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. 2011 Dec 23;334(6063):1706-10.
doi: 10.1126/science.1211222. Epub 2011 Nov 24.

Mouse B-type lamins are required for proper organogenesis but not by embryonic stem cells

Youngjo Kim  1 , Alexei A Sharov, Katie McDole, Melody Cheng, Haiping Hao, Chen-Ming Fan, Nicholas Gaiano, Minoru S H Ko, Yixian Zheng
Affiliations

Mouse B-type lamins are required for proper organogenesis but not by embryonic stem cells

Youngjo Kim et al. Science. .

Abstract

B-type lamins, the major components of the nuclear lamina, are believed to be essential for cell proliferation and survival. We found that mouse embryonic stem cells (ESCs) do not need any lamins for self-renewal and pluripotency. Although genome-wide lamin-B binding profiles correlate with reduced gene expression, such binding is not directly required for gene silencing in ESCs or trophectoderm cells. However, B-type lamins are required for proper organogenesis. Defects in spindle orientation in neural progenitor cells and migration of neurons probably cause brain disorganizations found in lamin-B null mice. Thus, our studies not only disprove several prevailing views of lamin-Bs but also establish a foundation for redefining the function of the nuclear lamina in the context of tissue building and homeostasis.

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Figures

Fig. 1
Fig. 1
Lamin-B1–chromatin interactions and gene silencing in ESCs and TE. (A) Binding of lamin-B1 around the Nanog gene locus as an example of differential lamin-B1 binding to genes in ESCs and TE cells. ZHBTc4 ESCs expressing the tetracycline (Tc)-regulated Oct4 transgene were used for efficient TE differentiation (21).x axis, the positions of individual orange and blue bars at chromosome 6. Each bar is plotted at ~250–base pair (bp) intervals. y axis, lamin-B1 binding plotted as the log10 ratio of methylation values of Dam–lamin-B1 over Dam alone. Orange bars, positive values; blue bars, negative values. (B) Correlation of lamin-B1 binding with low expression of genes with or without CpG islands in ESCs. y axis, the log10 ratio of methylation values of Dam–lamin-B1 over Dam alone; x axis, the log10 gene expression level. (C) Correlation of lamin-B1 binding with decreased gene expression in TE cells relative to ESCs. x axis, the log10 ratio of gene expression in TE to that in ESCs; y axis, the log10 ratio of lamin-B1 binding in TE to that in ESCs. Each dot is the average of 100 genes with similar expression (B) or ratio of expression (C). There was no correlation between DNA methylation of CpG islands and lamin-B1 binding.
Fig. 2
Fig. 2
Characterization of Lmnb1−/−Lmnb2−/− ESCs. (A and B) Protein immunoblotting analyses of lamin-B1, -B2, and -B3 (B3 shares the same C terminus as B2). Wild-type testes lysates were used as positive controls for lamin-B3. β-Actin was used as a loading control. wt, LB1−/−, LB2−/−, and DKO represent Lmnb1+/+Lmnb2+/+, Lmnb1−/−Lmnb2+/+, Lmnb1+/+Lmnb2−/−, and Lmnb1−/−Lmnb2−/−, respectively. (C) Immunofluorescence staining of ESCs with antibodies to lamin-B1 (LB1), lamin-B2 (LB2), or emerin. DNA was counterstained with Hoechst dye. Scale bars, 10 µm. (D) Electron micrographs of ESCs. N, nucleus; C, cytoplasm; NUP, nuclear pore; INM, inner nuclear membrane; ONM, outer nuclear membrane. Scale bars, 1 µm (whole-cell views) and 0.1 µm (enlarged views).
Fig. 3
Fig. 3
Effects of lamins on gene expression. (A) Lmnb1+/+Lmnb2+/+ and Lmnb1−/−Lmnb2−/− ESCs exhibit similar changes in protein abundance during differentiation toward the TE lineage. ESCs were treated with tamoxifen (Tx) and fibroblast growth factor (FGF) for 2 days to induce differentiation followed by terminal differentiation in the absence of FGF and Tx for 6 days. Protein immunoblotting was used to examine ESC proteins associated with pluripotency (Oct4, Sox2, and Nanog), the TE-specific protein (Krt8), and lamin-A and -C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B to D) Microarray analyses. Gene expression was compared between Lmnb1+/+Lmnb2+/+ and Lmnb1−/−Lmnb2−/− ESCs (B), TE cells (C), and between Lmnb1+/+Lmnb2+/+ ESCs and Lmnb1+/+Lmnb2+/+ TE cells (D). Increased or decreased transcription (by twofold) is represented by red or green dots, respectively. The Lmnb1 and Lmnb2 genes are indicated in (B) and (C). (E) Ratios of differentially expressed genes between Lmnb1+/+Lmnb2+/+ and Lmnb1−/−Lmnb2−/− cells (x axis) were plotted against the lamin-B1 binding of these genes measured by Dam-ID in Lmnb1+/+Lmnb2+/+ cells (y axis). Each point indicates a single gene that was differentially expressed between Lmnb1+/+Lmnb2+/+ and Lmnb1−/−Lmnb2−/− cells in (B) or (C).
Fig. 4
Fig. 4
Requirements for lamin-Bs in brain development. (A) E18.5 embryos. (B to D and I) E18.5 brains with genotypes indicated at the top. (B) E18.5 brains. (C) Nissl staining of coronal sections of the neocortex. MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. (D) Layer-specific marker staining of the neocortex. Tbr1 (green) labels early-born deep layer (V and VI) neurons, whereas Brn1 (red) labels late-born outer neuronal layers (II to IV). DNA (blue) was counterstained with Hoechst dye. Three different color channels are merged. Images of individual channels are in fig. S11B. (E) Representative confocal images of mitotic cells at VZ of E14.5 neocortices stained with anti-Pericentrin (red, centrosome), anti-Nestin (green, NPC marker), and Hoechst dye (blue, DNA). Yellow dashed lines indicate the orientation of cleavage planes deduced from positions of anaphase chromosomes and centrosomes, whereas white dashed lines delineate the ventricular surface. (F and G) Quantifications of spindle orientation at E9.5 (F) and E14.5 (G). +/+;+/−, Lmnb1+/+Lmnb2+/− (E9.5, 78.12 ± 4.49°; E14.5, 73.12 ± 2.17°); −/−;+/+, Lmnb1−/−Lmnb2+/+(E14.5, 61.46 ± 2.51°); +/+;−/−, Lmnb1+/+Lmnb2−/− (E14.5, 62.17 ± 2.69°); −/−;−/−, Lmnb1−/−Lmnb2−/− (E9.5, 46.25 ± 5.31°; E14.5, 57.39 ± 2.68°). Error bars, SEM. *, P < 10−4, t test (F); *, **, ***, P < 0.002 (G). (H) Cell cycle exit rates. BrdU was injected at E14.5. BrdU+ and Ki67+ cells in neocotices were analyzed 24 hours later (see fig. S18B). Cell cycle exit rates were calculated by dividing the number of BrdU+Ki67− cells (no longer proliferating) by the number of BrdU+Ki67+ cells (still cycling). Error bars, SEM. *, P < 0.001, t test, n = 6 sections (>60 cells per section). (I) BrdU birth dating indicates that neurons from lamin-B mutants have migration defects, whereas neurons from Lmnb1+/+Lmnb2+/+ littermates have migrated into the basal surface. BrdU (green) was injected at E14.5 to label mid-to-late–born neurons, and mice were dissected at E18.5. Scale bars, 5 mm (A and B), 200 µm (C, D, and I), and 5 µm (E).
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