Acta Physiol Scand 1998, 163, S7±S10
The role of hedgehog proteins in vertebrate slow
and fast skeletal muscle patterning
S . M . H U G H E S , C . S . B L A G D E N , X . L I and A . G R I M A L D I
Developmental Biology Research Centre and MRC Muscle and Cell Motility Unit, The Randall Institute, King's College London, UK
Adult muscle is characterized by a large number of
distinct ®bre types, specialized for particular kinds of
contraction and patterns of use (for reviews see
Schiaf®no & Reggiani 1996, Pette & Staron 1997). The
origins of these diverse ®bre types during development
have been chie¯y studied by following the initiation of
expression of distinct myosin heavy chain genes, either
in vivo or in vitro. In this review, we focus on the major
distinction between fast and slow muscle that exists in
all vertebrates so far examined. Our analysis will ignore
the possibility of multiple origin of both slow and fast
®bres, and the further diversi®cation of ®bres into
distinct subtypes, for example the type IIA, IIX and IIB
®bres that differ both in contractile and metabolic
properties. Clearly, a full description of ®bre type di-
versi®cation will include many in¯uences, particularly
that of innervation. However, as ®bre type diversity can
arise in the absence of innervation (Butler et al. 1982,
Condon et al. 1990) the initial patterning events do not
appear to require either electrical activity or trophic
factors provided by neurones.
Vertebrate muscle is thought to develop from a
variety of distinct myoblast populations, some of which
arise during embryonic development of the somite.
Somatic muscle development is regulated by signals
from neighbouring tissues (reviewed in Lassar &
Munsterberg 1996). On the other hand, clonally heri-
table myoblast diversity has been best characterized in
developing avian limb muscle (Stockdale 1992). To
understand the diverse routes to myogenesis in verte-
brates we and others have examined myogenesis of
distinct ®bre types in the somite of a non-amniote
vertebrate, the zebra®sh.
During the generation of slow and fast muscle in
zebra®sh embryos slow ®bres arise ®rst, adjacent to the
notochord (Devoto et al. 1996, Blagden et al. 1997).
Keywords: ®bre type, hedgehog, muscle, myoblast, notochord,
somite
Correspondence: Dr Simon M. Hughes, MRC Muscle and Cell
Motility Unit, King's College London, 26±29 Drury Lane, London
WC2B 5RL, UK. email: s.hughes@kcl.ac.uk.
Ó 1998 Scandinavian Physiological Society
These cells, identi®able by expression of slow myosin,
elongate and become arrayed at the medial surface of
the somite, before migrating laterally past the undif-
ferentiated somitic cells to lie super®cially close to the
epidermis (Devoto et al. 1996). As the slow cells mi-
grate, many remaining somite cells differentiate into fast
muscle, and are detected using an antibody to fast
myosin. The initial location of slow cells adjacent to the
notochord suggested that a notochord-derived signal
such as Sonic hedgehog (Shh) might induce slow
muscle formation (Blagden et al. 1997). Bozozok mutant
zebra®sh that lack a notochord, and hence shh expres-
sion, fail to form slow muscle, but do form fast muscle.
Similarly, the posterior of no tail (ntl) embryos lack the
shh expression, and these ®sh also show disruption of
slow muscle formation in the posterior tail. Thus, in the
absence of Shh, slow muscle fails to form, suggesting
that this factor might induce slow muscle formation. To
address this issue, we injected RNA encoding shh into
zebra®sh embryos at the 2±4 cell stage. Ectopic ex-
pression of shh, either in wild type or bozozok mutant
embryos, leads to ectopic slow muscle at the expense of
fast muscle. Thus, Shh is a signal from the mid-line that
is suf®cient to induce slow muscle formation in somitic
tissue. To rule out the possibility that arti®cially high
levels of Shh are necessary to induce slow muscle for-
mation, we examined the ¯oating head (¯h) mutant in
which the notochord degenerates shortly after its for-
mation leaving a residual source of Shh in the form of
patches of ¯oor plate in the ventral neural tube. Slow
muscle is formed in the ¯h mutant, but only in the mid-
line where somitic cells are close to residual shh ex-
pression (Blagden et al. 1997). These data indicate that
shh acts to induce slow muscle in the zebra®sh embryo.
Muscle is classically regarded as arising in a two step
process: a mesodermal cell ®rst commits to the pro-
liferative myoblast, which subsequently undergoes ter-
minal differentiation leading to contractile muscle
®bres. In the zebra®sh pre-somitic mesoderm and so-
mite, there is little proliferation prior to muscle ®bre
formation (Kimmel & Warga 1987, Kanki & Ho 1997).
Yet shh appears to induce an equivalent of the myoblast
S7
Hedgehog and muscle pattern
S M Hughes et al.
state because, prior to their terminal differentiation,
adaxial cells express the zebra®sh myoD muscle marker
from early stages and over-expression of shh leads to
ectopic early myoD expression in cells of the lateral pre-
somitic mesoderm (Weinberg et al. 1996). Moreover,
we observed that in ¯h embryos the location of terminal
differentiation of adaxial myoblasts does not correlate
with regions of highest shh mRNA accumulation, sug-
gesting that once somitic cells have received the Shh
signal, the continued presence of the protein may not
be required for these committed myoblasts to differ-
entiate as slow muscle ®bres. Such data suggest that
Shh acts to induce myoblasts committed to slow muscle
differentiation directly from uncommitted pre-somitic
mesoderm.
Sonic hedgehog is not the only hedgehog protein
likely to in¯uence muscle development in the zebra®sh.
Echidna hedgehog (Ehh), another member of the
hedgehog family, is expressed in the notochord of de-
veloping zebra®sh embryos at stages slightly later than
Shh (Currie & Ingham 1996). Over-expression of Ehh
in wild type embryos can trigger the ectopic appearance
of additional muscle pioneer cells (Currie & Ingham
1996). Muscle pioneers are a subset of the slow adaxial
cells which form in the dorsoventral mid-line adjacent
to the notochord at around the time of somitogenesis.
They can be recognized by their location, their ex-
pression of transcription factors of the engrailed family
(Hatta et al. 1991) and their early terminal differentia-
tion into elongated striated muscle cells (Felsenfeld
et al. 1991). Moreover, several zebra®sh mutants, for
example ¯h, that retain adaxial slow cells and shh ex-
pression, lack ehh expression and fail to form muscle
pioneers. Taken together, these data suggest a model in
which successive hedgehog signals could induce distinct
subpopulations of myoblasts during development.
The relevance of these results in zebra®sh to the
formation of fast and slow muscle in other vertebrate
embryos is currently under investigation. We have ob-
served distinct ®bre populations in early Xenopus
embryos; although the relative position of fast and slow
muscle differs in the two species (Fig. 1). In Xenopus,
®bres reactive with slow myosin antibodies are located
medially, whereas fast-type ®bres are super®cial. Dis-
tinct ®bre types have not been extensively studied in
early myotomes of amniotes, although diverse ®bre
types are thought to be present (Dhoot 1994). That
hedgehog proteins may be involved in regulating
myogenesis in amniotes is suggested by a wide range of
in vivo and in vitro studies (for reviews see Cossu et al.
1996, Lassar & Munsterberg 1996). Firstly, either
notochord explants or cell pellets expressing shh are
capable of inducing myogenic markers in explanted
somites or pre-somitic mesoderm segments (Buf®nger
& Stockdale 1994, Buf®nger & Stockdale 1995, Mun-
S8
Acta Physiol Scand 1998, 163, S7±S10
Figure 1 Distinct muscle ®bre populations in early Xenopus
embryos. Muscle ®bres reactive with an antibody to slow myosin (A)
are present in the medial somite near the notochord (not), whereas an
antibody to fast myosin (B) detects a super®cial population of muscle
®bres next to the dermis. A single transverse cryosection from around
somite 20 of a methanol-®xed stage 41 Xenopus embryo was
incubated with antibodies that recognize myosin heavy chain isoforms
that distinguish slow and fast muscles in other species, BA-D5 and
EB165, respectively (Schiaf®no et al. 1989, Gardahaut et al. 1992,
Blagden et al. 1997). Each antibody was detected with subclass
speci®c secondary antibodies (Serotec) followed by tertiary reagents
conjugated to FITC or Cy3 (Jackson) as described (Blagden et al.
1997).
sterberg et al. 1995, Munsterberg & Lassar 1995, Stern
et al. 1995). Secondly, manipulations of chick embryos
in ovo suggest that mid-line and notochord signals
control somitic myogenesis (Teillet & Le Douarin 1983,
Rong et al. 1992, Goulding et al. 1994, Pownall et al.
1996, Dietrich et al. 1997). Thirdly, mice in which the
shh gene has been ablated by homologous recombina-
tion show defects in expression of the early muscle
marker myf-5, in addition to defects in sclerotomal de-
rivatives (Chiang et al. 1996). Fourth, chick embryos
over-expressing shh from a retroviral vector show in-
creased myoD expression (Johnson et al. 1994). Taken
together, these data are suggestive of a role for Shh in
amniote myotomal muscle formation.
However, as in zebra®sh, shh does not appear to be
required for all muscle formation. It is clear that not all
skeletal muscles in amniotes require mid-line signals as
chick muscle of both the lateral somite and limb can
form in the absence of mid-line structures (Rong et al.
1992). Furthermore, mice in which shh has been deleted
by homologous recombination fail to form a normal
medial population of myf-5-expressing myoblasts, yet
lateral myoD expression is unaffected (Chiang et al.
1996). Preliminary results from our own laboratory
suggest that cultured mouse myoblasts can be induced
to express increased levels of slow myosin by treatment
with Shh (data not shown). Together, this evidence
suggests that hedgehog proteins may be involved in
generating muscle cell diversity in amniote somites,
rather than simply inducing some `generic' myoblast.
The situation in developing limbs is more complex
to understand as it is thought that several myoblast
Ó 1998 Scandinavian Physiological Society
Acta Physiol Scand 1998, 163, S7±S10
populations migrate into the early limb bud (Seed &
Hauschka 1984, Van Swearingen & Lance-Jones 1995).
Moreover, shh is required in the zone of polarizing
activity (ZPA) to permit proper limb outgrowth, so the
possibility of exposure of myogenic cells to limb-de-
rived Shh cannot be excluded. However, slow muscle
does not appear to be located preferentially close to the
ZPA, so this early distal source of Shh does not appear
to pattern fast and slow muscle. On the contrary, slow
muscle ®bres frequently form in medial regions of de-
veloping limbs near the developing bone. It is striking
that the cartilaginous growth plate expresses Indian
hedgehog (ihh) (Bitgood & McMahon 1995, Vortkamp
et al. 1996), the sole residual known location of
hedgehog protein in limbs at the time of muscle ter-
minal differentiation. Analysis of this possibility will
await the manipulation of ihh expression in vivo.
A further interesting facet of this issue is the func-
tion of shh expression in developing motor neurones
(Marti et al. 1995, Stone et al. 1996). As motor neurones
innervate nascent muscle ®bres and are known to have
a strong in¯uence on ®bre type in adult life (Buller et al.
1960), it is tempting to speculate that Shh could be
transported along motor neurone axons and in¯uence
muscle development, as has been observed in the
Drosophila visual system, where hedgehog expression in
retinal neurones controls the maturation of their central
target tissue (Huang & Kunes 1996). It is striking that a
growing body of data suggests that both motor neurone
(Ericson et al. 1996) and muscle ®bre diversity may be
dependent on hedgehog signals from the notochord or
¯oor plate. These observations suggest that during
evolution fundamental distinctions in each cell popu-
lation may have co-evolved to permit coordination of
distinct kinds of movement.
Although hedgehog proteins are likely to be in-
volved in regulating muscle development, that is not to
say that these factors will be the only, or indeed the
dominant, regulators of muscle ®bre type pattern. It is
clear that many other signalling molecules, such as
members of the TGFb, FGF, IGF and Wnt families
(reviewed in Hughes, S.M., in preparation), can in¯u-
ence the commitment and terminal differentiation of
myoblasts. It will be important to investigate the effects
of these diverse signalling molecules, many of which are
expressed in or close to the developing somitic and
limb musculature, on muscle patterning.
In conclusion, a growing body of evidence suggests
that hedgehog proteins are able to in¯uence muscle
differentiation at various stages of development. Un-
derstanding how hedgehog and other signals are inte-
grated during development to provide an appropriate
balance of muscles specialized for particular kinds of
movement in each species remains a major challenge.
Given that distinct muscles and ®bre types show dif-
Ó 1998 Scandinavian Physiological Society
S M Hughes et al.
Hedgehog and muscle pattern
ferent responses to similar external stimuli (for example
electrical stimulation), it is tempting to speculate that
the developmental origins of ®bres may underlie at least
some of these differences. Elucidation of the funda-
mental differences between the muscle ®bre types
generated during development will permit more in-
sightful study of ®bre type adaptation dependent upon
demands on the individual in later life.
We thank Stefano Schiaf®no and Everett Bandman for antibodies and
the MRC and EU Biomed grant BMH4-CT96±0174 for ®nancial
support.
R E F E RE N CE S
Bitgood, M.J. & McMahon, A.P. 1995. Hedgehog and Bmp
genes are coexpressed at many diverse sites of cell- cell
interaction in the mouse embryo. Dev Biol 172, 126±138.
Blagden, C.S., Currie, P.D., Ingham, P.W. & Hughes, S.M.
1997. Notochord induction of zebra®sh slow muscle
mediated by Sonic Hedgehog. Genes Dev 11, 2163±2175.
Buf®nger, N. & Stockdale, F.E. 1994. Myogenic speci®cation
in somites: induction by axial structures. Development 120,
1443±1452.
Buf®nger, N. & Stockdale, F.E. 1995. Myogenic speci®cation
of somites is mediated by diffusible factors. Dev Biol 169,
96±108.
Buller, A.J., Eccles, J.C. & Eccles, R.M. 1960. Interactions
between motoneurones and muscles in respect of the
characteristics speeds of their responses. J Physiol 150,
417±439.
Butler, J., Cosmos, E. & Brierley, J. 1982. Differentiation of
muscle ®ber types in aneurogenic brachial muscles of the
chick embryo. J Exp Zool 224, 65±80.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J.L.,
Westphal, H. & Beachy, P.A. 1996. Cyclopia and defective
axial patterning in mice lacking Sonic hedgehog gene
function. Nature 383, 407±413.
Condon, K., Silberstein, L., Blau, H.M. & Thompson, W.J.
1990. Differentiation of ®ber types in aneural musculature
of the prenatal rat hindlimb. Dev Biol 138, 275±295.
Cossu, G., Tajbakhsh, S. & Buckingham, M. 1996. How is
myogenesis initiated in the embryo? Trends Genet 12,
218±222.
Currie, P.D. & Ingham, P.W. 1996. Induction of a speci®c
muscle cell type by a hedgehog-like protein in zebra®sh.
Nature 382, 452±455.
Devoto, S.H., Melancon, E., Eisen, J.S. & Wester®eld, M.
1996. Identi®cation of separate slow and fast muscle
precursor cells in vivo, prior to somite formation.
Development 122, 3371±3380.
Dhoot, G.K. 1994. Mammalian myoblasts become fast or
slow myocytes within the somitic myotome. J Muscle Res
Cell Motil 15, 617±622.
Dietrich, S., Schubert, F.R. & Lumsden, A. 1997. Control of
dorsoventral pattern in the chick paraxial mesoderm.
Development 124, 3895±3908.
Ericson, J., Morton, S., Kawakami, A., Roelink, H. & Jessell,
T.M. 1996. Two critical periods of Sonic Hedgehog
S9
Hedgehog and muscle pattern
S M Hughes et al.
signaling required for the speci®cation of motor neuron
identity. Cell 87, 661±673.
Felsenfeld, A.L., Curry, M. & Kimmel, C.B. 1991. The fub-1
mutation blocks initial myo®bril formation in zebra®sh
muscle pioneer cells. Dev Biol 148, 23±30.
Gardahaut, M.F., Fontaine-Perus, J., Rouaud, T., Bandman,
E. & Ferrand, R. 1992. Developmental modulation of
myosin expression by thyroid hormone in avian skeletal
muscle. Development 115, 1121±1131.
Goulding, M., Lumsden, A. & Paquette, A.J. 1994. Regulation
of Pax-3 expression in the dermamyotome and its role in
muscle development. Development 120, 957±971.
Hatta, K., Bremiller, R., Wester®eld, M. & Kimmel, C.B.
1991. Diversity of expression of engrailed-like antigens in
zebra®sh. Development 112, 821±832.
Huang, Z. & Kunes, S. 1996. Hedgehog, transmitted
along retinal axons, triggers neurogensis in the
developing visual centers of the Drosophila brain. Cell 86,
411±422.
Johnson, R.L., Laufer, E., Riddle, R.D. & Tabin, C. 1994.
Ectopic expression of Sonic hedgehog alters dorsal-ventral
patterning of somites. Cell 79, 1165±1173.
Kanki, J.P. & Ho, R.K. 1997. The development of the
posterior body in zebra®sh. Development 124, 881±893.
Kimmel, C.B. & Warga, R.M. 1987. Cell lineages generating
axial muscle in the zebra®sh embryo. Nature 327,
234±237.
Lassar, A.B. & Munsterberg, A.E. 1996. The role of positive
and negative signals in somite patterning. Curr Opin
Neurobiol 6, 57±63.
Marti, E., Takada, R., Bumcrot, D.A., Sasaki, H. & McMahon,
A.P. 1995. Distribution of Sonic hedgehog peptides in the
developing chick and mouse embryo. Development 121,
2537±2547.
Munsterberg, A.E., Kitajewski, J., Bumcrot, D.A., McMahon,
A.P. & Lassar, A.B. 1995. Combinatorial signalling by sonic
hedgehog and Wnt family members induces myogenic
bHLH gene expression in the somite. Genes Dev 9, 2911±
2922.
Munsterberg, A.E. & Lassar, A.B. 1995. Combinatorial signals
from the neural tube, ¯oor plate and notochord induce
myogenic bHLH gene expression in the somite. Development
121, 651±660.
S10
Acta Physiol Scand 1998, 163, S7±S10
Pette, D. & Staron, R.S. 1997. Mammalian skeletal muscle
®ber type transitions. Int Rev Cytol 170, 143±223.
Pownall, M.E., Strunk, K.E. & Emerson, C.P.J. 1996.
Notochord signals control the transcriptional cascade of
myogenic bHLH genes in somites of quail embryos.
Development 122, 1475±1488.
Rong, P.M., Teillet, M.A., Ziller, C. & Le Douarin, N.M. 1992.
The neural tube/notochord complex is necessary for
vertebral but not limb and body wall striated muscle
differentiation. Development 115, 657±672.
Schiaf®no, S., Gorza, L., Sartore, S. et al. 1989. Three myosin
heavy chain isoforms in type 2 skeletal muscle ®bres.
J Muscle Res and Cell Motil 10, 197±205.
Schiaf®no, S. & Reggiani, C. 1996. Molecular diversity of
myo®brillar proteins: Gene regulation and functional
signi®cance. Physiol Reviews 76, 371±423.
Seed, J. & Hauschka, S.D. 1984. Temporal separation of the
migration of distinct myogenic precursor populations into
the developing chick wing bud. Dev Biol 106, 389±393.
Stern, H.M., Brown, A.M.C. & Hauschka, S.D. 1995.
Myogenesis in paraxial mesoderm: Preferential induction
by dorsal neural tube and by cells expressing Wnt-1.
Development 121, 3675±3686.
Stockdale, F.E. 1992. Myogenic cell lineages. Dev Biol 154,
284±298.
Stone, D.M., Hynes, M., Armanini, M. et al. 1996. The
tumour-suppressor gene patched encodes a candidate
receptor for Sonic hedgehog. Nature 384, 129±134.
Teillet, M.A. & Le Douarin, N.M. 1983. Consequences of
neural tube and notochord excision on the development of
the peripheral nervous system in the chick embryos. Dev
Biol 98, 192±211.
Van Swearingen, J. & Lance-Jones, C. 1995. Slow and fast
muscle ®bres are preferentially dervied from myoblasts
migrating into the chick limb bud at different
developmental times. Dev Biol 170, 321±337.
Vortkamp, A., Lee, K., Lanske, B., Segre, G.V., Kronenberg,
H.M. & Tabin, C.J. 1996. Regulation of rate of cartilage
differentiation by Indian Hedgehog and PTH-related
protein. Science 273, 613±622.
Weinberg, E.S., Allende, M.L., Kelly, C.S. et al. 1996.
Developmental regulation of zebra®sh MyoD in wild-type,
no tail and spadetail embryos. Development 122, 271±280.
Ó 1998 Scandinavian Physiological Society