To evaluate histone-tagged fluorescent protein fusions in embryonic stem (ES) cells and mice, we generated constructs comprising an N-terminally positioned human H2B sequence followed at the C-terminus by sequences for various fluorescent proteins both GFP and DsRed-based. We previously reported that DsRed1 was not amenable to use in ES cells or mice [22
], however several improved DsRed variants have recently become available [10
]. We therefore chose to evaluate DsRed2 and DsRedExpress as part of this study.
The H2B-fluorescent protein fusions we generated were introduced into vectors utilizing the CAG promoter [19
] designed to drive high-level constitutive gene expression in ES cells, embryos and adult mice [20
]. Standard protocols were used to establish stable lines of ES cells constitutively expressing an H2B fusion [20
]. Several transgenic ES cell lines were generated each expressing H2B-EGFP at strong homogenous levels [23
]. However, even though we did recover lines with H2B-DsRed2 and H2B-DsRedExpress expression [24
], subsequent maintenance of these lines in culture revealed a continued reduction and heterogeneity in fluorescence. We were unable to establish lines with sustained homogenous H2B-DsRed2 or H2B-DsRedExpress fluorescence. Moreover our recent data suggest that mRFP1 [10
], a rapidly-maturing monomeric form of DsRed, is amenable to use in mice, both in its native form and as a part of functional fusion proteins (AKH unpublished observations).
H2B-EGFP expressing ES cells are shown in Fig. . It noteworthy that with this histone fusion we observed a high signal-to-noise ratio and so could achieve high-resolution imaging of mitotic chromosomes (pink arrowheads), various states of interphase chromatin and nuclear debris (yellow arrowheads). Moreover for cells undergoing mitosis we could also discern the stage of mitosis and the plane of cell division (Fig. b inset). Previous work indicated that a similar fusion protein expressed in HeLa cells did not affect cell cycle progression [15
], and accordingly not only could we visualize nuclear dynamics and identify the various phases of mitosis in live ES cells [26
] (Fig. ), but in doing so, we did not observe any change in growth rate or mitotic index in the transgenic ES cells compared to non-transgenic parental ES cells (data not shown). By imaging several CAG::H2B-EGFP transgenic ES cells undergoing mitosis (n
= 30) we calculated the progression from early prophase to cytokinesis to take less than one hour (Fig. ). Furthermore imaging of embryoid bodies demonstrated that individual nuclei could be discerned from a three-dimensional population of densely packed cells all of which were expressing the H2B-EGFP marker (Fig. c). No loss of fluorescence was observed with prolonged in vitro
passage of the ES cells expressing the H2B-EGFP fusion in the absence of positive selection in the presence or absence of LIF (t
> 3 months in the presence of LIF).
Figure 1 Imaging chromatin in living transgenic ES cells constitutively expressing a H2B-EGFP fusion protein. (a) Bright-field and (b) dark-field micrographs of a CAG::H2B-EGFP ES cell colony. The inset shows a detail with three nuclei in metaphase (pink arrowheads) (more ...)
Figure 2 Live imaging the progression through mitosis. Laser scanning confocal x-y images taken at a single z-plane at five minute intervals for one hour. Note that not all green fluorescence (corresponding to nuclear material) will be represented in the plane (more ...)
We next tested the effects of widespread expression of an H2B fusion protein in mice. We generated germ line chimeras and established transgenic lines of mice constitutively expressing H2B-EGFP. We were able to breed this transgene to homozygosity, resulting in viable and fertile animals exhibiting widespread expression with no overt morphological abnormalities. The transgene has been maintained for over three years in a breeding colony of homozygous mice with no apparent effect on viability, breeding performance or lifespan. We therefore infer that this fusion protein is developmentally neutral and does not interfere with either mitosis or meiosis. Wide-field microscopic analysis of both mouse embryos and adult organs demonstrates widespread expression of the H2B-EGFP fusion in all types of nucleated cells.
We used laser scanning confocal microscopy [10
] to image this constitutively expressed transgenic reporter at subcellular resolution in live mouse embryos. Such non-invasive visualization of chromatin in living preparations allowed us to acquire high-magnification sequential optical sections (z
-stacks) that can be used to generate high-resolution anatomical volumetric (3-dimensional) images with details of interphase chromatin in addition to mitotic chromosomes and fragmenting nuclei. To do this, stacks of sequential optical sections are reconstructed into 3-dimensional projections. This methodology can be used to generate 3-dimensional (3D) image sets not only of cells propagated in culture but also of cells in situ
in living animals and is illustrated here by imaging whole mouse embryos at the 4-cell stage, the blastocyst stage, and the pre-gastrula stage (Fig. and Additional Files 1
). These data sets can be computationally manipulated in various ways, for example for the visualization of individual xy
slices from a z
-stack, rendered images from the full, or part of a z
-stack, and color-coded depth projections of a z
-stack (Fig. ).
Figure 3 Live embryo imaging of preimplantation and early postimplantation mouse embryos hemizygous for a constitutively expressed H2B-EGFP fluorescent fusion. (a) Single confocal optical section fluorescence overlay on a bright-field image of a 5-cell stage pre-implantation (more ...)
Data on older embryos and adult organs illustrates that larger specimens can be imaged, however not in their entirety given current limitations in optical imaging capabilities. Instead of imaging the whole specimen, larger samples are positioned so that data can be acquired from regions of interest, which can then be acquired in a tiled manner and computationally re-aligned in image acquisition and processing software.
Our data demonstrates that nuclear morphology afforded by the H2B-EGFP fusion can be used to identify different cell types. In both the raw data, and a rotated rendered stack of an embryonic day (E) 7.5 embryo, cells of the definitive endoderm, mesoderm and embryonic ectoderm can be distinguished solely on the basis of nuclear morphology and orientation in addition to their expected position (Fig. b–h and Additional File 3
). Low magnification rendered z
-stacks taken from a transversely cut section through the head of an E10.5 embryo (Fig. i) reveal the stereotypical 3D organization of nuclei within the region imaged (Fig. j), and electronically magnified views of this image illustrate a characteristic apposition of nuclei both in and around the notochord, and within the mesenchyme and endoderm of the pharyngeal region (Fig. k and l and Additional Files 4
), in addition to providing information on cell division and cell death (pink and yellow arrowheads, respectively in Fig. l).
Figure 4 Live imaging H2B-EGFP in postimplantation mouse embryos. (a) Lateral view of the embryonic region of an E7.5 embryo (anterior to the left) with box depicting the region imaged in b and double-headed arrow depicting the x-y layering of the z-stack. (b (more ...)
Wide-field microscopic examination of organs from adult animals revealed widespread fluorescence as has been reported for animals expressing native fluorescent proteins under the regulation of the CAG promoter [20
]. Laser scanning confocal imaging of various organs obtained from adult animals was used to generate high-resolution images revealing stereotypical nuclear positions, reflecting different cell types and revealing other subcellular details, such as mitosis and nuclear fragments, also observed in embryos (Fig. and Additional Files 3
Figure 5 High resolution live imaging of the organs of CAG::H2B-EGFP adult mice. Confocal images of freshly isolated organs from a 6 week old adult male hemizygous CAG::H2B-EGFP Tg/+ animal illustrate the widespread nuclear localized expression of the histone (more ...)
Finally we investigated whether we could follow cell movement, division, and death in time-lapse experiments using various imaging modalities. We cultured ES cells and embryos on the stages various each of which had been modified to permit culture under physiological conditions. The different types of data routinely generated using different optical imaging modalities that are widely used and commercially available are illustrated in Figure . Spinning disc confocal microscopy [1
] was used for short-term high-resolution 4D imaging of CAG::H2B-EGFP ES cells (Fig. and Additional File 6
), wide-field microscopy was used for long-term low-resolution imaging of CAG::H2B-EGFP preimplantation stage embryos (Fig. and Additional File 7
). Note that development proceeds normally in most embryos, and that some of the embryos imaged are undergoing cavitation to form blastocysts [28
] (arrowheads). Two-photon excitation microscopy [10
] was used to image cells in a whole gastrula-stage mouse embryo without perturbing the morphogenetic movements associated with gastrulation (Fig. and Additional File 8
). Cells can clearly be followed through the successive time points in each of these experimental situations ranging from a few minutes (short-term) to 24 hours (long-term) time-lapse duration. These studies reflect the range of resolutions at which information can be acquired using a marker of this type. We observed normal cell proliferation throughout the course of these imaging experiments and no excessive nuclear fragmentation. Also, because the on-stage cultures were comparable to parallel cultures maintained in a tissue culture incubator, we conclude that the outcome of the cultures was not affected by the various imaging modalities.
Figure 6 Dynamic time-lapse imaging of mouse CAG::H2B-EGFP transgenic ES cells, preimplantation and postimplantation embryos using different imaging modalities. (a) Rendered confocal stacks of transgenic ES cells constitutively expressing a CAG::H2B-EGFP transgene (more ...)