Generation and characterization of a lamin C2-deficient mouse line.

<p>(A) Murine somatic lamins A and C are composed of exons 1 through 12 and 1 through 10 of the <i>Lmna</i> gene, respectively, both excluding exon 1a. Lamin C2 is encoded by exons 1a through 10 with exon 1a serving as an alternative starting exon specific for lamin C2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003261#pgen.1003261-Nakajima1" target="_blank">[18]</a>. Exon 1a was targeted for construction of a knockout mouse model deficient for lamin C2. SmaI restriction sites and the probe used for southern blot analyses are indicated. (B) Southern blot analyses of genomic mouse DNA digested by SmaI distinguishing wildtype, heterozygous and homozygous lamin C2-deficient animals. (C, C′) RT-PCR and immunoblot analyses showing presence of lamin C2 in the testes of wildtype and heterozygous mice, but absence in <i>lamin C2^−/−</i> testes: expression of lamin A/C remained unaffected in all three genetic backgrounds. (C″, C′″) Immunohistochemical analyses using anti-A-type lamin antibodies on liver and testis tissues showing unaffected expression of A-type lamins in somatic cells, while in meiotic cells of <i>lamin C2^−/−</i> testes A-type lamins are completely absent. (D) Histological sections of wildtype and <i>lamin C2^−/−</i> testes demonstrating the absence of post-meiotic cells in <i>lamin C2^−/−</i> tissue. Scale bars 100 µm. As shown in the insets, testes size of <i>lamin C2^−/−</i> males is significantly reduced. Scale bars 1 mm. S somatic cells, Sg spermatogonia, Sc spermatocytes, Sp spermatids.</p

Loss of lamin C2 has no effect on meiotic telomere attachment.

<p>(A) 3D-preserved swab preparations showing wildtype (A) and knockout (A′) spermatocytes simultaneously labelled with anti-TRF1 and SUN1 antibodies. As in the wildtype, in <i>lamin C2^−/−</i> spermatocytes virtual all telomeres appear to be attached to the NE as indicated by co-localisation of TRF1 and SUN1 signals. Scale bars 5 µm. (B) Quantifications of co-localised and non-co-localised TRF1/SUN1 signals (see A) revealed that ratios of co-localised to non-co-localised spots comparing wildtype and knockout spermatocytes show no significant difference (wildtype <i>n</i> = 33; <i>lamin C2^−/− n</i> = 45; Pearson's Chi² test p-value: 0.799). (C,D) Chromosome spread preparations of pachytene-like <i>lamin C2^−/−</i> spermatocytes showing that all telomeres are associated with SUN1. In (C) TeloFISH and in (D) anti-SUN1 staining in co-localisation with SYCP3. Scale bars 10 µm.</p

Defective synapsis of the homologous chromosomes in <i>lamin C2^−/−</i> meiocytes.

<p>Representative chromosome spreads of spermatocytes (A) and oocytes (17.5 dpf) (B) labelled with anti-SYCP3 and anti-SYCP1 antibodies. Heterologous associations (red box in A′) are observed in <i>lamin C2^−/−</i> spermatocytes only. Incomplete pairing of homologs (white boxes in A′ and B′) as well as univalent chromosomes (arrowheads in A″,A′″ and B″) occur in both <i>lamin C2^−/−</i> spermatocytes and oocytes. <i>Lamin C2^−/−</i> spermatocytes also display cells with X and Y chromosomes as the only univalents (arrowheads in A′″) or with linear telomere-telomere associations between non-homologous chromosomes (arrow in A″″). Scale bars 10 µm. (C) Quantification of meiocytes with defective synapsis in <i>lamin C2^−/−</i> males and females. For both sexes, differences between mutants and controls are highly significant (Pearson's Chi² test p-value<0.0001 each) (D) Synaptic pairing defects in males were further categorised and quantified. Interestingly, sex chromosomes were univalent in the vast majority of mutant spermatocytes. See text for further discussion.</p

LNT decreases the pathological changes of lymphedema.

<p>A) <i>Left panel</i>: Representative H&E stain of the hindlimbs of mice with or without LNT. Cross-sections were obtained 2 mm proximal to the tarsal joint. The dotted black line indicates the area of fibroadipose deposition. The area highlighted by the blue dotted box is shown in high-power view in part B. <i>Right panel</i>: Quantification of the percentage of fibroadipose deposition area of hindlimbs of mice with and without LNT. B) <i>Left panel</i>: Representative high-power view of the areas indicated in the blue dotted boxes in part A. Note the decreased hyperkeratosis and dermal thickness in mice treated with LNT (+LNT). <i>Right panel</i>: Quantification of epidermal and dermal thickness in hindlimbs of mice with and without LNT. C) <i>Left panel</i>: Representative immunofluorescent images of hindlimbs stained for type I collagen (red) and nuclear DAPI (blue). Note decreased type I collagen deposition in mice treated with LNT (+LNT). <i>Right panel</i>: Quantification of type I collagen deposition (measured as a percentage of the total slide stained area) after surgery with and without LNT.</p

LNT improves T cell-mediated immune responses.

<p>A) Schematic diagram of experimental protocol. Ten weeks after surgery with or without LNT, mice were sensitized with topical 0.5% DNFB to the ipsilateral hindlimb once daily for three days. T cell response was elicited by challenging the contralateral ear with topical 0.3% DNFB 5 days later. The ears were harvested for analysis 3 days later. B) <i>Left panel</i>: Representative H&E stain of the ear skin from mice with and without LNT. Note the increased inflammatory reaction in mice treated with LNT (+LNT). <i>Right panel</i>: Quantification of epidermal and dermal thickness of ear skin. Note significant increase in both epidermal and dermal thickness in mice treated with LNT (+LNT). C) <i>Left panel</i>: Representative images demonstrating immunofluorescent localization of CD45⁺ cells (red) in ear skin of mice with and without LNT. <i>Right panel</i>: Quantification of CD45⁺ cells per HPF (80x) in mice treated with and without LNT. Note the more robust inflammatory response as indicated by the greater amount of CD45⁺ cells in mice treated with LNT (+LNT).</p

LNT decreases perilymphatic accumulation of inflammatory cells.

<p>A) <i>Left panel</i>: Representative images of CD45⁺ cells (red) surrounding subdermal lymphatic vessels (green) in hindlimbs of mice with or without LNT. <i>Right panel</i>: Quantification of CD45⁺ cells located within a 50 μm radius of lymphatic vessels in the hindlimbs of mice with and without LNT. B) <i>Left panel</i>: Representative images of CD3⁺ cells (red) surrounding the subdermal lymphatic vessels (green) in hindlimbs of mice in the hindlimbs of mice with and without LNT. <i>Right panel</i>: Quantification of perilymphatic CD3⁺ cells located within a 50 μm radius of lymphatic vessels in the hindlimbs of mice with and without LNT.</p

LNT promotes regeneration of collecting lymphatic vessels.

<p>A) <i>Upper panel</i>: Representative images of ICG lymphangiograms of normal mice (i.e., Cre-Lox mice without DT ablation), mice that had undergone local DT ablation followed by PLND but no LNT (-LNT), and mice that had undergone local DT ablation followed by PLND then LNT (+LNT). Main lymphatic collecting vessels can be seen in the normal hindlimb as white parallel linear structures, whereas no visible collecting vessels are noted in mice with ablated lymphatic circulation and no LNT (-LNT). Also note the dermal reflux as represented by accumulation of white dye diffusely in the mice without LNT (-LNT). In contrast, mice treated with LNT after lymphatic ablation (+LNT) have abnormal hyperplastic lymphatic vessels that converging toward the transplanted node, as indicated by the bright white spot in the blue dotted box. <i>Lower panel</i>: Immunofluorescent image of the transplanted lymph node indicated by the blue dotted box in the upper panel. Lymphatic collecting vessels indicated by podoplanin (green) and α-SMA (red). The inset in the lower left corner represents the magnified view of the lymphatic vessel adjacent to the transplant lymph node as indicated by the white dotted line. B) Representative immunofluorescent images of collecting lymphatics in mice with and without LNT. Note the collapsed lumen and proliferation of α-SMA⁺ cells in mice without LNT (-LNT). <i>C)</i> Quantification of collecting vessels in a 0.25 mm² area, collecting vessel diameter, and α-SMA thickness in mice with and without LNT. Note the significant increase in number of collecting vessels and luminal diameter with a corresponding decrease in α-SMA thickness in mice treated with LNT (+LNT).</p