle progression through inhibition of cyclin D-dependent kinases [45] and is a hallmark of cells entering senescence. The expression of p16Ink4a was also increased in ct9E cells at t = 5 compared with t = 0, but to a much lesser extent. In addition, at both stages ct8E cells also exhibited a significant reduction in expression of Smc2, which encodes a core component of the condensin I and II complexes that play key roles in chromosome condensation during mitosis [46,47]. We conclude that changes in expression of cell cycle-associated proteins are consistent with reduced cell cycle progression in cells expressing the Lmnb18E variant, compared with those expressing the wild- type Lmnb19E variant. Finally, we tested whether the Lmnb1 variants were also associated with differences in cellular proliferation rate in the developing embryo. Analysis was performed on the neural folds and hindgut at the axial level of the closing PNP, at the stage at which the underlying defect in proliferation in the hindgut of affected curly tail embryos was reported [12,13]. Consistent with the findings in cultured cells, the EdU labelling index was lower in ct8E than in ct9E embryos, particularly in the hindgut (Figure 6D, Table S3). Mitotic index was similar in the sub-strains (Table S3). The diminished S-phase progression of cells in the hindgut of ct8E embryos corresponds with the proliferation defect that is known to underlie spinal NTDs in curly tail embryos. PLOS Genetics | www.plosgenetics.org 7 November 2012 | Volume 8 | Issue 11 | e1003059