Lamin B1 and Neural Tube Defects Figure 2. Lamin B1 shows variation in length of glutamic acid repeat, which significantly affects mobility within the nuclear envelope. (A) Lamin B1 protein sequence contains a series of Glu (E) residues beginning at amino acid 553, encoded at the genomic level by a GAG repeat. (B) The wild-type (+ct) sequence encodes nine E residues, whereas the curly tail sequence encodes only eight E residues (one fewer GAG). (C– D) Fluorescence loss in photobleaching (FLIP) was performed to examine the mobility of NLS-YFP-Lamin B1 tail domain fusion proteins, containing eight or nine Glu residues. (C) Fusion proteins are shown diagrammatically and localised to the nuclear envelope as expected. (D) The relative intensity of fluorescence in an unbleached region of the nuclear envelope compared with pre-bleaching (time 0) was determined. Mean intensity 6 SEM is shown for five nuclei with each construct. There is a more rapid decline in intensity in cells expressing the 8E variant (red circles) compared with the 9E variant (open circles), and differences persist throughout the experiment. Statistical analysis shows a significant difference between the 8E and 9E variants at both the 100 s and 200 s time points (p,0.001, t-test). doi:10.1371/journal.pgen.1003059.g002 defect. To test this idea, we inter-crossed ct/ct and +ct/+ct mice to generate sub-strains of mice carrying different combinations of the Lmnb1 variant (i.e. Lmnb18E and Lmnb19E; abbreviated hereafter as L8E and L9E) and the Grhl3 mutant allele (Grhl3ct or Grhl3+; abbreviated as Gct and G + ). Each sub-strain was maintained in homozygous form for both Lmnb1 and Grhl3 alleles, that is: (i) L8E/ 8E; Gct/ct (denoted ct8E); (ii) L8E/8E; G+/+ (denoted +ct;8E); (iii) L9E/9E; Gct/ct (denoted ct9E); (iv) L9E/9E; G+/+ (denoted +ct;9E). In the +ct strain the genetic background is approximately 97% curly tail [9] and in each sub-strain it is predicted to be 99.5% curly tail (see Figure S2 for breeding scheme). Embryos were collected at E11.5– 15.5 and analysed for the presence or absence of NTDs. Among embryos of the ct8E sub-strain, the range and frequency of phenotypes was closely similar to that observed in the curly tail (ct) strain which has the same genotype at the Lmnb1 and Grhl3 loci. Defects included spina bifida, tail flexion defects and exencephaly (Figure 3B–3D), while other embryos appeared normal (Figure 3A). Importantly, however, varying the Lmnb1 genotype produced a striking difference in frequency of NTDs (Figure 3E). Thus, spina bifida occurred at significantly lower frequency in the ct9E sub- strain (5.8%) than in curly tail (14.2%) or in the ct8E sub-strain (15.8%). Therefore, homozygosity for the Lmnb18E variant confers approximately three-fold higher risk of spina bifida in Gct/ct embryos, compared with homozygosity for the Lmnb19E variant. Interestingly, although cranial NTDs occur at lower frequency than spina bifida in the curly tail strain, we also observed the rate of exencephaly to be significantly reduced among ct9E embryos (3.0%) compared with curly tail (6.4%) or ct8E embryos (8.2%). As expected, the frequency of exencephaly in the latter two strains did not differ significantly (Figure 3E). Although the genetic background of each sub-strain was predicted to be approximately 99.5% curly tail we could not exclude a possible effect of the region of DNA that is tightly linked and inherited with lmnb1. We therefore examined the possibility that a neighbouring gene to lmnb1 could vary in expression between the ct9E and ct8E sub-strains. Using a list of genes that are located within a 41 Mb interval of chromosome 18 centred on lmnb1, we interrogated microarray data generated from RNA of the caudal region of stage-matched ct/ct and +ct/+ct embryos (E10.5; 28–29 somite stage). Among 11 differentially expressed genes (p,0.05; fold-change 1.5-fold or greater), 4 showed a similar trend of differential expression on qRT-PCR analysis of indepen- dent ct/ct and +ct/+ct samples. However, none of these genes varied in expression when analysed by qRT-PCR in stage-matched ct9E and ct8E samples (Table S2), suggesting that the phenotypic difference between the sub-strains does not result from differential expression of genes located in proximity to lmnb1. Instead, variation in expression between ct/ct and +ct/+ct samples seem likely to be due to downstream effects of the Grhl3ct mutation in ct/ ct embryos. Embryos of the wild-type congenic curly tail strain (+ct;9E, genotype: L9E/9E; G+/+) do not develop exencephaly, spina bifida or tail flexion defects (Figure 3E) [9]. However, when the Lmnb18E variant was bred onto the Grhl3 wild-type genetic background, to produce mice of L8E/8E; G+/+ genotype (+ct;8E), we observed a low frequency of tail flexion defects, indicative of delayed PNP closure (Figure 3E). Exencephaly was also occasionally observed (Figure 3E). These data demonstrate that the presence of the Lmnb18E variant can predispose to defects of cranial and spinal neural tube closure, even in the absence of the Grhl3 mutation. Although curly tail NTDs are partially penetrant, affected embryos can be recognised on the basis of an enlarged PNP at E10.5 [40]. In order to examine the effect of Lmnb1 variants on the progress of spinal neural tube closure directly, PNP length was measured in a series of embryos at E10.5 (Figure 4). Among embryos that were wild-type at the Grhl3 locus (+ct and +ct;8E), PNP length diminished rapidly between the 26 and 31 somite stages and, by the 30–31 somite stage, the PNP was very small (12 out of 37 embryos) or closed (25 of 37 embryos). There was no detectable difference between embryos with Lmnb18E/8E and Lmnb19E/9E genotypes. In contrast, mean PNP lengths were significantly larger in the Grhl3ct/ct sub-strains, reflecting an overall delay in closure. Although mean PNP length did not differ between curly tail and the ct8E sub-strain, embryos of the ct9E sub-strain exhibited a more rapid reduction in PNP length from the 28–29 somite stage onwards (Figure 4), indicative of an overall normalisation of spinal neural tube closure. The distribution of PNP lengths in embryos of the ct9E sub-strain was shifted towards smaller values, with a significantly lower mean PNP length. Moreover, only a few ct9E embryos showed very large PNPs, whereas a greater proportion of embryos had completed PNP closure by the 30–31 somite stage (8 of 30 compared with 1 out of 20 among the ct8E sub-strain; p,0.05, z-test; Figure S3). These observations on PNP length correlate with the diminished frequency of spina bifida in the ct9E sub-strain later in development. Generation of the curly tail sub-strains provided an opportunity to test directly whether the variation in number of Glu residues is responsible for the 2-DE migration difference of lamin B1 in curly tail and wild-type samples. 2D gels were generated from mouse strains expressing the Lmnb18E (ct, ct8E, +8E) or Lmnb19E (+ct, ct9E) variants. In each case the migration pattern corresponded with the number of Glu residues (Figure S4), confirming that the characteristic strain-dependent 2-DE pattern reflects the Lmnb1 polymorphism. We hypothesised that the mechanism by which variation in lamin B1 sequence affects risk of NTDs could relate to the apparent effect on stability of the nuclear lamina, as shown by FLIP. In order to further examine the effects of the lamin B1 variants, we examined nuclear morphology in MEFs derived from embryos of differing strains. We previously showed that Grhl3 is expressed in MEFs and that the expression deficit is observed in cells derived from curly tail embryos, as in the embryos themselves [41]. Immunostaining for lamin A and lamin B1 allowed visualisation of nuclear shape and, among curly tail MEFs, many nuclei showed a high degree of irregularity in shape, including herniations and/or lobulations (Figure 5A). Moreover, in a proportion of curly tail cells, lamin B1 staining was discontinuous. Lamin A showed a similar distribution to lamin B1, suggesting that PLOS Genetics | www.plosgenetics.org 5 November 2012 | Volume 8 | Issue 11 | e1003059