Characterization of a Dmd EGFP reporter mouse as a tool to investigate dystrophin expression
Generation and validation of the Dmd EGFP
reporter mice
For generation of the Dmd EGFP
mice, the murine dystrophin locus on the X-chromosome was modified using a targeting
vector (Fig. 1a). This resulted in a modification of dystrophin exon 79 whose natural termination
codon was removed and a C-terminal fusion protein generated with a FLAG- and an EGFP-L221K
coding sequence. The 3?UTR further contained a loxP flanked neomycin (neo) cassette for ES-cell selection (Fig. 1a). After germline transmission, the neo cassette was successfully removed by transient crossbreeding with ubiquitous Cre-deleter
mice. Dmd EGFP
transgenic animals could be identified by allele-specific PCR (Fig. 1b). Hemizygous males, as well as hetero- and homozygous females were viable and fertile
and were analyzed in comparison with their wild-type littermates.
An important consideration for our targeting strategy was the site for EGFP insertion
into the endogenous dystrophin locus. The aim was (i) to target the major dystrophin
isoforms that are expressed in most of the relevant tissues and (ii) to find out whether
the natural endogenous dystrophin-EGFP expression would be strong enough for detection
without further amplification by immunofluorescence. Since the FLAG-EGFP tag was appended
to the C-terminus of dystrophin, to a protein domain where several interacting proteins
bind, we had to investigate (iii) whether the genetic manipulation might cause a dystrophic
phenotype per se and whether (iv) dystrophin and its binding partners from the DAPC
would have the correct subcellular localization. (v) Finally, we wanted to investigate
whether the published data in the literature on tissue-specific dystrophin expression
patterns would correspond to those seen in our Dmd EGFP
mice.
The aim of our study was to generate a reporter mouse, in which dystrophin expression
can be visualized and tracked in different tissues by means of EGFP fluorescence.
Dystrophin and its numerous isoforms and splice variants share the C-terminal domain,
which is very important for the interaction of the protein with its binding partners
and might thus interfere with proper function; however, previous studies had already
shown that C-terminally EGFP-tagged mini- or micro-dystrophins in transgenic mice
or cells would be functional 64]–66]. Therefore, we inserted the FLAG-EGFP sequence downstream of exon 79, the last exon
present in the major dystrophin isoforms (Additional file 1: Figure S1). Insertion of the FLAG-EGFP downstream of exon 79, would theoretically
tag the C-terminus and express the following proteins as fusions: Dp427 (B, M, P),
Dp260, Dp140, Dp116, Dp71, Dp71d, and Dp71c. The alternative hydrophobic C-terminus
of Dp71f and Dp71
?110
would not be targeted in our model since skipping of exon 78 shifts the reading frame
of the last exon 79 due to alternative splicing (Additional file 1: Figure S1) 31], 67]. In addition, Dp40 expressing another alternative C-terminus would not be tagged
as well.
In order to investigate the expression pattern of the fusion protein, we first analyzed
the skeletal muscle of Dmd EGFP
mice. The presence of the dystrophin-EGFP fusion protein was confirmed by Western
blot analysis (Fig. 1c) using dystrophin antibodies against the rod and C-terminal domains in TA protein
lysates. The bands from wild-type dystrophin and from dystrophin-EGFP had similar
expression intensities as detected by the rod-domain-specific Dys1 antibody (Fig. 1c). Moreover, using MANDYS19 as another rod-domain-specific antibody, we compared dystrophin
expression in protein extracts from the TA muscle of normal controls and of Dmd EGFP
mice over 3 orders of magnitude and found comparable protein concentrations of the
wildtype Dp427 and the Dp427-EGFP band (Additional file 1: Figure S12). However, the band intensity of the Dmd EGFP
samples was lower if the C-terminal-specific (H4, Fig. 1c) antibody was used. A possible explanation could be a reduction of binding affinity
of the H4 antibody through steric hindrance by the FLAG-EGFP tag. A similar difference
was found in the Western blot of brain samples (Additional file 1: Figure S13). The molecular sizes of the proteins detected in both wild-type and
Dmd EGFP
samples were comparable since differences in molecular weight of 27.9 kDa between
full-length dystrophin and dystrophin-EGFP would not be easily detectable on Western
blot at molecular weights beyond 400 kDa. We detected an EGFP band in the molecular
size range of dystrophin only in the muscle and brain of Dmd EGFP
mice (Fig. 1c, Additional file 1: Figure S13B). Furthermore, the strong expression of dystrophin-EGFP could be directly
observed under the microscope via the natural green fluorescence localized at the
sarcolemma (Fig. 1d), while the EGFP signal showed exact superposition with the immune signal from the
anti-FLAG antibody on TA sections (Fig. 2, first column).
Fig. 2. Correct localization of the EGFP-tagged dystrophin at the sarcolemma. Immunofluorescent
staining of cryosections from the tibialis anterior of Dmd EGFP
mice with antibodies against the FLAG-tag, C-terminal dystrophin (Dys2), rod-domain
dystrophin (MANDYS19), cytoskeletal ?-spectrin, and the basement membrane protein
laminin (all colored in red). Exact colocalization was observed between the natural EGFP fluorescence (green) and the signals deriving from the two anti-dystrophin antibodies. Merged images
were counterstained with DAPI (blue)
Dystrophin-EGFP expression in skeletal muscle and normal muscle morphology in Dmd EGFP
reporter mice
The correct localization of native EGFP fluorescence was further confirmed in TA,
soleus, and gastrocnemius muscles by co-immunostaining with anti-dystrophin antibodies against the rod (MANDYS19)
and C-terminal (Dys2) domains (Fig. 2, Additional file 1: Figure S2), which showed an exact superposition of the signals. MANDYS19 binds to
the amino acid sequence encoded by dystrophin exons 21-22 and thus recognizes only
the full-length protein 68]. All the alternative dystrophin promoters that control the expression of shorter
dystrophin isoforms are located downstream of intron 29, and dystrophin isoforms generated
from these promoters would not be picked up by the MANDYS19 antibody. Hence, the signal
from the MANDYS19 antibody, which was similar to the signal of the Dys2 C-terminal
antibody, confirmed the correct tagging and subcellular localization of the skeletal
muscle Dp427 isoform. Correct expression of the ECM component laminin as well as of
the subsarcolemmal ?-spectrin was also confirmed on skeletal muscle sections (Additional
file 1: Figure S3), where ?-spectrin colocalized with dystrophin-EGFP in the myofibers.
Importantly, a comparison between the EGFP and laminin signals exhibited, as expected,
distinct differences, with EGFP being present at the cytoplasmatic side of the sarcolemma
and excluded from the capillaries. Notably, the endothelial cells of the endomysial
capillaries were also stained by the anti-?-spectrin antibody where dystrophin-EGFP
expression was absent. This suggests that dystrophin expression in the vascular system
is confined to larger vessels that possess a tunica media (e.g., arteries and larger veins) 69].
The C-terminal domain of dystrophin plays an important role in the binding and assembly
of the DAPC, which has a crucial role for muscle function and sarcolemmal stability
70]. Adding a 27.9-kDa EGFP tag to the C-terminus of dystrophin might hence interfere
with proper protein folding, its subcellular localization, and protein function, thereby
provoking a muscular dystrophy such as seen in the mdx mouse model for Duchenne muscular dystrophy (DMD). We thus investigated the skeletal
muscle phenotype. HE staining of cross sections from different skeletal as well as
from heart muscle did not show any differences between the adult reporter mice and
their wild-type littermates (Fig. 3a, Additional file 1: Figure S4). Moreover, no dystrophic changes could be detected in 10-month-old Dmd EGFP
mice, which were well beyond the age when dystrophic changes are commonly encountered
in mdx mice. For comparison, we analyzed aged-matched mdx mice, which exhibited strong signs of fibrosis, immune cell infiltration, and central
nucleation—all hallmarks of an advanced dystrophic process (Fig. 3a). Thus, histological analysis confirmed that EGFP tagging of the dystrophin C-terminus
did not cause overt muscle pathology. This notion was further confirmed by measurement
of normal CPK activities in the serum of Dmd EGFP
mice, which were largely elevated only in the mdx mice (Fig. 3c). Moreover, analysis of fiber size (minimal Feret diameters) of EDL and soleus muscles
from Dmd EGFP
in comparison to wild-type mice did not reveal any significant differences (Fig. 3d).
Fig. 3. Dmd EGFP
mice do not show signs of muscle dystrophy. a Hematoxylin and eosin (HE) staining of tibialis anterior (TA) muscle sections from adult (8- to 12-week-old) and aged (10-month-old) wild-type
(WT) and Dmd EGFP
mice show normal morphology. For comparison, HE staining of an aged-matched mdx TA muscle shows the morphological characteristics of muscle dystrophy comprising
a large variation of fiber size, mononuclear cell infiltrates, fibrosis, and abundant
centrally located myonuclei. b Wild-type and Dmd EGFP
mice show normal localization and expression of the DAPC components ?-dystroglycan,
?-sarcoglycan, and nNOS in TA muscle cross sections. c Serum creatine phosphokinase (CPK) activities in the serum of wild-type, Dmd EGFP
, and mdx mice (n?=?3 of each genotype). Values are shown as means?±?S.E.M., p values were calculated using one-way ANOVA. d The histograms depict the minimal Feret diameter of muscle fibers from EDL muscles
(upper panel) and soleus muscles (lower panel) from adult wild-type and Dmd EGFP
mice. Values are shown as means?±?S.E.M. (n?=?3 mice from each genotype were analyzed)
Finally, we screened different skeletal muscles for the expression of representative
members of the various DAPC sub-complexes. We confirmed normal sarcolemmal/subsarcolemmal
expression patterns for ?-, ?-, ?-sarcoglycan, ?-dystroglycan, as well as for nNOS
suggesting proper function of the fusion protein (Fig. 3b, Additional file 1: Figures S5-S9). We conclude that no deleterious changes of the dystrophin protein
structure might have occurred that would disturb those interactions. However, for
some components, it appeared as if the expression levels would differ between Dmd EGFP
and wild-type mice. However, the DAB-mediated visualization of immune signals is not
really quantitative and subject to too many confounding factors in order to pick up
subtle differences. Here, it would be preferable in the future to employ mass spectrometric
techniques to identify and quantify proteins that bind to the dystrophin-EGFP, which
could be easily immunoprecipitated with an anti-FLAG or anti-GFP antibody 71].
Dystrophin-EGFP expression in cardiac and smooth muscle
Dystrophin expression in the heart is controlled by the muscle- and brain-specific
promoters, while expression in the smooth muscles of the gastrointestinal (GI) tract
is controlled by the muscle-specific promoter. We confirmed correct dystrophin-EGFP
expression in the heart (Fig. 4a) as well as in the ileum (Fig. 4b) as a representative part of the GI tract through verification of colocalization
between the natural EGFP signal and signals generated by anti-dystrophin antibodies.
Moreover, intramuscular cardiac blood vessels showed an EGFP signal as well, which
partially colocalized with the anti-CD31 signal serving as a marker for endothelial
cells (Fig. 4a, right column). Dystrophin is known to be expressed in the walls of large arteries
and veins as well as in small arteries 69], in all those vessels that are able to regulate their vascular tone through contraction
of the smooth muscle cells in their tunica media. In contrast, dystrophin was absent from small veins (not shown), whose diameter
is only passively regulated by the blood volume and elastic fibers.
Fig. 4. Dystrophin-EGFP expression in heart and smooth muscle. a Colocalization at the sarcolemma of the immunofluorescent signal from dystrophin
(Dys2) and natural EGFP fluorescence (green) in heart muscle. Blood vessel endothelium is stained with anti-CD31 (red) and also exhibits, at least, partial colocalization with the EGFP signal. b Immunofluorescent staining with a C-terminal rabbit anti-dystrophin antibody (red) of ileum sections verifies colocalization with natural EGFP fluorescence (green). Sections were counterstained with DAPI (blue)
The localization of dystrophin near the plasma membrane of smooth muscle cells via
its natural EGFP fluorescence could be readily observed in cryosections of other intestinal
organs as well. In cross sections of stomach, ileum, and duodenum of Dmd EGFP
mice, we observed bundles of longitudinal and circular layers of smooth muscle cells
with dystrophin located at their periphery (Additional file 1: Figure S10A). The distribution of the EGFP signal at the membrane of smooth muscle
cells was discontinuous, which is in line with previous studies using dystrophin antibodies
24]. This phenomenon is due to the exclusion of dystrophin from the adherens junctions between smooth muscles 72].
Dystrophin-EGFP expression in non-muscle tissue
In order to investigate whether the different dystrophin isoforms were tagged in non-muscle
tissues as well, we analyzed the EGFP expression in the brain and retina of Dmd EGFP
mice. Western blot analysis of whole brain lysates from Dmd EGFP
mice and wild-type littermates (Additional file 1: Figure S13) revealed only very low levels of the full-length dystrophin Dp427 isoform
using the rod domain-specific Dys1 antibody. Similar low levels of Dp427 could be
detected with an anti-GFP antibody in Dmd EGFP
samples. The Dp140 and Dp71 isoforms could be detected with the C-terminal specific
H4 antibody only in wild-type but not in Dmd EGFP
samples, which further confirms our observation from Western blot analysis on TA lysates
that the FLAG-EGFP-tag causes steric hindrance against binding of the H4 antibody.
In the Dmd EGFP
samples, the corresponding bands, which run at a higher molecular weight (of 170 and
100 kDa, respectively), were detected using the anti-GFP antibody, confirming the
correct targeting of the Dp427, Dp140, and Dp71 isoforms in the brain of the Dmd EGFP
mice. Furthermore, we confirmed the natural EGFP expression in cryosections of the
brain where the EGFP signal showed exact colocalization with the immune signal from
staining with an anti-dystrophin antibody (Fig. 5, left column).
Fig. 5. Dystrophin-EGFP expression in the brain. Immunofluorescent staining of brain sections
with the Dys2 antibody (red) shows colocalization of the natural EGFP signal (green) with the dystrophin signal in the cerebellum of Dmd EGFP
mice. Partial colocalization of the EGFP signal with the anti-CD31 (red) signal confirmed the presence of dystrophin expression also in the blood vessels
of the brain (second column). Open arrowheads show the EGFP signal surrounding the signal from the CD31 marker.
Immunofluorescent staining with anti-glial fibrillary acidic protein (GFAP, red) shows colocalization with the EGFP signal at the glial endfeet in the cerebellum (third column). A higher magnification of the anti-GFAP signal is shown in the fourth column. EGFP expression is confined to the glial endfeet (asterisk), whereas the green fluorescent tag is not expressed in the glial cell body (closed arrowheads)
In the retina of Dmd EGFP
reporter mice, EGFP expression was observed at the outer plexiform layer (OPL), the
inner limiting membrane (ILM), and sporadically at the inner nuclear layer (INL) (Fig. 6a). A strong punctate EGFP signal colocalized with dystrophin immunofluorescence and
was detectable in the OPL of the retina (Fig. 6b). Exact colocalization of the natural EGFP signal was detected when using a C-terminal
anti-dystrophin antibody (Dys2). However, the rod domain-specific MANDYS19 antibody
did not recognize all EGFP-positive structures confirming the targeting of different
isoforms in the retina. Although it is widely accepted that dystrophin is expressed
at the photoreceptor terminals 25], a recent in-depth study of dystrophin expression in the retina suggested the presence
of dystrophin also in the OPL layer, where rod and cone bipolar cells of the ON-type
are located. Of note, the authors could not exclude artifacts by fluorescent signals
from neighboring regions 53]. Hence, using the Dmd EGFP
mice, a better characterization of this region and the distribution of dystrophin
in the retina could be achieved without the use of antibodies and potentially associated
staining artifacts.
Fig. 6. Dystrophin-EGFP expression in the retina. a Presence of the natural EGFP signal in the different layers of the retina: outer
plexiform layer (OPL), inner nuclear layer (INL), and inner limiting membrane (ILM)
(left image). These regions are depicted on a corresponding HE stained cross section from the
retina of a wild-type mouse (right image). A higher magnification of the EGFP-positive regions highlighted by the squares
in a is presented in b and c. b Immunofluorescent staining with antibodies against dystrophin (Dys2, MANDYS19, red) and CD31 (red) showed differential colocalization of natural EGFP signal (green) in the retinal OPL of Dmd EGFP
mice. Arrows indicate EGFP-positive retinal blood vessels that were detected with Dys2 and anti-CD31
antibody, but not with the rod-specific antibody. Strong punctuated expression of
the natural EGFP was observed in the OPL region corresponding to the photoreceptor
terminals. In this region, an exact superposition with the Dys2 signal and some overlay
with the MANDYS19 signal were observed, whereas no anti-CD31 signal was detected.
c Immunofluorescent staining with antibodies against the glial fibrillary protein (GFAP,
red) showed partial colocalization with the natural EGFP signal at the ILM. Arrowheads indicate colocalization between GFAP and EGFP at the glial endfeet. The asterisk marks the GFAP positive region, which did not express EGFP, corresponding to the
glial cell body
In the brain and retina of Dmd EGFP
mice, we observed natural dystrophin-EGFP expression around blood vessels that were
stained with the anti-CD31 endothelial cell marker (Figs. 5 and 6b). Moreover, staining with an antibody against the glial fibrillary acidic protein
(GFAP) revealed partial colocalization of the dystrophin-EGFP with the Bergmann glia
in the cerebellum (Fig. 5), the glial cells of the hippocampus (Additional file 1: Figure S10B), and the Müller cells at the inner limiting membrane of the retina
(Fig. 6c). In these regions, the EGFP signal only colocalized with the glial endfeet, which
confirms a characteristic expression pattern of the Dp71 isoform 73]. On the other hand, dystrophin is also expressed in the blood vessels, mainly in
the wall surrounding the endothelial cells. The EGFP signal was at some locations
exactly encircling the CD31 immunosignal (Fig. 5), suggesting expression of the dystrophin in the outer walls of the vessels, but
not in the vascular endothelium. However, from our immunofluorescent images, it did
not become entirely clear, whether the green fluorescence really derived from the
vessel wall or from the glial endfeet. Here, high-resolution imaging techniques like
STED microscopy could resolve the issue.
It was reported in the literature that the full-length dystrophin isoform in the brain
was expressed in neurons such as in Purkinje cells of the cerebellum or in neurons of the hippocampus21], 74], 75]. Unfortunately, we were unable to detect any natural EGFP fluorescence in the aforementioned
regions using our standard fluorescence EGFP-imaging techniques. However, via immunostaining
using an anti-GFP antibody, we detected neuronal dystrophin-EGFP expression in CA1/CA2
regions of the hippocampus and in Purkinje cells (Additional file 1: Figure S11). The apparent absence of natural EGFP fluorescence in the neurons of
Dmd EGFP
mice could be explained by the much lower expression levels of the full-length Dp427
(B) dystrophin in the brain in contrast to the Dp71 isoform, which gave much stronger
signals in the Western blot analysis (Additional file 1: Figure S13). Moreover, immunofluorescence staining with an anti-GFP antibody shows
large differences in the GFP signal intensities between Dp71-expressing blood vessels
and Dp427-expressing neurons. For the detection of dystrophin-EGFP in the neurons,
we had to use high exposure times that led to massive overexposure of the EGFP-positive
blood vessels (Additional file 1: Figure S11). Even after immunostaining with an anti-GFP antibody, using lower exposure
times to image the blood vessels would fail to visualize the neurons.
EGFP fluorescence is detectable on the level of single muscle fibers and in satellite
cell-derived myotubes
On the level of single myofibers, we wanted to explore whether the presence of dystrophin-EGFP
would allow live-cell imaging of dystrophin expression in cultures of isolated myofibers.
Indeed, freshly isolated myofibers from Dmd EGFP
mice were strongly EGFP-fluorescent and stood in stark contrast to the dark wild-type
fibers (Fig. 7a). In addition, strong dystrophin-EGFP expression was detected at the neuromuscular
junction (NMJ) of single fibers that had been co-stained with AlexaFluor568-labeled
?-bungarotoxin, as a specific marker for acetylcholine receptors (Fig. 7b). Hence, the strong EGFP expression at the NMJ makes our model suitable for longitudinal
in vivo studies on the dynamics of dystrophin at the NMJ.
Fig. 7. Dystrophin-EGFP expression of single fibers. a Isolated single EDL muscle fibers from Dmd EGFP
mice and wild-type littermates. The natural EGFP fluorescence (green) permits easy distinction between Dmd EGFP
and wild-type fibers without necessity for immunostaining. Fibers were fixed and stained
with DAPI (blue). b Natural EGFP expression at the neuromuscular junction of a single fiber co-stained
with AlexaFluor568 labeled ?-bungarotoxin (BTX, red). The higher magnification shows colocalization of natural dystrophin EGFP with the
acetylcholine receptor. Fibers were counterstained with DAPI
Finally, we wanted to find out whether freshly isolated satellite cells cultured in
vitro would express dystrophin once they formed myotubes. Unfortunately, after 8 days
of culture, the natural EGFP signal in the myotubes was too weak to be picked up by
epi-fluorescence microscopy, possibly due to the still too low numbers of available
dystrophin molecules. Interestingly, using a well-established anti-GFP antibody (Additional
file 2: Table S1), the dystrophin-EGFP fusion protein was detected with a much stronger
signal then using any of the anti-dystrophin antibodies (Fig. 8). This further confirms the usefulness of our animal model to study dystrophin expression
during myogenesis or muscle regeneration. The ability of ex vivo satellite cells from
Dmd EGFP
reporter mice to produce dystrophin EGFP once differentiating could serve as a valuable
tool to be exploited for transplantation and cell tracing experiments.
Fig. 8. Dystrophin-EGFP expression of satellite cell-derived myotubes. Dystrophin-EGFP expression
of in vitro differentiated satellite cells; isolated single fibers with their attached
satellite cells from wild-type (WT) and Dmd EGFP
mice were cultured for 4 days and differentiated for a further 8 days. Forming myotubes
were fixed and stained with anti-GFP (green) and Dys2 (red) antibodies and counterstained with DAPI (blue)