30 min (5% heat-inactivated goat serum, PBS-0.1% Triton, 0.15% glycine, 2 mg/ml BSA) prior to immunohistochemistry for phospho-histone H3. Primary and secondary antibodies were anti-phospho histone H3 (1:250, Millipore) and Alexa Fluor 488- conjugated anti-rabbit (1:500, Invitrogen). For nuclear staining, cells were incubated with Hoechst (1:2,000 in PBS). Ten random fields were analysed per cover slip using Image J software (U.S. National Institutes of Health, Bethesda, Maryland, USA). Cells in mitosis were scored by visual inspection of pH 3-positive cells. The experiment was repeated three times, each using an independent cell line. For in vivo proliferation analysis, mice were injected with 150 mg EdU at E10.5. Embryos were collected after 90 minutes, fixed in 4% PFA and processed for embedding in paraffin wax. Transverse 7 mm sections at the axial level of the closing neural folds were used for proliferation analysis (5–7 sections per embryo), as described previously [12]. Detection of EdU (Click-It EdU Imaging Kit) was followed by immunohistochemistry for phos- pho-histone H3 (as above) as described [13]. Fluorescent images were collected on an Axiophot microscope (Zeiss) with a DC500 camera (Leica), using FireCam software (Leica). Images were analysed using the Cell Counter plugin in Image J. Statistical analysis All statistical analysis was carried out using SigmaStat (version 3.5; Systat Software Inc). Supporting Information Figure S1 Expression of Lmnb1 mRNA in curly tail and wild-type embryos. Whole mount in situ hybridisation at E10.5 shows intense expression of Lmnb1 throughout most of the embryo, with the exception of the heart (shown at higher magnification in F–G) and dorsal surface (arrowheads in A and E). On sections (C, D, H, I; cut at the level of the dotted lines in A and E) the diminished or absent staining in the heart is also evident. Diminished expression at the dorsal surface in whole mounts appears to correspond to lack of staining in the dorsal neural tube and surface ectoderm, particularly evident in sections through the PNP region (D, I). We did not observe any consistent differences in staining pattern between strains. A sense control probe did not give signal (B). Scale bars represent 1 mm (A, B, E), 0.5 mm (F, G) or 0.1 mm (C, D, H, I). Abbreviations: H, heart; Hg, hindgut; NF, neural folds. (TIF) Figure S2 Breeding scheme for generation of curly tail sub-strains carrying different combinations of Grhl3 and Lmnb1 alleles. The key strains of interest were ct8E (same genotype as ct/ct at Grhl3 and Lmnb1), and ct9E which both carry the Grhl3 mutation, but differ in Lmnb1 sequence. A third strain, +ct;8E, is wild-type for Grhl3 but carries the 8E Lmnb1 variant. The predicted frequency of each genotype is indicated. The genetic background of the +ct/+ct strain is approximately 97% curly tail. Therefore, following the two further backcrosses to ct/ct the genetic background of the resultant ct8E and ct9E sub-strains is predicted to be 99.5% curly tail. (TIF) Figure S3 Posterior neuropore length of embryos from curly tail sub-strains during spinal neural tube closure. The data for individual embryos is shown, with the mean PNP length (6 SEM) indicated for each strain at each stage. From the 28 somite stage, a large range of values is observed, particularly within the curly tail and ct8E strains. Thus, at the 30–31 somite stage, PNPs ranged from closed to as much as 1 mm long, reflecting the range of possible outcomes from normal closure to spina bifida. The Lamin B1 and Neural Tube Defects overall distribution of PNP lengths in embryos of the ct9E sub- strain was shifted towards smaller values. (TIF) Figure S4 Migration of lamin B1 protein on 2-DE correlates with number of glutamic acid residues. Two dimensional protein gels were generated using embryo samples from wild-type (+ct), curly tail (ct), Grhl3-BAC-transgenic curly tail (ctTgGrhl3) and ct9E strains. (A) Differential migration of lamin B1 (major spot arrowed) was observed in comparison of aligned gels for ct/ct and +ct/+ct samples. (B) In strains expressing the 8E lamin B1 variant (ct and ctTgGrhl3), alignment of lamin B1 spots was evident (software- generated spot outline is shown), whereas the corresponding spot was absent in strains expressing the Lmnb19E variant (+ct and ct9E). (C) Conversely, the major lamin B1 spot (outlined) aligned in strains expressing the Lmnb19E variant, but was absent in strains expressing the Lmnb18E variant. (TIF) Table S1 Identification of lamin B1 spots by LC-MS/MS. Three spots found to migrate differentially on 2-DE of curly tail and wild-type samples were excised from gels and subjected to liquid chromatography coupled to electrospray tandem mass spectrom- etry (LC-MS/MS). Spots are numbered 1–3 from basic to acidic (right to left on 2-DE images in Figure 1), with spot 1 the most abundant in each case. The identified peptides are listed with the MASCOT score and p-value for confidence of identification. For the most abundant spot on curly tail gels 40% coverage of the protein was achieved. (DOC) Table S2 Expression analysis of genes located in proximity to Lmnb1 on chromosome 18. A gene-list was generated that corresponds to genes located in a 41 Mb interval surrounding Lmnb1 on chromosome 18:32.47–73.80 Mb (between markers D18Mit88 and Dev1), using UCSC Genome Browser (assembly NCBI37/mm9). This list was used to interrogate a list of genes that were found by microarray analysis to be differentially expressed (p,0.05; fold-change 1.5 or greater) in +ct/+ct and ct/ ct embryos (caudal region of embryos at the 28–29 somite stage). This analysis identified 11 genes, whose relative level of expression in +ct/+ct compared with ct/ct microarray samples is indicated. Expression was evaluated by qRT-PCR (n = 5 of each genotype, repeated twice). Four genes (underlined) showed the same trend in expression in both microarray and qRT-PCR analysis, two of which (indicated in bold) were also found to significantly differ in expression between ct and +ct (*p,0.05; **p,0.001) by qRT-PCR. 1500015A07Rik and Grpel2 also showed significantly altered expression on both microarray and qRT-PCR analysis but the direction of altered expression was not consistent. None of the genes showed a significant difference in expression in comparison of ct9E and ct8E samples (n = 5 of each genotype, repeated twice; NS indicates non-significant difference). NT, indicates not tested. Note: Lack of correlation between microarray and qRT-PCR for some genes may relate in several cases to low level expression of these genes, which may have given rise to a false positive on the microarray. (DOCX) Table S3 Analysis of cellular proliferation rate in embryos of the ct9E and ct8E sub-strains. The proportion of cells labelled with EdU (following 90 minute treatment) and the mitotic index (based visual inspection of phospho-histone H3 positive cells) was determined at the axial level of the closure point of the neural folds in ct9E (n = 6) and ct8E (n = 5) embryos at E10.5 (mean number of somites = 28.560.8 and 27.660.5, respectively). The PLOS Genetics | www.plosgenetics.org 12 November 2012 | Volume 8 | Issue 11 | e1003059