Basic helix-loop-helix (bHLH) transcription factors are crucial
regulators, in some instances referred to as ‘master regulators’, of
cell-type identities. Among the bHLH proteins, the Myod family
drives skeletal myogenesis, similar to the regulation of
neurogenesis by Neurod proteins and lymphocyte development by
E proteins (Tapscott, 2005; Lee, 1997; Quong et al., 2002).
Homeodomain transcription factors have also been termed
‘master regulators’, but typically of organ or positional identities.
For example, the Hox proteins control segmental identities, and
Pax6 controls eye development (Pearson et al., 2005; Gehring,
1996). How are positional-identity programs linked with cell-
type-identity programs, so that neurons in the eye acquire a
different phenotype to neurons in the brain, or that neurons in one
hindbrain segment differ from those in another segment?
Although the mechanism is not known, one possibility is that
positional-identity factors modulate the activity of cell-type-
identity factors. As suggested by Westerman et al. (Westerman et
al., 2003), homeodomain proteins might act in their region of
expression to directly modulate the activity of bHLH proteins and
thus confer specific characteristics on a cell-type identity.
Although synergistic interactions of bHLH and homeodomain
proteins have been described at some promoters (Westerman et
al., 2003), the model in which homeodomain proteins can instruct
the bHLH proteins to modulate a cell-type program to generate,
for example, a particular type of neuron or muscle cell has not yet
Previous demonstrations of molecular interactions between the
bHLH Myod protein and the homeodomain proteins Pbx and Meis
suggest that skeletal myogenesis might be a good system to directly
test the role of homeodomain proteins in modulating bHLH-driven
cell-type programs (Knoepfler et al., 1999; Berkes et al., 2004).
Furthermore, myogenesis is a model system in which to study the
acquisition of cellular phenotype diversity; for example, the
generation of different fiber types. Skeletal myogenesis in vertebrate
embryos is coordinated by the bHLH transcription factors Myod,
Myf5, Myog and Mrf4 (Buckingham, 2001). Myod is sufficient to
convert fibroblasts and other non-muscle cells into skeletal muscle
(Weintraub et al., 1989). Myod directly activates the expression of
multiple additional transcription factors, including Myog, and acts
in a feed-forward mechanism in cooperation with those factors to
directly activate muscle genes expressed later in the differentiation
program (Penn et al., 2004; Cao et al., 2006). At a portion of these
later target genes, Myod appears necessary to initiate chromatin
remodeling before other transcription factors, such as Myog, can
bind to the promoters (Cao et al., 2006). Therefore, Myod has a
crucial function of identifying and remodeling the target genes that
will subsequently be available for activation by other factors.
Because Myod directly regulates a broad suite of muscle
differentiation genes, one question that arises is how does Myod
correctly regulate promoters used in different skeletal muscle types,
such as fast- or slow-twitch muscle?
Mutations of the histidine- and cysteine-rich domain or the C-
terminal helix III domain of Myod prevent full activation of a subset
of Myod target genes and also prevent cooperative DNA binding
with Pbx and Meis proteins on specific Myod target promoters,
including that ofMyog(Berkes et al., 2004). Because Pbx and Meis
proteins are present on some of these promoters prior to Myod
expression, we suggested that a Pbx-Meis complex might mark a
subset of genes for Myod activation (Berkes et al., 2004; de la Serna
et al., 2005). However, mutations in the Myod protein might alter
interactions with additional factors, and a requirement for Pbx-Meis
in skeletal myogenesis has not yet been demonstrated. Thus, it
Pbx homeodomain proteins direct Myod activity to promote
Lisa Maves1,*, Andrew Jan Waskiewicz2, Biswajit Paul1, Yi Cao1, Ashlee Tyler1, Cecilia B. Moens3and
Stephen J. Tapscott1
The basic helix-loop-helix (bHLH) transcription factor Myod directly regulates gene expression throughout the program of skeletal
muscle differentiation. It is not known how a Myod-driven myogenic program is modulated to achieve muscle fiber-type-specific
gene expression. Pbx homeodomain proteins mark promoters of a subset of Myod target genes, including myogenin (Myog); thus,
Pbx proteins might modulate the program of myogenesis driven by Myod. By inhibiting Pbx function in zebrafish embryos, we show
that Pbx proteins are required in order for Myod to induce the expression of a subset of muscle genes in the somites. In the absence
of Pbx function, expression of myog and of fast-muscle genes is inhibited, whereas slow-muscle gene expression appears normal. By
knocking down Pbx or Myod function in combination with another bHLH myogenic factor, Myf5, we show that Pbx is required for
Myod to regulate fast-muscle, but not slow-muscle, development. Furthermore, we show that Sonic hedgehog requires Myod in
order to induce both fast- and slow-muscle markers but requires Pbx only to induce fast-muscle markers. Our results reveal that Pbx
proteins modulate Myod activity to drive fast-muscle gene expression, thus showing that homeodomain proteins can direct bHLH
proteins to establish a specific cell-type identity.
KEY WORDS: Myod, Pbx, Muscle, Zebrafish
Development 134, 3371-3382 (2007) doi:10.1242/dev.003905
1Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle,
Washington 98109, USA. 2Department of Biological Sciences, University of Alberta,
Edmonton, Alberta, T6G2E9, Canada. 3Howard Hughes Medical Institute and
Division of Basic Science, Fred Hutchinson Cancer Research Center, Seattle,
Washington 98109, USA.
*Author for correspondence (e-mail: email@example.com)
Accepted 13 July 2007
remains to be determined whether a Pbx-Meis complex is necessary
for Myod activity at Myog or other specific genes in vivo and
whether this subset of genes regulates a specific aspect of muscle
Zebrafish embryos provide an attractive biological system in
which to test the role of Pbx proteins in skeletal myogenesis. Pbx
(and Meis) proteins are TALE (three amino acid loop extension)-
class homeodomain proteins that are best characterized as cofactors
for Hox proteins. In mice, overlapping expression and functional
redundancy among the Pbx genes complicates the analysis of their
requirements, and skeletal muscle defects have not been reported for
knock-outs of individual Pbx genes (Moens and Selleri, 2006).
Zebrafish have five Pbx genes, but only pbx2and pbx4are expressed
during the period of early myogenesis (Pöpperl et al., 2000;
Waskiewicz et al., 2002). Knock-down of both pbx2 and pbx4 in
zebrafish results in severe hindbrain-patterning defects that reflect
the role of Pbx proteins as Hox cofactors (Waskiewicz et al., 2002),
but skeletal muscle development was not assessed. The Pbx-Meis
binding site in the mouse Myog promoter is conserved in the
zebrafish myoggene (Berkes et al., 2004), providing an opportunity
to test the developmental role of Pbx in myogenesis. Here, we
demonstrate that Pbx proteins are necessary for the timely activation
of myog by Myod, demonstrating that Pbx has a necessary role in
marking the myoggene for efficient activation during development.
Furthermore, we demonstrate that Pbx proteins facilitate Myod
activity to drive the expression of a subset of genes necessary for
fast-muscle differentiation, thus showing that homeodomain
proteins can direct bHLH proteins to establish a specific cell-type
MATERIALS AND METHODS
Zebrafish (Danio rerio) were raised and staged as previously described
(Westerfield, 2000). Time (hpf) refers to hours post-fertilization at 28.5°C.
In some cases, embryos were raised for periods at room temperature, at
approximately 25°C. The wild-type stock used was an AB/WIK hybrid. For
microarray analyses, the AB stock was used. The pbx4mutant line has been
described (Pöpperl et al., 2000).
Morpholino and mRNA injections
Morpholino injections were performed, and working concentrations were
determined, as previously described (Maves et al., 2002). Morpholinos
(shown 5? to 3?) were used at the following working concentrations: pbx2-
MO2 (Waskiewicz et al., 2002), CCGTTGCCTGTGATGGGCTGCTGCG,
0.25 mg/ml; pbx2-MO3, GCTGCAACATCCTGAGCACTACATT, 0.5
mg/ml; pbx2-MO3 five-base mismatch control (mismatched bases shown in
lowercase), GCTcCAAgATCCTcAcCAgTACATT, 0.5 mg/ml; pbx4-MO1,
CGCCGCAAACCAATGAAAGCGTGTT, 0.5 mg/ml; myod-splMO E1I1,
AATAAGTTTCTCACAATGCCATCAG, 5 mg/ml; myod-splMO E2I2,
TTTCGAGCAAACTTACCATTTGGTG, 2.5 mg/ml; myod-MO1,
GCAAGAAATGTACTTGAATGTTTCC, 0.5 mg/ml; myod-MO2,
GATTGGTTTGGTGTTGAAGGTTTCT, 0.25 mg/ml; myf5-MO2,
GATCTGGGATGTGGAGAATACGTCC, 0.25 mg/ml. pbx2-MO2, pbx2-
MO3, pbx4-MO1 and pbx4-MO2 were combined, myod-MO1 and myod-
MO2 were combined, and myf5-MO1 and myf5-MO2 were combined in
order to knock down their respective gene products. Embryos that are
knocked-down for pbx2 and pbx4 function show a developmental delay of
approximately two somites during somitogenesis stages, comparable to
maternal-zygotic pbx4–/–; pbx2-MO embryos (see Fig. S2 in the
supplementary material; C.B.M., unpublished observations). We somite-
stage-matched sibling control and MO-treated embryos when collecting
embryos for staining or for RNA or protein analyses.
Injections of exd or shh (shha – ZFIN) mRNA were performed as
previously described (Pöpperl et al., 2000; Barresi et al., 2000).
RNA in situ hybridization and immunocytochemistry
RNA in situ hybridizations were performed as previously described
(Maves et al., 2002). The following cDNA probes were used: myod
(Weinberg et al., 1996); krox-20 [egr2b – Zebrafish Information Network
(Oxtoby and Jowett, 1993)]; myog (Weinberg et al., 1996); myhc4 (EST
fb27a08); aldh1a2 (Begemann et al., 2001); mylz2 (Xu et al., 2000);
chrna1 (Sepich et al., 1998); tmem161a (IMAGE:7149790); ttnl (EST
eu247); atp2a1 (EST fc22f07); srl (EST fb94b10); vmhc (Yelon et al.,
1999); smyhc1 (Bryson-Richardson et al., 2005); and cxcr4a (EST cb824,
Zebrafish International Resource Center). We subcloned the desmin EST
cb290 (Zebrafish International Resource Center) into pCRII-TOPO, which
was linearized with BamHI and transcribed with T7 to make the antisense
Whole-embryo immunostaining was performed with the following
primary antibodies: anti-pan zebrafish Pbx, 1:500 (Pöpperl et al., 2000);
anti-Myf5, 1:100 [Santa Cruz, C-20, sc-302 (Hammond et al., 2007)]; anti-
myosin heavy chain F59, 1:10 [supernatant (Devoto et al., 1996)]; anti-
myosin heavy chain MF20, 1:10 [supernatant (Bader et al., 1982)]; and anti-
fast myosin light chain F310, 1:10 [supernatant (Hamade et al., 2006)]. F59,
F310 and MF20 antibodies were developed by F. E. Stockdale and D. A.
Fischman and were obtained from the Developmental Studies Hybridoma
Bank, maintained by the University of Iowa. Stainings were performed as
previously described (Feng et al., 2006) with the following modifications:
for anti-Pbx staining, embryos were fixed in 4% PFA at 4°C for 4 hours and
methanol dehydration was omitted; for anti-Myf5 (Myod) staining,
embryos were fixed in 4% PFA for 1 hour at room temperature, methanol
dehydration was omitted, and washes and incubations were done in
PBS+1% Triton-X; for F59, F310 and MF20 staining, secondaries
(Southern Biotech) used were goat anti-mouse IgG1-FITC (1:100, F59 and
F310) and goat anti-mouse IgG2b-TRITC (1:100, MF20); for SYTOX
Green staining, embryos were rinsed in PBS following antibody staining,
and then were incubated in a 1:10,000 dilution of SYTOX Green
(Invitrogen) overnight at 4°C.
Embryos were photographed using a Zeiss Axioplan2 microscope, a
SPOT RT digital camera (Diagnostic Instruments) and MetaMorph software
or imaged using a Zeiss Pascal confocal microscope. Images were
assembled using Adobe Photoshop.
Western analysis was performed as previously described (Waskiewicz et al.,
2001). Samples were run on NuPage 4-12% BIS-TRIS gels (Invitrogen). The
equivalent of approximately one embryo was loaded per lane. Antibodies
were used at the following dilutions: anti-pan zebrafish Pbx (Pöpperl et al.,
2000), 1:2000; and anti-Histone H4 (Upstate Biotechnology), 1:1000.
Quantitative real-time RT-PCR
Dechorionated embryos were homogenized in TRIzol using a 1 cc insulin
syringe, and RNA was isolated following the TRIzol protocol (Invitrogen).
1 ?g of RNA plus random hexamers were used in a reverse-transcriptase
(RT) reaction with SuperScriptII Reverse Transcriptase (Invitrogen). Real-
time PCRs were performed using an Applied Biosystems 7900HT System
according to the manufacturer’s instructions. The relative expression levels
were normalized to those of ornithine decarboxylase 1 (odc1) (Draper et
al., 2001). We used the following probe and primer sets (sequences given
are 5? to 3?): myogL1 forward, CTGGGGTGTCGTCCTCTAGT; myogR1
reverse, TCGTCGTTCAGCAGATCCT; myogP1 probe, TGGAGCA GC -
GCGTCTGATCA; myodL2 forward, TCAGACGAGAAGACGGAACA;
myodR2 reverse, CACGATGCTGGACAGACAAT; myodP2 probe, CAC-
CAAATGCTGACGCACGG; odcL2 forward, GACTTTGACTTCGCC -
TTCCT; odcR2 reverse, GAGGTGCTTCTTCAGGACATC; odcP1 probe;
CGCGCGATATCGTGGAGCAG; desminL1 forward, GAGGCCGAG-
GACTGGTATAA; desminR1 reverse, GGTGTAGGACTGGAGCTGGT;
desminP1 probe, AGCCAAGCAGGAGACCATGCAA.
cDNA microarray analysis
pbx2-MO; pbx4-MO embryos as well as their control siblings (pbx2-MO3
mismatch control) were collected at the 10 somite (10s) or 18s stage from
three independent sets of injections. A subset of embryos from each set of
injections were used for in situ hybridization to confirm somite staging
Development 134 (18)
fates in Caenorhabditis elegans (Alper and Kenyon, 2001).
However, in these cases, the mechanism by which these modulations
occur is not known.
Our results provide a novel demonstration of how interactions
between different types of ‘master regulatory’ factors control cell-
type diversity. Homeodomain factors control positional identities,
whereas bHLH proteins control cell-type identities. We propose that
these identity programs merge together such that homeodomain
proteins modulate the set of bHLH-activated genes to achieve
region-specific cellular phenotypes. Recent studies are continuing
to underscore the role of homeodomain proteins in modulating
cellular diversity within a cell type, because Hox proteins are now
understood to modulate skin-cell phenotype in different regions of
the body (Rinn et al., 2006). We propose that homeodomain
proteins, by instructing bHLH proteins to regulate a subset of their
target genes, provide competence for a cell to execute a region-
specific differentiation program.
We thank the following colleagues for generously providing reagents: Peter
Currie, Stephen Devoto, Zhiyuan Gong, Randall Moon, Heike Pöpperl, Charles
Sagerstrom, Monte Westerfield and Debbie Yelon. The Zebrafish International
Resource Center (supported by grants RR12546 and RR15402-01 from the
NIH) provided cDNA clones. We thank Daniel Osborn and Simon Hughes for
advice on use of the anti-Myf5 antibody. We also thank Erwin Analau and
Sean Rhodes for technical assistance; and Stephen Devoto, Clarissa Henry,
Estelle Hirsinger and members of the Moens and Tapscott labs, especially
Laurie Snider, for advice. B.P. was supported by an HHMI Undergraduate
Research Internship and by the Washington NASA Space Grant Consortium.
C.B.M. is an investigator with the Howard Hughes Medical Institute. This work
was supported by a Research Development Grant from the Muscular
Dystrophy Association (L.M.) and by NIH AR45113 (S.J.T.).
Supplementary material for this article is available at
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