Lmnb18E variant compared with Lmnb19E. Abnormalities in lamin immunostaining and nuclear shape are reminiscent of cells with nuclear envelope abnormalities, such as from progeria models [42], lamin B1 mutant mice [33,34] and following shRNA- mediated silencing of lamin B1 [48]. Using contour ratio analysis, abnormal nuclei were observed in around 47% of primary embryonic curly tail fibroblasts (current study), compared with 68% of primary dermal fibroblasts derived from a patient with Hutchison-Gilford progeria syndrome [42]. Only 7–15% of nuclei among control fibroblasts exhibited such abnormalities. In these previously reported examples, abnormalities of cell proliferation, chromosome position, transcription factor localisation and gene expression have all been noted [22,26,33]. We found a strong correlation between frequency of dysmor- phic nuclei in MEFs derived from embryos of the ct strain, and frequency of NTDs. For example, among mice homozygous for the Grhl3ct hypomorphic allele, presence of the wild-type Lmnb19E led to a reduced NTD frequency and an increased proportion of ‘normalised’ nuclei in MEFs. These findings suggest that nuclear lamina function plays a contributory role to the efficiency of neural tube closure during embryogenesis. Whether altered nuclear structure directly affects NTD risk or is a secondary marker of altered lamin B1 function is not known. To investigate the cellular mechanism by which lamin B1 affects embryonic development we focussed on a possible effect on cell cycle progression, in view of the known tissue-specific cell cycle defect that underlies spinal NTDs in curly tail mice [8]. Lamin B1 functions in nuclear envelope breakdown/assembly and mitotic spindle formation [23,25,49]. In addition, lamin B types are spatially associated with and required for DNA synthesis during S-phase [50]. Effects of lamin B1 dysfunction on cell cycle regulation could also be mediated through altered regulation of gene expression. For example, sequestration of the transcription factor Oct-1 at the nuclear periphery is lost in cells expressing a truncated form of lamin B1, resulting in mis-expression of target genes, including cell cycle mediators [27,51]. In ct fibroblasts expressing the Lmnb18E variant, analysis of growth curves and cell cycle markers revealed diminished proliferative capacity and premature senescence, accompanied by characteristic changes in expression of cell cycle mediators. Cell labelling experiments suggest that the reduced proliferation rate of ct8E cells does not result from a defect at the S-phase/G2 transition but more likely from impairment of G1 or G1/S transition. Such an idea is consistent with the reduced expression of cyclin D1, which promotes progression through G1/S. The proliferative crisis that occurs in ct8E following extended culture is accompanied by reduced expression of cyclins A2 and B1, which function at G2/M [52], and increased expression of p16Ink4a. Although our study addresses an amino acid change rather than reduced expression, these observations are consistent with recent studies showing that silencing of Lmnb1 expression reduces proliferation rate and induces premature senescence in fibroblast cell lines [53]. Altered cell cycle exit is also thought to be responsible for reduced thickness of the cortex in lmnb1/lmnb2 knockout embryos [29,34]. Rather than a generalised growth retarding effect of the lamin B1 variant, it appears that there is an additive effect with the Grhl3ct mutation. Cell cycle differences between ct8E and ct9E cells therefore suggest a mechanism by which Lmnb1 genotype affects the morphogenetic movements of neural tube closure in curly tail mutant embryos. It was previously found that: (i) the cellular basis of spinal NTDs in curly tail mutant embryos involves a proliferation defect in cells of the hindgut which causes excessive axial curvature [12]; and (ii) inhibition of proliferation by anti-mitotics or experimental growth retardation increases frequency of cranial Lamin B1 and Neural Tube Defects NTDs [18,54]. Therefore, the reduction in cellular proliferation rate resulting from the combination of diminished Grhl3 expression together with perturbation of lamin B1 function, would be predicted to exacerbate both spinal and cranial neurulation, as we observe. In support of this model, and correlated with prevention of NTDs in embryos, reinstatement of Grhl3 expression in cultured cells that express the Lmnb18E variant partially normalises nuclear morphology (e.g. in ctTgGrhl3) and proliferative capacity (e.g. growth curves of +ct;8E and ct9E cells do not differ). Moreover, in vivo analysis confirmed that proliferation is diminished in the hindgut of ct8E compared with ct9E embryos, which suggests an explanation for their greater susceptibility to spinal NTDs. Overall, our findings show that Lmnb1 is a modifier gene that has a significant influence on the risk of NTDs in curly tail (Grhl3ct) embryos. We propose that the Lmnb18E polymorphism and Grhl3ct mutation interact genetically to influence nuclear morphology and prolifera- tion, and hence susceptibility to NTDs. The influence of gene-gene interactions on susceptibility to NTDs in the curly tail model parallels the apparent multigenic etiology of the corresponding human conditions. Thus, it appears possible that some individuals carry ‘risk’ alleles that are insufficient to cause NTDs when present in isolation, but confer susceptibility to NTDs when co-inherited with other predisposing alleles. We speculate that variation in human lamin B1, either in the Glu repeat or elsewhere in the protein, would be worthy of investigation in the context of human NTDs. Methods Maintenance of mice and genotyping Curly tail (ct/ct), genetically-matched (partially congenic) wild- type (+ct/+ct) and transgenic curly tail mice carrying a Grhl3- expressing BAC (Grhl3ct/Grhl3;Tg(Grhl3)1NDEG, here referred to as ctTgGrhl3) were as described previously [8,9]. A two-step breeding programme (Figure S2) was used to generate mice carrying different combinations of the Grhl3 alleles (referred to as Grhl3+ or Grhl3ct) and Lmnb1 variants (referred to as Lmnb19E and Lmnb18E). Mice of genotype Grhl3ct/ct; Lmnb18E/8E, Grhl3ct/ct; Lmnb19E/9E and Grhl3+/+; Lmnb18E/8E were selected and inter-crossed to establish independent colonies. The Grhl3ct allele was genotyped on the basis of the putative mutation, C-21350T, upstream of Grhl3 by PCR amplification of genomic DNA with restriction digest of PCR products [9]. Genotyping was confirmed by PCR amplification of polymorphic microsatellite markers, D4Bwg1551e and D4Mit204, downstream of Grhl3. The Lmnb1 GAG repeat variant (Deletion, 18: 56909394) was genotyped by PCR amplification of genomic DNA using primers that encompass the repeat (59-GACCACCATACCC- GAGGAG and 59- TCCACAGCCACTCCGATG), with separa- tion of products on 5% agarose gels. The C612T SNP (18: 56868078) creates a HindIII restriction site, allowing genotyping by PCR amplification of exon 1 (using primer pair 59-GGC CTGTGGTTTGTACCTTC-39 and 59-GGCACCCCTGTT- CAGTTCTA-39), followed by restriction digest of the PCR product. Collection of embryos Experimental litters were generated by timed matings. Pregnant females were killed at embryonic day by cervical dislocation and embryos were dissected from the uterus in Dulbecco’s Modified Eagle’s Medium (Invitrogen) containing 10% fetal calf serum (Sigma). At E10.5, the caudal regions of individual embryos at the 30–31 somite stage were excised at the level of somite 15, rinsed in phosphate buffered saline (PBS) and stored at 280uC prior to analysis by 2-DE or Western blot. For in situ hybridisation embryos PLOS Genetics | www.plosgenetics.org 10 November 2012 | Volume 8 | Issue 11 | e1003059