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Non Conservation of Function for the Evolutionarily
Conserved Prdm1 Protein in the Control of the Slow Twitch
Myogenic Program in the Mouse Embryo
Stéphane D. Vincent,*, ,1 Alicia Mayeuf,1 Claire Niro,2 Mitinori Saitou,à,3 and Margaret Buckingham1
1
Department of Developmental Biology, CNRS URA 2575, Institut Pasteur, Paris, France
Department of Genetics and Development, Institut Cochin, Université Paris Descartes, CNRS UMR 8104, INSERM U567, Paris, France
3
Laboratory for Mammalian Germ Cell Biology, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan
Present address: Department of Development and Stem Cells, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS
UMR 7104, Inserm U964, Université de Strasbourg, Illkirch, France
àPresent address: Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho,
Sakyo-ku, Kyoto, Japan
*Corresponding author: E-mail: [email protected].
Associate editor: Claudia Kappen
2
Muscles are composed of multinucleated muscle fibers with different contractile and physiological properties, which result
from specific slow or fast gene expression programs in the differentiated muscle cells. In the zebra fish embryo, the slow
program is under the control of Hedgehog signaling from the notochord and floor plate. This pathway activates the
expression of the conserved transcriptional repressor, Prdm1 (Blimp1), which in turn represses the fast program and
promotes the slow program in adaxial cells of the somite and their descendants. In the mouse embryo, myogenesis is also
initiated in the myotomal compartment of the somite, but the slow muscle program is not confined to a specific subset of
cells. We now show that Prdm1 is expressed in the first differentiated myocytes of the early myotome from embryonic day
(E)9.5–E11.5. During this period, muscle formation depends on the myogenic regulatory factors, Myf5 and Mrf4. In their
absence, Prdm1 is not activated, in apparent contrast to zebra fish where Prdm1 is expressed in the absence of Myf5 and
MyoD that drive myogenesis in adaxial cells. However, as in zebra fish, Prdm1 expression in the mouse myotome does not
occur in the absence of Hedgehog signaling. Analysis of the muscle phenotype of Prdm1 mutant embryos shows that
myogenesis appears to proceed normally. Notably, there is no requirement for Prdm1 activation of the slow muscle
program in the mouse myotome. Furthermore, the gene for the transcriptional repressor, Sox6, which is repressed by
Prdm1 to permit slow muscle differentiation in zebra fish, is not expressed in the mouse myotome. We propose that the
lack of functional conservation for mouse Prdm1, that can nevertheless partially rescue the adaxial cells of zebra fish Prdm1
mutants, reflects differences in the evolution of the role of key regulators such as Prdm1 or Sox6, in initiating the onset of
the slow muscle program, between teleosts and mammals.
Key words: mouse, embryonic myogenesis, myotome, Prdm1, slow muscle gene expression, myogenic regulatory factors.
Introduction
Cellular processes such as tissue differentiation are coordinated by the controlled expression of regulatory proteins
that trigger specific gene expression programs. Implementation of a program depends on both transcriptional activators and also on transcriptional repressors. The Prdm1
gene encodes a transcriptional repressor, containing five
zinc fingers and a PR/SET domain, that binds to a specific
DNA sequence and recruits co-repressors such as class I
histone deacetylases, Groucho family members or histone
methyl transferases (for a review, see Bikoff et al. 2009).
Prdm1 was first identified as a master gene controlling
the terminal differentiation of B lymphocytes into plasmocytes (Turner et al. 1994). In this context, Prdm1 regulates
the expression of hundreds of genes (Shaffer et al. 2002;
Sciammas and Davis 2004), by direct repression of targets,
such as c-Myc (Lin et al. 1997), and by indirect activation of
secondary target genes via the repression of the gene coding for Pax5, that is, itself, a repressor (Lin et al. 2002).
Interestingly, Prdm1 is dynamically expressed during mouse
embryonic development (de Souza et al. 1999; Chang et al.
2002; Vincent et al. 2005) and has been shown to play major roles in a number of differentiation programs. It is the
earliest marker of primordial germ cells (Ohinata et al. 2005;
Vincent et al. 2005). In Prdm1 mutants, these cells are not
specified correctly (Vincent et al. 2005) and those that can
be detected show failure to repress the somatic program
(Ohinata et al. 2005). Prdm1 is also expressed in the endoderm, ectoderm, and mesoderm of the branchial arches
and is required for their development (Vincent et al.
2005) and for normal heart morphogenesis, reflecting its
expression in the Second Heart Field (Vincent et al.
2005; Robertson et al. 2007). In limb buds, Prdm1 is
expressed in the posterior domain (Vincent et al. 2005)
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Mol. Biol. Evol. 29(10):3181–3191. 2012 doi:10.1093/molbev/mss125
Advance Access publication April 20, 2012
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Research article
Abstract
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Vincent et al. · doi:10.1093/molbev/mss125
and is required for the maintenance of the zone of polarizing
activity cells that express Sonic hedgehog, an essential signaling molecule during limb development (Robertson et al.
2007). The embryonic expression of Prdm1 is mainly conserved among vertebrates, as illustrated by branchial arch
and limb/fin bud patterns (Chang et al. 2002; Baxendale
et al. 2004; Vincent et al. 2005; Wilm and Solnica-Krezel 2005).
During the early formation of skeletal muscle, Prdm1 expression has been reported in the myotomal compartment
of somites of mouse and Xenopus and in the adaxial myogenic cells of zebra fish somites (de Souza et al. 1999; Chang
et al. 2002; Baxendale et al. 2004; Vincent et al. 2005; Wilm
and Solnica-Krezel 2005). A recent report on the chick embryo has also shown Prdm1 expression in myogenic cells, in
keeping with the conservation of its expression in the muscle lineage, with only subtle differences between different
species (Beermann et al. 2010). Moreover, the Prdm1 protein is also very well conserved among vertebrates. It has
been estimated that the Prdm1 proteins of Xenopus and
mouse share 70% of overall identity (de Souza et al.
1999). The conservation of the expression pattern and of
the protein are in favor of a conserved role during evolution.
Indeed, the mouse Prdm1 protein can rescue the phenotype of a zebra fish Prdm1 mutant (Ubo) (Liew et al. 2008).
In zebra fish, Prdm1 is expressed in adaxial cells
(Baxendale et al. 2004) that are fated to give rise to slow
muscle (Devoto et al. 1996). This pathway is regulated by
Hedgehog signaling from the adjacent axial structures
(notochord, ventral neural tube) (Blagden et al. 1997).
Ubo (Prdm1) mutants are characterized by U-shaped
somites (van Eeden et al. 1996) and lack of slow-twitch
fibers (Roy et al. 2001). Detailed analysis of the phenotype
has shown that adaxial cells still differentiate into muscle
but fail to activate the slow program and express fast muscle markers (Roy et al. 2001). Prdm1, acting downstream of
Hedgehog signaling (Baxendale et al. 2004), has been shown
to directly repress some fast muscle genes (Liew et al. 2008;
von Hofsten et al. 2008). It also promotes the onset of the
slow program by repressing the expression of the transcriptional repressor Sox6 (von Hofsten et al. 2008) through an
indirect mechanism (Wang et al. 2011). Sox6, in zebra fish,
directly represses the expression of slow muscle genes. Interestingly, Sox6 mouse mutants are characterized by the
failure to repress the expression of the slow myosin heavy
chain in fetal fibers, indicating that in the mouse Sox6 also
acts as a repressor of the slow program at later stages
(Hagiwara et al. 2005, 2007).
In the mouse embryo, it has been shown recently that
Six1 and Six4 homeodomain transcription factors, that regulate muscle progenitor cell behavior at the level of Pax3
and the myogenic regulatory factors, are required for the
correct expression of fast muscle genes in the myotome
(Niro et al. 2009). In the absence of Six1 and Six4, the slow
program is normally activated, but fast genes fail to be expressed. In the mouse myotome, unlike the zebra fish, distinct fast and slow muscle cells are not initially specified
and differentiated muscle cells express both fast and slow
muscle markers (Niro et al. 2009; this study).
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Contrary to the fast muscle program, it is not clear what
controls the onset of the slow program in Amniotes, but
Sonic hedgehog (Shh) plays a role. Shh promotes terminal
differentiation of slow muscle fibers in chick embryos and
Shh mouse mutant embryos, which have defective myotomes (Borycki et al. 1999; Kruger et al. 2001), lack slow
myosin heavy chain expression in the remaining differentiated muscle cells (Li et al. 2004). Given its function in slow
myogenesis and dependence on Hedgehog signaling in
zebra fish, and because of its expression pattern in the
mouse myotome, Prdm1 is a potentially good candidate
to control the onset of the slow muscle program in the
mouse embryo.
In this study, we analyzed in detail the expression pattern of Prdm1 and show that it is expressed in the first
differentiating myocytes of the early myotome. As in the
zebra fish embryo, this expression is dependent on Hh signaling. However, contrary to the zebra fish embryo, mutant
analysis shows that Sox6 expression is not controlled by
Prdm1 and slow as well as fast muscle markers continue
to be expressed in the myotome. In conclusion, our results
demonstrate that despite the conserved biochemical
properties of Prdm1 between mouse and zebra fish, Prdm1
function in the control of the slow muscle program is not
conserved during vertebrate radiation. Furthermore, this
lack of functional conservation for Prdm1 is explained,
at least in part, by further evolutionary differences in slow
program regulation between zebra fish and mouse, with
respect to other important regulators, such as Sox6.
Materials and Methods
Generation of the pCAG Prdm1b Vector and pCAG
CAT 1b8 Transgenic Line
The coding sequence of the murine truncated form of
Prdm1 (Prdm1b) (Gyory et al. 2003) cloned by rapid amplification of cDNA-ends by polymerase chain reaction
(RACE PCR) (Vincent SD, unpublished data) linked to
an IRES green fluorescent protein (GFP) was cloned into
the conditional pCAG CAT vector (kindly provided by
Y. Saga, National Institute of Genetics, Japan) (Watanabe
et al. 2006). Efficient conditional expression of the Prdm1b
protein was checked by co-transfection in C2C12 cells with
a plasmid expressing Cre followed by green fluorescent protein (GFP) expression and western blot analysis. Linearized
vector was microinjected by the Centre d’Ingénierie Génétique Murine platform (Institut Pasteur, France) to generate
transgenic mouse lines. Three independent lines were established, only one of which showed bright GFP expression
after Cre recombination. This line, Tg(pCAG CAT 1b8), was
employed in this study, using the Myf5Cre/þ line.
Mouse Strains and Genotyping
The Blimp1-mEGFP, Mrf4 (Myf6tm1Eno), Myf5Cre, Myf5lox,
Myf5nlacZ, Myod1, Pax3Cre, Prdm1BEH, Prdm1CA, Shh,
Smo alleles have been described elsewhere (Rudnicki
et al. 1992; Zhang et al. 1995; Tajbakhsh, Bober, et al.
1996; Tallquist et al. 2000; Zhang et al. 2001; Shapiro-Shelef
Prdm1 Function Is Not Conserved During Myogenesis · doi:10.1093/molbev/mss125
et al. 2003; Kassar-Duchossoy et al. 2004; Engleka et al. 2005;
Ohinata et al. 2005; Vincent et al. 2005). The Myog (Myogenin) allele was obtained from the Myogflox allele (Knapp
et al. 2006) by crossing with the PGKCre transgenic line
(Lallemand et al. 1998). Mouse care and procedures were
in accordance with institutional and national guidelines.
Embryonic day (E) 0.5 was counted from the appearance
of a vaginal plug.
Whole-Mount In Situ Hybridization and
Immunolocalization
Whole-mount in situ hybridization was performed according to standard protocols (Nagy et al. 2003). Prdm1
(Vincent et al. 2003) and Myog (Sassoon et al. 1989) probes
have been previously described. One of the Sox6 probes was
produced from a plasmid template kindly given by N. Hagiwara (University of California, Davis): The template was
linearized with NotI and the probe was transcribed using
SP6 RNA polymerase (Promega). The second Sox6 probe
was transcribed using SP6 RNA polymerase from a template
generated by PCR (GCA GCC ACA CGG AGT TGA TG and
CAT TTA GGT GAC ACT ATA GTG ATG GTG TGG TCG
TTG CC primers). Both probes gave similar results.
Embryos were dissected at E10.5 and then fixed for 2 h in
4% paraformaldehyde at 4 °C. After three rinses, embryos
were transferred to 15% sucrose/phosphate buffered saline
and embedded in 7% gelatin and 15% sucrose. Embryos
were sectioned on a Leica cryostat, to give 20-lm thick
slices (or 50-lm thick slices for Sox6 and Myog wholemount in situ hybridized embryos). As primary antibodies,
a chick polyclonal for GFP (Invitrogen), rabbit polyclonal
antibodies; Myogenin (Santa Cruz), and Mrf4 (Invitrogen),
mouse monoclonal antibodies; MF20 (all MyHC) (DSHB),
Pax3 (DSHB), and Ki67 (BD Biosciences), and a rat monoclonal antibody for Prdm1 (6D3; Santa Cruz) were used. As
secondary antibodies, conjugated Alexa 488/546 against
the different species (Invitrogen) were used. Immunolocalization was performed as described and analyzed with
a Zeiss ApoTome system.
Quantitative Reverse Transcription–PCR Analysis
Total RNA was prepared from E10.5 and E11.5 embryos.
Briefly, individual embryos were staged by counting the
somite number and lysed in TRIzol (Invitrogen). After
genotyping, total RNA was prepared according to the manufacturer’s protocol (Applied Biosystem). RNA was purified
on PureLink RNA mini kit columns (Invitrogen). RNA was
then quantified using a nanodrop spectrophotometer. Five
hundred nanograms of RNA were retro-transcribed with
random hexamers and Superscript II (Invitrogen). Quantitative PCR was performed using the Power SYBR Green
PCR Master Mix on a StepOne Plus Real-Time PCR system
(Applied Biosystem), with RNA extracted from embryos
with the same number of somites. Three mutant and
two control embryos with the same somite number were
analyzed as triplicates. Primer sequences have been published elsewhere (Niro et al. 2009). Results were standardized to GAPDH and Myog transcripts. Data were analyzed
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using the Mann–Whitney non parametric test (http://
www.anastats.fr/).
Results
Prdm1 Is Expressed in Differentiated Myocytes of
the Early Myotome
We investigated the expression of Prdm1 in somites of the
developing mouse embryo. Transcripts are first detectable
in more mature anterior somites, from about somite 14, at
embryonic day (E) 9.5 (fig. 1A). At E10.5 (fig. 1B), Prdm1 is
expressed in all somites except the most posterior, whereas
by E11.5, as somite maturation proceeds in an anterior/posterior direction, transcripts are mainly detected in more
posterior somites (fig. 1C). Prdm1 therefore has a dynamic
expression pattern in maturing somites, in a central band of
cells extending from the epaxial to the hypaxial domain at
E10.5 and also at E11.5, despite myotomal expansion by this
stage. Such expression is characteristic of the early
myotome and is very reminiscent of the first wave of expression of Mrf4 (Bober et al. 1991). In order to examine
this further, we analyzed Prdm1 expression on sections,
using co-immunohistochemistry. Since Prdm1 antibodies
are difficult to use (fig. 1I), we employed a transgenic line
carrying an engineered BAC that contains the Prdm1 locus,
with a membrane bound GFP reporter (Blimp1-mEGFP)
(Ohinata et al. 2005). This faithfully recapitulates the
expression pattern of Prdm1. Pax3, that marks myogenic
progenitor cells in the dermomyotome at E10.5 (Goulding
et al. 1991), does not overlap with GFP (fig. 1D), whereas coimmunolocalization is seen with an antibody to striated
muscle myosin heavy chains (fig. 1G), that marks differentiated muscle cells in the myotome. No co-localization with
Ki67 (fig. 1F), expressed in proliferating cells in the dermomyotome, confirms that Prdm1/GFP is present only in the
post-mitotic cells of the myotome. Some overlapping
expression is seen with the myogenic differentiation factor,
Myogenin, (fig. 1E) and with Mrf4 in the myotome (result
not shown). In more immature posterior myotomes at
E10.5, Mrf4 is present in centrally located differentiated
myocytes which have a characteristic elongated shaped
nucleus (fig. 1H), whereas Myogenin is strongly expressed
in more peripheral myotomal cells at this stage. Using the
Prdm1 antibody to confirm the myotomal location seen
with the transgene, we observe nuclear expression
of Prdm1 in Mrf4-positive nuclei of myocytes (fig. 1I).
Co-immunolocalization in a central subgroup of Mrf4positive cells suggests that Prdm1 is present in fully
differentiated cells of the early myotome.
Prdm1 Expression Is Restricted to the Early
Myotome and Depends on the Myogenic
Determination Factors Myf5 and/or Mrf4
Since it has been proposed that Prdm1 expression in the
adaxial cells of zebra fish somites does not depend on myogenic regulatory factors (Hinits et al. 2009), we investigated
this relationship in the myotome of mouse embryos, on
different mutant backgrounds. In embryos that lack
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FIG. 1. Prdm1 expression in the early myotome. (A–C) Wholemount in situ hybridization with a Prdm1 probe, showing Prdm1
transcripts in more mature anterior somites, at E9.5 (A), in the
central myotome domain of somites, at E10.5 (B) and mainly in this
domain of more posterior somites, at E11.5 (C). (D–G) Transverse
sections in the interlimb region of Prdm1GFP(Blimp1mEGFP) BAC
transgenic embryos, at E10.5, treated for co-immunohistochemistry
with a GFP antibody, reflecting Prdm1 expression, and a Pax3
antibody that marks progenitor cells in the dermomyotome (D) or
with an antibody directed against all striated muscle myosin heavy
chains (MyHC) that marks differentiated skeletal muscle cells (G)
shows that membrane bound GFP labeling co-localizes with myosinpositive cells in the myotome. No co-localization is seen with an
antibody to Ki67, which marks proliferating Pax3-positive cells (F),
whereas there is co-localization with Myogenin (E), in keeping with
expression of the Prdm1 transgene in differentiating post-mitotic
cells of the myotome. (H and I) Transverse sections of less mature
posterior somites, at E10.5, show that Mrf4 is principally expressed
in elongated nuclei of myocytes, whereas Myogenin is present in the
nuclei of more peripheral myotomal cells (H). Nuclear accumulation
of Prdm1 is detected in Mrf4-positive myocytes (I).
Myf5 (Myf5lox/lox), the onset of myogenesis is slightly delayed but is initiated by Mrf4, also acting as a myogenic
determination factor in this context (Kassar-Duchossoy
et al. 2004). In these mutant embryos, Prdm1 is expressed
in the myotome, although less strongly than in the control
at E10.5 (fig. 2A and B), reflecting the delayed onset of myogenesis. In the absence of the other myogenic determination factor, MyoD, which begins to be expressed at this
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FIG. 2. The dependence of Prdm1 expression on the myogenic
regulatory factors. (A–I) Whole-mount in situ hybridization with
a Prdm1 (A–F, H, and I) or a Myog (Myogenin) probe (G), on control
(A and D) or MRF mutant embryos at E10.5 (A–C), E10.75 (D–F), or
E11.75 (G–I). In the absence of Myf5 (Myf5lox/lox) (B), Prdm1
transcripts are still detectable (arrowhead in A indicates the central
domain of the myotome where Prdm1 is expressed, arrowhead in B
indicates the epaxial, and arrow the hypaxial, extent of the
myotome); in the absence of MyoD (Myod1/) (C), there is robust
Prdm1 expression in the myotome. In the absence of the myogenic
differentiation factor, Myogenin (Myog/), Prdm1 transcripts are
present (E) and are still detectable, although reduced in the absence
of Mrf4 (Mrf4/) (F). In Myf5nlacZ/nlacZ embryos (G–I), in the
absence of both Myf5 and Mrf4, when Myog (Myogenin) transcripts
have begun to accumulate in the myotome (G), Prdm1 expression is
not detectable (H, with interlimb somites enlarged in I).
time, Prdm1 expression is unaffected (fig. 2C). Prdm1 is also
expressed in the myotome of Myogenin (Myog) (fig. 2E) and
Mrf4 (fig. 2F) mutant embryos. In the Myf5nlacZ/nlacZ mutant, both Myf5 and Mrf4 are absent (Kassar-Duchossoy
et al. 2004) and cells do not enter the myogenic program
until Myod1 is expressed independently, from about E11
(Tajbakhsh, Rocancourt, et al. 1996). Under these conditions, the myotome is initially absent and the myogenic
differentiation gene, Myog, is only expressed after Myod1
activation. As shown in figure 2G, at E11.75 myotome formation is rescued. However, Prdm1 transcripts are not detectable (fig. 2H and I), showing that Prdm1 is not
expressed in the MyoD-dependent myogenic program, despite the presence of Myogenin. In the double Mrf4/Myf5
mutant (Myf5nlacZ/nlacZ), undifferentiated cells that have
activated the Myf5 gene (visualized by nlacZ expression)
mislocate or accummulate adjacent to the edges of the
dermomyotome (Tajbakhsh, Rocancourt, et al. 1996).
Prdm1 Function Is Not Conserved During Myogenesis · doi:10.1093/molbev/mss125
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FIG. 3. Prdm1 is expressed in the absence of Shh but not detected in the absence of Hedgehog signaling. (A–D) Whole-mount in situ
hybridization for Prdm1 transcripts on wild-type (A) and Shh/ mutant (B–D) embryos at E10 (A and B) and E10.75 (C and D). Prdm1
expression in the myotome (arrowhead) is maintained in the remaining myotomal cells of Shh/ mutant embryos (B and C, with magnification
of C in D). (E–H) Whole-mount in situ hybridization for Myf5 (E and F) and Prdm1 (G and H) transcripts on wild-type (E and G) and Smo/
mutant (F and H) embryos at E9.5. In the absence of Hedgehog signaling, Myf5 is still transcribed in the somites with a characteristic segmented
pattern (arrowhead), contrary to Prdm1, which does not show any somitic expression. As in Shh/ mutant embryos, the Prdm1 expression
pattern is still observed in the limb buds (asterisk), gut endoderm and branchial arch region (bracket) in Smo/ mutant embryos.
Prdm1 is not expressed in these cells. Prdm1 expression in
the mouse embryo therefore depends on the formation of
the early myotome and hence on Mrf4 or Myf5. Consistent
with the relationship between Prdm1 expression and Myf5/
Mrf4 regulation of the onset of myogenesis, later expression
of Prdm1 at E11.5 is confined to a narrow band of cells in
the central domain of the myotome (fig. 1C), which by this
stage has expanded due to the on-going contribution of
MyoD-positive myogenic cells which do not express Prdm1
(fig. 2H and I). MyoD normally takes over the role of myogenic determination during later embryonic development,
when Myf5 is downregulated. Consistent with the dependence of Prdm1 expression on Myf5/Mrf4, it is no longer expressed in skeletal muscle at later developmental stages nor
in the adult myofibers (data not shown). As Prdm1 expression is associated only with the formation of the early myotome, and it is not re-expressed later during muscle
development, contrary to Mrf4, this suggests that it is also
controlled by environmental and temporal cues.
Prdm1 Expression in the Myotome Requires
Hedgehog Signaling
Sonic hedgehog (Shh) coming from the notochord and the
floor plate of the neural tube is one of the crucial signals implicated in the onset of myogenesis (Munsterberg et al. 1995).
During myogenesis in the zebra fish embryo, abrogation of
Hedgehog signaling results in the absence of Prdm1 expression in adaxial cells of the myotome (Baxendale et al. 2004). In
order to see whether Shh signaling affects Prdm1 expression
in the myotome of the mouse embryo, we analyzed Shh/
mutant embryos. In these mutants, the somites are severely
affected and the initiation of myogenesis is impaired. However, some cells still activate the myogenic determination
gene, Myf5, independently of Shh, leading to partial myotomal muscle formation (Borycki et al. 1999; Kruger et al. 2001;
Zhang et al. 2001). In situ hybridization for Prdm1 transcripts
in Shh/ mutant embryos shows that these are still detectable in anterior somites at E10 (fig. 3A and B) and in interlimb
somites at E10.75 (fig. 3C and D), despite perturbations due
to the lack of Shh signaling. In other domains, such as in the
branchial arches or early limb buds (fig. 1A and B and fig. 3A),
Prdm1 expression is not affected. However, in Shh/ mutant embryos, the Shh target Ptc1 is still detected, suggesting
that Indian hedgehog (Ihh) is able to functionally compensate (Zhang et al. 2001). In the somites of Smoothened mutants (Smo), in which all Hedgehog signaling is absent, Myf5
expression is observed as in Shh/ mutant embryos (Zhang
et al. 2001). At E9.5, Myf5 is still expressed in the absence of
Hedgehog signaling (fig. 3E and F); however, we failed to detect Prdm1 expression in the somites despite normal expression in the other embryonic domains (fig. 3G and H). These
results suggest that, as in the zebra fish, Hedgehog signaling is
required for Prdm1 expression, in cells already engaged in the
myogenic program as a result of Myf5/Mrf4 expression in the
mouse embryo.
Normal Onset of Myogenesis and of Slow Myosin
Heavy Chain Expression in the Absence of Prdm1
In order to investigate a potential role for Prdm1 at the
onset of myogenesis in the mouse myotome, we
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FIG. 4. Expression of slow and fast embryonic myosin heavy chains in the myotomes of mouse embryos in the presence and absence of Prdm1.
(A–L) Co-immunolocalization with antibodies to myogenic differentiation factors Mrf4 (A and B) and Myogenin (C–E, H–J) and to all striated
myosin heavy chains (MyHC) (A, B, C, F, G, H, K, and L), slow MyHC (D, F, I, and K), and fast embryonic MyHC (embMyHC) (E, G, J, and L) on
transverse sections of control (A and C–G) and conditional Myf5Cre/þ;Prdm1CA/CA mutant (B) or Pax3Cre/þ;Prdm1CA/CA mutant (H–L) embryos
at E10.25 in less mature posterior somites (A and B) or interlimb somites at E10.5 (C–L), showing co-localization of fast and slow myosin
isoforms in Mrf4-positive and in some Myogenin-positive cells in the myotome, in the presence (A and C–G) or absence (B and H–L) of Prdm1.
investigated the expression of myosin heavy chain isoforms.
Prdm1 null mutant embryos (Prdm1BEH/BEH) die around
E10.5 because of placental defects (Vincent et al. 2005;
Robertson et al. 2007); however, some embryos are viable
at this stage. In these embryos, although somite development is retarded and the morphology of the myotome is
abnormal, both slow and embryonic fast myosin heavy
chains are expressed (supplementary fig. 1, Supplementary
Material online). In order to overcome the embryonic lethality of the null mutation, we used a conditional Prdm1
line (Shapiro-Shelef et al. 2003), crossed onto a Myf5Cre/þ
(Tallquist et al. 2000) or a Pax3Cre/þ (Engleka et al. 2005)
genetic background to delete Prdm1 in myogenic cells or
myogenic progenitor cells, respectively. Both Cre lines led
to efficient deletion of Prdm1 in the myotome (results not
shown) and gave similar results. As shown in figure 4A and
B and supplementary figure 2 (Supplementary Material online), the formation of the early myotome is not affected in
the absence of Prdm1, with myosin heavy chains expressed
in Mrf4-positive cells. Both slow myosin heavy chain and
fast embryonic myosin heavy chain are present in most myosin-positive differentiated cells of the myotome, some of
which are also Myogenin-positive at E10.5 (fig. 4C–G). In
the conditional Prdm1 mutant, differentiation of cells in
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the myotome is normal, with expression of both slow
(fig. 4I and K) and fast embryonic (fig. 4J and L) myosin heavy
chains. Furthermore, conditional deletion of Prdm1 with the
Myf5Cre allele on different Myf5, Mrf4 or Myog mutant backgrounds did not affect the expression of slow, or fast embryonic myosin heavy chains in the myotome (results not
shown). In supplementary figure 3 (Supplementary Material
online), this is shown with a Myf5Cre/lox;Prdm1CA/BEH cross
in which Myf5 is absent and Mrf4, produced by the Myf5lox
allele, is responsible for early myotome formation. We therefore conclude that, unlike the zebra fish, Prdm1 is not required for the slow skeletal muscle program in the mouse
myotome. This is further supported by reverse transcription–
quantitative polymerase chain reaction (RT-qPCR) analysis
(fig. 5), which shows that, although there are some minor
differences with the control (statistically not significant),
a range of slow as well as fast skeletal muscle genes are
transcribed in the myotome of conditional Prdm1
mutant embryos.
Expression of a Dominant Negative Truncated
Prdm1 in the Myotome Does Not Affect Myogenesis
We cannot exclude that the absence of phenotype in the
Prdm1 conditional mutant embryos is the result of a
Prdm1 Function Is Not Conserved During Myogenesis · doi:10.1093/molbev/mss125
MBE
to the myotome since when this transgene is expressed in
heart progenitors, it has a strong phenotype (Vincent SD,
Buckingham M, unpublished data). We conclude that the
slow and fast programs are not under the control of Prdm1
in the mouse myotome.
Sox6, a Repressor of the Slow Muscle Program in
Zebra Fish, Is Not Expressed in the Myotome and Is
Not Repressed by Prdm1 in the Mouse Myotome
FIG. 5. Analysis of transcripts of slow and fast skeletal muscle genes
expressed in the myotome in the absence of Prdm1. Quantitative PCR
analysis of transcripts, expressed relative to Gapdh and Myog
transcripts taken as 1, of Myog (Myogenin), Pax3, and slow muscle
genes, slow myosin heavy chain (slow MyHC), slow myosin light
chain 4 (Myl4), slow myosin light chain 6b (Myl6B), TroponinT1
(TnnT1), ATPase, Caþþ transporting, cardiac muscle, slow twitch 2
(Atp2a2), or fast muscle genes, myosin light chain one (Myl1),
phosphorylatable myosin light chain (Mylpf), or ATPase, Caþþ
transporting, cardiac muscle, fast twitch 1 (Atp2a1), shows that these
are expressed in both control (Myf5Cre/þ;Prdm1CA/þ, n 5 2, triplicates)
and Prdm1 conditional mutant (Myf5Cre/þ; Prdm1CA/CA, n 5 3,
triplicates) embryos, at E11.5. There is no statistically significant
difference in the expression of these markers between mutant and
control embryos (ns, not significant; brackets indicate the standard
error of the mean).
functional compensation. Whole-mount in situ hybridization of several members of the Prdm family did not reveal
any obvious expression in the myotome (data not shown).
In order to indirectly assess this question, we constructed
a transgenic line Tg(pCAG CAT 1b8) conditionally expressing a truncated form of Prdm1 (Prdm1b) and GFP. This
truncated form does not have a PR/SET domain and displays dramatically reduced repressive activity, although still
able to bind to its DNA targets (Gyory et al. 2003), therefore,
it should interfere with the endogenous full length Prdm1
(Angelin-Duclos et al. 2002). The endogenous transcript encoding the Prdm1b protein, which could be potentially generated from an internal promoter as seen in human myeloma
cell lines (Gyory et al. 2003), is not expressed in the embryo at
the time of myotome formation (Vincent SD, unpublished
data). As shown in figure 6A and B, expression of Prdm1b
depends on the expression of Cre. Embryos carrying the
transgene and one copy of the Myf5Cre allele display the expected GFP expression profile in the somite, although this is
mosaic (fig. 6C). Expression of Pax3 in the dermomyotome is
not affected in cells that express the transgene (fig. 6D). Expression of myosin heavy chain (all MyHC, slow MyHC and
emb MyHC) in cells of the myotome is also not perturbed by
the expression of the transgene (fig. 6E–G), suggesting that
interference with the repressive activity of Prdm1 does not
affect the expression of the slow or fast programs in the
mouse myotome. The absence of a phenotype is specific
A key repressor of the slow muscle program in zebra fish is
Sox6, which is transcriptionally repressed by Prdm1 (von
Hofsten et al. 2008). In the mouse also, Sox6 has been
shown to inhibit slow muscle myosin heavy chain expression at fetal stages by directly binding to the promoter
region of this gene (Hagiwara et al. 2007). At E17.5,
Sox6 accumulates in the myonuclei of secondary fibers
(data not shown). In order to assess whether Sox6 has
a role in the control of the slow muscle program in
the mouse myotome, we performed whole-mount in situ
hybridization at E10.75. Sox6 transcripts are not detectable in the myotome marked by Myogenin (Myog) expression (fig. 7A and B, sections in E and F, respectively). In
immature caudal somites, where the myotome has not
yet formed, Sox6 is transcribed in the ventral sclerotome
that will give rise to cartilage and bone (fig. 7A and B, sections in I and J, respectively). This dynamic expression pattern in the somites is not the result of Prdm1 repressor
activity since in the conditional Prdm1 mutants, the
Sox6 expression pattern is not affected (fig. 7C and D
section in G and K and H and L, respectively). These results
demonstrate that in the mouse embryo, contrary to the
zebra fish (Wang et al. 2011), Sox6 is not detected in the
myotome. This suggest first, that, in the mouse myotome,
Prdm1 expression does not impact on Sox6 expression,
and second, that Sox6 repression of slow muscle gene expression may not be relevant to the initial stages of mouse
myogenesis, as suggested by the absence of an early myogenic phenotype in Sox6 null mutant embryos (Hagiwara
et al. 2005, 2007).
Discussion
We have investigated the expression and role of Prdm1 at
the onset of skeletal muscle differentiation in the mouse,
relative to its important function in promoting slow muscle
formation in the zebra fish embryo. First, we show that
Prdm1 is expressed in the differentiated cells of the early
mouse myotome, where the myogenic factor, Mrf4, is also
present. Consistent with its myogenic expression, Prdm1
activation depends on Myf5 and/or Mrf4, which are required for early myotome formation. In the absence of
these factors, myogenic progenitor cells do not differentiate and are mislocated (Tajbakhsh, Rocancourt, et al. 1996).
Prdm1 is not expressed in these cells. In contrast, in the
zebra fish embryo where the onset of myogenesis in adaxial
cells depends on Myf5 and/or MyoD, in Myf5 mutants, in
which Myod1 expression has been downregulated with
morpholinos, Prdm1 is still expressed, in the absence of
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Vincent et al. · doi:10.1093/molbev/mss125
MBE
FIG. 6. Overexpression of Prdm1b does not affect myogenesis. (A and B) Co-transfection of C2C12 cells with the pCAG CAT 1b8 IRES GFP
together with the Cre expression pMC13 plasmids. After Cre expression, GFP (A) and expression of the truncated protein Prdm1b as shown
by western blot (B), are detected. (C) The GFP expression pattern of a Myf5Cre/þ;Tg(pCAGCAT1b8/þ) embryo at E11 marks the myotome
(C1,2). A longitudinal section indicates that the expression of the transgene is mosaic in the tail somites (C3). (D–G) Co-immunolocalization on
transverse sections of Myf5Cre/þ;Tg(pCAGCAT1b8/þ) embryos at E10.5 using antibody to GFP and Pax3 (D), all MyHCs (E), slow MyHC (F), and
fast embryonic (emb) MyHC (G) indicating that expression of these markers in the somites is not affected by the expression of the transgene.
Arrows point to examples of cells that express the transgene.
muscle formation (Hinits et al. 2009). The Myf5/Mrf4 mutant analysis indicates that, in the mouse embryo, these
myogenic factors are either directly required or that the
location of cells in respect to the signaling environment
of the myotome is essential for Prdm1 activation. Later
specification of myogenic cells that contribute to the
formation of skeletal muscle in the myotome depends
on MyoD, in the mouse embryo. Prdm1 activation is
MyoD-independent and its downregulation from E11.5 is
in accordance with its activation during the earlier wave
of myogenesis in the myotome. This is further supported
by the analysis of Six1/;Six4/ mouse mutant embryos
(Grifone et al. 2005). In the absence of these two important
upstream myogenic regulators, Prdm1 is still expressed in
the residual early myotome formed as a result of Myf5
activation (Niro et al. 2009). In this case, Mrf4 expression
is not detected and myogenic differentiation depends on
low levels of Myogenin (Grifone et al. 2005). In these double
mutants, Prdm1 expression correlates with the expression
of Myf5 during early myotome formation.
In the zebra fish embryo, Prdm1 activation depends on
Hedgehog signaling to adaxial cells from the adjacent notochord and floor plate of the neural tube (Baxendale et al.
2004). In Shh/ mouse mutants, however, Prdm1 activation
3188
is still observed in the remaining myogenic cells. It is possible
that other Hedgehog signaling pathways are important for
activation, in particular, Ihh has been shown to be responsible for the Hedgehog signaling in Shh/ mutant embryos
(Zhang et al. 2001). However, in the Smoothened mutant, in
which all Hedgehog signaling is abrogated, Myf5 expression is
still induced as in the Shh/ mutant (Zhang et al. 2001).
However, we do not detect somitic expression of Prdm1
in Smo/ mutant embryos. Hedgehog-dependent activation of Prdm1 is, therefore, comparable to zebra fish. If
one makes the analogy between adaxial cells and the epaxial
cells of the early mouse somite, then Myf5 activation and
subsequent early epaxial myogenesis, with accompanying
Prdm1 expression in the differentiated epaxial myotome,
are mainly Shh dependent in the mouse embryo also, as
indicated by Shh and Gli2/3 mutant phenotypes (Borycki
et al. 1999; McDermott et al. 2005) and the importance of
the Gli binding site for activation of the early epaxial enhancer of Myf5 (Gustafsson et al. 2002; Borello et al. 2006).
Early hypaxial myogenesis is also affected by the absence of
Shh (Kruger et al. 2001); however, some skeletal muscle formation takes place (Borycki et al. 1999) and in these myotomal cells, Prdm1 continues to be expressed, probably
under the control of Ihh. A Gli binding region has been
Prdm1 Function Is Not Conserved During Myogenesis · doi:10.1093/molbev/mss125
MBE
FIG. 7. Sox6 is not expressed in the myotome of normal and conditional Prdm1 mutant embryos. (A and C) Sox6 whole-mount in situ
hybridization of control (A, transverse sections in E, I with plane of section indicated in dotted red line in A) or conditional Prdm1 mutant
(Myf5Cre/þ;Prdm1CA/CA) (C, transverse sections in G and K with plane of section indicated in dotted red line in B) embryos at E10.75. (B and D).
Whole-mount in situ hybridization for Myogenin (Myog) transcripts of control (B, transverse sections in F and J) or conditional Prdm1 mutant
(Myf5Cre/þ;Prdm1CA/CA) embryos (D, transverse sections in H and L). No expression of Prdm1 is detected (with two independent probes) in the
myotome (My) as shown on section (F), only rostro-caudal lips of the dermomyotome (Dm) display weak Sox6 expression. Faint expression is
detected in the posterior dorsal root ganglia (A and C arrowhead). In the newly formed somites (I), Sox6 expression is detected in the
notochord (arrowhead) and in the sclerotome (asterisk), in the absence of the myotome that has not yet begun to form, as shown by the lack
of Myogenin expression (J). In the absence of Prdm1, the expression pattern of Sox6 is not affected (C, G, and K).
identified in the 5# of the Prdm1 locus by chromatin immunoprecipitation from E11.5 limb buds (Vokes et al.
2008). Prdm1 expression in the limb buds is not affected
in Smo/ mutant embryos, suggesting that this region
is not active in the limb buds, but could be responsible
of its expression in the somites.
In mouse, in comparison with zebra fish, the single early
myotomal compartment expresses both slow and fast muscle genes within the same cells. In zebra fish, initial expression domains of smyhc1 (slow) and myhc4 (fast) show
some overlap in the adaxial cell region at the 10 somite
stage (Bryson-Richardson et al. 2005). Expression of myhc4
is much weaker and its domain more restricted compared
with smyhc1 expression, suggesting that this co-expression
is only transient, due to the subsequent activity of Prdm1.
In the mouse embryo, however, divergence of slow and fast
muscle phenotypes occurs later, after the onset of innervation during fetal development. In zebra fish, as in Xenopus, embryos innervation occurs much earlier in the
myotome (Blackshaw and Warner 1976; Westerfield
et al. 1986), in response to the requirement for rapid muscular movement, mainly mediated by myotomal muscle,
already, at the larval/tadpole stage. In contrast, during
mammalian development, there is no such early deployment of muscular activity (Deries et al. 2008). Perhaps,
as a consequence of this premature requirement for efficient fast/slow muscle function, the zebra fish embryo
has implemented a different regulatory strategy to that operational in the mammalian myotome where fast and slow
muscle genes are co-expressed. Contrary to the situation in
zebra fish, Sox6, that encodes a repressor of the slow program, is not expressed in the mouse myotome and is not
de-repressed in the absence of the murine Prdm1. Moreover, in the mouse myotome, Prdm1 does not act as a repressor of the fast muscle program. Therefore, regulatory
factors, that in the zebra fish promote the slow skeletal
muscle program, are not functional in this context in
the mouse embryo. Mouse Prdm1 has conserved a functional
role, as indicated by its capacity to replace the zebra fish
protein during a limited time window in adaxial cells of the
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Vincent et al. · doi:10.1093/molbev/mss125
Ubo mutant (Liew et al. 2008). It remains to be seen
whether Prdm1, which can therefore potentially function
as a repressor in the mouse myotome, actually has a role.
This was not revealed by our analysis of conditional Prdm1
myogenic mutant embryos or of transgenic embryos expressing a dominant negative form of Prdm1. It also
remains unclear how the slow skeletal muscle genes are
activated in the early mouse myotome. Neither Prdm1
nor Sox6 appear to be implicated. Our study clearly indicates
divergences in Prdm1 regulation and function during early
myogenesis in the zebra fish compared with the mouse
embryo.
Supplementary Material
Supplementary figures 1–3 are available at Molecular Biology
and Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
We thank S. Tajbakhsh, Y. Lallemand, and Y. Ohinata for
reagents and Emmanuel Pecnard, Sabrina Coqueran, and
Catherine Bodin for technical help. We are grateful to
Kathryn Calame for generously giving us the conditional
Prdm1 mouse line. We thank Emmanuel Perret and Pascal Roux (Imagopole, Institut Pasteur, Paris) for advice on
the imaging. We thank the Centre d’Ingénierie Génétique
Murine (CIGM) plateform (Institut Pasteur, Paris) for micronuclear injections to make transgenic lines. We especially thank Pascal Maire and Phil Ingham for helpful
discussion. Work in MB’s laboratory on Prdm1 was
funded by the Institut Pasteur and the Centre National
de la Recherche Scientifique (CNRS) with grants from the
Association Franc
xaise contre les Myopathies (AFM), the
European Union consortia Cells into Organs (NoE, 6th
framework programme) and CardioCell (7th framework
programme). S.D.V. is an Institut National de la Santé et
de la Recherche Médicale (INSERM) investigator.
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