Lamin B1 is required for mouse development and nuclear integrity

Abstract

Lamins are key structural components of the nuclear lamina, an intermediate filament meshwork that lies beneath the inner nuclear membrane. Lamins play a role in nuclear architecture, DNA replication, and gene expression. Mutations affecting A-type lamins have been associated with a variety of human diseases, including muscular dystrophy, cardiomyopathy, lipodystrophy, and progeria, but mutations in B-type lamins have never been identified in humans or in experimental animals. To investigate the in vivo function of lamin B1, the major B-type lamin, we generated mice with an insertional mutation in Lmnb1. The mutation resulted in the synthesis of a mutant lamin B1 protein lacking several key functional domains, including a portion of the rod domain, the nuclear localization signal, and the CAAX motif (the carboxyl-terminal signal for farnesylation). Homozygous Lmnb1 mutant mice survived embryonic development but died at birth with defects in lung and bone. Fibroblasts from mutant embryos grew under standard cell-culture conditions but displayed grossly misshapen nuclei, impaired differentiation, increased polyploidy, and premature senescence. Thus, the lamin B1 mutant mice provide evidence for a broad and nonredundant function of lamin B1 in mammalian development. These mutant mice and cell lines derived from them will be useful models for studying the role of the nuclear lamina in various cellular processes.

Fig. 1.

Structure and expression of lamin B1 in gene-trap mice. (A) Structure of lamin B1 in wild-type and Lmnb1^Δ/Δ mice. The amino-terminal half of lamin B1 consists primarily of the rod domain (1A, 1B, 2A, and 2B) and flanking phosphorylation sites (P-site). The C-terminal half contains the nuclear localization signal (NLS) and the CAAX motif. In Lmnb1^Δ/Δ cells, a portion of the rod domain and the entire carboxyl-terminal domain are replaced by βgeo. (B) RT-PCR analysis of mRNA expression levels for lamins B1 (5′ and 3′ regions), B2, A, and C in Lmnb1^+/+, Lmnb1^+/Δ, and Lmnb1^Δ/Δ MEFs from two embryos of each genotype. Hprt (hypoxanthine phosphoribosyl transferase) was used as a normalization control. (C) Immunoblot analysis of lamins and LAP2 in protein extracts from Lmnb1^+/+, Lmnb1^+/Δ, and Lmnb1^Δ/Δ MEFs. (D) RT-PCR analysis of mRNA expression levels for lamins B1 and B2 in 7.5 dpc whole embryos.

Fig. 2.

Appearance of Lmnb1^Δ/Δ embryos. (A) Reduced size and abnormal shape of Lmnb1^Δ/Δ embryos at 18.5 dpc. (B) Sagittal sections of embryos stained with hematoxylin/eosin. (C) Lung sections obtained before inflation, stained with hematoxylin/eosin.

Fig. 3.

Abnormal skeleton and skull shape in Lmnb1^Δ/Δ mice. (A) Alizarin red (bone) and Alcian blue (cartilage) staining of Lmnb1^Δ/Δ skeleton (Left) shows a flattened skull, an abnormal vertebral column, and reduced ossification in phalanges, talus, and calcaneus (arrows). (B) Dorsal view of cranial vault. Alizarin red stain shows closure of the coronal sutures between parietal and frontal bones (arrows) and overlapping of the parietal (p) bones at the sagittal suture (arrowhead) in the Lmnb1^Δ/Δ embryo (Left). (C) Removal of scalp reveals obscured view of underlying blood vessels due to suture closure in the Lmnb1^Δ/Δ embryo (Left).

Fig. 4.

Abnormal nuclear architecture in Lmnb1^Δ/Δ MEFs. (A) Confocal micrographs of MEFs from Lmnb1^+/+ or Lmnb1^Δ/Δ embryos stained with antibodies for nuclear proteins (Left) and merged with DNA stain for nuclei (Right). A- and B-type lamins, LAP2, and nuclear pore complex (NPC) proteins were detected with specific antibodies; the lamin B1-βgeo fusion in cells was detected with an antibody against β-gal. (B) Higher magnification of lamin A/C and LAP2 staining showing nonuniform distribution in Lmnb1^Δ/Δ cells.

Fig. 5.

Cell proliferation and differentiation defects in Lmnb1^Δ/Δ MEFs. (A) Lmnb1^+/+ and Lmnb1^Δ/Δ MEFs were grown to confluence and, 2 days later, induced to differentiate into adipocytes. Six days later, cells were stained with Oil Red O to detect lipid accumulation. Representative cultures are shown at ×6 (Upper) and ×200 (Lower) magnification. (B) MEFs were continuously passaged at 95% confluence and replating at 30% confluent density. Passage number was plotted against the number of days between passages, averaged for three independent MEF isolates of each genotype. Passage 1 denotes the initial isolation of MEFs. (C) Analysis of chromosome number in metaphase spreads of Lmnb1^Δ/Δ and Lmnb1^+/+ MEFs at passage 4. Scatterplot points show the distribution of chromosome numbers; horizontal lines show mean values. ***, P < 0.00001. Photomicrographs at right show representative diploid Lmnb1^+/+ and polyploid Lmnb1^Δ/Δ cells (magnification, ×800).