Download Signaling networks that regulate muscle development: Lessons from

Develop. Growth Differ. (2007) 49, 1–11
doi: 10.1111/j.1440-169x.2007.00905.x
Review
Blackwell Publishing Asia
Signaling networks that regulate muscle development:
Lessons from zebrafish
Haruki Ochi and Monte Westerfield*
Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA
Locomotion mediated by skeletal muscle provides a basis for the behavioral repertoire of most animals.
Embryological and genetic studies of mouse, bird, fish and frog embryos are providing insights into the
functions of the myogenic regulatory factors (MRFs) and the signaling molecules that regulate activity of
MRFs. Nevertheless, our understanding of muscle development remains somewhat limited. Fundamental
goals are to elucidate how mesodermal cells are induced during gastrulation to form muscle precursor cells
and how muscle precursor cells acquire specific cell fates, such as slow and fast muscle cells. In this review,
we focus on studies of zebrafish muscle development that have advanced our understanding of the molecular
genetics of muscle cell induction and specification.
Key words: Fibroblast growth factor, Hedgehog, skeletal muscle, t-box gene, zebrafish.
Introduction
Recent studies of muscle development in zebrafish
provide exciting insights into the molecular genetic
mechanisms that regulate induction, specification and
differentiation of muscle cells. In addition, the ability
to perform both genetic and embryological analyses
with single cell resolution makes the zebrafish a
powerful tool for the study of muscle development.
Genetic screens provide an opportunity to identify
genes not previously implicated in myogenesis.
Analysis of gene expression provides an opportunity
to evaluate the timing and lineage specificity of myogenic regulatory factors in relation to the processes
of muscle linage specification and differentiation. The
present review focuses on the cellular lineage that
gives rise to skeletal muscle and the genetic network
that controls specification and differentiation of this
lineage. In the first section, we summarize the fate
map and lineage of zebrafish muscle precursor
cells. In the second section, we describe the genes
and molecular mechanisms that regulate muscle
*Author to whom all correspondence should be addressed.
Email: [email protected]
Received 12 October 2006; revised 3 November 2006;
accepted 6 November 2006.
© 2007 The Authors
Journal compilation © 2007 Japanese Society of
Developmental Biologists
development. In the third section, we propose a genetic
network model of the signal pathways and transcription
factors that regulate induction of muscle precursor
cells from multipotent mesodermal cells, specification
of muscle subtypes and terminal differentiation.
Fate map of muscle precursor cells
Cell types in zebrafish
Zebrafish axial skeletal muscles contain four fiber types;
slow muscle cells, muscle pioneer cells, fast muscle
cells and medial fast fiber cells (Fig. 1e). Each fiber
type has distinct morphological and developmental
properties (Bone 1978; Devoto et al. 1996; Wolff et al.
2003) and occupies a distinct region of the axial
muscle (Devoto et al. 1996; Wolff et al. 2003).
Slow and fast muscle cells have distinct physiological
and biochemical properties. Slow muscle cells comprise the red muscle whereas fast muscle cells
comprise the white muscle. In most fish, including
zebrafish, slow muscle fibers are located superficially,
just under the skin, with fast muscle fibers located
deeper (Fig. 1e) (Devoto et al. 1996). Muscle pioneer
cells, a subset of the slow muscle, are located medially in the developing somite and express members
of the Engrailed (Eng) homeobox protein family
(Fig. 1e) (Hatta et al. 1991). The muscle pioneers are
thought to serve as intermediate targets for the
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H. Ochi and M. Westerfield
Fig. 1. Schematic illustrations of zebrafish muscle development. (a) Slow and fast muscle precursors occupy a distinct region in the
marginal zone at the gastrula stage. Magenta dots indicate the locations of slow muscle or muscle pioneer cell precursors. Green
dots indicate the locations of fast muscle cell precursor. Muscle precursor cells are not yet committed at this stage. Muscle precursor
cells in the marginal zone undergo involution (arrow). (b) Myogenic gene expression starts during the mid-gastrulation stages. Blue
indicates myod expression. Magenta dots indicate the location of slow muscle and muscle pioneer cell precursors. Green dots
indicate the location of fast muscle cell precursors. Presumptive notochord (N) and muscle precursors extend anteriorly (arrow) due
to convergence extension movements. (c) Slow muscle precursors are specified by signaling from the notochord. Magenta indicates
the location of adaxial cells, precursors of muscle pioneer and other slow muscle cells. Green indicates the location of fast muscle
precursors. Upper panel shows the dorsal view, lower panel shows a cross-section. (d) Adaxial cells incorporate into the somites.
Upper panel shows the dorsal view. Magenta indicates the location of slow muscle cell precursors. Green indicates the location of
fast muscle precursors. Lower panel shows a cross-section; magenta and green indicate the myotome and yellow indicates the
sclerotome. (e) Four types of zebrafish muscle cells towards the end of the segmentation period (24 hpf, hours postfertilization stage).
A cross-section through the trunk of a late segmentation stage embryo. Adaxial cells migrate to the lateral surface of the somite where
they differentiate into slow muscle cells (magenta). A subset of the adaxial cells remains deep within the somite and differentiates into
muscle pioneer cells (dotted magenta). Fast muscle cells differentiate in the central part of the somite (meshed green). A subset of
Eng positive fast muscle cells, medial fast muscle cells, differentiate next to the notochord (meshed blue). The external cell layer,
dermomyotome, appears during the late segmentation stages (meshed orange). (Modified from Devoto et al. 1996; Holley & NussleinVolhard 2000; Hirsinger et al. 2004; Devoto et al. 2006).
growth cones of the earliest motor axons, because
ablation of muscle pioneers affects growth of the Cap
and Mip primary motoneuron axons (Melançon et al.
1997). It is unclear if muscle pioneer cells persist or
have a later role in mature zebrafish. By the end of
the first day of development, Eng antibodies label
not only muscle pioneers but also lower level Eng
expression in a cloud of nuclei surrounding the muscle
pioneer cells (Hatta et al. 1991; Wolff et al. 2003).
Because these cells that express low levels of Eng
do not express slow myosin heavy chain, which is
expressed by slow muscle and muscle pioneer cells,
they are classified as medial fast fiber cells (also called
Eng positive fast muscle cells; Fig. 1e) (Wolff et al.
2003). To date, no specific role for the medial fast
muscle cells has been reported.
Fate map and lineage analyses
Fate-mapping studies revealed that before the onset
of gastrulation, cells that ultimately give rise to skeletal
muscle occupy the marginal zone of the zebrafish
embryo (Kimmel et al. 1990). In the marginal zone,
slow and fast muscle precursor cells already occupy
distinct locations (Fig. 1a) (Hirsinger et al. 2004).
Slow muscle precursors are located close to the shield
(Fig. 1a), the organizer and future dorsal side, whereas
fast muscle precursors are located farther around the
margin toward the ventral side (Fig. 1a). Slow muscle
and muscle pioneers can arise from the same marginal cell, indicating that they share a common linage
at the shield stage (Fig. 1a). Even though slow and
fast muscle precursors occupy distinct locations at
the shield stage, they readily change fate when transplanted into the other domain, indicating that they
are not yet committed to form a particular skeletal
muscle subtype (Hirsinger et al. 2004).
Muscle precursor cells in the marginal zone then
undergo involution and convergence extension movements as part of gastrulation (Fig. 1a,b, arrow). During
this period, they begin myogenesis, as indicated by
initial transcription of the myogenic genes, myod and
myf5 (Fig. 1b) (Weinberg et al. 1996).
By the end of gastrulation, muscle precursor cells
position themselves in the segmental plate on either
side of the nascent notochord (Fig. 1c). These cells
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
Muscle development in zebrafish
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Fig. 2. Expression of genes involved in skeletal muscle development. Schematic illustrations of
gene expression patterns during
muscle development. Expression
of fibroblast growth factors (fgf )
and t-box genes is detectable at
about the 30% epiboly stage in
the blastoderm margin. hhip and
smoothened (not shown) are maternally provided, whereas expression
of hh genes begins at about the
50% epiboly stage. myod, an
early myogenic marker, becomes
detectable at the 75% epiboly
stage. prdm1 is detectable in the
blastoderm margin and is then
restricted to adaxial cells at the
bud stage.
continue to express myod and form a monolayer of
pseudo-epithelial cells, called adaxial cells (Fig. 1c
and Fig. 2). Adaxial cells are precursors of slow
muscle cells including muscle pioneers (Devoto et al.
1996) and, like the fast muscle precursors, they
become committed to their specific fates later, just
before they become incorporated into a somite
(Hirsinger et al. 2004). The somite is patterned into
myotome and sclerotome (Fig. 1d, lower panel)
(Holley & Nusslein-Volhard 2000). After the somite
forms, adaxial cells leave the pseudo-epithelium and
migrate to the lateral surface of the somite where
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
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H. Ochi and M. Westerfield
they differentiate into slow muscle fibers (Fig. 1e,
upper panel). A subset of adaxial cells remains next
to the notochord and differentiates into muscle pioneer
cells (Fig. 1e). Lateral, nonadaxial muscle precursors
in the segmental plate differentiate into fast muscle
cells after the somite forms (Fig. 1e, lower panel)
(Devoto et al. 1996). The origin of the medial fast
muscle cells is currently unknown.
Toward the end of the segmentation period, lateral
cells between the slow muscle and dermis that have
been termed external cells (Devoto et al. 2006) express
the paired-type homeobox proteins, Pax3 and Pax7.
They also express the myogenic regulatory factors
(MRFs), Myf5 and Myogenin, which are expressed
by myogenic precursor cells in other vertebrates, but
they do not express myogenic differentiation markers
such as Myosins (Fig. 1e) (Devoto et al. 2006). This
suggests that the external cell layer includes myogenic precursors that differentiate into muscle cells
somewhere else. Because the dermomyotome of
amniotes expresses a similar set of genes, it has been
proposed that these external cells in zebrafish are
homologous to the amniote dermomyotome (Devoto
et al. 2006). During larval growth, slow muscle fibers
are added in growth zones near the dorsal and ventral
extremes of the myotome, immediately adjacent to
the presumptive dermomyotome (Barresi et al. 2001).
Thus, in zebrafish, after initial patterning of slow and
fast muscle by cells from the segmental plate, the
dermomyotome may provide a continuing source of
slow muscle cells.
Genetic regulation of zebrafish muscle
development
In zebrafish, mutants have been isolated from gamma
ray and chemical (ethylnitorosourea, ENU) mutagenesis
screens (Fritz et al. 1996; van Eeden et al. 1996) that
have provided new insights into our understanding
of the molecular genetics of muscle development.
Analysis of gene expression using in situ hybridization
also provides an opportunity to evaluate the timing
and lineage specificity of MRFs. These types of analyses have implicated additional genes in zebrafish
muscle development (Fig. 2) that we describe below.
Myogenic regulatory factors, Myod, Myf5 and
Myogenin
Myod, Myf5, Myogenin and MRF4 are MRFs of the
basic helix loop helix family. In mouse, Myf5 and
Myod are expressed in proliferative myoblasts, which
implicates these MRFs in the establishment and
maintenance of muscle progenitor cells (Pownall et al.
2002). In contrast, Myogenin and Mrf4 are activated
during myoblast differentiation (Pownall et al. 2002).
In zebrafish, myod expression is first detectable as
small triangular patches on each side of the embryonic
shield at about the 70–75% epiboly stage (Fig. 2)
(Weinberg et al. 1996). Slightly later, at 80% epiboly,
myf5 transcripts appear in two stripes adjacent to
the notochord (Fig. 2) (Coutelle et al. 2001). myogenin
transcripts are detectable much later during the
segmentation period (Fig. 2) (Weinberg et al. 1996).
myod is also expressed in the somites during the
segmentation period (Fig. 2) (Weinberg et al. 1996).
To date, expression of zebrafish mrf4 has not been
reported. There have been few functional studies of
zebrafish MRFs, so their roles in the specification and
differentiation of muscle lineages are still unclear.
Fibroblast growth factor signaling in muscle
development
Fibroblast growth factor (Fgf) is a signaling molecule
that acts through Fgf receptors and an intracellular
MAP kinase signaling pathway to regulate various
aspects of development (Gotoh & Nishida 1995).
Studies of cultured myoblasts suggest that Fgf
promotes myoblast proliferation and represses myogenic differentiation (Winter et al. 1993; Yoshida et al.
1996; Edom-Vovard et al. 2001). Recent analysis has
shown that Fgf signaling directly activates Xmyod
expression (Fisher et al. 2002). Studies in chick indicate instead that Fgf signaling regulates muscle
progenitor differentiation, rather than modifying their
proliferative capacities (Marics et al. 2002). Thus, it
is still somewhat unclear how Fgf signaling regulates
myogenesis.
In zebrafish, fgf8 and fgf24 function together in
mesoderm formation (Draper et al. 2003). fgf8 expression becomes detectable at the 30% epiboly stage
in the blastoderm margin, and by 50% epiboly forms
a dorsal-ventral gradient with highest levels of expression in the dorsal embryonic shield (Fig. 2) (Reifers
et al. 1998). In contrast, fgf24 expression is first
detectable somewhat later by 50–60% epiboly, also
in the blastoderm margin (Fig. 2) (Draper et al. 2003).
During segmentation stages, fgf8 expression extends
into the posterior presomitic mesoderm and appears
in the somites (Fig. 2), whereas fgf24 expression is
not detectable in somites (Draper et al. 2003; Thisse
et al. 2004).
In zebrafish acerebellar (fgf8) mutant embryos,
myod expression is reduced in adaxial cells but is
maintained in somites (Reifers et al. 1998). Inhibition
of Fgf24 function in fgf8 mutants suppresses formation of posterior mesoderm and essentially eliminates
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
Muscle development in zebrafish
all myod expression (Draper et al. 2003), indicating
that Fgf8 and Fgf24 together regulate mesoderm
formation and myod expression.
T-box transcription factors, No tail and Spadetail,
in muscle development
Screens of gamma ray induced mutations led to the
discovery of no tail (ntl) (Kimmel et al. 1991) and
spadetail (spt, tbx16) (Kimmel et al. 1989) mutations
that affect muscle development. spt (tbx16) embryos
fail to form trunk somites and have a shortened,
spade-like tail while ntl embryos lack both the notochord and the tail. Both genes encode T-box transcription factors, spt encodes Tbx16 and ntl encodes
Brachyury (Halpern et al. 1993; Griffin et al. 1998).
In either ntl or spt mutant embryos, the onset of
myod expression is delayed until the end of gastrulation but partially recovers during segmentation
stages (Amacher et al. 2002; Weinberg et al. 1996).
In contrast, myod expressing cells are never detected
in ntl –/–; spt –/– double mutant embryos (Amacher et al.
2002). Thus, Ntl and Spt are essential together for
expression of myogenic genes (Amacher et al. 2002;
Goering et al. 2003).
Hedgehog signaling in muscle development
Hedgehog (Hh) is a secreted signaling protein that
acts through at least two proteins, Patched (Ptc) and
Smoothened (Smo). Ptc negatively regulates Hh signaling by inhibiting Smo. Hh binding to Ptc relieves
this inhibition and allows Smo to transduce the signal
(Ingham & McMahon 2001). The primary biochemical
mechanisms of the Hh pathway are conserved from
Drosophila to vertebrates, although vertebrates have
multiple Hh proteins (Ingham & McMahon 2001). In
zebrafish, Hh has been shown to play critical roles
in muscle development. Hh activity is necessary and
sufficient to induce slow muscle and muscle pioneer
cells both in vivo and in vitro (Weinberg et al. 1996;
Du et al. 1997; Norris et al. 2000; Wolff et al. 2003).
shha expression begins around 60% epiboly in the
shield and then appears later in the notochord and
floor plate (Krauss et al. 1993). The zebrafish shh
duplicate, shhb, is expressed in the embryonic shield
from 50% epiboly and then later in the floor plate
and ventral brain. ihhb expression starts slightly later
than shha, during late gastrulation, in the notochord
(Krauss et al. 1993; Currie & Ingham 1996).
From ENU screens, you-type mutants have been
identified, including you-too (yot), sonic-you (syu),
you (you), u-boot (ubo) and chameleon (con). In youtype mutants, somites have a U-shape and reduced
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formation of the horizontal myoseptum and muscle
pioneers (van Eeden et al. 1996). detour (dtr) and
iguana (igu) mutants also have somite defects,
although they were originally isolated because of
errors in retinal axon guidance (Karlstrom et al. 1996).
The genes disrupted by these mutations have now
been cloned and shown to encode components of
the Hh signaling pathway that are conserved between
Drosophila and vertebrates, including syu that encodes
Shha (Schauerte et al. 1998), yot that encodes Gli2
(Karlstrom et al. 1999), con that encodes dispatched
(Nakano et al. 2004), dtr that encodes Gli1 (Karlstrom
et al. 2003) and ubo that encodes Prdm1 (van Eeden
et al. 1996; Baxendale et al. 2004).
In shha (syu) and gli2 (yot) mutant embryos, myod
expression in adaxial cells is strongly reduced or
absent (Karlstrom et al. 1996; Schauerte et al. 1998).
Because Shhb and Ihhb are still expressed in shha
(syu) mutants, Eng positive muscle pioneer cells are
formed (Schauerte et al. 1998). In contrast, almost
all muscle pioneer cells are lacking in gli2 (yot)
mutants, consistent with the downstream role of Gli2
in the Hh pathway (Karlstrom et al. 1996). Mutations
in Smo were isolated from an ENU screen for morphological defects (Barresi et al. 2000; Varga et al.
2001) and from a retroviral insertion screen (Chen
et al. 2001). In smo mutants, myod expression in
adaxial cells is missing and essentially all muscle
pioneers and other slow muscle cells fail to form
(Barresi et al. 2000). Later, however, expression of
myod recovers in the somites and fast muscle cells
form (Barresi et al. 2000; Chen et al. 2001; Varga
et al. 2001). Thus, interrupting the Hh signaling
pathway with mutations in smo or gli2 reduces myod
expression in adaxial cells and blocks formation of
adaxial derived muscle pioneer and slow muscle
cells, but leaves fast muscle cells, which derive from
the paraxial mesoderm, relatively unaffected.
Discovery of novel genes that regulate muscle
development
The ability to perform both genetic and embryological analysis makes the zebrafish a powerful tool for
discovering novel genes that regulate muscle development. Molecular cloning has shown that you encodes
Scube2 and igu encodes Dzip, both of which regulate
Hh signaling, although neither appears to have a
homologue in Drosophila (van Eeden et al. 1996;
Sekimizu et al. 2004; Wolff et al. 2004; Kawakami
et al. 2005; Woods & Talbot 2005; Hollway et al.
2006). Hedgehog interacting protein (Hhip) functions
in the formation of muscle pioneers and has been
shown to be another vertebrate-specific Hh activity
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
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H. Ochi and M. Westerfield
regulator (Ochi et al. 2006). Based on gain and loss
of function experiments, we originally suggested that
different levels of Hh signaling may specify different
muscle subtypes (Du et al. 1997). Together, these
findings suggest that in vertebrates, multiple regulators
of Hh activity may be required to fine tune Hh activity
during muscle cell development.
Vertebrate specific Hedgehog regulator, You/Scube2,
in muscle development
The secreted matrix protein, Scube2, has conserved
EGF and CUB domains and a secretory pathway signal
sequence (van Eeden et al. 1996; Kawakami et al.
2005; Woods & Talbot 2005; Hollway et al. 2006).
scube2 transcripts appear to be maternally deposited and distributed widely throughout the embryo
until the late gastrulation stages (Fig. 2) (Kawakami
et al. 2005; Hollway et al. 2006). During segmentation
stages, scube2 expression is detected in the eye field,
in stripes in the midbrain and the midbrain–hindbrain
boundary, in a complex pattern in the hindbrain, and
in paraxial stripes along the anterior-posterior axis
(Woods & Talbot 2005).
The molecular function of Scube2 is still controversial
and at least three different models have been proposed. First, Scube2 could function in the transport
or stability of Hh in the extracellular space (Woods
& Talbot 2005). Second, Scube2 may mediate Hh
signaling and may act indirectly via a long-range
regulator of Hh signaling (Kawakami et al. 2005), as
it apparently does to modulate the long-range action
of Bmp-dependent signaling in the neural tube and
somites (Kawakami et al. 2005). Third, because Sube2
has homology to Cubilin, which encodes an endocytic receptor involved in protein trafficking, Scube2
might function in protein trafficking during Hh signal
transduction (Hollway et al. 2006). Further analysis
should reveal which of these roles Scube2 plays in
the Hh pathway.
In the you (scube2) zebrafish mutants, myod expression is disrupted in adaxial cells and muscle pioneer
cells fail to form (Woods & Talbot 2005). In contrast,
cells in the somites express myod later during the
segmentation stages (Kawakami et al. 2005; Woods
& Talbot 2005). Thus, Scube2 is required for the Hh
dependent development of adaxial cell derived slow
muscle and muscle pioneers.
Vertebrate specific Hedgehog regulator, Hhip,
in muscle development
Hhip, a type I transmembrane protein, was originally
identified as a molecule that binds Hh (Chuang &
McMahon 1999). Genetic and biochemical analyses
suggest that Hhip acts as a negative regulator of Hh
signaling by sequestering Hh at the cell surface
(Treier et al. 2001; Chuang et al. 2003; Kawahira et al.
2003), by being released from cells where it can bind
Hh extracellularly (Coulombe et al. 2004) and by
modulating Smo localization (Ochi et al. 2006). Hence,
the major function of Hhip is to attenuate Hh signaling
locally.
Zebrafish hhip is initially expressed by adaxial cells
and later by muscle pioneer cells and a subset of
fast muscle cells (Fig. 3) (Ochi et al. 2006). Experimentally induced gain and loss of Hhip function
reveals that Hhip is required for restricted expression
of myod in adaxial cells and subsequent slow muscle
and muscle pioneer development (Ochi et al. 2006).
Mutations in hhip, ukkie (uki ), were isolated in a
screen for mutants with altered levels of cellular proliferation (Koudijs et al. 2005). In hhip (uki ) mutants,
proliferation of cells in the ciliary marginal zone of
the retina is increased (Koudijs et al. 2005). In addition,
the pectoral fins, which are reduced in shha (syu)
and dispatched (con) mutants (van Eeden et al. 1996;
Schauerte et al. 1998), are enlarged in hhip (uki ) mutants,
indicating that Hh signaling is increased in hhip (uki )
mutants. Consistent with this interpretation, a slightly
increased number of muscle pioneer cells form in
hhip (uki ) mutants (Ochi et al. 2006).
Vertebrate specific Hedgehog regulator, Dzip1,
in muscle development
DAZ interacting protein 1 (dzip1) mRNA is maternally
deposited and abundant in newly fertilized eggs.
Throughout segmentation stages, dzip1 is ubiquitously
expressed throughout the embryo (Sekimizu et al.
2004; Wolff et al. 2004), like smo (Varga et al. 2001).
Functional analyses have suggested that Dzip1 shuttles between the cytoplasm and nucleus in an Hh
dependent manner (Sekimizu et al. 2004; Wolff et al.
2004) and that Dzip1 may both positively and negatively control Hh activity (Sekimizu et al. 2004; Wolff
et al. 2004). Hh dependent muscle cell types form
aberrantly in dzip1 (igu) mutants (Wolff et al. 2004),
contributing further support to the notion that Dzip
functions in the Hh signaling pathway.
Transcriptional repressor, Prdm1, in muscle
development
Prdm1 is a SET domain containing transcriptional
repressor considered to be a master regulator of
terminal B-cell differentiation (Yu et al. 2000). During
B-cell differentiation, transcription of the c-myc gene
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
Muscle development in zebrafish
7
Fig. 3. Model of skeletal muscle
development in zebrafish. (a)
Schematic illustration of the genetic
regulation of early myogenesis in
zebrafish. Both fibroblast growth
factor (Fgf) signaling and T-box
genes are required for early myod
expression. (b) Molecular mechanisms that specify the formation
of slow and muscle pioneer cells
from adaxial cells. Expression of
Patched (Ptc) may allow adaxial
cells to respond to high levels
of Hh activity and, together with
Hedgehog interacting protein
(Hhip), prevent the lateral diffusion
of Hh proteins. (c) Specification of
fast muscle cells. A combination
of Retinoic acid (RA) and Fgf8
regulates myod expression in the
somites and promotes fast muscle
differentiation. (d) Model explaining how Fgf and Hh signal pathways function in the specification
of zebrafish skeletal muscle cell
types. NT, neural tube; N, notochord.
is repressed by Prdm1 via association with histone
deacetylase (HDAC) (Yu et al. 2000).
In zebrafish, expression of prdm1 starts in adaxial
cells after the onset of myod and myf5 expression
(Fig. 2) (Baxendale et al. 2004; Wilm & Solnica-Krezel
2005). Because prdm1 expression is absent in smo
mutants and overexpression of shha induces expres-
sion of ectopic prdm1, prdm1 expression in adaxial
cells is apparently regulated by Hh activity (Baxendale
et al. 2004). During segmentation stages, expression
of prdm1 in adaxial cells decreases gradually in more
anterior somites, but is maintained in the posterior,
youngest somites (Baxendale et al. 2004; Wilm &
Solnica-Krezel 2005).
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
8
H. Ochi and M. Westerfield
In prdm1 mutants, slow muscle cells fail to express
the slow muscle marker, Prox1, and transfate into fast
myosin heavy chain expressing cells that resemble
fast muscle cells (Roy et al. 2001). Thus, it has been
suggested that Prdm1 functions at a critical choice
point in the slow and fast differentiation program (Roy
et al. 2001), although the targets of Prdm1 have not
yet been identified.
Genetic network model of skeletal muscle
development
The formation of skeletal muscle can be divided into
a series of steps. Initially, naïve, unspecified and
uncommitted mesodermal cells are induced to form
muscle precursors that then differentiate into specific
skeletal muscle cell subtypes. Both extrinsic signaling factors and intrinsic transcription factors regulate
each of these steps.
Induction
In mouse and chicken, skeletal muscle cells originate
from the dermomyotome in the somite (Pownall et al.
2002). The paired-type homeobox gene, pax-3 (and
related gene pax-7), which is expressed in the
paraxial mesoderm before the somites form, is a key
regulator of myogenesis because it activates Myod
expression in the somites (Rawls & Olson 1997). The
induction of Pax3 expression in the myotome is a
consequence of the combinational effects of the Wnt,
Shh, Fgf and BMP signaling factors (Pownall et al.
2002).
Zebrafish myogenesis is first indicated by the onset
of myod expression at about the 70% epiboly stage
(Fig. 2). The roles of Pax3 and Pax7 in the induction
of myod and myf5 expression in zebrafish are currently unknown. Instead, studies in zebrafish have
identified Fgf signaling and the T-box transcription
factors, Ntl and Spt, as key regulators of myod
expression in zebrafish (Weinberg et al. 1996; Reifers
et al. 1998; Amacher et al. 2002). As described above,
compromised Fgf signaling or T-box gene expression
results in reduced expression of myod. In addition,
Fgf signaling regulates expression of T-box transcription factors (Amaya et al. 1993; Griffin et al. 1995;
Granato et al. 1996) and Ntl regulates fgf8 expression
(Draper et al. 2003), suggesting that interactions
between Fgf signaling and T-box gene expression
are essential for induction of myogenesis in zebrafish
(Fig. 3a). However, both fgf and ntl are already
expressed in the marginal zone by 30% epiboly
(Fig. 2), much earlier than the onset of myod expression, and both genes are essential for mesoderm
formation (Griffin et al. 1998; Rodaway et al. 1999;
Schier & Talbot 2005). Thus, it is unclear if Fgf and
T-box genes function directly in the induction of
myogenesis or if they act permissively in muscle
induction by providing the necessary mesodermal
precursors.
Specification and differentiation of muscle pioneer
and slow muscle cells
Previous work in zebrafish has shown that expression
of both myod and myf5 normally begins in the rostral
presomitic mesoderm in mutants lacking shha (Coutelle et al. 2001), suggesting that Shh is not required
for initial induction of myogenesis. Instead, numerous
studies have implicated Hh activity in the induction
of the slow muscle cell type (Du et al. 1997; Norris
et al. 2000; Wolff et al. 2003). More recently, however,
we showed that slow muscle precursors form independently of Hh signaling and that Hh, instead, acts
later when postmitotic muscle cells become committed
to the slow fate (Hirsinger et al. 2004). Transplantation
experiments demonstrated that muscle precursors
are uncommitted to slow or fast fates at the shield
stage, and become committed to the slow fate after
they converge toward the midline and join the pseudoepithelial adaxial cell layer next to the notochord
(Hirsinger et al. 2004). In smo mutants, which lack
Hh signaling, adaxial cells form muscle fibers, but
unlike wild-type adaxial cells they fail to develop into
slow muscle or muscle pioneer cells. Together, these
studies indicate that Hh signaling is required for
muscle precursors to commit to the slow muscle fate
(Hirsinger et al. 2004).
How then is the choice between muscle pioneers
and non-muscle pioneer slow muscle cells made?
Our early studies suggested that differences in the
level of Hh signaling specify different skeletal muscle
cell subtypes (Du et al. 1997). More recent analyses
using mutants and pharmacological inhibition of
Smo support this model that Hh signaling acts in a
dose-dependent manner to specify cell fates in the
zebrafish myotome (Wolff et al. 2003). At the highest
concentrations of Smo inhibitor, most muscle pioneers, other slow muscle cells and Eng positive
medial fast muscle cells are eliminated (Wolff et al.
2003). However, at intermediate concentrations,
muscle pioneer and Eng positive medial fast muscle
cells are absent, whereas other slow muscle cells
are unaffected (Wolff et al. 2003). At lowest concentrations, only muscle pioneer cells are disrupted
(Wolff et al. 2003). Thus, immediately adjacent to the
notochord, high levels of Hh activity commit muscle
precursors to form muscle pioneer cells and Eng
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
Muscle development in zebrafish
positive medial fast fibers, whereas lower levels of
Hh activity regulate slow muscle cell commitment
(Fig. 3d) (Wolff et al. 2003). Such a gradient of Hh
activity and threshold responses to specific levels
of Hh might be provided by a combination of the
spatial locations of precursor cells and the functions
of proteins that interact with Hh in precursor cells
(Fig. 3b,d).
Hh proteins are produced by midline cells and the
Hh receptor, Ptc, is expressed at high levels by
adaxial cells at the segmentation stage (Fig. 2)
(Lewis et al. 1999a; Lewis et al. 1999b). Hhip, a negative regulator of Hh activity, is also expressed at
high levels by adaxial cells (Fig. 2) (Ochi et al. 2006).
Expression of Ptc may allow adaxial cells to receive
high levels Hh signaling and, together with Hhip, may
prevent lateral diffusion of Hh proteins, thus producing a sharp gradient of Hh activity. Adaxial cells
respond to this local high concentration of Hh and
become committed to form slow muscle and muscle
pioneer cells (Fig. 3b,d).
Fused (Fu), a putative serine/threonine kinase, is
required for cells to respond to maximal levels of
Hh activity in Drosophila (Ingham & McMahon 2001).
In zebrafish, inhibition of Fu activity causes a loss of
muscle pioneer cells but not other slow muscle cells
or medial fast muscle (Wolff et al. 2003), suggesting
that Fu functions at a choice point in the differentiation
program of muscle pioneer cells (Fig. 3d) (Ingham &
Kim 2005). Dzip has unique properties in the Hh regulatory pathway (Sekimizu et al. 2004; Wolff et al.
2004). Muscle pioneer cells, which are committed by
high levels of Hh activity, and the other slow muscle
cells that require low levels of Hh are reduced in
igu mutants (Wolff et al. 2004), whereas the later Hh
dependent medial fast fibers that require intermediate levels of Hh are substantially increased (Wolff
et al. 2004). Thus, Dzip may act as a positive regulator of Hh activity for early developing slow muscle
cells and as a negative regulator for later developing
medial fast fiber cells (Fig. 3d) (Sekimizu et al. 2004;
Wolff et al. 2004). Prdm1, expressed in adaxial cells,
is apparently required for adaxial derived muscle
cells to form slow muscle, because loss of Prdm1
in u-boot mutants causes slow muscle cells to transfate into fast myosin heavy chain expressing cells
(Roy et al. 2001). Thus Prdm1 may function at a
critical choice point in the slow muscle differentiation
program (Fig. 3d) (Roy et al. 2001). These various
factors, together with the spatial location of precursor cells, may produce a gradient of Hh activity
and differential responses to specific levels of Hh
activity along the gradient (Fig. 3b,d) (Ingham & Kim
2005).
9
Specification and differentiation of fast muscle cells
After muscle precursor cells are specified to form
slow muscle or muscle pioneer cells, slow muscle
precursors migrate to the lateral surface of the
somite and fast muscle cells begin differentiation
within the somite (Devoto et al. 1996). As we described
earlier, fgf8 is initially expressed in the germ ring at
the 30% epiboly stage and is dispensable for induction
of myogenesis. During segmentation stages, however,
Fgf8 signaling apparently regulates fast muscle cell
differentiation, because cells in the somites express
Fgf8 that regulates myod expression and terminal differentiation of a subset of fast muscle cells (Groves
et al. 2005). Retinoic acid (RA) has been shown to
inhibit proliferation and to promote differentiation of
myoblasts (Alric et al. 1998). In zebrafish, Retinaldehyde dehydrogenase 2 (Raldh2), the main RA synthesizing enzyme, is expressed in the somites where
it activates fgf8 expression and consequently fast
muscle cell differentiation (Fig. 3c–d) (Hamade et al.
2006). Thus, Fgf8 in the somites specifies fast
muscle cell development under the control of RA.
Conclusions
In zebrafish, the Fgf, Hh and RA signaling pathways
as well as T-box genes and numerous other factors
are required for the development of skeletal muscle.
Several of these factors have been described first in
zebrafish and their functions in other vertebrates are
yet to be determined. In many vertebrates, myogenic
precursors express Pax3 and Pax7 and downregulate
these genes as they begin to express MRFs. It is still
unclear, however, if Pax3 and Pax7 function similarly
in zebrafish myogenesis. Thus, additional studies in
both zebrafish and other vertebrates will be required
for us to gain a holistic understanding of the mechanisms that regulate skeletal muscle development.
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