Biphasic Myopathic Phenotype of Mouse DUX, an ORF
within Conserved FSHD-Related Repeats
Darko Bosnakovski1,2,4, Randy S. Daughters3, Zhaohui Xu4, Jonathan M. W. Slack3, Michael Kyba1,3,4*
1Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, United States of America, 2Faculty of Technology and Technical
Science, University St. Kliment Ohridski, Veles, Republic of Macedonia, 3Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, United States of America,
4Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
Facioscapulohumeral muscular dystrophy (FSHD) is caused by contractions of D4Z4 repeats at 4q35.2 thought to induce
misregulation of nearby genes, one of which, DUX4, is actually localized within each repeat. A conserved ORF (mDUX),
embedded within D4Z4-like repeats, encoding a double-homeodomain protein, was recently identified on mouse
chromosome 10. We show here that high level mDUX expression induces myoblast death, while low non-toxic levels block
myogenic differentiation by down-regulating MyoD and Myf5. Toxicity and MyoD/Myf5 expression changes were
competitively reversed by overexpression of Pax3 or Pax7, implying mechanistic similarities with the anti-myogenic activity
of human DUX4. We tested the effect of mDUX expression on Xenopus development, and found that global overexpression
led to abnormalities in gastrulation. When targeted unilaterally into blastomeres fated to become tail muscle in 16-cell
embryos, mDUX caused markedly reduced tail myogenesis on the injected side. These novel cell and animal models
highlight the myopathic nature of sequences within the FSHD-related repeat array.
Citation: Bosnakovski D, Daughters RS, Xu Z, Slack JMW, Kyba M (2009) Biphasic Myopathic Phenotype of Mouse DUX, an ORF within Conserved FSHD-Related
Repeats. PLoS ONE 4(9): e7003. doi:10.1371/journal.pone.0007003
Editor: Joanna Mary Bridger, Brunel University, United Kingdom
Received June 6, 2009; Accepted August 19, 2009; Published September 16, 2009
Copyright: ? 2009 Bosnakovski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: DB was supported by a Muscular Dystrophy Association Development Grant (MDA 4361) and a fellowship supplement from the Facioscapulohumeral
Muscular Dystrophy (FSHD) Society. RSD was supported by Training Grant NIH T32 AR050938 ‘‘Musculoskeletal Training Grant’’. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Facioscapulohumeral muscular dystrophy (FSHD), one of the
most common inherited myopathies, is caused by a contraction
within a subtelomeric array of D4Z4 repeats 4q35.2. FSHD-
affected individuals carry from 1 to a maximum of 10 repeats,
while unaffected individuals have from 11 to 150 copies of 3.3 kb
D4Z4 elements [1,2]. The D4Z4 repeat is very GC rich and
contains an intronless double homeobox gene named DUX4
(double homeobox, chromosome 4). It also contains a heterochro-
matic LSau sequence and sequence elements that bind the
repressor elements YY1, HMGB2 and nucleolin . Several
molecular mechanisms have been proposed to link the D4Z4
contraction to up-regulation of FSHD candidate genes, including
multimerization of YY-1-containing transcriptional repressor
complexes , hypomethylation of the contracted D4Z4 allele
, differential looping interactions  or loss of a barrier to an
enhancer distal to the repeats .
A C-terminally-truncated inverted D4Z4 element is localized
42 kb centromeric to the array and referred as DUX4c . A
number of other FSHD candidate genes, including FRG1,
TUBB4Q, FRG2 and ANT1 [2,8,9,10] are also localized to the
centromeric side of the D4Z4 array. Recent publications have
documented low but detectable levels of DUX4 transcript and
protein in biopsy samples and myoblast cultures from FSHD
patients but not unaffected controls [11,12]. We have recently
shown that DUX4, but not DUX4c, increases susceptibility to
oxidative stress due to downregulation of the genes involved in the
glutathione redox cycle, and that both DUX4 and DUX4c rapidly
downregulate MyoD expression resulting in impaired myogenic
differentiation [13,14]. Both changes in glutathione redox
potential and effects on MyoD have been identified in studies on
FSHD myoblasts and biopsies [15,16,17,18,19]. We also demon-
strated that the key myogenic regulators Pax3 and Pax7, two
proteins with homeodomains similar to DUX4 and predicted to
compete for the same DNA recognition sequences, did in fact
compete for regulation of MyoD and Myf5, and significantly
reversed the toxicity of DUX4 to myoblasts .
Recently, Clapp et. al. analyzed DNA sequence data from
primates, rodents, afrotherian and other species and concluded
that a D4Z4-like repeat family containing an ORF encoding a
double homeodomain, is evolutionally conserved and arose over
100 million years ago . The mouse representative (named
mDUX) contains a 2 kb ORF embedded within a 5 kb repeat unit
on chromosome 10 . Using specific partial primer sets and in
situ hybridization, bidirectional transcription from different tissues
including brain, heart, lung and muscle was documented. The
possibility of bidirectional transcription, (as well as various splice
forms, and potential miRNAs within D4Z4) has also been
demonstrated by Snider et al. .
Although the double homeodomain sequences are strongly
conserved between mDUX and human DUX4, the remainder of
the proteins are highly divergent. We therefore tested whether
mDUX expression could affect viability, myogenic gene expres-
sion, or myogenic differentiation potential both in vitro using a cell-
based assay, and in vivo by microinjection of mDUX RNA into
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developing Xenopus embryos. We find remarkable molecular
similarities between mouse DUX and human DUX4 or DUX4c
and discuss the value of engendering animal models based on
altering the regulation of endogenous D4Z4-like repeats, or
conditionally expressing of the ORF they encode.
Results and Discussion
Generation of mDUX inducible cell lines and evaluating
We wished to use a conditional gain of function approach to
analyze the effect of mDUX, because we reasoned that mDUX
could have toxic characteristics similar to human DUX4 [13,14].
For this purpose, we used C2C12 myoblasts, 3T3 fibroblasts, and
murine ES cells modified for inducible cassette exchange, a
method that enables rapid gene targeting to a locus that is tightly
and conditionally regulated. In these cell lines, mDUX can be
turned on and off, and expression can be regulated over a 3-log
range of mRNA levels by titrating the concentration of the
inducer, doxycycline . We refer to the derivative inducible cell
lines as iC2C12-mDUX, i3T3-mDUX and iES-mDUX, respec-
mDUX expressed at high level in iC2C12-mDUX myoblasts
induced rapid cell death within 24 hours (Fig. 1A). Detached cell
aggregates floating in the culture medium were visible after
18 hours of induction. Using propidium iodide staining and FACS
analyses we confirmed that floating cells are dead and not live but
detached cells (data not shown). Remarkably, we did not observe
significant structural or morphological changes in the induced cells
prior to lifting and dying (Fig. 1A) as we have observed with
DUX4 which, before dying, stretch out to acquire an elongated
cell shape and ovoid nucleus . Necrotic phenotypes have been
observed in myoblasts cultured from FSHD patients . We next
used an ATP assay to analyze viability of the mDUX expressing
myoblasts over a wide range of levels of induction with
doxycycline. A significant decrease of cell viability was detected
in the cultures induced with as little as 32 ng/mL doxycyline in the
first 24 hours (Fig. 1B). This trend increased in the following
24 hours, where toxicity became obvious even in the cells induced
with lower doses (16 ng/mL). We did not detect any significant
effect of doxycycline on the parental iC2C12 nor C2C12 (grand-
parental) cells (Figure 1H). By using annexin V/7-AAD staining
we demonstrated that the cells undergo apoptosis and did not just
decrease the proliferation rate (Figure 1C). At high concentrations
of doxycycline (500 ng/mL), the first signs of increased apoptosis
and cell death were evident after 12 hours of induction. By
24 hours, 30% of cell-sized events were apoptotic and 44% were
dead (Fig. 1C). This data demonstrates a clear deleterious effect of
mDUX on myoblasts, very much like that exhibited by DUX4
when expressed at high levels [12,14]. FSHD myoblasts have been
reported to be highly susceptibile to oxidative stress  and
several studies analyzing RNA and protein expression in FSHD
biopsy samples confirmed misregulation of a number of factors
involved in glutathione redox buffering [16,18,22]. We observed
similar effects when DUX4 was expressed in iC2C12-DUX4 cells
and showed that antioxidants were able to moderate toxicity of
even very high levels of DUX4 expression . To test whether
antioxidants could rescue the toxic phenotypes of mDUX, we
treated iC2C12-mDUX cells with ascorbic acid, b-mercaptoeth-
anol and monothioglycerol. iC2C12-mDUX were induced with 32
and 125 ng/mL doxycyline, treated with serial dilutions of these
antioxidants and analyzed by ATP assay after 24 hours.
Surprisingly, we did not observe any beneficial effect of the
antioxidants even in the cells which were induced with low levels
of doxycyline (32 ng/mL) (Fig. 1D and E), demonstrating that key
aspects of mDUX toxicity are distinct or perhaps more potent that
those of DUX4.
To determine whether mDUX was exclusively toxic for
myoblasts or would be toxic in other cell types, we evaluated
fibroblasts (i3T3-mDUX)  and murine embryonic stem cells
(iES-mDUX) . mDUX expressed at high levels in fibroblasts
and ES cells also induced rapid cell death (Fig. 1F and G).
Together these results show that mDUX, like human DUX4, is
generally toxic for various cell types when expressed at high levels.
However, unlike DUX4, oxidative stress is not the principal reason
for death of cells expressing mDUX. Given this toxicity, it is
remarkable that transcription of mDUX has been reported in
various tissues, including robust expression in CNS, lung and liver
. Which cell types within those organs, and at which stages
they express mDUX, was not shown, however splicing variants
were suggested, and these could theoretically result in non-toxic
versions of the protein. We used the whole mDUX ORF sequence
(2 kb) as a template to generate in situ hybridization probes and
were not able to detect any robust and convincing mDUX
expression (from the sense strand) in embryonic neural tube, adult
brain or muscle tissue (data not shown).
mDUX and myogenic regulators
mDUX, DUX4 and DUX4c each contain two homeoboxes of
the Pax family, each with significant sequence similarity to the
Pax3 and Pax7 homeoboxes . These Pax factors are the
master myogenic regulators of embryonic and adult myogenesis,
respectively (for review see ). In our previous work, we
demonstrated that DUX4 and DUX4c both interfere with the
expression of myogenic regulators and suggested that this was due
to competitive binding between DUX and Pax proteins for the
same regulatory sites in these genes. The apical targets of Pax3/
Pax7 in the cascade of myogenesis are MyoD and Myf5 [25,26].
One interesting difference between DUX4 and DUX4c in this
regard is that DUX4 upregulates Myf5 about two-fold in
myoblasts (more in fibroblasts) whereas DUX4c represses Myf5
[13,14]. For this reason we analyzed the expression of MyoD and
Myf5 in mDUX-expressing myoblasts. As predicted, transcription
of MyoD was rapidly downregulated (seen by 4 hours post-
induction) with 500 ng/mL doxycyline (Fig. 2A). We confirmed
these rapid kinetics at the protein level using immunofluorescence
(Fig. 2B). For Myf5 we detected a slight downregulation after
4 hours and a more significant downregulation after 8 hours of
induction (Fig. 2A). As a consequence of the MyoD suppression,
some of its target genes including myogenin and m-cadherin were
also downregulated (Fig. 2A). MyoD binds to noncanonical E
boxes in the myogenin gene through interactions with Pbx1 and
Meis1 . In mDUX-induced cells, we detected a slight
repression of Pbx1 (Fig. 2A). On the other hand Pbx3, Pbx4,
Meis1 and Meis2 remained unchanged (data not shown).
Interestingly, we found that Pax7 was also suppressed, suggesting
the possibility of interference with a Pax7 positive autoregulatory
loop. Three lines of evidence suggest that these changes are not
simply due to cells becoming apoptotic and downregulating gene
expression generally. First, DUX4 has many upregulated targets
, and we discovered at least one upregulated target of mDUX,
namely MEF2C (Fig. 2A). Second, genes downstream of MyoD
are also repressed, but with correspondingly delayed kinetics. For
example, desmin expression is not significantly affected until
12 hours (Fig. 2A), suggesting a secondary effect resulting from
MyoD depletion. Third, MyoD was downregulated by mDUX
induced with as little as 8 ng/ml doxycyline for 24 hours (Fig. 2C).
At that level we did not observe any significant toxic effect even
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Figure1.ToxicityofmDUX.(A)MorphologyofiC2C12-mDUXcellsinducedfor24 hours(Dox)with500 ng/mldoxycycline.Themajorityofinducedcells
weredetachedandfloatingafter24 hours.(B)ATP assay foranalysis ofviability in iC2C12-mDUX cellsinducedwith variousconcentrationsofdoxycylinefor
24and48 hours. Decreasedcellviabilitywas significant inthecellsinducedwith aslittleas32 ng/mldoxycyclineinthefirst24 hours.Resultsarepresented
as fold difference compare to untreated cells at 24 hours. (C) FACS analysis of annexin V/7-AAD stained cells for determination of apoptosis and cell death.
Single annexin V positive cells (x-axis, bottom right corner) represent cells undergoing apoptosis, and double positive cells (annexin V+and 7-AAD+, right
top population) represent dead cells. A slight increase of apoptotic and dead cells was detected at 12 hours which progressed to significant after 24 hours
of induction. (D) ATP assay on the cells induced for 24 hours demonstrated that antioxidants (AsAc: ascorbic acid (21.25 mM), B-MET: b-mercaptoethanol
(0.5 mM), MTG: monothioglycerol (2.25 mM)) did not have any beneficial effect on cell viability even in cells treated with the low dose of doxycycline
(32 ng/ml). (E)Morphology of cells,either uninduced(Control), mDUX-induced(Dox, 125 mg/ml) or induced andtreatedwith antioxidants.(F) Morphology
of mDUX inducible fibroblasts (i3T3-mDUX) and inducible mDUX embryonic stem cells (iES-mDUX) (G) after 24 hours of induction with 500 ng/ml
doxycyline. mDUX expressed at high levels induces cell deathin fibroblasts and embryonic stem cells. (H) ATP assay for effectsof doxycycline on viability of
control C2C12 and iC2C12 cells after 48 hours of treatment. Results are presented as fold difference compare to untreated C2C12 cells.
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Figure 2. mDUX and myogenic regulators. qRT-PCR for mDUX and myogenic genes in iC2C12-mDUX cells evaluated at different times (A, using
500 ng/mL doxycycline) or doses (C, at 12 hours). Results are presented as fold difference compared to uninduced cells (0 ng/ml) except for the
expression of mDUX in which 12 hours of induction was taken as the group for comparison. Error bars represent the STDEV. Induction with 8 ng/mL
of doxycycline was sufficient for significant down-regulation of MyoD. (B) Immunofluorescence for detection of MyoD (red) in iC2C12-mDUX cells
induced during the time course of 12 hours. Nuclei were stained with DAPI (blue). A notable decrease in the number of the positive-staining nuclei
and the intensity of the staining was detected as early as 4 hours after induction. (C) Expression of mDUX, MyoD, and Pax7 when mDUX is induced
with various concentrations of doxycycline.
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after 48 hours of induction. MyoD and Myf5 are critical myogenic
factors. In order for progenitors to be able to undergo myogenic
differentiation, they must at one point express MyoD and/or
Myf5; accordingly, mice null for both MyoD and Myf5 lack all
skeletal muscles . MyoD has been suggested to be a factor in
the pathogenesis of the FSHD by studies demonstrating
misregulation of MyoD and its target genes in myoblasts from
the patients [15,16].
mDUX and myogenic differentiation
To evaluate whether downregulation of MyoD and it target
genes lead to the functional abnormalities, we analyzed the
potential of mDUX expressing myoblasts to fuse and form
myotubes. Differentiation was induced by culturing confluent
iC2C12-mDUX cells in nutrition/growth factor-poor medium
(DMEM supplemented with 2% horse serum). During the course
of differentiation, mDUX was induced with 2.5, 10 and 25 ng/ml
of doxycyline. In the presence of 10 or 25 ng/ml doxycycline,
differentiation was visibly impaired, while non-treated cells fused
and formed typical elongated myotubes (Fig. 3A). At the terminal
stages of the experiment (after day 6 of differentiation) some dead
cells were observed at 10 and 25 ng/mL dox, but the majority
were alive and still attached to the plate, and at 25 ng/mL, even
sporadic myotubes were not seen (Fig. 3A). Differentiation and
formation of mature myotubes was confirmed by immunofluores-
cence staining for MyHC (Fig. 3B) and measuring the myotube
fusion index (percent of nuclei within myotubes). The fusion index
in the control and 2.5 ng/mL-induced cells was slightly over 50%.
However the number of the nuclei within myotubes in the 10 ng/
mL-induced group was much decreased, and the myotubes that
did form were smaller and shorter. Immunostaining and qRT-
PCR for MyoD revealed reduced expression in the induced cells
which was proportional to the levels of inhibition of differentiation
(Fig. 3B and D). Gene expression analyses of markers of
differentiation, myogenin, MCK and desmin further confirmed
diminished differentiation in the mDUX-induced cells (Fig. 3D).
To eliminate the influence of doxycyline by itself on myogenic
differentiation we conducted the same experiments on the iC2C12
parental and C2C12 (grand-parental) cell lines. We did not find
any significant doxycyline-related inhibition of differentiation by
immunofluorescence for MyHC, calculation of myotube fusion
index, or analysis of gene expression (Figure 3E, F and G) [13,14].
Since mDUX inhibited differentiation at non-toxic levels of
induction, we assume that this is due to interference with myogenic
pathways. In support of this explanation, other non-toxic versions
of DUX4, for example DUX4c  or certain mutations in the c-
terminus of DUX4 (our unpublished data) display potent
inhibition of differentiation as well as inhibition of MyoD and
Myf5 expression. It was previously reported that increased
numbers of D4Z4 repeats transfected into C2C12 myoblasts
impairs their differentiation ability . Furthermore, morpho-
logical differentiation abnormalities were reported in myoblasts
and mesangioblasts isolated from FSHD patients [17,30]. The
ability of low levels of mDUX to interfere with the differentiation
of myoblasts, together with its toxicity at high levels, suggests that
its mechanism of action is conserved with DUX4 and relevant to
mDUX expression in Xenopus laevis
Previous results on the in vitro effect of DUX4 and the data
presented in this paper suggest that mDUX would interfere with
myogenesis in vivo. Therefore we tested the effects of in vivo
expression of mDUX in a whole animal model: embryos of the
frog Xenopus laevis. Embryos were microinjected with mDUX plus
GFP (1 ng+100 pg) or GFP (1 ng) mRNA alone at the four-cell or
sixteen-cell stage (NF) embryos. At the four-cell stage, mDUX
(n=120) or GFP (n=120) mRNA was injected into one
blastomere of the dorsal animal side and the embryos were
allowed to develop under normal conditions at room temperature.
They were observed under a dissecting microscope at 1, 3, and 7
days post injection and those embryos expressing mDUX or
control RNA were identified by positive GFP fluorescence and
were separated for further analysis. 89% of embryos expressing
mDUX (GFP+) were observed to have gastrulation defects
compared to only 7% of GFP control injected embryos one day
post injection (Fig. 4A). All embryos expressing mDUX died prior
to day 7 (stage 45) showing severe defects in morphology consistent
with the initial defects in gastrulation. No large scale cell death was
apparent, which suggests that at this concentration, the main
deleterious effect of mDUX is to interfere with cell and tissue
movements, in addition to any effects it may have on myogenesis.
To bypass the effects of early mDUX expression on gastrulation
movements, we then performed injections into blastomeres V2.1
and V2.2 on one side of a sixteen-cell stage embryo. Previously
described cell fate maps have shown these blastomeres to
contribute primarily to cells of the dorsal and ventral somite and
the corresponding musculature of the Xenopus tadpole tail [31,32].
mDUX (n=120) embryos were observed to develop normally
through gastrulation one day post-injection but had significant
defects in tail development at three days post-injection compared
to GFP controls (n=120). At seven days (stage 45, NF) 72% of
mDUX tadpoles had truncated or reduced tails compared to only
6% of controls. Whole mount immunostaining of tadpoles with
12/101 antibody, which identifies skeletal muscle, showed a delay
in myogenic differentiation and a decrease in the number of
muscle fibers in mDUX tadpoles on the injected side, compared to
the contralateral and uninjected controls (Fig. 4B). Together these
results are consistent with mDUX having an inhibitory effect on
muscle differentiation, but also showing significant other effects
manifested by the derangement of gastrulation movements.
Competition with Pax factors
The DUX4 homeodomain showed very high similarity to the
homeodomains of Pax3 and Pax7 . This similarity was the key
factor in our hypothesis that DUX proteins, including mDUX,
interfere with Pax3 and Pax7 by competing for the same DNA
targets. One prediction of this hypothesis is that excess Pax3 or
Pax7 in cells should block or reduce the toxicity of DUX proteins,
and we found this to be the case for DUX4 . We therefore
transduced iC2C12-mDUX cells with Pax3 or Pax7 using
retroviral vectors bearing ires-GFP reporters (allowing identifica-
tion of transduced cells) or GFP alone. Transduced cells (60–70%
infection rate) were FACS-sorted to obtain a homogeneous cell
population. Subsequent FACS analyses confirmed that almost all
of the sorted, expanded cells were GFP+
immunofluorescent staining confirmed that the GFP+
expressed Pax3 or Pax7 (Fig. 5B). To evaluate the potential
competitive interaction, we induced mDUX at different levels and
cell viability was followed over 48 hours. This experiment
demonstrated that cells overexpressing Pax3 or Pax7 are resistant
to the toxicity of mDUX induced by 32 ng/mL (Fig. 3C). The
rescue was complete in the first 24 hours and still significant after
48 hours. Importantly, within 48 hours, the induced cells (32 ng/
mL) did not merely survive latently, but proliferated as indicated
by a higher ATP content at 48 hours compared to 24 hours
(Fig. 5C). On the other hand, cell death was rapid in the control
cells expressing only GFP. The competitive effect of Pax3 and
Pax7 was also evident on the expression of myogenic regulators.
(Fig. 5A) and
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MyoD and its target genes, which were rapidly downregulated by
mDUX even with low levels of doxycyline (Fig. 2), were resistant
to low levels (32 ng/ml) of mDUX in the Pax3 or Pax7 transduced
populations but not in the GFP-only controls (Fig. 4D). This
interaction was indeed competitive, because at higher levels of
induction, the mDUX repressive effect dominated. This compe-
tition reveals that mDUX and DUX4 act in similar mechanistic
In this study we show that mDUX provokes myopathic
molecular and physiological cellular changes related to those
caused by human DUX4. Specific differences between mDUX
and DUX4 were noted: mDUX downregulated Myf5 whereas
DUX4 upregulated Myf5, mDUX-expressing cells did not stretch
before dying whereas DUX4-expressing cells did, and mDUX-
mediated death could not be rescued by antioxidants, as it could to
Figure 3. mDUX and myogenic differentiation. (A) Phase-contrast microscopy of iC2C12-mDUX cells induced with doxycyline through 6 days of
differentiation. (B) Immunofluorescence for detection of MyHC (red, upper panels) and MyoD (red, lower panels) in cells induced with low levels of
doxycycline. Nuclei were counterstained with DAPI (blue). iC2C12-mDUX cells were in differentiation medium for 6 days when myotube fusion index
was calculated (C). Significantly diminished myogenic differentiation was observed in the cells induced with 10 ng/mL doxycyline. (D) Inhibition of
differentiation was confirmed by qRT-PCR. Results are presented as fold difference compared to uninduced cells (0 ng/mL) and the error bars
represent the STDEV. (E) Immunofluorescence for detection of MyHC (red) in C2C12 and iC2C12 control cells after 6 days of differentiation and
treatment with different concentrations of doxycycline. (F) Calculated fusion index and (G) gene expression analyses of differentiated C2C12 and
iC2C12 control cells. Doxycycline by itself did not have any significant effect on myoblast (C2C12 and iC2C12) differentiation.
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a significant degree for DUX4. With regard to Myf5, mDUX
actually behaves as DUX4c, which downregulated both MyoD
and Myf5. With regard to cellular morphology and oxidative
stress, it should be noted that DUX4 provokes multiple deleterious
pathways, and although antioxidants could fully rescue from low
levels of DUX4, it could only partially protect against high levels of
DUX4, and then only for a few days. The distinction between
mDUX and DUX4 in this regard may be more a distinction of the
degree to which various similar pathways are affected, rather than
a qualitatively different mechanism of cell death. Like DUX4,
mDUX shows a biphasic response: high levels were toxic while low
levels perturbed differentiation and myogenic gene expression.
Considering the toxic effects of mDUX expression, its interaction
with myogenic regulators and inhibition of myogenic differentia-
tion in both cell- and organism-based assays, we propose that the
mouse D4Z4-like arrays represent a genetic unit functionally
orthologous to the DUX4-bearing repeats at human 4q35.2.
Although the normal function of these repeats remains unknown,
the abnormal expression of the double homeodomain protein they
encode results in clear myopathic effects, with several lines of
evidence suggesting that these effects are relevant to FSHD. We
therefore propose that intervention in the normal expression or
repression of mDUX through either deleting integral numbers of
murine repeats, insertion of a strong enhancer into the vicinity of
the repeat array, or placing sequences from the repeat array, for
example the mDUX ORF, under the control of a tissue-specific,
Figure 4. mDUX expression in Xenopus. (A) Neurula stage embryos following injection of GFP mRNA alone (control) or GFP+mDUX mRNA. The
green color indicates the domain filled by the RNA injection. The control has a normal neural plate while the mDUX case is very abnormal due to
deranged gastrulation movements. (B) Tail muscle pattern in stage 45 tadpoles. The green color shows immunostaining with 12/101 antibody.
Control shows the normal pattern of myotomes. Two examples (mDUX) demonstrate how injection into blastomeres V2.1 and 2.2 results in an
inhibition of muscle differentiation on the injected (left) side.
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regulated promoter would represent physiologically relevant ways
to model FSHD in mice.
Because the mechanism linking the D4Z4 deletion to myopathy
has not been established, there is an urgent need for animal
models that incorporate D4Z4 sequences. We have developed a
model based on expression of the mDUX ORF during Xenopus
development. This model makes evident the myopathic nature of
D4Z4 itself. Furthermore, it is experimentally tractable and
amenable to a medium-throughput approach. It represents an
example of a vertebrate model based on sequences encoded by the
repeats affected by the FSHD deletion itself, and highlights the
utility of using rapidly developing lower vertebrates to model
human genetic disease.
Materials and Methods
Cloning of mDUX
The mDUX ORF was amplified from 100 ng mouse genomic
DNA by 0.4 uM forward (atggcagaagctggcag) and 0.4 uM reverse
PCRmixture(5 ul106bufferwith1.8 mMMgCl2,200 uMofeach
dNTP and 2% DMSO; all from Roche). Amplification was done with
35 cycles, each consist of 30 s denaturation at 95uC, anneling for 30 s
at 60uC and elongation for 140 s at 72uC. Sequencing confirmed that
the PCR product corresponded to mDUX clone C1 (accession
No. AM398151). The PCR products were subcloned into p2lox 
and used for gene targeting.
C2C12 cells and NIH 3T3 fibroblasts were cultured in high
glucose Dulbecco’s Modified Eagle Media (DMEM) with L-
glutamine and sodium pyruvate (Gibco), penicillin and strepto-
mycin (P/S, Gibco) and 10% fetal bovine serum (FBS, HyClone)
at 37uC in 5% O2/5% CO2. For myotube formation, C2C12 cells
were cultured on gelatin-coated plates until confluence, and then
washed with serum-free DMEM and differentiated with DMEM
supplemented with 2% horse serum (HS, Invitrogen) and insulin
(Sigma) for 4–6 days. Mouse embryonic stem (ES) cells were
cultured on irradiated MEFs in ‘‘Knockout’’ DMEM/15% FBS,
P/S, glutamine, nonessential amino acids, 0.1 mM b-mercapto-
Figure 5. Pax3 and Pax7 compete with mDUX. (A) FACS analysis of iC2C12-mDUX cells transduced with MSCV retroviral constructs caring GFP,
Pax3-ires-GFP or Pax7-ires-GFP. Almost all of the cells at the time of the experiment stably express GFP (x-axis). (B) Immunofluorescence for Pax3 or Pax7
(red) and GFP (green) reveals that Pax3 and Pax7 are expressed in GFP+cells. Cell number is decreased in induced samples due to toxicity of mDUX. (C)
ATP assay for determination of cell viability in iC2C12-mDUX cells transduced with MSCV-ires-GFP, MSCV-Pax3-ires-GFP or MSCV-Pax7-ires-GFP. Cells
were induced with various concentrations of doxycycline for 24 and 48 hours. Pax3- and Pax7-expressing cells are largely resistant to the toxicity of
mDUX induced by 32 ng/mL dox even after 48 hours of induction. (D) qRT-PCR analyses for MyoD and Myf5 in the cells shown in (C), induced for
18 hours. Expression of MyoD and Myf5 is strongly repressed at 32 ng/mL induction in the control cells, but not the Pax3 or Pax7 expressing cells.
Myopathic Effects of Mouse DUX
PLoS ONE | www.plosone.org8 September 2009 | Volume 4 | Issue 9 | e7003
ethanol, and 100 U/ml LIF (Peprotech). iC2C12-mDUX, i3T3-
mDUX and iES-mDUX were generated as previously we
Retro-virus production and generation of Pax3/Pax7-
expressing iC2C12-mDUX cell lines
Retroviral supernatant was produced in 293T cells cultured in
DMEM/10% FBS. Retroviral constructs (1 ug of pMSCV-ires-
GFP to generate pMSCV-Pax3-ires-GFP and pMSCV-Pax7-ires-
GFP ) were co-transfected with 1 ug pCL-Eco packaging
constructs using Fugine 6 (6 ul, Roche). Medium was changed
after 24 hours and the viral supernatant was collected at 48 hours
post-transfection. Filtered supernatant (0.45 mm) was applied to
the cells with polybrene (4 mg/mL). Spin-infection was performed
at 2000 g at 33uC for 90 min. Cells were incubated overnight at
37uC in 5% O2/5% CO2 after which the supernatant was
replaced with fresh medium. Several days after infection iC2C12-
mDUX cells were trypsinized and analyzed for GFP expression
using a FACS Aria (BD). GFP+cells were sorted to obtain ,100%
positive expressing cell lines using FACS Aria (BD).
Cells were plated in a 96 well plate (1200 cells/well). The genes
were induced the following day with various doxycycline
concentrations for 24 or 48 hours. To assay ATP levels, medium
from the wells was removed and cells were lysed by 50 ul ATPlite
for 1 minute (ATP determination Kit, Molecular Probe, Eugene,
OR). The intensity of luminescence was assayed on a POLARstar
Optima Microplate Reader (BMG LABTECH, Offenburg,
Germany). Data is presented as fold difference compare to the
control and the error bars represent SDEV (n=8).
Cells were cultured on gelatin coated 24 well plates, fixed with
4% paraformaldehyde for 20 min, permeablized by 0.3%Triton
X-100 for 30 min and blocked by 3% BSA for 1 hour at room
temperature. The cells were incubated with the primary antibodies
(mouse monoclonal anti mouse MyoD (1:250, BD Biosciences),
mouse monoclonal anti-Pax3 and anti-Pax7 (1:250, R&D
Systems), mouse anti-myosin heavy chain (1:20, Developmental
Studies Hybridoma Bank, U Iowa), polyclonal chicken anti-GFP
antibody (1:500, AbCam) diluted in PBS/3% BSA at 4uC
overnight. The secondary antibodies Alexa Fluor 488 and/or
555 (1:500, Invitrogen) were applied at room temperature for
45 min. Cells were counterstained using 49,6-diamidino-2-pheny-
lindole (DAPI, Invitrogen) to visualize nuclei.
Whole mount immunohistochemistry was carried out as
described previously . Briefly, stage 46 (NF) tadpoles were
fixed in Zamboni’s fixative (40 mM NaH2PO4, 120 mM
Na2HPO4, 2% PFA, 0.1% saturated picric acid) overnight at
4uC. Tadpoles were washed in PBSA three times for 10 minutes
each and bleached in 5% (w/v) H2O2(Sigma) in PBSA for 1 hour
under direct light. Samples were washed in BBT (PBSA+1% (w/v)
BSA+0.1% (w/v) Triton X-100), blocked in BBT containing 5%
horse serum for 1 hour and incubated overnight at 4uC in
blocking solution containing a mouse monoclonal antibody 12/
101 (1:100; grown in the lab, original gift from Liz Jones).
Tadpoles were incubated overnight at 4uC in BBT with 5% horse
serum containing Alexa Fluor 488 anti-mouse IgG (1:200;
Invitrogen) labeled secondary antibody. Tadpoles were visualized
using a Leica Fluo III fluorescent dissecting microscope with a
GFP2 filter set and photographed with Spot RT Camera
Quantitative Real Time RT-PCR (qRT-PCR)
Total RNAwasextractedwith Trizol(Invitrogen)andcDNA was
TermoScript (Invitrogen) following the manufacture instruction.
Quantitative PCR was performed by using pre-made primers/
probes (GAPDH Mm99999915_g1, MyoD1 Mm00440387_m1,
Myf5Mm00435125_m1, myogenin Mm00446194_m1, desmin
Mm00802455_m1 and MCK Mm00432556_m1 all from Applied
Biosystems) and TaqMan PCR premixture on 7500 real time PCR
System (Applied Biosystems). Glyceraldehyde phosphate dehydro-
genase(GAPDH) wasused as the internal standard. Fordetection of
mDUX, SyberGeeen (Applied Biosystems) premixture and the
following f: gcactcaagcagacagcaca and r: gtgtccatttcgtcccatgt
primers were used. Gene expression was normalized and analyzed
using the delta Ct method by 7500 System Software (Applied
Biosystems). Results were presented as fold difference of the mean
compared to the control (DDCt). All reactions were performed at
least in triplicate and the error bar is STDEV.
Annexin V Staining
iC2C12-mDUX cultured in 6 well plates were induced with
500 ng/ml dox for 4, 8, 12 and 24 hours. Annexin V/7-AAD
stainig was done using Staining BD Bioscience kit and
following the manufacturer’s instructions. In brief, from each
group 105trypsin-detached cells were stained with APC-coupled
Annexin V antibody and 7-AAD dye in binding buffer (BD
Bioscience). Dead and apoptotic cells were detected by FACS
Embryos, tadpoles and microinjection
Animal care was provided in accordance with NIH guidelines
with oversight provided by the University of Minnesota Animal
Care and Use Committee. Xenopus laevis embryos were obtained
by in vitro fertilization and staged according to the Nieuwkoop
and Faber (NF) tables . Embryos were dejelled with 2%
cysteine (Sigma) (pH 7.8) and then cultured in 0.1X Normal
Amphibian Medium (NAM; ) for approximately 5 days to NF
stage 46/47. RNA used for microinjections was transcribed in
vitro and capped using mMessage Machine (Ambion) from
plasmids containing either the mouse DUX4 CDS or nuclear
GFP (pcDNA 3.1 NucGFP) and was injected into early Xenopus
laevis embryos using a Nanoject injector (Drummond Scientific
company). mDUX (1 ng mDUX+100 pg nucGFP) or nucGFP
(1 ng nuclear GFP) mRNA was injected in a volume of 2.3 nl into
either one blastomere of dorsal animal side at the four-cell stage
or into V2.1 and 2.2 blastomeres of at the sixteen-cell stage .
The embryos were incubated in half strength NAM (NAM/
2)+3% Ficoll during injection and kept in this medium for 2–
3 hours post injection before being transferred to NAM/10
solution until staining.
All experiments were done at least 3 times. Data shown for Real
Time PCR are the mean6STDEV. Difference between means
was compare by the two-tailed Student test and was considered
significantly different at P,0.05.
We thank the Dr. Bob and Jean Smith Foundation and the Pacific
Northwest Friends of FSH Research. We thank Matthew Struck, Nathan
Zaidman and Jessica Klassen for calculation of myotube fusion index,
Naoko Koyano and Ondine Cleaver for in situ hybridization analyses and
members of Kyba laboratory for technical help and discussion.
Myopathic Effects of Mouse DUX
PLoS ONE | www.plosone.org9September 2009 | Volume 4 | Issue 9 | e7003
Author Contributions Download full-text
Conceived and designed the experiments: DB RSD JS MK. Performed the
experiments: DB RSD ZX. Analyzed the data: DB RSD JS MK.
Contributed reagents/materials/analysis tools: MK. Wrote the paper: DB
RSD JS MK.
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