Lamin B1 Polymorphism Influences Morphology of the Nuclear Envelope, Cell Cycle Progression, and Risk of Neural Tube Defects in Mice Sandra C. P. De Castro1, Ashraf Malhas2, Kit-Yi Leung 1 , Peter Gustavsson 3 , David J. Vaux2, Andrew J. Copp 1 , Nicholas D. E. Greene1* 1 Neural Development Unit, UCL Institute of Child Health, University College London, London, United Kingdom, 2 Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom, 3 Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden Abstract Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects whose complex multigenic causation has hampered efforts to delineate their molecular basis. The effect of putative modifier genes in determining NTD susceptibility may be investigated in mouse models, particularly those that display partial penetrance such as curly tail, a strain in which NTDs result from a hypomorphic allele of the grainyhead-like-3 gene. Through proteomic analysis, we found that the curly tail genetic background harbours a polymorphic variant of lamin B1, lacking one of a series of nine glutamic acid residues. Lamins are intermediate filament proteins of the nuclear lamina with multiple functions that influence nuclear structure, cell cycle properties, and transcriptional regulation. Fluorescence loss in photobleaching showed that the variant lamin B1 exhibited reduced stability in the nuclear lamina. Genetic analysis demonstrated that the variant also affects neural tube closure: the frequency of spina bifida and anencephaly was reduced three-fold when wild-type lamin B1 was bred into the curly tail strain background. Cultured fibroblasts expressing variant lamin B1 show significantly increased nuclear dysmorphology and diminished proliferative capacity, as well as premature senescence, associated with reduced expression of cyclins and Smc2, and increased expression of p16. The cellular basis of spinal NTDs in curly tail embryos involves a proliferation defect localised to the hindgut epithelium, and S-phase progression was diminished in the hindgut of embryos expressing variant lamin B1. These observations indicate a mechanistic link between altered lamin B1 function, exacerbation of the Grhl3-mediated cell proliferation defect, and enhanced susceptibility to NTDs. We conclude that lamin B1 is a modifier gene of major effect for NTDs resulting from loss of Grhl3 function, a role that is likely mediated via the key function of lamin B1 in maintaining integrity of the nuclear envelope and ensuring normal cell cycle progression. Citation: De Castro SCP, Malhas A, Leung K-Y, Gustavsson P, Vaux DJ, et al. (2012) Lamin B1 Polymorphism Influences Morphology of the Nuclear Envelope, Cell Cycle Progression, and Risk of Neural Tube Defects in Mice. PLoS Genet 8(11): e1003059. doi:10.1371/journal.pgen.1003059 Editor: Bogi Andersen, University of California Irvine, United States of America Received February 6, 2012; Accepted September 14, 2012; Published November 15, 2012 Copyright: 2012 De Castro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Medical Research Council (NDEG, DJV, K-YL, AJC) and the Wellcome Trust (AJC, NDEG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: n.greene@ucl.ac.uk Introduction Modifier genes have been ascribed significant influence in determining susceptibility to disease in complex traits, as well as partial penetrance and variable expressivity of monogenic conditions [1]. Moreover, modifier genes are considered largely responsible for the phenotypic variation observed when mutations are bred onto different genetic backgrounds in mice. However, identification of modifier genes and determination of their functional effects presents a considerable challenge. Understand- ing the genetic basis of neural tube defects (NTDs), such as spina bifida and anencephaly, typifies these difficulties. NTDs are common, severe congenital malformations resulting from failure of closure of the neural tube during embryonic development [2]. In humans, they are among the commonest birth defects, affecting around 1 per 1000 pregnancies worldwide. However, the causes are not well understood owing to their multigenic inheritance and the potential influence of environmen- tal factors, either predisposing or ameliorating [3,4]. The potential complexity of NTD genetics is illustrated by the fact that more than 200 different genes have been implicated as potential contributors to the overall burden of NTDs, with neural tube closure phenotypes in mouse strains carrying naturally occurring or targeted mutations [5–7]. Additionally, in many of these models penetrance is influenced by genetic background, indicating the presence of modifier genes. The curly tail (ct) mouse mutant is among the most extensively characterised models of NTDs [8]. Approximately 5–10% of homozygous ct/ct embryos develop cranial NTDs (exencephaly), while 15–20% exhibit spinal NTDs (spina bifida), due to failure of closure of neural folds in the prospective brain and low spinal region, respectively. The major ct gene corresponds to a hypomorphic allele of the transcription factor grainyhead-like-3 (Grhl3), mutants of which display spina bifida with 100% penetrance [9–11]. Expression of Grhl3 is diminished in the hindgut of ct mutant embryos, due to an upstream regulatory mutation, resulting in a diminished cellular proliferation rate in the hindgut endoderm [12,13]. The consequent dorso-ventral growth PLOS Genetics | www.plosgenetics.org 1 November 2012 | Volume 8 | Issue 11 | e1003059