Research Article 505

Cited3 activates Mef2c to control muscle cell

differentiation and survival
Gnanapackiam Sheela Devakanmalai, Hasan E. Zumrut and Ertuğrul M. Özbudak*
Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA
*Author for correspondence ([email protected])

Biology Open 2, 505–514

doi: 10.1242/bio.20132550
Received 18th July 2012
Accepted 25th March 2013

Summary
Vertebrate muscle development occurs through sequential genes in fast myofibers. Restoring expression of cited3 or
differentiation of cells residing in somitic mesoderm – a mef2c rescued all the cited3 loss-of-function phenotypes.
process that is largely governed by transcriptional regulators. Protein truncation experiments revealed the functional
Our recent spatiotemporal microarray study in zebrafish has necessity of C-terminally conserved domain of Cited3,
identified functionally uncharacterized transcriptional which is known to mediate interactions of Cited-family
regulators that are expressed at the initial stages of proteins with histone acetylases. Our findings demonstrate
myogenesis. cited3 is one such novel gene encoding a that Cited3 is a critical transcriptional coactivator
transcriptional coactivator, which is expressed in the functioning during muscle differentiation and its absence
precursors of oxidative slow-twitch myofibers. Our leads to defects in terminal differentiation and survival of
experiments placed cited3 into a gene regulatory network, muscle cells.
where it acts downstream of Hedgehog signaling and myoD/
Biology Open

myf5 but upstream of mef2c. Knockdown of expression of ß 2013. Published by The Company of Biologists Ltd. This is
cited3 by antisense morpholino oligonucleotides impaired an Open Access article distributed under the terms of the
muscle cell differentiation and growth, caused muscle cell Creative Commons Attribution Non-Commercial Share Alike
death and eventually led to total immotility. Transplantation License (http://creativecommons.org/licenses/by-nc-sa/3.0).
experiments demonstrated that Cited3 cell-autonomously
activates the expression of mef2c in slow myofibers, while it Key words: Myogenesis, Muscle cell death, Zebrafish, mef2c, Cited,
non-cell-autonomously regulates expression of structural Differentiation

Introduction transcriptional regulator genes as cells commit to muscle

Skeletal muscle is a major tissue type comprising around 40 differentiation.
percent of the adult mass, controlling locomotion and body- cited3, which is a member of the CITED (CBP/p300-interacting
support, and accounting for most of an individual’s daily energy transactivator with glutamic acid/aspartic acid-rich tail) family
consumption. Loss of skeletal muscle mass and/or strength transcriptional coactivator, was among the differentially expressed
occurs during aging, disease, or inactivity (Evans, 2010). When transcriptional regulators. Zebrafish cited3 is orthologous with
the function of the muscle tissue is impaired it results in various mammalian Cited4. Expression of Cited4 was previously detected
metabolism- and motility-related defects. One of the therapeutic in human skeletal muscle tissue (Tews et al., 2007). Although the
avenues for treating muscle diseases is cell-based therapies, functional roles of Cited genes in skeletal muscle development
where functional muscle cells will be generated and amplified in have not been studied, they are important for mammalian
culture, and finally delivered into patient tissue. However, development and have been implicated in cardiac (Sperling et
generation of functional muscle cells requires a detailed al., 2005; Boström et al., 2010; MacDonald et al., 2012) and
understanding of how muscle cells differentiate in the first place. skeletal muscle (Pescatori et al., 2007; Sáenz et al., 2008;
As in other tissue types, specification of muscle cells does not Tobimatsu et al., 2009) defects. Cited2 RNA levels were
rely on a linear regulatory pathway but rather on a network of significantly reduced in Duchenne muscular dystrophy and limb-
interlinked signaling pathways and transcriptional regulators girdle muscular dystrophy 2A human patients (Pescatori et al.,
(Parker et al., 2003; Berkes and Tapscott, 2005; Ochi and 2007; Sáenz et al., 2008) and overexpression of Cited2 counteracts
Westerfield, 2007; Bryson-Richardson and Currie, 2008). Hence, glucocorticoid-induced muscle atrophy in C2C12 myotubes in cell
elucidating this process can serve as a model for understanding culture (Tobimatsu et al., 2009). On the other hand, an increased
development of other tissues. To identify the transcriptional level of Cited4 was previously associated with cardiomyocyte
regulators that are upregulated during the initial stages of muscle growth and proliferation in mice (Boström et al., 2010). Therefore,
development in vivo, we microdissected zebrafish paraxial we decided to investigate the functional role of cited3 in skeletal
mesoderm into different spatiotemporal domains and performed muscle development.
microarrays (Ozbudak et al., 2010). Our recent study, for the first An elegant lineage-tracing study in zebrafish previously
time, revealed the in vivo temporal induction order of demonstrated that slow muscle cells differentiate from their
 Cited3 activates Mef2c 506

progenitor adaxial cells residing next to the midline axis in the Immunostaining
somites (Devoto et al., 1996; Ochi and Westerfield, 2007; The following primary antibodies were used: F59 IgG (1:10 dilution) mainly
detects slow muscle specific myosin heavy chain, S58 (1:10 dilution) detects slow
Stellabotte and Devoto, 2007; Bryson-Richardson and Currie, muscle specific myosin heavy chain, MF20 (1:10 dilution) labels all differentiated
2008). By in situ hybridization, we identified that cited3 is muscle fibers and F310 (1:10 dilution) labels fast muscle specific myosin heavy
expressed in the precursors of slow myofibers and this expression chain were obtained from DSHB. Anti-Mef2 (1:100 dilution) was obtained from
Santa Cruz. The following secondary antibodies were used at 1:800 dilutions:
is depended on Hedgehog signaling via MyoD and Myf5. Alexa Fluor 488 or 555 goat anti-mouse IgG1 or IgA, Alexa Fluor 568 or 647 goat
We knocked down the expression of cited3 by injecting two anti-rabbit IgG, Alexa Fluor 647 goat anti-mouse IgG2b (Molecular Probes).
different antisense morpholino oligonucleotides that block Immunostaining was performed following the method of Bird et al. (Bird et al.,
splicing of cited3; this impaired muscle cell differentiation and 2012).
growth, increased the number of apoptotic muscle cells and
eventually led to total immotility. Overexpression of cited3 BrdU and Tunel staining
Embryos were pulsed for different time periods in 1 mM BrdU (Sigma), fixed in
mRNA rescued all the cited3 loss-of-function phenotypes. In situ 4% PFA and stained using anti-BrdU antibody (Sigma) following the protocol of
hybridization for various muscle-specific genes placed cited3 Barresi et al. (Barresi et al., 2001) with minor modification for whole mount and
into a gene regulatory network, where it acts downstream of on sections. To identify apoptotic cells, in situ cell death detection kit (Roche) was
used. Embryos fixed (4% PFA) at different stages were treated with Proteinase K
myoD/myf5 but upstream of mef2c. We identified that mef2c is 1 mg/ml according to the stage and washed with PBST and stained with TMR red
the main target of Cited3 as its phenotype is rescued by restoring for 30–60 minutes at 37 ˚C.
expression of mef2c. These data make a new and unprecedented
connection between Cited3 and Mef2c – a crucial transcription Motility test
factor for both heart and skeletal muscle tissues (Lin et al., 1997; Embryos that are injected with control MO, cited3 MO or coinjected with cited3
RNA were tested for motility. Touch evoked response was recorded using live
Nakagawa et al., 2005; Hinits and Hughes, 2007; Potthoff et al.,
imaging of embryos from 2 days-post-fertilization (dpf) to 5 dpf using a Leica
2007; Potthoff and Olson, 2007). This is the first report microscope and the speed of movements were analyzed using Sony Vegas movie
demonstrating a Cited-family transcriptional coactivator Studio HD 9.0 software.
functioning in the early stages of muscle development.
Imaging
Materials and Methods In situ hybridized embryos and immunostained embryos were scanned under a
Leica 205MA microscope and Carl Zeiss Imager Z2 equipped with apotome and
Zebrafish strains axiovision 4.8 Rel. Serial sections of fluorescent images of antibody staining were
Biology Open

Wild-type AB and TB fish strains were raised in the fish facility. Loss-of-function taken at 1–3 mm intervals with Apotome and then 2D images were created by
experiments of FGF signaling was performed using transgenic fish with heat- stacking all pictures taken at different focal planes.
shock-driven expression of dn-fgfr1 Tg (hsp70: dn-fgfr1-gfp) (Lee et al., 2005).
Loss of function of Wnt signaling was accomplished by using transgenic fish with
heat-shock-driven expression of Dtcf: Tg (hsp70: Dtcf-gfp), (Lewis et al., 2004). Quantitation and statistical analysis
Gain of function of Wnt signaling was performed by using transgenic fish with Mef2, Tunel and BrdU positive cells were counted using Image J. Cell counts were
heat-shock driven expression of wnt8: Tg (hsp70: wnt8-gfp) (Weidinger et al., performed on z-stacks spanning an entire segment on one side of the embryo or on
2005). myf5hu2022 mutant were obtained from the Wellcome Trust Sanger Institute. sections. Somites in 5–10 animals were scored and the experiments were
Fish were bred and maintained at 28.5 ˚C on a 14–10 h light/dark cycle as reproduced minimum three times. Fluorescent images of different samples were
described (Westerfield, 1993). taken on the same microscope with the same objective and exposure duration. The
fluorescent intensity was quantified on the unmodified original images and the
background intensity was subtracted by using Image J. F59 staining was
Microinjection
diminished significantly after 2 dpf, while S58 staining was reduced at a slower
Capped RNA for microinjection was synthesized by in vitro transcription
pace and lost after 5 dpf. Therefore, we used S58 staining to count the number of
according to Manufacturer’s protocol (Ambion, the RNA Company, Texas, US)
slow fibers. Nevertheless, as S58 immunostaining was reduced in the morphants,
from linearized plasmid of pCS2+-cited3-254 (coding for full-length 254 amino
we pseudo increased the intensity of staining in order to count the number of
acids), pCS2+-cited3-200 and pCS2+-Mef2c (Mouse cDNA, kind gift from Stephen
fibers. Fibers were counted on whole mount embryos on one hemi-segment in the
Tapscott). Antisense cited3 (S1) morpholino oligonucleotide (MO) ‘TCTCCC-
anterior 10–12 somites and posterior 16–18 somites. Total number of fibers were
TTCATTAAAACACACAAGA’ and cited3 (S2) MO ‘GCATCATCATGTGC-
counted from the 3D images acquired using axiovision software and verified with
TCTGCCATAGA’, are synthesized to block the proper splicing of cited3 at two
fluorescent intensity plots from plot profile using Image J (Barresi et al., 2001). All
different domains, were injected at a concentration of 0.8 or 12 ng/embryo,
P-values were calculated by performing two-tailed Student’s t-test in Excel. A
respectively, and both produced similar phenotypes. myoD MO ‘ATATCCGACA-
P-value below 0.05 was considered statistically significant.
ACTCCATCTTTTTTG’, was injected at 0.4 ng/embryo according to Hinits et al.
(Hinits et al., 2009). Equivalent concentration of control MO (Gene Tools) was
injected as a control. Fertilized eggs at one- to four-cell stages were microinjected Cell transplantation
with 80–200 pg RNA/embryo. Embryos were fixed at the desired stages in 4% Cell transplantation was done following the protocol of Zeller and Granato et al.
paraformaldehyde fixative overnight at 4 ˚C and washed with phosphate-buffered (Zeller and Granato, 1999). Donor embryos were injected with fixable tetramethyl
saline and stored in methanol at 220 ˚C until use. rhodamine dextran fluorescent dye (Molecular Probes, D7162) with or without
morpholinos. Cells from donor embryos were transplanted at shield stage into host
Gene cloning and in situ hybridization embryos; chimeric embryos were identified, fixed, immunostained with F310 or
The coding sequence of cited3 was cloned into pDrive (Qiagen) and pCS2+ vectors F59 and Mef2 antibodies, and imaged.
by using cited3F: ATGGCAGAGCACATGATGATGCC and cited3R:
TCAGCAGCTAACGGTGCTCGG primers. The C-terminally truncated cited3 Results
variant is created by placing a stop codon after the first 199th amino acids. Whole-
mount in situ hybridization was carried out as previously described (Thisse and
cited3 is expressed in the slow muscle precursors in somites
Thisse, 2008). Antisense RNA probes were synthesized by in vitro transcription and its expression is activated by Hedgehog signaling and
from linearized plasmids using RNA polymerase T7 or T3 enzyme and myod/myf5
digoxigenin RNA labeling mix (Roche). The following in situ hybridization
probes were used: myod, myf5 (cb641 from ZIRC), mef2ca, prox1, stnnc and
cited3 is expressed in the polster (arrow), which is the precursor
smyhc1 (kind gift from Phillip Ingham), alpha actinin, myogenin, myhz1, myhz2, of hatching gland, and in the notochord (red arrow) and chordo-
mylc2, tnnt1 and tnnt3b (kind gift from Monte Westerfield), mef2d (kind gift from neural hinge (green arrowhead) at 10 hpf (Fig. 1A). At 13 hpf,
Simon Hughes), cmlc2 (kind gift from Deborah Yelon). Mouse Mef2c mRNA cited3 is expressed in the slow muscle precursor adaxial cells
(kind gift from Stephen Tapscott) was injected in rescue experiments. Double
fluorescent in situ hybridization was done following the protocol of Brend and (black arrowhead in Fig. 1B) on either side of the notochord.
Holley (Brend and Holley, 2009). Subsequently, cited3 transcripts become abundant in slow muscle
 Cited3 activates Mef2c 507

atrium and ventricle heart precursors (Fig. 1I–K) overlapping

with cmlc2 expression (Yelon et al., 1999).
In order to identify the signaling pathways regulating cited3
expression, we blocked the hedgehog signaling by treating
embryos with hedgehog inhibitor cyclopamine or with ethanol as
a control from 5 hpf to 15 hpf. As a control, we checked the
transcription of myod, which is known to be lost only in the
adaxial region upon loss of Hedgehog signaling (Lewis et al.,
1999; Osborn et al., 2011). Transcription of both myod and cited3
was lost in cyclopamine treated embryos in the adaxial region
(Fig. 1M,O) but myod transcription was retained in precursors of
fast myofibers and that of cited3 was retained in the chordo-
neural hinge, compared to ethanol treated embryos respectively
(Fig. 1L,N). These results indicate that cited3 is activated in
response to Hedgehog signaling only in slow muscle precursors.
Then we blocked the activity of Hedgehog signaling for a shorter
duration starting at 13 hpf (when transcription of cited3 is already
turned on in the adaxial cells) until 18 hpf. This resulted in loss of
myod transcription in the adaxial cells throughout the axis
(Fig. 1P,Q), while cited3 transcription is reduced only in the
posterior-most part (presomitic mesoderm) of the adaxial region
Fig. 1. Expression of cited3 during segmentation stage. (A) cited3 is
expressed in the polster (black arrow) and in the notochord (red arrow) and but not in the somites (Fig. 1R,S). Incubation of embryos with
chordo-neural hinge (green arrowhead) at 10 hpf. (B) At 13 hpf, cited3 is cyclopamine only for 2 hours from 13 hpf to 15 hpf reduced the
expressed in the adaxial cells (slow muscle precursor; black arrowhead) on transcription levels of myod drastically but not that of cited3 (data
either side of the notochord. (C) Expression of cited3 becomes abundant in the not shown). The results suggest that myod is a direct target of
slow muscle precursors at 14 hpf. (D) cited3 is also expressed in the
differentiated slow muscle cells as indicated by black arrowhead and in the
Hedgehog signaling, while cited3 might be indirectly regulated
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hatching gland as indicated by black arrow and neural crest cells (red by Hedgehog signaling.
arrowhead) at 24 hpf. (E–H) cited3 is expressed in the differentiating slow Then we investigated the regulatory relationship between
muscle cells. (E) Cross section of 24 hpf embryo depicting Hoechst staining in cited3 and muscle-regulatory-factors myod and myf5. We
the nucleus; (F) cited3 expression in the slow muscle domain; (G) myod knocked down the expression of myod with the myod MO
expression in the slow and fast muscle domain; (H) merge of cited3 and myod
expression in the slow muscle domain (indicated by a white arrowhead). (Hinits et al., 2009). Expression of cited3 was not affected in the
(I–K) cited3 is expressed in the heart precursors. (I) cited3 is expressed in the myod morphants (Fig. 1T,U). Similarly, we checked the
heart precursors both in the atrium and ventricle (J) cmlc2 expression in the expression of cited3 in embryos from the mating of
heart precursors (K) merge (ventral view of embryos). (L–S) Expression of heterozygous myf5hu202 mutants and found that its expression
cited3 is regulated by the Hedgehog signaling (L,N) Expression of myoD,
cited3 was not perturbed in ethanol treated control embryos, (M,O) but
was not affected (data not shown). MyoD and Myf5 often
expression of myod and cited3 is lost in the adaxial cells in cyclopamine treated regulate their targets redundantly. To investigate this possibility,
(from 5–15hpf) embryos (indicated by arrowhead) whereas myod expression is therefore, we knocked down the expression of myod in embryos
still retained in the fast muscle (M) and cited3 expression in the chordo-neural- from myf5hu202 heterozygous incross and observed that
hinge (arrow) (O). In comparison to control ethanol treatment, cyclopamine expression of cited3 was not affected in 75% of the embryos
treatment from 13 to 18 hpf blocked transcription of myod in all adaxial cells
(P,Q), but that of cited3 only in adaxial cells located in the presomitic (Fig. 1V,V9) but was lost in the remaining 25% of embryos
mesoderm (R,S). (T–W) Myod and Myf5 redundantly regulate the expression (Fig. 1W,W9). Hence, these results demonstrate that Myod and
of cited3. (U) Expression of cited3 was not perturbed in the embryos that were Myf5 redundantly activate transcription of cited3. Furthermore,
injected with myoD MO compared to those injected with the control MO (T) at they also suggest that Hedgehog signaling might activate the
17 hpf. Similarly, expression of cited3 was not affected in 75% of the embryos
that were obtained from mating of heterozygous myf5hu202 mutant fishes and
expression of cited3 via activating the expression of myod and
injected with the myod MO (22/28) (V,V9). However, expression of cited3 was myf5 (Lewis et al., 1999; Osborn et al., 2011) in the adaxial cells.
reduced or lost in the remaining 25% of the embryos from the same clutch As Wnt and Fgf signaling are known to regulate muscle
injected with myod MO (6/28) (W,W9), while the remaining expression of development, we tested the effect of these signaling factors on
cited3 is in the neural crest cells. Scale bars: 100 mm in D,L,P,T, and 50 mm in the expression of cited3. We perturbed the activities of Wnt and
V9.
Fgf signaling in a time-resolved fashion by applying a pulse of
heat-shock to transgenic embryos, where heat shock-responsive
cells at 14 hpf to 18 hpf (Fig. 1C) when they are known to hsp70 promoter activates the expression of various transgenes
differentiate and migrate to the periphery. Expression of cited3 is (supplementary material Fig. S1). However, we found that
detected in the hatching gland (black arrow), neural crest cells neither Wnt nor Fgf signaling regulate transcription of cited3
(red arrowhead), brain and branchial arches at 24 hpf (Fig. 1D). (supplementary material Fig. S1).
We performed double fluorescent in situ hybridization for cited3
and myod on the cross-sections of embryos that are at 24 hpf. At Transcriptional coactivator domain of Cited3 is necessary for its
this stage, differentiating slow myofibers are located lateral to the function
differentiating fast myofibers. We demonstrated that cited3 is We performed loss-of-function experiments for cited3 to
expressed only in the slow myofibers, while myod is expressed in investigate its functional role during development. We designed
both myofiber types (Fig. 1E–H). Double fluorescent in situ two splice-blocking morpholino oligonucleotides (MO) targeting
hybridization also revealed that cited3 is expressed both in the the first intron-exon boundary (S1-MO and S2-MO) of cited3. As
 Cited3 activates Mef2c 508

a control, we used the standard control MO that does not target Cited3 protein depends on this conserved domain, we coinjected
any zebrafish gene. Subsequently, we confirmed the efficacy of C-terminally truncated cited3 mRNA with cited3 MO. The
two cited3 MOs by RT-PCR: injection of each cited3 MO truncated cited31–200 mRNA could not rescue the morphant
prevented the splicing of the first intron significantly, whereas it phenotype; it also failed to rescue the F59 staining (Fig. 2D,H,L).
is efficiently spliced out in the embryos injected with the control Hence, we conclude that the transcriptional coactivator domain of
MO (supplementary material Fig. S2). Cited3 protein is necessary for its function during development.
Knockdown of cited3 resulted in obvious morphological Fig. 2M is the graphical representation of intensity measurements
phenotypes compared to wild-type embryos or embryos from F59 staining. Intensity of F59 staining was significantly
injected with the control MO starting after 1 dpf. In the cited3 reduced in cited3 morphants when compared to that in wild-type (P-
morphants, the heart was dilated, the body axis did not grow as value,261024); the staining was significantly rescued when the
much as it should (Fig. 2A,B), and the myosin heavy chain full-length cited3 (P-value,261022) but not the truncated cited31–
200
staining that is detected with F59 was reduced (Fig. 2E,F,I,J). RNA was co-injected (P-value.0.7). Interestingly, injection of
The specificity of the phenotype was verified by coinjection of full-length cited3 RNA alone into wild-type embryos increased the
full-length spliced mRNA of cited3 along with cited3 MOs. Full- myosin heavy chain expression significantly compared to that in
length cited3 mRNA rescued the cited3 morphant phenotype wild-type embryos (data not shown).
(Fig. 2C,G,K).
Cited proteins are transcriptional coactivators. They interact Cited3 stimulates the growth and maintenance of muscle fibers
with histone acetylases with their conserved C-terminal CR2 Knockdown of cited3 resulted in shorter embryos; this led us to
domain (Bragança et al., 2002). To assess whether the function of test whether muscle cell growth is impaired and/or muscle cells
are dying in the absence of cited3. First, we investigated muscle
cell death in cited3 morphants by using in situ cell death
detection kit, TMR Red. These embryos were stained with anti-
Mef2 antibody to identify muscle cells. cited3 morphants had
increased cell death in the muscle (Fig. 3D–F) compared to
control MO injected embryos (Fig. 3A–C) and this phenotype
was rescued in the embryos coinjected with cited3 RNA
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(Fig. 3G–I). We quantified the number of apoptotic non-muscle

versus muscle cells in all three treated groups. At 1 dpf, apoptotic
cells were not found in the muscle of all three groups of embryos
(data not shown). However, we found significantly higher
number of apoptotic muscle cells in cited3 morphants at 2 dpf
and 3 dpf (Fig. 3M). Simultaneously, we did BrdU pulses in
these embryos from 30 hpf to 42 hpf and 54 hpf to 66 hpf. We
found that the numbers of BrdU positive cells were not
significantly different in all the three treated groups (Fig. 3J–L;
data not shown). Correlated with this finding, we still detect
generation of thin secondary myofibers due to cell proliferation
in cited3 morphants after 1 dpf (data not shown). Hence, these
results demonstrate that the absence of cited3 leads to muscle cell
apoptosis but it does not affect proliferation of the cells.
Next, we quantified the number of slow myofibers through 1–4
dpf by using slow muscle specific S58 antibody staining. cited3
knockdown resulted in statistically significant reduction in the
number of slow myofibers starting at 3 dpf, which was rescued by
coinjecting cited3 RNA (Fig. 4). cited3 knockdown also
Fig. 2. Knock down of cited3 disrupts muscle differentiation. (A,B) Knock
down of cited3 with two splice-blocking morpholino oligonucleotides (MO), prevented the growth of myofibers; the width and length of
S1-MO and S2-MO, resulted in thinner and shorter trunk, and dilated heart (B) individual fibers and the width of somites were reduced
compared to wild-type embryos (A) or embryos injected with the control MO significantly at 5 dpf (supplementary material Fig. S3).
(data not shown). Coinjection of full length cited3 RNA (codes for 254 amino Correspondingly, the heights of somites were shorter in embryos
acid) rescued cited3 morphant phenotype (C). (D) In contrast, coinjection of a
C-terminally truncated form of cited3 RNA (coding for the first 200 amino
that were injected with the cited3 MO compared to those that
acids) did not rescue the morphant phenotype. (E) The expression of myosin were injected with the control MO (supplementary material Fig.
heavy chain as evidenced by F59 antibody in an area covering somite 16th–18th S3). Altogether, these defects resulted in reduced motility in
in a wild-type embryo at 2 dpf. (F) The staining of F59 is reduced in embryos cited3 morphants, which became totally immotile at 5 dpf as
injected with cited3 MO, whereas the expression of myosin heavy chain was
assessed by touch-evoked escape response test (supplementary
restored when cited3 RNA was co-injected with cited3 MO (G) but not rescued
when cited31–200 RNA was coinjected (H). (I–L) Magnified images of F59 material Fig. S4). The motility defect was significantly rescued
staining. (M) The graphical representation of the average fluorescent intensity by the coinjection of cited3 RNA together with the cited3 MO
measurement of F59 immunostaining (of 10–18 embryos from three different (supplementary material Fig. S4).
experiments). Intensity of F59 staining was significantly reduced in cited3
morphants when compared to that in wild-type (P-value,261024), rescued
when cited3 RNA was co-injected (P-value,261022) but not rescued when Cited3 regulates expression of mef2ca and myogenin
cited31–200 RNA was coinjected (P-value.0.7). Scale bars: 100 mm in A and In order to identify where cited3 fits in the gene regulatory
50 mm in E. network controlling muscle cell differentiation, we assessed
 Cited3 activates Mef2c 509

Fig. 3. Knockdown of cited3 results in increased cell death but it does not affect proliferation. Embryos that are injected with the control MO (A–C), cited3 MO
(D–F), cited3 MO + cited3 RNA (G–I) were fixed at 2 dpf and immunostained with anti-Mef2 antibody (A,D,G) and TMR red in situ cell death detection kit (B,E,H).
Tunel positive cells were counted in non-muscle cells as well as in muscle cells in each group of embryos from 50–100 sections obtained from trunk and tail somites.
More apoptotic muscle cells were detected in embryos that are injected with the cited3 MO and fixed at 2 dpf and 3 dpf (M). Values were normalized compared to
cited3 morphant values. Similarly, embryos injected with the control MO (J), cited3MO (K), cited3 MO + cited3 RNA (L) was pulsed with BrdU from 30 hpf to
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42 hpf, and were fixed and immunostained with anti-BrdU and MF20 antibodies (images of 16th–18th somites). There is no significant difference between the
three treated groups (J,K,L) when the BrdU-positive muscle cells were counted (data not shown). Scale bars: 25 mm in A and 50 mm in K.

expression levels of a set of muscle genes via in situ staining, we quantified the intensity of Mef2 staining in both the
hybridization. We found that knockdown of cited3 does not transplanted and 8–10 closest neighboring slow myofibers that are
affect transcriptions of muscle regulatory genes myod located in the same somite. We transplanted presumptive slow
(Fig. 5A,B) or myf5 (Fig. 5C,D). But, differentiation marker muscle cells from donor wild-type embryos that are injected with
myogenin transcripts were retained in cited3 morphants at a stage the rhodamine dextran fluorescent dye into control MO injected host
when it is downregulated in control embryos (Fig. 5E,F). A embryos of similar stage (Fig. 5P–R9). There was no significant
similar effect on myogenin expression was previously detected difference in Mef2 protein levels between the transplanted wild-type
when the expression of zebrafish mef2 genes were lost (Hinits et slow myofiber and the slow myofibers in host embryos (Fig. 5Q; see
al., 2009). Therefore, we checked the expressions of zebrafish supplementary material Fig. S5 for quantification). Similarly, we
mef2 genes. Transcription of mef2ca was reduced in the cited3 transplanted presumptive slow muscle cells from wild-type donors
morphants compared to the control MO-injected embryos into cited3 morphant hosts (Fig. 5S–U9) and found that Mef2
(Fig. 5G,H). Coinjection of cited3 RNA along with cited3 MO protein levels were retained in the wild-type transplanted cells that
restored the mef2ca transcription (Fig. 5I). On the other hand, the are surrounded by the morphant cells in which Mef2 protein levels
transcription of mef2d was not perturbed (Fig. 5J–L). were lower (Fig. 5T and quantified in Fig. 5Y). Whereas donor
We also validated our findings by immunostaining with the fibers that are transplanted from cited3 MO injected embryos into
general Mef2 antibody that recognizes both Mef2c and Mef2d wild-type hosts (Fig. 5V–X9) depicted a reduction in Mef2 protein
proteins in zebrafish (Hinits and Hughes, 2007). We found a levels (Fig. 5W and quantified in Fig. 5Z). Thus, these data
significant reduction in the levels of total Mef2 proteins in cited3 demonstrate that cited3 activates the expression of mef2c cell-
morphants and this was rescued by coinjection of cited3 RNA autonomously in slow myofibers.
(Fig. 5M–O). We also detected a similar phenotype in the heart,
where Mef2 protein levels were reduced in heart cardiomyocytes
of the cited3 morphants. This phenotype was also rescued by Expression of muscle structural genes are perturbed in cited3
coinjection of cited3 RNA (Fig. 5M9–O9). Altogether, these results morphants
demonstrate that cited3 functions upstream of mef2ca in the heart Expression levels of prox1, smyhc1, alpha actinin, myhz1, myhz2
and skeletal muscle. and mylc2 were not affected at 19 hpf (supplementary material Fig.
S6). As differentiation proceeds further, transcription levels of
Cited3 cell-autonomously activates the expression of mef2c in smyhc, stnnc, tnnt1, myhz2, mylc2, tnnt3b and myhz1 are declining
slow muscle cells in wild-type embryos at 2 dpf. However, their transcription levels
In order to identify whether Cited3 activates the expression of mef2c were retained in cited3 morphants at the same stage (Fig. 6A–N).
cell-autonomously or not, we carried out cell transplantation Interestingly, fast-muscle specific F310 myosin heavy chain
experiments. As the anti-Mef2 antibody recognizes both Mef2c expression is reduced after 2 dpf in cited3 morphants (Fig. 6P)
and Mef2d proteins and there are cell-to-cell variations in its compared to control MO injected embryos (Fig. 6O).
 Cited3 activates Mef2c 510

Fig. 4. The number of slow

myofibers is reduced in cited3
morphants starting at 3 dpf. The
number of slow fibers was counted on
whole mount embryos on one
hemisegment from 10th–12th somites in
the anterior region and 16th–18th somite
in the posterior region of embryos at 1–
4 dpf. Slow myofiber number is
reduced significantly starting from 3
dpf in the morphant compared to WT
(P-value,261022 at 3 dpf and P-
value,261023 at 4 dpf) and was
restored when cited3RNA was
coinjected (P-value,461022 at 3 dpf
and P-value,561023 at 4 dpf).

Cited3 non-cell-autonomously activates expression of fast- Discussion

myofiber specific myosin heavy chain expression cited3 is placed in a regulatory network downstream of myod/
We detected transcription of cited3 only in the slow fiber cells myf5 and Hedgehog signaling but upstream of mef2ca
both by regular and fluorescent in situ hybridization. However, The muscle regulatory factors (MRFs) MyoD, Myf5, Myogenin
starting after 2 dpf, we have detected disturbances in the and Mrf4 initiate myogenesis (Parker et al., 2003; Berkes and
expression of slow myofiber specific genes smyhc, stnnc and Tapscott, 2005; Hinits et al., 2009). Mef2 proteins are expressed
tnnt1 (Fig. 6A–F), fast myofiber specific genes myhz2, mylc2 and during terminal differentiation of muscle cells (Black and Olson,
tnnt3b (Fig. 6G–L), and myhz1 (which is expressed in both fiber 1998). Expression of Mef2c is activated by the MRFs both in
types) (Fig. 6M,N). Expression of fast myosin heavy chain that is mice and zebrafish (Wang et al., 2001; Hinits et al., 2009). Later
Biology Open

detected by F310 antibody staining is also reduced in cited3 on, Mef2 proteins interact with the MRFs to cooperatively
morphants at the same stage (Fig. 6O,P). In order to identify activate late-stage muscle differentiation genes (Penn et al.,
whether Cited3 regulates expression of fast-myofiber specific 2004). Mef2c is required for heart looping morphogenesis and
myosin heavy chain cell-autonomously or not, we transplanted formation of the right ventricle in mice (Lin et al., 1997). mef2c
presumptive muscle cells from donor wild-type embryos that are gene is duplicated in zebrafish. Morpholino-mediated knockdown
injected with rhodamine dextran fluorescent dye into host of mef2ca (Ghosh et al., 2009) or mef2cb (Lazic and Scott, 2011)
embryos of similar stage that are injected with the cited3 MO are reported to result in pericardial edema or cell addition defects
and stained embryos with F310 at 2 dpf. The transplanted wild- from the secondary-heart field, respectively. mef2ca2/2;
type fast myofibers express F310 at low levels that are mef2cb2/2 double mutants had reduced expression of
comparable to their neighboring morphant fast myofibers sarcomeric genes in zebrafish heart (Hinits et al., 2012).
(supplementary material Table S1; Fig. S7). This result suggests Skeletal muscle-specific deletion of Mef2c in mice results in
that fast-fiber myosin heavy chain expression is non-cell- pups that were slightly smaller than the wild-type littermates and
autonomously upregulated by cited3. We performed the opposite the mutant mice died within the first day after birth. At earlier
experiment by transplanting cells from cited3 morphants into stages of development, Mef2c-deficient myocytes differentiate
wild-type hosts. The transplanted morphant cells expressed fast and fuse normally; however, muscles gradually become
myosin heavy chain at comparable levels to their neighboring disorganized at later stages (Potthoff et al., 2007). Double
wild-type fast myofibers. Altogether, this result suggests that knockdown of mef2ca and mef2d did not affect the initial
Cited3 acts non-cell-autonomously to promote terminal differentiation and fusion of myoblasts, however, the myoblasts
differentiation of fast myofibers. failed to complete sarcomere assembly and double morphant fish
larva also exhibit motility defects starting at 1 dpf (Hinits and
Restoring the expression of mef2c rescues the phenotypes of Hughes, 2007). This phenotype exhibits similarities to the
loss-of-function of cited3 skeletal muscle specific loss of Mef2c in mice.
To determine whether mef2c transcription factor is the main Here, we showed that expression of cited3 is activated by
target of Cited3, we coinjected mouse Mef2c RNA together with MyoD/Myf5 and Hedgehog signaling (Fig. 1). As transcription of
cited3 MO into embryos. We found that Mef2c rescues the myod switches off much faster than that of cited3 upon blockage
morphological phenotypes of the cited3 morphants (Fig. 7A–C). of Hedgehog signaling, it is highly likely that Hedgehog
Similarly, Mef2c also rescued expression of myosin heavy chain signaling activates expression of cited3 via activating myod and
as detected by F59 staining (Fig. 7D–F). Furthermore, restoring myf5 (Lewis et al., 1999; Osborn et al., 2011). Although the short
the expression of Mef2c also prevented the increased cell death time interval between reductions of transcription levels of myod
phenotype that was observed upon knockdown of cited3 as to that of cited3 in cyclopamine treated embryos suggests that
revealed by the tunel staining (Fig. 7G–O). Quantification of Myod (and Myf5) directly activates transcription of cited3, we do
apoptotic muscle cells confirmed that cited3 maintains the not have a proof of a direct regulation at the moment. In turn,
muscle cells by activating the expression of mef2c as Mef2c Cited3 activates expression of mef2ca (Fig. 5G–I, Fig. 8), which
could rescue the cell-death phenotype of cited3 morphants results in retainment of myog transcription at 22 hpf as previously
(Fig. 7P). observed in zebrafish (Hinits and Hughes, 2007). This results in
 Cited3 activates Mef2c 511
Biology Open

Fig. 5. Cited3 regulates expression of mef2c and myogenin and it acts cell-autonomously. (A–D) Expression of myoD and myf5 was not perturbed in embryos injected
with the cited3 MO at 19 hpf. (E,F) Myogenin transcripts were retained in mature somites in the cited3 morphants at 22 hpf (F) where it was usually downregulated in the
control MO-injected embryos (E). Transcription of mef2ca was reduced in cited3 morphants (H) compared to control MO-injected embryos (G) and it was restored to normal
levels when coinjected with cited3 RNA (I) at 19 hpf. (J–L) Mef2d expression was not affected in embryos injected with the cited3 MO or cited3 MO + cited3 RNA at 19 hpf.
(M–O) Immunostaining with the general anti-Mef2 antibody (recognizes all Mef2 variants) revealed that cumulative Mef2 protein levels were reduced in the cited3
morphants (N compared to M) and were restored to normal levels when cited3 RNA was coinjected (O) at 22 hpf (images spanning somites 16th–18th are shown) . Similarly,
Mef2 protein levels were reduced in the heart of cited3 morphants (N9) compared to the control MO-injected embryo (M9) and rescued in the cited3 RNA coinjected embryos
(O9) at 30 hpf. (P–U9) Slow muscle fibers labeled with rhodamine dextran dye were transplanted from wild-type donor embryos into control MO-injected (P–R9) or cited3
MO-injected host embryos (S–U9). Anti-Mef2 immunostaining revealed that Mef2 protein levels in wild-type transplanted myofibers (arrowhead) were not different from
those in the control MO injected host myofibers (Q), but were brighter compared to those in the cited3 MO containing host slow myofibers (T). (V–X9) Mef2 staining in the
transplanted cited3 morphant myofibers is fainter than their wild-type host counterparts. (Y,Z) The normalized intensity of Mef2 protein level is plotted for different
transplantation conditions. The number of quantified transplanted slow myofibers ranged from 23 to 141. P-values are 1027 in Y and 10212 in Z. Scale bars: 100 mm in A and
25 mm in M,P. Images in (P–U9) are from trunk somites, while those in (V–X9) are from anterior somites.

retainment of expression of muscle structural genes encoding failed to complete muscle differentiation, had muscle growth and
various thin and thick filaments in myofibers at 2 dpf (Fig. 6). maintenance defects and impaired motility (Figs 1–4;
supplementary material Figs S3, S4). The onset of muscle
mef2c is the main target of cited3 both in the skeletal muscle differentiation and motility defects in the cited3 morphants was at
and heart 2 dpf (Fig. 2; supplementary material Fig. S4), which is later
Knockdown of cited3 did not prevent initial differentiation and compared to those in the double knockdown of mef2ca and mef2d
fusion of myoblasts at 1 dpf; it also did not perturb expression (Hinits and Hughes, 2007). Likewise, expression levels of various
levels of various muscle structural genes at 19 hpf muscle structural genes were perturbed at later stages (F59
(supplementary material Fig. S6). However, cited3 morphants staining at 30 hpf, F310 staining and transcription of other genes
 Cited3 activates Mef2c 512
Biology Open

Fig. 6. Expression of various muscle structural genes were affected in cited3

morphants starting at 2 dpf. As differentiation proceeds, the expression levels
of smyhc, stnnc, tnnt1, myhz2, mylc2, tnnt3b and myhz1 in wild-type embryo were
down regulated at 2 dpf (A,C,E,G,I,K,M). However, their respective expression
in cited3 morphants were retained (B,D,F,H,J,L,N). Expression of fast muscle
myosin heavy chain is reduced after 2 dpf in cited3 morphants (P) compared to
control MO injected embryos (O) as evidenced by F310 immunostaining
(somites 15th–18th). Scale bars: 100 mm in B and 25 mm in P.
Fig. 7. Restoring expression of Mef2c rescues functional loss of cited3 and
prevents cell death. Coinjection of mouse Mef2c RNA rescues the
at 2 dpf) in cited3 morphants (Figs 2,6). This could be due to the morphological phenotypes (A–C) and the reduction in the expression of myosin
persistence of mef2d transcripts in the cited3 morphants (Fig. 5J– heavy chain (15th–18th somites) (D–F) in the cited3 MO-injected embryos.
K). Strikingly, expression of mouse Mef2c rescued the cited3 Coinjection of mouse Mef2c RNA also reduced the number of apoptotic muscle
cells in cited3 MO-injected embryos. (H,K,N) The number of apoptotic muscle
morphant phenotype (Fig. 7) suggesting that mef2c is the main cells are increased in cited3 MO-injected embryos and it was restored to normal
transcriptional target of Cited3 in skeletal muscle differentiation. levels by the coinjection of mouse Mef2c RNA (as seen in I,L,O) as compared
Interestingly, transcription of mef2ca starts at 14 hpf (Hinits and to control MO injected embryos (G,J,M). (P) Quantification of the apoptotic
Hughes, 2007), which is soon after cited3 is expressed in the cells revealed that mouse Mef2c RNA significantly prevented the cell death in
muscle cells of embryos that are coinjected with cited3 MO. These are average
adaxial cells (Fig. 1B) suggesting that mef2ca might be a direct values from 97 sections from trunk and tail of 5–10 embryos from three
target of Cited3. different experiments. Values were normalized compared to cited3 morphant
Cited3 morphants also had a dilated heart. Due to its small values. Scale bars: 100 mm in A and 25 mm in E,G.
size, fish embryos can develop and survive in the absence of a
functional heart and blood circulation as oxygen is obtained via As cited3 is expressed in the heart (Fig. 1) and regulates the
diffusion (Bakkers, 2011). Therefore, the skeletal muscle levels of Mef2 proteins in the heart (Fig. 5), and mouse Mef2c
phenotype is unlikely to result from a defect in circulation. rescues loss of function phenotype of cited3 in the heart
 Cited3 activates Mef2c 513

(Fig. 7), mef2c is likely to be the major target of Cited3 also in fast myofibers even after their initial migration (Henry and
the heart. Amacher, 2004).
As cited3 is functionally important for both heart and skeletal
muscle development, Cited3 protein might interact with different This is the first report demonstrating the importance of the
tissue-specific transcription factors in each tissue or interact with cited3 gene in terminal muscle cell differentiation and
the same transcription factor that is expressed in both tissues. At maintenance
this point, we do not know which transcription factor Cited3 Muscle diseases develop due to genetic mutations, metabolic
interacts with and also do not have a proof that Cited3 directly disorders and aging. Development of cellular therapies in which
activates transcription of mef2ca. We will investigate these functional muscle cells are introduced into the patient muscles is
important questions and the detailed characterization of the heart one of the most promising avenues for treating muscle diseases.
phenotype in cited3 morphants in our future studies. Recent progress in the efficiency of direct transdifferentiation of
cells into a different cell fate (Davis et al., 1987; Takeuchi and
cited3 non-cell-autonomously regulates differentiation and Bruneau, 2009; Forsberg et al., 2010; Harvey, 2010; Ieda et al.,
maintenance of fast myofibers 2010; Vierbuchen et al., 2010) demonstrated the importance of
Although cited3 is expressed only in the precursors of slow identifying gene clusters functioning during embryonic
myofibers, loss of its expression also resulted in defects in fast development of each tissue type. Discovering functionally
myofibers. In addition to slow myofibers, apoptotic cells are also significant genes in muscle development, likewise, has the
detected among fast myofibers after 2 dpf (Fig. 3). Expression potential to result in significant cell-based therapeutic
levels of fast-fiber specific genes are also perturbed starting at 2 advancement (Parker et al., 2003; Snider and Tapscott, 2003;
dpf (Fig. 6). Our cell transplantation experiments revealed that Tajbakhsh, 2009). Strikingly, Mef2c, which we now have
Cited3 activates expression of Mef2 proteins cell-autonomously identified to be the target of Cited3, was previously
in the slow myofibers (Fig. 5). However, expression of fast fiber demonstrated to cooperate with MyoD and Myogenin to
specific myosin heavy chains (detected by F310) is non-cell- enhance transdifferentiation of fibroblasts into muscle cells
autonomously regulated by Cited3 (supplementary material Fig. (Molkentin et al., 1995).
S7; Table S1). Altogether, this result suggests that Cited3 acts The functional roles of Cited-family genes in skeletal muscle
non-cell-autonomously to promote terminal differentiation and development have so far not been investigated in any organism.
Biology Open

maintenance of fast myofibers. Slow fibers are the first However, expression of Cited2 was reduced in Duchenne and
differentiating fiber type in zebrafish and their differentiation limb-girdle muscular dystrophy patients (Pescatori et al., 2007;
trigger timely differentiation of the progenitors of fast myofibers Sáenz et al., 2008). In contrast, overexpression of Cited2
(Henry and Amacher, 2004). Our results suggest that slow prevented drug-induced muscle atrophy in cell culture
myofibers play an instructive role in terminal differentiation of (Tobimatsu et al., 2009). Interestingly, loss of Mef2 genes was
also implicated in muscular atrophy in mammals (Yamakuchi et
al., 2000; Uozumi et al., 2006; Tessier and Storey, 2010). This is
the first report, where we demonstrate that expression of cited3 is
critical for muscle cell differentiation and maintenance and it acts
through activating the expression of mef2ca.

Acknowledgements
We thank Dr Monte Westerfield, Simon Hughes, Stephen Devoto,
Stephen Tapscott, Deborah Yelon and Phillip Ingham for plasmids;
Bruce Appel (for the hsp70:gal4VP16), José Campos-Ortega (for
the UAS: myc-notch1a-intra‘kca3), Kenneth Poss (for the
hsp70:dnfgfr1-EGFP), and Randall Moon (for the hsp70:wnt8a-
GFP and hsp70l: DTcf-GFP) transgenic lines; Dr Stemple and the
Wellcome Trust Sanger Institute for the myf5hu2022 mutant line.
We also thank Burcu Guner-Ataman, Xin Li, Becky Weiss, Aaron
Sarvet, Abdulvahap Sahin, Betul Sisman, Adem Demir, Rini
Dcunha, Murat Isbilen, Cagri Kurt, Zeynep Ulupinar and Fatih
Keles for technical help; Dr Florence Marlow for her support during
the start-up phase of our lab; and members of Özbudak, Marlow and
Jenny labs at the Albert Einstein College of Medicine for helpful
discussions.

Competing Interests
The authors have no competing interests to declare.
Fig. 8. Schematic representation of a genetic pathway that connects cited3
to muscle cell differentiation and survival. Hedgehog signaling activated the
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