Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle

Abstract

Current approaches to monitor and quantify cell division in live cells, and reliably distinguish between acytokinesis and endoreduplication, are limited and complicate determination of stem cell pool identities. Here we overcome these limitations by generating an in vivo reporter system using the scaffolding protein anillin fused to enhanced green fluorescent protein, to provide high spatiotemporal resolution of mitotic phase. This approach visualizes cytokinesis and midbody formation as hallmarks of expansion of stem and somatic cells, and enables distinction from cell cycle variations. High-resolution microscopy in embryonic heart and brain tissues of enhanced green fluorescent protein-anillin transgenic mice allows live monitoring of cell division and quantitation of cell cycle kinetics. Analysis of cell division in hearts post injury shows that border zone cardiomyocytes in the infarct respond with increasing ploidy, but not cell division. Thus, the enhanced green fluorescent protein-anillin system enables monitoring and measurement of cell division in vivo and markedly simplifies in vitro analysis in fixed cells.

Figure 1. eGFP–anillin expression is a mitotic marker in mouse pluripotent cells.

(a) Schemes of subcellular localization of the eGFP–anillin fusion protein during the cell cycle and of the CAG–eGFP–anillin fusion construct: Anillin is fused to the C terminus of eGFP and its expression is under control of the CAG promoter. APC, anaphase-promoting complex; C, cytokinesis; Cdc20, cell-division cycle protein 20; Cdh1, Cdc20 homologue-1; K, karyokinesis; SCF, Skp, Cullin, F-box-containing complex. (b) Picture of a living colony of a stably transfected mESC line expressing eGFP–anillin under control of the CAG promoter. eGFP–anillin (green) localization in the cytoplasm (open arrowhead), cell cortex (solid arrowhead) and midbody (arrow). Scale bar, 25 μm. (c) Analysis of fixated transgenic mESCs; nuclei are stained with Hoechst nuclear dye (blue, upper left picture). Differential localization of eGFP–anillin (green) in the nucleus (solid arrowhead), cortex/contractile ring (open arrowhead) or midbody (arrow). Scale bars, 25 (upper left picture) and 5 μm (all other pictures). (d) Time-lapse images taken from a CAG–eGFP–anillin mESC clone. The arrows mark a cell with changing localization of the eGFP–anillin fluorescence signal during M-phase. The arrowheads point to the nuclei of the daughter cells. Scale bar, 5 μm. (e) Stainings for the proliferation markers Ki-67, pHH3 and Aurora-B kinase (all red) reveals co-labelling with the eGFP–anillin (green) fluorescence signal. Note localization in cytoplasm/cortex (open arrowheads), contractile rings (solid arrowheads) and midbodies (arrows); nuclei are stained with Hoechst dye (blue). Scale bar, 25 μm. (f) Flow-cytometric profiles of eGFP–anillin mESCs and non-transfected G4 mESCs stained for Ki-67 or pHH3, respectively. The majority of eGFP–anillin⁺ cells is Ki-67⁺, a smaller fraction of eGFP–anillin⁺ mESCs is positive for the M-phase marker pHH3.

Figure 2. eGFP–anillin marks proliferating cells in human iPS- and ES cell lines.

(a) Scheme of the lentiviral vector: black triangle, mutation in 3′ LTR, leading to self inactivation (SIN vector); CAG, combination of early enhancer element of CMV (cytomegalovirus) promoter and chicken β-actin promoter; cPPT, central polypurine tract; eGFP-anillin, enhanced green fluorescent protein fused to anillin; gag, truncated group-specific antigen sequence; SA, splice acceptor sites; LTR, long terminal repeat; Psi, packaging signal; RRE, rev response element; SD, splice donor sites; WPRE, post-transcriptional regulatory element of woodchuck hepatitis virus. (b) Stainings of lentivirus CAG–eGFP–anillin-transduced human H9 ESCs and human iPS cells for the proliferation markers Ki-67, pHH3 and Aurora-B kinase (all three red). eGFP–anillin (green) has M-phase-specific localization in the nucleus, cytoplasm (open arrowhead), contractile ring (solid arrowhead) and midbodies (arrows); Ki-67 stains nucleoli in human cells; nuclei are stained with Hoechst dye (blue). Scale bars, 10 μm. (c) Analysis of cell cycle kinetics of human iPS and ESCs with time-lapse microscopy. Data are shown as mean±s.e.m. from n=22 (iPS) and n=21 (H9) cells.

Figure 3. eGFP–anillin is a mitotic marker in mESC-derived differentiating cells.

(a) Percentage of Ki-67⁺ and eGFP–anillin⁺ cells in dissociated EBs at days 0 to 9 of differentiation; Ki-67 staining matches eGFP–anillin expression over the time course of differentiation. Data shown are mean±range of one differentiation experiment with n=2 or n=3 (day 4) different eGFP–anillin mESC clones. (b) Sections of transgenic murine eGFP–anillin EBs after 3 and 9 days of differentiation. On day 3, eGFP–anillin⁺ (green) cells are found throughout the EB; at day 9, proliferation is restricted to the cortical areas of the EB; nuclei are stained with Hoechst dye (blue). Scale bar, 200 μm. (c) Staining for Ki-67 (red) of plated EBs at day 16 of differentiation revealed a high overlap with eGFP–anillin (green) expression; Ki-67⁻ and eGFP–anillin⁻ cells are marked by arrows. Note that some of the weak eGFP–anillin⁺ cells have a very faint, but visible Ki-67 signal (arrowheads); nuclei are stained with Hoechst nuclear dye (blue). Scale bar, 25 μm. (d) EB-derived cells (day 12 of differentiation, 2 days after plating) from an eGFP–anillin mESC line are isolated and stained with subtype-specific markers: α-actinin for mesodermal, Tubb3 for neuroectodermal, and Krt8 for endodermal cells (all white). eGFP–anillin⁺ cells (green) co-stain for Ki-67 (red); nuclei are stained with Hoechst nuclear dye (blue). Scale bars, 10 μm. (e) eGFP–anillin (green) fluorescence marked proliferating cardiomyocytes due to its specific position in contractile rings and midbodies; the cells are identified as cardiomyocytes based on α-actinin⁺ staining (white) and co-labelled with Ki-67 (red); nuclei are stained with Hoechst dye (blue). Scale bars, 5 μm.

Figure 4. The eGFP–anillin system distinguishes between cell division and endoreduplication in TS cells.

(a) Epifluorescence pictures of eGFP–anillin-transfected TS cells 4 (upper and lower left pictures) and 48 h (lower right picture) after addition of RO3306. eGFP–anillin (green) is localized in the midbody of undifferentiated TS cells (arrowhead), but in the nucleus of differentiating, endoreduplicating TS cells (arrows). After 48 h of differentiation, typical TGCs (dotted line in DIC overlay) with nuclear eGFP–anillin localization can be observed; nuclei are stained with Hoechst nuclear dye (blue). Scale bars, 10 μm. (b) Percentage of distribution of nuclear DNA content of TS cells without (green line, n=200) or 48 h after addition of RO3306 (black line, n=54). C=C-value. (c) Time-lapse images taken from a differentiating TGC (dotted line in DIC image) expressing eGFP–anillin (green) at 0 and 47:30 h of differentiation. Scale bar, 20 μm. (d) Stainings of differentiating TGCs transfected with eGFP–anillin for cyclin A (red) and p57^Kip2 (white). The eGFP–anillin signal (green) co-localizes with cyclin A, but not p57 (arrows); nuclei are stained with Hoechst nuclear dye (blue). Scale bar, 20 μm. (e) TGC in placenta of transgenic mice display exclusively nuclear eGFP–anillin localization (green, TGC, arrow). Scale bar, 25 μm. (f) Chorionic plate of an E13.5 eGFP–anillin placenta. M-phase typical localizations of eGFP (arrowheads) are seen. White box illustrates the area shown in g; autofluorescence is red. Scale bar, 25 μm. (g) Chorionic plate cell with eGFP–anillin (green) localization to the contractile ring. Nuclei are stained blue; scale bar, 5 μm.

Figure 5. eGFP–anillin is a mitotic marker in vivo during embryonic development.

(a) Epifluorescence picture taken from an E10.5 eGFP–anillin transgenic embryo showing eGFP fluorescence (green); autofluorescence is red. Scale bar, 1 mm. (b) Sagittal sections of an E10.5 eGFP–anillin embryo reveals eGFP expression (green) in all tissues; cardiomyocytes are identified by α-actinin staining (magenta, dashed square). Scale bar, 200 μm. (c) Sagittal sections through an E10.5 eGFP–anillin heart evidence that atria (A) and ventricle (V) are strongly eGFP–anillin⁺ (green). Ki-67 staining (red) of same section proves overlap between both fluorescence signals; nuclei are blue in the merged image. Scale bar, 100 μm, (d) Magnification of boxed area in c. Ventricular cardiomyocytes display eGFP–anillin (green) expression and are Ki-67⁺ (red); nuclei are blue in the merged image. Scale bar, 10 μm. (e) Proliferating α-actinin⁺ (white) cardiomyocytes in sections from an E18.5 eGFP–anillin heart are identified by eGFP labelling (green) of contractile rings or midbodies; Ki-67 staining is red; nuclei are blue. Scale bar, 10 μm. (f) Co-stainings for eGFP (green) and Ki-67 (red) in cross sections of a P3 eGFP–anillin⁺ heart reveal restriction of the eGFP signal to cardiomyocytes (arrows). Arrowheads mark Ki-67⁺/eGFP⁻ cells; nuclei are blue. Scale bar, 25 μm. (g) Sections of E10.5 and E13.5 hearts from eGFP–anillin/αMHC–mCherry mice. Proliferating cardiomyocytes (arrows) are identified by co-expression of mCherry (red) and eGFP–anillin (green). Scale bars, 50 (E10.5), 20 and 5 μm (E13.5). (h) Optical section from an eGFP–anillin/αMHC–mCherry E11.5 heart showing eGFP–anillin⁺ (green) and mCherry⁺ (red) cells. The white box illustrates the area shown in i and j. Scale bar, 40 μm. (i) The eGFP–anillin⁺ nucleus (arrow) can be attributed to a αMHC–mCherry⁺ cell. The illustrated single optical section (x,y) is located in the centre of 13 planes. The corresponding x- (top) and y-projections (right) are given. Scale bar, 10 μm. (j) Time series of the same individual myocyte (white arrow in c and at t=0) imaged without optical filtering. A complete M-phase is visualized based on eGFP–anillin fluorescence during 6 h. Two nuclei of daughter cells become visible starting at 114 min (white arrows). Scale bar, 10 μm.

Figure 6. eGFP–anillin specifically marks proliferating radial glia (RG) and basal progenitor (BP) cells at mid neurogenesis.

(a) Cross sections through the brain of an E10.5 fixed embryo show eGFP–anillin signals in the neuroepithelium (arrows). Scale bar, 200 μm. (b) Cross section of E14.5 cerebral cortex from eGFP–anillin mice immunostained for Ki-67 (red), Tubb3 (white) and Hoechst (blue). Notice overlap of Ki-67 and eGFP–anillin (green) fluorescence in the merged image and loss of proliferative marker Ki-67 in differentiated neurons (Tubb3). IZ=intermediate zone, SVZ=subventricular zone, VZ=ventricular zone. Scale bar, 50 μm. (c) eGFP–anillin⁺ midbodies and contractile rings indicative for cell division are observed in neuroepithelial (arrows) but not Tubb3⁺ (white) cells. Ki-67 staining (red) correlates with eGFP–anillin expression (green). Nuclei are stained with Hoechst dye (blue). Scale bar, 10 μm. (d) At E14.5 eGFP staining co-localizes with brain lipid-binding protein (BLBP; red), a known marker for RG cells in the VZ (single optical section). Scale bar, 10 μm. (e) BP cells of the cortical SVZ from eGFP–anillin mice co-stain for eGFP and the transcription factor Tbr2, a marker for BPs (red). Scale bar, 10 μm.

Figure 7. eGFP–anillin enables live monitoring of neural progenitor cell division in acute brain slices.

(a,b) Cross sections of the cerebral cortex of an E14.5 embryo reveal co-staining for anti-eGFP and anti-p-vimentin or anti-pHH3 in the neuroepithelium. Close-ups of stainings of apical progenitor cells for pHH3 and p-vimentin (both red), identifies cells in M-phase. eGFP–anillin⁺ contractile rings and midbodies are clearly visible (arrows). Nuclei are stained with Hoechst dye (blue). Scale bars, 5 (a) and 10 μm (b). (c) Cross section of the cerebral cortex of an E14.5 embryo shows eGFP–anillin expression (green) in the neuroepithelium and co-localization with Ki-67 staining (red). Close-ups depict apical progenitor cells, these are Ki-67⁺ (red) and eGFP–anillin is detected in contractile rings (arrows) or in cortical/cytoplasmatic localization (arrowheads), proving different stages of M-phase. Nuclei are stained with Hoechst dye (blue). Scale bars, 5 μm. (d) Time-lapse images taken from an acute brain slice of an E14.5 eGFP–anillin embryo. Representative apical (white arrow) and basal (yellow arrow) progenitor cells are shown. Formation of contractile rings is indicated by red arrows. Scale bar, 20 μm. (e) Time-lapse images from an acute brain slice of an E14.5 eGFP–anillin/CAG–H2B–eGFP double transgenic embryo with nuclear H2B–eGFP signal in all cells. The eGFP–anillin signal becomes bright green and markedly visible during cytokinesis and formation of the midbody (white arrows). After cell division, the midbody remains close to the cell body of one daughter cell (red arrows), whereas the cell body of the other daughter cell migrates towards the basal side of the neuroepithelium (green arrows), possibly indicating asymmetry in inheritance of the midbody. Scale bar, 10 μm.

Figure 8. Transgenic eGFP–anillin mice reveal endoreduplication in cardiomyocytes after cardiac injury.

(a) eGFP–anillin expression (green) can be observed in cardiomyocytes of P3 mice; in 2-month-old mice, no eGFP–anillin signals can be detected in the heart. Scale bar, 25 μm. (b) Sections through a heart of an adult eGFP–anillin transgenic mouse 4 days after cryoinfarction (CI). eGFP–anillin fluorescence (green) reveals numerous proliferating cells in the infarcted area (IA); the myocardium adjacent to the lesion is identified based on its strong autofluorescence (yellow, BZ (border zone)); nuclei are stained with Hoechst dye (blue). Scale bars, 100 (overview) and 25 μm (magnification). (c) Positive staining for asmac (white) identifies the eGFP–anillin-expressing cells (green) as myofibroblasts (arrows). High magnification of such cells reveals eGFP–anillin localization in contractile rings (open arrowhead) and midbodies (solid arrowhead) proving cell division; nuclei are blue; autofluorescence is red. Scale bars, 5 μm. (d) Some of the cardiomyocytes in the BZ (LAD d12) show nuclear localization of eGFP–anillin (green, arrow); none displays a contractile ring or midbody. Nuclei of eGFP–anillin⁻ cardiomyocytes (white dotted lines) are blue. Scale bar, 20 μm. (e) Co-stainings of cardiomyocytes against eGFP and Ki-67 in the BZ of the lesion revealed double-positive nuclei; scale bar, 10 μm. (f) Quantitation of nuclear area of cardiomyocytes in the BZ of d12CI and LAD hearts reveals that eGFP–anillin⁺ nuclei (n=13 hearts) are significantly larger than eGFP–anillin⁻ nuclei (n=6 hearts). Data are shown as mean±s.e.m., *P<0.05 (Student's t-test). (g) Three-dimensional (3D) confocal reconstruction of a 50-μm section from a d12 CI heart displaying an eGFP–anillin⁺ nucleus (green) in a BZ cardiomyocyte, Two nuclei of eGFP–anillin⁻ cardiomyocytes are shown (red arrows and red dotted lines). Nuclei are stained with TO-PRO 3 iodide (white). Scale bar, 20 μm. (h) Scatter plot showing mean DNA content of eGFP–anillin⁺ cardiomyocytes (n=33), eGFP–anillin⁻ cardiomyocytes (n=37) and non-cardiomyocytes (PECAM⁺ endothelial cells, n=33) from sections (n=6) of a d12 CI heart (n=1). (i) Percentage of distribution of nuclear DNA content in eGFP–anillin⁺ cardiomyocytes (green line, n=33), eGFP–anillin⁻ cardiomyocytes (black line, n=37) and endothelial cells (blue line, n=33). C=C-value.