Lipid-modified fluorescent protein fusions that label the secretory pathway including the plasma membrane were developed to provide dynamic information on 1) cell morphology and cell behavior, and 2) secretory pathway itineraries. To construct the GFP-GPI fusion, the N-terminal signal sequence from the sperm secretory protein acrosin and the C-terminal GPI-anchor sequence of Thy-1 were added to the N- and C-terminus of enhanced green fluorescent protein (EGFP; Cormack et al., 1996
), respectively, as described previously (Kondoh et al., 1999
). The myr-fluorescent proteins fusion were generated by adding the sequence encoding the N-terminal nine amino acids from avian c-Src (Thomas and Brugge, 1997
) to the 5′ end of EGFP and Venus (Nagai et al., 2002
). Venus was chosen over enhanced yellow fluorescent protein (EYFP) as it is currently the brightest and fastest-maturing GFP-variant exhibiting a red-shifted yellow fluorescence (Nagai et al., 2002
). The resulting GFP-GPI, myr-GFP, and myr-Venus fusions were then placed under the regulation of the CAG
promoter (Niwa et al., 1991
) to generate the plasmids pCX::GFP-GPI, pCX::myr-GFP,
. The CAG
promoter comprises the CMV immediate early enhancer, chicken beta actin promoter, 5′ UTR, and first intron and has previously been shown to drive robust widespread transgene expression in ES cells, embryos, and adult mice (Niwa et al., 1991
; Hadjantonakis et al., 1998
Plasmid constructs were tested for fluorescent fusion protein expression and subcellular localization by transient transfection of Cos-7 cells. Since myr-GFP and myr-Venus were shown to exhibit an identical subcellular localization (data not shown), and since Venus is spectrally distinct from GFP and can therefore be combinatorially imaged with GFP, we chose pCX::myr-Venus
for further analysis in ES cell-mediated transgenesis experiments. Plasmids pCX::GFP-GPI
were used to generate lines of CAG:: GFP-GPI
transgenic ES cells as described previously (Hadjantonakis and Papaioannou, 2004
). Fluorescent colonies were identified and picked under an epifluorescence stereo dissecting microscope. Clones were passaged in 96-well plates in the absence of drug selection and monitored for maintenance of transgene-encoded fluorescence. ES cell lines exhibiting constitutive expression of either GFP-GPI or myr-Venus while retaining normal morphology when grown either in the presence of LIF or when induced to differentiate into embryoid bodies were further analyzed for level and heterogeneity of fluorescence by flow cytometry as described previously (Hadjantonakis and Nagy, 2000
). Only clones satisfying these criteria were used for further investigation.
To determine the subcellular distribution of each fluorescent fusion at high resolution, two different ES cell clones for each transgenic construct were examined using confocal laser scanning microscopy. The lipid-modified fluorescent protein fusions retained both fluorescence and subcellular localization after fixation of specimens in paraformaldehyde. No differences in level of expression or subcellular compartmentalization were observed between ES cell clones expressing any given fusion. Furthermore, treatment of transgenic ES cells with cycloheximide did not alter the localization of fluorescence, suggesting that the observed fluorescent protein distribution was likely due to targeted localization of the fusion rather than buildup of excess newly synthesized protein (data not shown). The cellular distributions for each construct in live ES cells are shown in . GFP-GPI was localized on plasma membranes (yellow arrowhead, ), Golgi membranes (red arrowhead, ), and some secretory vesicles (blue arrowhead, ). myr-Venus is concentrated in perinuclear regions (green arrowhead, ) and vesicular structures located inside the nucleus (white arrowhead, ), in addition to the plasma membrane (yellow arrowhead, ), endosomes of the secretory pathway (blue arrowhead, ), and the Golgi (red arrowhead, ). With the exception of the nuclear localization of myr-Venus, which we attribute to either the increased resolution or live context of our analysis, these distributions are consistent with previous reports of GPI localization in F9 cells and Src localization in fibroblasts (Kaplan et al., 1992
FIG. 1 Localization of GPI- and myristoyl-tagged fluorescent proteins in live mouse ES cells. Live imaging of CAG::GFP-GPI and CAG::.myr-Venus hemizygous transgenic ES cells. The left column represents a single channel fluorescence image of either GFP-GPI or (more ...)
3D and 4D information was generated by computer-assisted rendering of the z-stacks of xy images. Rendered images of CAG::GFP-GPI and CAG::myr-Venus ES cells are shown in (blue, red, and yellow arrowheads highlight similar structures). Post-Golgi carriers fusing with the membrane were identified by comparison of the face-on (red arrowhead, ) and rotated () views of the rendered stacks. Higher density of myr-Venus-labeled structures in the cytoplasm () as compared to GFP-GPI was also evident. These images also provided data with respect to the direction of membrane secretion occurring within a single cell in a non-polarized cluster, also discerned with respect to the position of the nucleus (white arrowhead, ).
Having established the neutrality of widespread lipid-modified fusion protein expression in ES cells, we next investigated the extent of individual transgene expression in embryos. We generated 4n (tetraploid) wildtype ↔ CAG::GFP-GPI
and equivalent CAG::myr-Venus
chimeras, as described previously (Hadjantonakis et al., 1998
). We then documented transgene expression (as determined by fluorescence) at embryonic day (E)9.5. Three independent ES cell lines were tested for each transgenic construct. All CAG::GFP-GPI
ES cell-derived embryos exhibited widespread GFP-GPI expression, indicating that: 1) the level of expression was sufficiently strong to be visualized in vivo; 2) that the transgene was not silenced; and 3) development was able to proceed normally to midgestation (data not shown).
For CAG::myr-Venus transgenic ES cell clones, all embryos exhibited normal morphology, with two lines exhibiting widespread myr-Venus expression. However, one line consistently produced mosaic myr-Venus expression around E9.5, which became more widespread at later stages. Our past experience in imaging cells in vivo in mouse embryos has shown that mosaic reporter expression, for example, in diploid chimeras, yields more information about cell morphology, since cells can be imaged in relative “isolation,” providing information on plasma membrane projections and dynamics. Since we have also generated strains of mice, exhibiting widespread expression of myristoyl-monomeric red fluorescent protein variants throughout embryonic development (G.S. Kwon, C.S.L., and A.-K.H., unpubl. data), we chose to use the latter midgestational mosaic CAG::myr-Venus ES cell clone for generating germline transmitting chimeras.
ES cell-mediated transgenesis was used for the generation of equivalent transgenic strains of mice. Chimeras transmitted their transgenes to F1 progeny in a Mendelian fashion, suggesting that widespread transgene expression is compatible with normal development and fertility. Hemizygous (CAG::GFP-GPITg/+ or CAG::myr-VenusTg/+) and homozygous (CAG::GFP-GPITg/Tg or CAG::myr-VenusTg/Tg) animals were indistinguishable from each other and from wildtype littermates; they exhibited sustained widespread expression of the lipid-modified fusions and were viable and fertile, demonstrating the developmental neutrality of the lipid-modified fusion proteins. Homozygous animals exhibited identical sub-cellular distribution but increased fluorescence as compared to hemizygotes.
We examined the distribution of the two fusion reporters in vivo in hemizygous animals in different cells and at different developmental stages using laser scanning confocal microscopy. The onset of the respective zygotic transgene expression was observed in early (uncompacted) morulae and was easily visualized as morulae compacted (). Both transgenes were widely expressed and clearly labeled the plasma membrane, with a high signal-to-noise ratio, such that inner cells of the morula fated to form the inner cell mass and primitive endoderm could be distinguished from outer cells fated to form the trophectoderm. Widespread expression of both transgenes was maintained as development proceeded (). By E3.5 the blastocoelic cavity could be detected in both single optical sections () and in rendered stacks (). In addition, close inspection of GFP-GPI revealed localization to other subcellular organelles, including the Golgi of the mural trophectoderm (yellow arrowhead, ).
FIG. 2 GPI-linked and myristoylated fluorescent protein localization in live preimplantation mouse embryos. Living embryos hemizygous for the CAG::myr-Venus or CAG::GFP-GPI transgenes were imaged by confocal laser scanning microscopy. A single xy section taken (more ...)
Sagittal sections through embryos expressing the respective reporters demonstrated widespread expression at postimplantation stages (). Even though the CAG
regulatory elements drive widespread expression in ES cells, embryos, and adult mice (Niwa et al., 1991
; Okabe et al., 1997
; Hadjantonakis et al., 1998
), specific populations consistently expressed higher levels of the reporter (Hadjantonakis and Nagy, 2000
; Lickert et al., 2004
), including the heart (, arrowhead) and notochord (). The GFP-GPI fusion was predominantly located at the plasma membrane of cells in the ventral region of the neural tube (), whereas myr-Venus also exhibited additional diffuse cytosolic expression (), which most likely reflects inherent differences in localization to different subcellular organelles.
FIG. 3 Widespread expression and subcellular localization of GPI-linked and myristoylated fluorescent protein expression in postimplantation mouse embryos. a–h: E9.5 embryos expressing either GFP-GPI or myr-Venus. a: Sagittal section of CAG::GFP-GPI/ (more ...)
The C-terminal region of Thy-1 incorporated into the GFP-GPI fusion has previously been shown to be sufficient to target basolateral proteins to the apical surface in MDCK cells (Brown et al.
, 1989). Moreover, previous characterization of GFP-GPI localization in a transgenic mouse strain generated by pronuclear injection of DNA into zygotes revealed a tissue-inherent apical distribution of the protein (Kondoh et al., 1999
). Consistent with these reports, we observed increased GFP-GPI signal in the apical membranes of specific cells at different developmental stages in different tissues. For example, GFP-GPI expression was evident throughout the plasma membrane, but was more intense at the apical membranes of the endoderm (white arrowhead, ) at E9.5. In contrast, myr-Venus distribution was not polarized (white arrowhead, ), as it was present throughout the plasma membrane and cytosol, yet consistently excluded from the nucleus.
While our work supported and confirmed previous observations, previous work imaging GFP-GPI in a strain of transgenic mice generated by pronuclear DNA injection supports the apical localization of the GFP-GPI fusion that we observed in many tissues (Kondoh et al., 1999
). In contrast, we also detected basolateral localization. The apparent disparity in the localization of the GFP-GPI fusion between past and current investigations might be attributed to: 1) A difference in the level of transgene expression, such that if the current strain exhibits higher levels of protein expression a wider repertoire of localization may be observed. This is unlikely, since all three GFP-GPI ES cell lines evaluated in 4n chimeras displayed identical localization. 2) The difference in the method used for transgenesis (transfection of DNA into ES cells versus microinjection of DNA into zygotes). We believe that this is unlikely, as the product of both methodologies (the transgenic strain) should yield comparable results. Furthermore, our past experience has demonstrated that reporter localization does not correlate with mode of transgenesis. 3) A difference in the coding sequence of the transgene. Previously, the C-terminal sequence KLEN (where N is the omega-site) was used to encode the GPI attachment sequence, whereas in the fusion used here the lysine (K) was changed to a glutamine (Q), because it was found that the lysine residue was sensitive to some proteases (such as kallikrein), which may cause artificial release of GFP-GPI from the cell membrane. We therefore favor the latter hypothesis.
To determine the distribution of the respective lipid-modified fusion reporters at a later stage during embryogenesis and organogenesis, we examined their distribution at E13.5. Transverse sections through a CAG::GFP-GPI or CAG::myr-Venus hemizygous transgenic embryos revealed widespread distribution of the respective lipid-modified fluorescent fusions (). Higher-magnification views of the ventral region of the developing spinal cord () revealed high levels of expression in the floorplate extending into the ependymal layer in both CAG::GFP-GPI and CAG::myr-Venus animals. Furthermore, differences in cell morphology were easily visualized in the differentiating ventral motor and interneurons within the mantle layer at these later stages () compared to the E9.5 neural tube (). In addition, distinct staining throughout the marginal layer marking the future white matter was observed ().
In the developing lung, the bronchioles revealed a polarized localization of GFP-GPI that was enriched within apical regions of the plasma membrane (white arrowhead, ). This is in contrast to the nonpolarized distribution of myr-Venus. Moreover, stromal cells surrounding some, but not all, bronchioles consistently expressed high levels of the respective fusions (red arrowhead, ). Optical sections of transverse cuts through the developing heart () revealed an enrichment of fluorescent membranes within the myocardium. GFP-GPI was observed throughout the plasma membrane, including clusters of fluorescence present in some cells (yellow arrowhead, ), in the Golgi (red arrowhead, ), and intracellular secretory vesicles (blue arrowhead, ). In addition to these populations, myr-Venus was also observed in perinuclear regions (green arrowhead, ) and microtubule organizing centers (MTOCs, red arrowhead, ). These data demonstrate the dynamic and cell type dependent subcellular distribution of the GFP-GPI and myr-Venus fusions during embryogenesis.
Consistent with our previous findings using the CAG
regulatory elements (Hadjantonakis et al., 1998
; Hadjantonakis and Papaioannou, 2004
; Long et al., 2005
), low-magnification wide-field microscopic examination of organs obtained from hemizygous (Tg/+) 4-day-old (P4), 6-week-old, and 3-month-old animals of both sexes revealed widespread expression of both GFP-GPI and myr-Venus (data not shown). Confocal images were acquired from sections of organs isolated from postnatal day (P)4 animals. Low-magnification images of transgenic kidneys revealed widespread expression of both fusion proteins in respective transgenic embryos (). As previously reported (Hadjantonakis et al., 1998
), the CAG
promoter drives increased expression in the glomeruli (red arrowheads, ). Apical localization in arterioles (white arrowhead, ), nonpolarized membrane distribution in Bowman’s capsule (yellow arrowhead, ), and diffuse cytoplasmic staining in the collecting ducts (blue arrowhead, ) demonstrated the diverse, cell context dependent distribution of GFP-GPI. The distribution of myr-Venus was similar to that of GFP-GPI in the cells of Bowman’s capsule (yellow arrowhead, compare ) but was not apically localized in arterioles (white arrowhead, compare ).
FIG. 4 Widespread expression and subcellular localization of GPI-linked and myristoylated fluorescent protein expression in adult mouse tissues. Tissues from P4 animals hemizygous for either transgenic were used for all images. Venus fluorescence is depicted (more ...)
Strong fluorescence was detected within the P4 heart of both CAG::GFP-GPI and CAG::myr-Venus transgenic lines (). Optical sections revealed that GFP-GPI was localized at the plasma membrane (yellow arrowhead, ), perinuclear regions (blue arrowhead, ), and intracellular vesicles (red arrowhead, ). Higher-magnification views revealed the absence of cell borders in fused cardiomyocytes (). Fluorescence was observed in vesicles distributed throughout the syncytium, the sarcoplasmic reticulum (blue arrowhead, ), on the Golgi (red arrowhead, ), and on the membranes of presumably unfused cells (yellow arrowhead, ).
In polyploid cells of the P4 liver, neither of the two distinct reporter fusions were concentrated at cell boundaries (). Rather, they showed similar diffuse distributions throughout the cytoplasm. Furthermore, 3D rendering of a field of liver tissue revealed that GFP-GPI was present on the plasma membrane of mononuclear hepatocytes (yellow arrowhead, ), microsomes (blue arrowhead, ), and Golgi (red arrowhead, ). The position of the Golgi with respect to the nucleus (white arrowhead, center panel, ,) was illustrated by obscuring of the structure in the merged panel. myr-Venus was localized to the plasma membrane (yellow arrowhead, ), nucleus (red arrowhead, ), and throughout the cytoplasm of hepatocytes. However, myr-Venus was absent from vacuolar structures within these cells (white arrowhead, ). The nucleus of a cell expressing myr-Venus is marked with a red arrowhead to highlight its position in the merged panel (center panel, ).
Sections through the P4 small intestine demonstrated that GFP-GPI was strongly expressed on membranes of cells within the lamina propria (yellow arrowhead, ) and muscularis (white arrowhead, ). However, the signal was diffuse throughout the mucosal epithelium (blue arrowhead, ). In contrast, myr-Venus expression was clearly present in the lateral membranes of the mucosal epithelium (green arrowhead, ) and apical brush borders (red arrowhead, ), in addition to punctate nuclear staining (blue arrowhead, ). Similar to the pattern observed in hepatocytes, myr-Venus was excluded from vacuolar structures inside the cytosol of cells of the mucosal epithelium (white arrowhead, ). We also examined fixed coronal sections of P4 brains to demonstrate widespread distribution of both fluorescent proteins (). These fluorescent reporters were subcellularly localized, suggesting that these transgenic lines can be used for the study of neuronal cell fate and behavior, similar to previous reports (Rodriguez et al., 1999
; Feng et al., 1999).
To investigate the utility of combinatorially imaging GFP-GPI and myr-Venus for investigating mechanisms of subcellular organelle trafficking, we examined their combined distributions in cells of live double transgenic (CAG::GFP-GPI/
+) E9.5 embryos. Due to the similarity in GFP and Venus spectra, linear unmixing (Dickinson et al., 2001
; Hadjantonakis et al., 2003
) was used to separate GFP and Venus fluorescence in vivo (). As expected from examinations of single transgenic embryos, we found both distinct and overlapping domains of fluorescent protein localization. An example of a distinct myr-Venus domain is shown in an epithelial cell of a live transverse view of somites (yellow arrowhead, ). As observed in fixed sections of E9.5 embryos, GFP-GPI is concentrated in the apical plasma membrane domain of live double transgenic gut, whereas the myr-Venus reporter does not exhibit a polarized distribution (white arrowhead, ). Finally, an image of live intermediate mesoderm is presented to highlight overlapping distributions on the plasma membranes (blue arrowhead, ). These data confirmed our ability to segregate distinct lipid moieties (Munro, 2004
) in a single double transgenic animal in vivo, where cells exist in the context of their normal microenvironment (Bissell and Labarge, 2005
FIG. 5 Differential localization of GFP-GPI and myr-Venus fusion proteins in E9.5 CAG::GFP-GPI/+; CAG::myr-Venus/+ double transgenic embryo. Linear unmixing of the independent GFP-variant fluorophores was used to identify unique and overlapping regions of reporter (more ...)
To extend our observations of live ES cells and early embryos to verify labeling of the secretory pathway, and in particular the Golgi apparatus, the subcellular localization of the GFP-GPI and myr-Venus fusions was determined with respect to Golgi markers. Markers used included antibodies directed against a 58K microtubule binding protein associated with the cytoplasmic surface of the Golgi apparatus () and β-COP (data not shown). Cells were processed for indirect immunofluorescence, with fluorescent protein expression visualized directly. The protocol used required both fixation of cells and subsequent detergent permeabilization and resulted in a more diffuse localization of the lipid-modified fluorescent proteins compared to live cells.
FIG. 6 Colocalization GFP-GPI or myr-Venus fusion proteins with Golgi markers in ES cells. Undifferentiated GFP-GPI ES cells (a) were stained and visualized for DAPI to label nuclei (blue), GFP was visualized directly (green), 58K Golgi was visualized with indirect (more ...)
Since ES cells grow in clumps and have an unusual morphology, with a large nucleus and minimal cytoplasm, we were unable to unequivocally confirm colocalization of the lipid-modified fluorescent proteins with secretory pathway markers (). However, GFP-variant fluorescence was covisualized with indirect immunofluorescence using the 58K Golgi-specific antibodies in ES cells that had been induced to differentiate (regions appearing yellow in merged panels, ). The colocalization of GFP-GPI and myr-Venus fluorescence with Golgi markers confirmed the localization of these fusions to the Golgi apparatus, suggesting that these fusion reporters could be used to monitor Golgi dynamics. Furthermore, the ability to visualize the position of the Golgi within individual cells in vivo should permit the determination of polarity in certain cell types.
A current challenge in the fields of cell and developmental biology is to directly visualize the spatial and temporal organization of intracellular events, including the dynamic and polarized nature of vesicle transport (Rothman, 2002
). Vesicles represent the compartmentalization of modular components that are critical for the regulation of different processes, including cell polarity (Nelson, 2003
) and cell fate (LeBorgne, et al., 2005
; Wang and Struhl, 2005
). To illustrate the utility of the CAG::GFP-GPI
transgenic lines for this purpose, we examined individual vesicular populations in live ES cells and embryonic tissues ().
FIG. 7 Differential vesicular distributions of GFP-GPI and myr-Venus fusion proteins in ES cells and in the somites of E9.5 transgenic embryos. Hemizygous GFP-GPI ES cells (a), myr-Venus ES cells (b), E9.5 GFP-GPI (c,e,g) or E9.5 myr-Venus (d,f,h) embryos were (more ...)
To label recycling endosomal populations, we cultured ES cells and E9.5 embryos in media supplemented with Alexa-Fluor 568-conjugated 10K Dextran and imaged them. We observed both distinct (green or red arrowheads, ) and overlapping (yellow arrowheads, ) distributions of the lipid-modified fluorescent protein-tagged proteins and moieties in ES cells having taken up and therefore been tagged with 10K Dextran (red arrowheads, ). Furthermore, to demonstrate these properties in cells operating in the context of their normal microenvironment, we next examined these two distributions in somites (). Lateral views at low () or high () magnification are presented to show the gross and fine distributions of both the fluorescent protein fusions and 10K Dextran in live tissues. We found that 10K Dextran became trapped in the extracellular matrix of live tissue, resulting in high background fluorescence (red, ). High-magnification views of somites show that 10K Dextran is localized in discrete vesicles, which appear similar to those observed in ES cells. These Dextran-positive vesicles both overlap (yellow arrowheads, ) and are distinct (red arrowheads, ) with the respective fluorescent protein fusions (green arrowheads, ). Combined, these data suggest that GPI-GFP and myr-Venus localize to distinct vesicles and overlap with endosomes of the secretory pathway.