Lamin B1 and Neural Tube Defects Figure 3. The frequency of NTDs resulting from mutation of Grhl3 is affected by Lmnb1 genotype. Embryos were scored as (A) apparently normal with a straight tail (ST), or with (B) a tail flexion defect (i.e. curly tail; CT), (C) spina bifida (SB) plus tail flexion defect, and/or (D) exencephaly (arrowhead indicates open hindbrain). Note that exencephaly can occur in association with any of the spinal phenotypes. Embryos shown are from the ct9E sub-strain. The frequency of NTD phenotypes is tabulated (E). The frequency of SB is significantly lower in the ct9E than in the ct8E and ct strains (* p,0.02, x2 test). Spina bifida and tail flexion defects were never observed among +ct;9E embryos but tail flexion defects did occasionally occur among +ct;8E embryos (** +ct;9E versus +ct;8E; p,0.05; Z-test), although at significantly lower frequency than among ct mutant embryos 2 2 (# p,0.001; x ). There is significant variation in the frequency of exencephaly between the sub-strains (p,0.001; x ) with a significantly lower exencephaly rate among ct9E than ct8E embryos ({ significantly different from ct8E, p,0.01; Z-test). Exencephaly was not observed in the +ct;9E strain ({ indicates significant difference from ct and ct8E (p,0.001; Z-test) and ct9E (p,0.02) strains). Exencephaly was observed in the +ct;8E strain, albeit at a significantly lower frequency than in the ct8E strain ({ p,0.05; Z-test). doi:10.1371/journal.pgen.1003059.g003 the variant lamin B1 imposes a dysmorphic phenotype on the nuclear lamina as a whole. Abnormalities were much less frequent in nuclei of the +ct;9E and C57BL/6 strains, carrying wild-type alleles of Lmnb1 and Grhl3. To provide a quantitative measure of nuclear morphology, the contour ratio (4???6area/perimeter) of DAPI-stained nuclei was analysed (Figure 5B, 5C). The mean contour ratio was significantly lower for ct nuclei than for any of the other strains (Figure 5B). Consistent with these findings, compared with other strains examined, a significantly greater proportion of ct nuclei showed a contour ratio of less than 0.7 (Figure 5C), which is considered abnormal [42]. The curly tail nuclear dysmorphology phenotype was rescued by the presence of the Lmnb19E variant in the ct9E sub-strain (Figure 5), correlating with the apparent increased stability of the lamina when this variant is present, as observed by FLIP (Figure 2C). Interestingly, in MEFs from a transgenic ct strain, ctTgGrhl3 in which Grhl3 expression is reinstated by over-expression from a Grhl3-containing BAC [9], the nuclear morphology was intermediate between that of ct and ct9E MEFs (Figure 5). Thus, although ctTgGrhl3 mice are on an identical genetic background to ct, including the Lmnb18E variant, it appears that over-expression of Grhl3 is sufficient to partially ameliorate the nuclear dysmorphol- ogy phenotype. The mean contour ratio of nuclei was higher, and the proportion of abnormal nuclei was lower, for C57BL/6 than any of the other strains, including +ct;9E (Figure 5). Thus, in addition to lamin B1 sequence and Grhl3 expression, other factors associated with the curly tail genetic background may influence nuclear morphology. Overall, among strains with the curly tail genetic background, those that express the Lmnb19E variant (+ct;9E and ct9E) have a significantly higher mean contour ratio (Figure 5B) and fewer dysmorphic nuclei (Figure 5C) than those that express the Lmnb18E variant (ct and ctTgGrhl3). The effect of the Lmnb18E variant on nuclear morphology and the known function of lamins in nuclear function, including DNA PLOS Genetics | www.plosgenetics.org 6 November 2012 | Volume 8 | Issue 11 | e1003059