Author Summary Failure of early development of the central nervous system leads to severe malformations termed neural tube defects (NTDs), including spina bifida and anencephaly. Inherited genetic risk factors play a major role in determining susceptibility to NTDs, but causative genes have proven difficult to identify. In this study we investigated genetic factors that could alter the risk of NTDs in an established mouse model, curly tail, in which defects result from partial loss of function of the grainyhead-like-3 (Grhl3) gene. We identified a variant of lamin B1, a key protein component of the envelope that surrounds the cell nucleus. The protein alteration reduces the structural integrity of the nuclear envelope, causes the nuclei to have altered shape, and reduces the rate of cell division. Curly tail embryos that carry the ‘‘abnormal’’ lamin B1 variant develop NTDs at three times the rate of those that carry the normal version. We conclude that lamin B1 function influences risk of NTDs due to effects on cell proliferation. imbalance leads to excessive ventral curvature of the caudal region of the embryo and, hence, mechanical suppression of neural tube closure at the posterior neuropore [14]. The incidence of curly tail NTDs can be influenced by multiple environmental and genetic factors [8,15–18]. In addition, NTD frequency is also markedly affected by backcross to different strains, indicating the presence of modifier loci in the curly tail genetic background [19]. Thus, it is apparent that the genetic component of predisposition to NTDs is multifactorial in ct, as in humans. In the current study, we identified lamin B1 as a modifier gene for NTDs in curly tail mice. Lamins are intermediate filament proteins of which the A-type, lamins A and C, are encoded by LMNA while, among the B-type, lamin B1 is encoded by LMNB1 and lamins B2 and B3, are encoded by LMNB2. The nuclear lamina is a protein complex underlying the inner nuclear membrane and composed of a meshwork of lamin polymers and lamin-binding proteins [20–22]. In addition to a key structural role in assembly and maintenance of the nuclear envelope, it has become clear that lamins have multiple functions in a diverse range of cellular properties. Thus, lamins influence nuclear shape and size as well as anchoring of protein structures, including nuclear pore complexes, in the nuclear envelope [23,24]. Additionally, lamins function in DNA synthesis and transcriptional regulation both through interaction with chromatin, to mediate sub-nuclear chromosomal positioning, and by direct interactions with transcription factors [25–29]. Highlighting the importance of lamin function, a number of clinically distinct diseases, termed laminopathies, have been found to result from mutation of LMNA [20,30]. These include muscular dystrophy disorders (e.g. Emery-Dreyfus muscular dystrophy), lipodystrophies, progeria syndromes (e.g. Hutchinson-Gilford progeria syndrome and Atypical Werner syndrome) and periph- eral neuropathy (Charcot-Marie-Tooth disease type 2B1). In contrast to LMNA, coding mutations in LMNB1 have not so far been associated with human disease, although genomic duplica- tion of LMNB1 is thought to cause a progressive demyelinating disorder, adult-onset autosomal dominant leukodystrophy [31,32]. Mice homozygous for a loss of function allele of Lmnb1 die at birth with reduced growth, impaired lung development and cortical abnormalities in the brain [33,34], while Lmnb2 knockouts exhibit neuronal migration defects in the cerebral cortex and cerebellum [35]. Lmnb1/lmnb2 double knockouts exhibit a reduced thickness of the brain cortex, with altered cell cycle exit of neuronal Lamin B1 and Neural Tube Defects progenitors and neuronal migration defects [29,34]. Forebrain- specific deletion of lmnb1 or lmnb2, allowed study of brain phenotypes at post-natal stages and showed that both genes are individually required for normal development of the cortex [34]. In the current study we identified a polymorphic variant form of lamin B1, present on the genetic background of the curly tail strain. The reduction in length of a series of glutamic acid residues, from nine to eight, was found to cause significant reduction in the stability of the lamin B1 interaction within the nuclear lamina. Genetic analysis, involving generation of curly tail sub-strains carrying combinations of the lamin B1 variant and Grhl3ct mutation demonstrate a dramatic effect of lamin B1 on frequency of NTDs. In parallel, lamin B1 has a profound effect on nuclear morphology and proliferative capacity. Overall, our findings show that Lmnb1 can act as a modifier gene affecting risk of NTDs, an effect that appears to be mediated through impaired cell cycle regulation which summates with the effect of Grhl3 mutation. Results In a proteomic analysis of the curly tail mutant, two-dimensional protein gels were generated from samples at embryonic day (E) 10.5: the stage of spinal neural tube closure. Comparison of stage- matched embryos revealed differential migration of a series of three spots, which migrated to a more basic position in gels derived from ct/ct samples than the equivalent spots in congenic wildtype (+ct/+ct) control gels (Figure 1A–1C). This migration change was apparent by the complete absence of the three spots that were detected in the +ct/+ct gels from the ct/ct gels and vice versa. This difference was detected both in analysis of whole embryos and in isolated caudal regions that encompassed the posterior neuropore (PNP), the region of active neural tube closure. In both strains, these spots were identified by liquid chromatography tandem mass spectrometry as lamin B1 (Table S1). Variation in abundance of some other spots between genotypes was observed, however, no spots other than those corresponding to lamin B1 showed a difference in migration. Neither the abundance of Lmnb1 mRNA nor total lamin B1 protein abundance were found to differ between ct/ct and +ct/+ct embryos, by real time qRT-PCR or western blot respectively (Figure 1D–1F). Moreover, the sites of Lmnb1 expression at neurulation stages were also comparable between genotypes as determined by whole mount in situ hybridisation (Figure 1G–1H). Expression was apparent through- out most of the embryo with the exception of surface ectoderm and the heart (Figure S1), where staining intensity was much lower than in other tissues. Altered migration of lamin B1 during the isoelectric focussing step of 2-DE results from a charge difference between the protein in ct/ct and +ct/+ct samples. Such a difference could potentially result from an alteration in primary sequence and the Lmnb1 coding region was therefore sequenced in ct/ct and +ct/+ct genomic DNA and cDNA. A synonymous polymorphism, C612T (anno- tated as SNP 18: 56868078), was found in exon 1 of the ct/ct sequence. In addition, a three base-pair GAG deletion (annotated as Deletion 18: 56909394) was noted in exon 10. This deletion corresponds to one of a sequence of GAG nucleotides at position 1657–1683 of the coding sequence, encoding a stretch of nine glutamic acid (Glu) residues in the tail domain of the wild-type protein (Figure 2A, 2B). Thus, the curly tail Lmnb1 gene encodes eight Glu residues at amino acids 553–560 (here denoted Lmnb18E to indicate number of glutamic acids), as opposed to nine Glu (residues 553–561) encoded by the +ct/+ct sequence (denoted Lmnb19E). Since Glu carries a negative charge, it appeared likely that the difference in number of Glu residues is responsible for the PLOS Genetics | www.plosgenetics.org 2 November 2012 | Volume 8 | Issue 11 | e1003059