Hedgehog can drive terminal differentiation of amniote slow skeletal muscle
- Xiaopeng Li†1,
- Christopher S Blagden†1, 2,
- Heidi Bildsoe†1,
- Marie Ange Bonnin3,
- Delphine Duprez3 and
- Simon M Hughes1Email author
© Li et al; licensee BioMed Central Ltd. 2004
Received: 17 January 2004
Accepted: 06 July 2004
Published: 06 July 2004
Secreted Hedgehog (Hh) signalling molecules have profound influences on many developing and regenerating tissues. Yet in most vertebrate tissues it is unclear which Hh-responses are the direct result of Hh action on a particular cell type because Hhs frequently elicit secondary signals. In developing skeletal muscle, Hhs promote slow myogenesis in zebrafish and are involved in specification of medial muscle cells in amniote somites. However, the extent to which non-myogenic cells, myoblasts or differentiating myocytes are direct or indirect targets of Hh signalling is not known.
We show that Sonic hedgehog (Shh) can act directly on cultured C2 myoblasts, driving Gli1 expression, myogenin up-regulation and terminal differentiation, even in the presence of growth factors that normally prevent differentiation. Distinct myoblasts respond differently to Shh: in some slow myosin expression is increased, whereas in others Shh simply enhances terminal differentiation. Exposure of chick wing bud cells to Shh in culture increases numbers of both muscle and non-muscle cells, yet simultaneously enhances differentiation of myoblasts. The small proportion of differentiated muscle cells expressing definitive slow myosin can be doubled by Shh. Shh over-expression in chick limb bud reduces muscle mass at early developmental stages while inducing ectopic slow muscle fibre formation. Abundant later-differentiating fibres, however, do not express extra slow myosin. Conversely, Hh loss of function in the limb bud, caused by implanting hybridoma cells expressing a functionally blocking anti-Hh antibody, reduces early slow muscle formation and differentiation, but does not prevent later slow myogenesis. Analysis of Hh knockout mice indicates that Shh promotes early somitic slow myogenesis.
Taken together, the data show that Hh can have direct pro-differentiative effects on myoblasts and that early-developing muscle requires Hh for normal differentiation and slow myosin expression. We propose a simple model of how direct and indirect effects of Hh regulate early limb myogenesis.
Each muscle in a developing chick limb acquires a unique character from its inception . Fibres form by the terminal differentiation of dividing myoblasts that elongate in particular orientations to form specific attachments to the skeleton. Simultaneously, the fibres of each muscle take on gene expression patterns characteristic of their future function. For example, those muscles destined to maintain body posture express certain isoforms of slow myosin from their inception, whereas future fast muscle regions fail to express this slow myosin . It has been suggested that distinct cell lineages underlie the formation of slow and fast muscle fibres, and much evidence for myoblast heterogeneity has been obtained from studies both in vitro and in vivo [[3–7], reviewed in ]. Nevertheless, it is clear that for fibres to undergo differentiation at the appropriate time and place extrinsic cues must regulate muscle patterning.
Work on muscle patterning in somites over the past decade has shown that various protein factors secreted by adjacent tissues act as extrinsic signals regulating the formation and fate of myogenic cells [, reviewed in [10–12]]. One such factor is Sonic hedgehog (Shh), derived from the ventral midline, which is required for expression of markers of the earliest population of myogenic cells in the medial somite of both birds and mice [13–15]. These medial somitic cells contribute to the early-born muscle fibres of the myotome, but their subsequent fate is not known in amniotes [16, 17]. Ventral midline Hedgehog (Hh) signals are also required for formation of the earliest muscle cells in the zebrafish embryo, the adaxial slow cells [[18, 19], reviewed in ]. The fate of these cells is known, they generate a population of slow muscle fibres that migrate to form a layer of slow muscle that covers the lateral surface of the somite [21, 22]. In all vertebrates examined, a second myogenic cell population arises in the lateral somite by a distinct Hh-independent genetic pathway in response to signals from more lateral and dorsal tissues. Signals such as FGFs, BMPs and WNTs and their antagonists are prime candidates for patterning of lateral somitic cells, at least in amniotes [reviewed in [8, 9, 23, 24]]. Wnt proteins from dorsal tissues are also implicated in medial myogenesis [25–30].
In the somite, induction of precursor myoblast populations is occurring close in space and time to terminal differentiation of myoblasts into contractile fibres. This makes analysis of the precise effects of extrinsic signals hard to determine. For example, Shh can promote both primary myogenesis and subsequent cell survival in somitic explants and in vivo, but the precise target cell populations are unclear [13, 15, 31–33]. In contrast, in the limb bud myogenic induction and terminal differentiation are temporally and spatially separated. Myogenic cells of the limb derive from a population of precursors that migrates into the limb bud from the lateral somite [34–36]. These cells already express genes required for myogenesis prior to their migration [37, 38]. Evidence suggests that several distinct populations of myogenic cells enter the limb bud [5, 39, 40]. Thus, muscle formation within the limb bud omits some of the early steps that occur in the somites. Consequently, we chose the somewhat simpler and more accessible limb bud to analyse the effects of Hh on the differentiation and patterning of muscle fibre types.
Previous work has shown that early manipulations that alter limb anteroposterior axis formation and skeletal pattern, including Shh mis-expression, change muscle and fibre type pattern in parallel [41, 42]. Moreover, myoblast clones appear uncommitted to a particular character either early or late in myogenesis [43, 44] indicating that local signals control fibre pattern. Nevertheless, implantation of cloned myoblasts into limbs can alter fibre pattern, although in such experiments it is difficult to rule out implantation of cells that are already undergoing terminal differentiation [45, 46]. Myoblast implantation at later stages shows that local limb signals can re-programme myoblast differentiation [47, 48]. It is clear that Hh expression within the limb at these later stages does not have a spatial pattern that is well correlated with formation of individual muscles or fibre types. Nevertheless, augmenting Hh signalling in a way that does not affect anteroposterior axis formation severely disrupts muscle patterning and differentiation leading to enlarged but disorganised muscles [49–51]. Conversely, a dramatic loss of muscle is observed in Shh-deficient mouse limbs . Interpretation of these Hh results has differed, possibly because muscle is not the only tissue affected. Moreover, these studies do not distinguish direct effects of Hh on myogenic cells from indirect effects acting via non-myogenic cell populations in the limb. In the current paper, we use both in vitro and in vivo approaches to analyse the effect of Hh on muscle differentiation and slow fibre formation. We establish definitively using in vitro cultures that myoblasts can be directly induced to terminal differentiation by Shh. Moreover, Shh enhances slow myosin accumulation. With these in vitro results in mind, in vivo analysis of limbs with increased or decreased Hh signalling indicates that Hh is a muscle differentiation factor that promotes early slow myogenesis.
Shh promotes myogenesis in limb monolayer culture
Shh can directly induce muscle terminal differentiation
Members of the Gli family of transcription factors are both targets and mediators of Shh signalling . Thus, if C2 myoblasts respond directly to Shh they would be expected to show elevated levels of gli gene transcription. The addition of Shh up-regulates gli1 transcripts in over 50% of C2 myoblasts (Fig. 2D), but does not induce gli2 expression (data not shown). Thus, C2 cells appear to be responsive to Shh through the normal Shh response pathway.
To examine the mechanisms involved in the Shh response we determined the effect of Shh on C2 cells in growth factor-rich medium, which normally maintains C2 myoblasts in the cell cycle and inhibits terminal differentiation. Control cultures have low levels of nuclear MyoD protein, very little Myogenin and only rare MyHC-containing cells. However, after two days of Shh treatment, C2 cells show enhanced MyoD protein accumulation in many nuclei and a markedly higher frequency of groups of Myogenin immunoreactive cells, some of which also contain detectable MyHC (Fig. 2E). Shh, therefore, can drive nuclear MyoD accumulation and terminal differentiation of myoblasts even in the presence of growth factors found in serum.
Shh can directly promote slow fibre formation
When wing bud cultures are analysed for expression of slow MyHC, the vast majority of differentiated muscle cells fail to express detectable slow MyHC, just as nascent fibres do not express slow MyHC at the initiation of myogenesis in vivo (see below). In control low density cultures, a small proportion of differentiated cells (1–2%, defined as the proportion of nuclei within MyHC-containing cytoplasm that are in slow MyHC-containing cytoplasm) contain slow MyHC, detected by A4.840 immunoreactivity (Fig. 1D). Shh exposure doubles the proportion of myocyte/myotubes expressing slow MyHC to ~4%, which corresponds to a six-fold increase in absolute numbers of slow MyHC-reactive cells (Fig. 1D). However, in high density culture, slow MyHC is suppressed in controls and Shh fails to induce an increase in the proportion of cells expressing slow MyHC.
Initial slow myogenesis correlates with Hh signalling in vivo
To examine the role of endogenous Hh signalling in wing myogenesis we analysed expression of gli1 and ptc1, a gene encoding a Hh receptor. Both genes are themselves downstream targets of Hh signalling . Ptc1 and gli1 are highly expressed in posterior and distal limb regions from HH21-28, due to Shh deriving from the zone of polarizing activity (ZPA; Fig. 4O,4R). Ptc1 expression declines in wing cells as they leave the progress zone and commence histogenesis (Fig. 4O). Distal gli1 expression is more extensive but declines similarly (Fig. 4R). However, by HH27 new regions of Hh signalling arise around the posterior region of the DMM, perhaps because Indian hedgehog (Ihh) expression commences in cartilage anlage . Ptc1 and gli1 are expressed in both non-myogenic tissues such as perichondrium and limb mesenchyme, and in the myogenic zone surrounding muscle fibres (Fig. 4P,4S). It is striking that the muscle region with highest ptc1 and gli1 mRNA roughly corresponds to the early slow zone, although it is also clear that Hh signalling in this region is not restricted to myogenic cells and that there are many fibres not expressing slow MyHC in the region of strong Hh signalling (Fig. 4F,4G,4O,4P,4R,4S).
Shh over-expression delays myogenesis and induces ectopic slow muscle 48 hours after grafting
Similarly implanted limbs were serially-sectioned and analysed for expression of Shh protein, slow MyHC and pan MyHC. Control contralateral limbs show a broad DMM, with slow MyHC in the posterior portion above the ulna condensation (Fig. 5B,5C). Among treated wings, three classes of outcome are observed. In ~50% (8/15) wings, there was a complete loss of muscle tissue in the posterior region of the dorsal zone and reduced muscle in the anterior region (Fig. 5E,5H). In another ~30% (4/15) wings, the DMM had altered shape and a variable reduction in the total number of differentiated fibres in the region of Shh accumulation (Fig. 5E,5H). The three remaining Shh/RCAS wings revealed no phenotype, correlated with low ectopic Shh protein and young age (data not shown). In all affected wings, the anterior DMM that would not normally express any slow MyHC contained significant levels of slow MyHC, such that the proportion of all fibres that contained slow MyHC was doubled (Fig. 5F,5I,5J). In most operated wings showing a muscle phenotype, Shh was detectable in or close to the DMM (Fig. 5G, arrowhead). The location of highest ectopic Shh correlated with loss of muscle fibres in severely-affected wings (Fig. 5E,5F,5G; arrowheads). Thus, exposure of developing chick wing buds to Shh leads to a reduction in total muscle differentiation 48 hours after grafting, as already reported [50, 51]. Despite this reduced myogenesis, we found an increase of slow muscle.
Prolonged Shh over-expression enhances limb and muscle size without increasing slow
Concerning slow differentiation, Shh over-expression increases the number of slow fibres at HH30 (Fig. 6B,6E). This change may relate to the increase in total fibres as many fibres do not express slow MyHC despite proximity to the Shh source (Fig. 6A,6D). By HH32, no increase in slow fibres, as a proportion of total fibres, is detected and slow fibre pattern appears normal (data not shown). Thus, the induction of an increased proportion of slow fibres was not a continuing process within the chick wing bud, despite the continued presence of Shh.
Blockade of Hh reduces slow muscle differentiation
Murine Hh knockouts contain slow muscle fibres
In shh-/- mice limb outgrowth and muscle growth is severely curtailed preventing meaningful analysis of muscle pattern . So we examined slow MyHC expression in the reduced somitic muscle. In wild-type E9.5 mice, around 12 rostral somites contain differentiated muscle fibres expressing MyHC (Fig. 9C). Slow fibres were observed in rostral somites, but not in the two-three caudalmost MyHC-expressing somites (n = 6 mice). In rostral somites, many fibres lacked slow MyHC, but expressed embryonic MyHC (Fig. 9C and data not shown). This confirms that embryonic MyHC is acquired before slow MyHC in early muscle fibres . In sections from shh-/- mice, fewer differentiated fibres were present, but around eight somites contained differentiated muscle which was often mis-oriented (6/6 individuals; Fig. 9C). Among residual fibres slow MyHC was undetectable in most animals (5/6; Fig. 9C). The single animal containing slow MyHC was developmentally more advanced, based on the presence of MyHC in more somites. Thus lack of Shh in somites leads to reduced early differentiation and delayed slow MyHC accummulation, as observed in chick limbs treated with anti-Hh antibodies.
Hitherto, all studies of the actions of Hh on amniote muscle have failed to rule out indirect effects deriving, for example, from Hh eliciting secondary signals from adjacent non-muscle cells. Here we show that Hh can directly promote terminal differentiation and slow MyHC accumulation by at least some myoblasts in cell culture. We find that Hh is required for the earliest definitive slow myogenesis in chick limb buds and use this new understanding to develop a simple model of a role of Hh signalling in limb myogenesis.
Hh directly induces muscle differentiation
The results presented show that Shh can promote the terminal differentiation of muscle fibres both in vivo and in vitro. Our observation that Shh promotes terminal differentiation of C2 cells (Fig. 2) definitively demonstrates that Shh can be a myoblast differentiation factor, at least on this adult muscle-derived cell line. This effect occurs even in the presence of growth factors. Early limb myogenic cells in culture also respond to Shh exposure by increased differentiation (Fig. 1). We also observed increase of muscles in Shh-treated limb (Fig. 6). This is consistent with the numerous reports showing that Shh increases muscle differentiation in explant cultures or in vivo [14, 25, 33, 52, 53, 65]. Conversely, and crucially, we find that local reduction of Hh function in the chick wing reduces muscle differentiation (Figs 7,8). This suggests that lack of Shh-driven myoblast differentiation may contribute to the severe reduction of muscle in early limb and somites in shh deficient mice [14, 52]. It is, therefore, highly likely that one action of Hh is the direct promotion of myoblast differentiation in developing chick wing bud.
A direct action of Hh on myoblasts is also supported by the rapid accumulation of gli1 mRNA, a downstream target of Hh signalling, in C2 cells and in limbs. In unmanipulated chicks, significant levels of ptc1 and gli1 mRNA accumulate in muscle masses, being highest in the posterior DMM (Fig. 4). This suggests that myogenic cells in both wing DMM and somite are exposed to Hh signals around the time of their first differentiation [13, 49, 59, 66, 67].
Which Hh could promote muscle differentiation in limbs? As the anti-Hh antibody blocks the function of both Shh and Ihh , our in vivo manipulations do not address this issue. Ihh is an obvious candidate, as ptc1 and gli1 expression are up-regulated in the posterior DMM at the time and location of commencement of Ihh expression in cartilage anlage around HH27 . However, forelimb myogenesis in Ihh-/- mice appears relatively normal, although we can not rule out undetected transient defects. Even in Ihh-/- hindlimb, where muscle differentiation is greatly reduced, it is impossible to ascribe this reduction to a direct action of Ihh because failure of long bone elongation could prevent muscle growth through lack of stretch-induced hypertrophy signals. Similarly, Shh is not absolutely required for the initiation of some murine limb muscle differentiation . However, gli1 expression suggests that Shh signalling extends quite far into what we have called the pre-myogenic zone  in the distal limb until at least HH27/28 (Fig. 4). So differentiating myogenic cells may also be exposed to low levels of Shh. In zebrafish somites, distinct levels of Hh signalling elicited by the combined action of at least three Hh genes lead to different myogenic outcomes [18, 69–72]. So the additive effects of Ihh and Shh, perhaps having different effects at particular overall concentrations, likely contribute to the sculpting of muscle differentiation.
Hh and slow myogenesis
We found that Shh has a consistent positive effect on slow myoblast differentiation. Differentiating chick wing myocytes express slow MyHC more frequently after Shh exposure in vitro (Fig. 1) or Shh over-expression in vivo (Fig. 5). Conversely, an early effect of blocking Hh in the wing is failure of slow MyHC accumulation (Figs 7,8). And Shh-/- mice have delayed slow MyHC accumulation in somites, perhaps due to loss of an early fibre population (Fig. 9). Lastly, one line of C2 cells accumulates more slow MyHC after Shh exposure (Fig. 3). Other C2 lines that do not express significant levels of slow MyHC in control conditions fail to up-regulate slow MyHC in response to Hh, perhaps indicating that Hh is unable to open the slow MyHC genomic locus. Both zebrafish and Xenopus embryos require Hh signalling to make some early populations of slow fibres, but not others [73, 74]. This argues strongly that Hh-driven slow myogenesis is an ancestral character of amniotes. Nevertheless, as in lower vertebrates, slow fibre formation does eventually occur in amniotes with defective Hh signalling. Indeed, considering the complex pattern of slow and fast fibres in older muscle, it is clear that many factors in addition to Hh must be involved in establishing the pattern. Our evidence suggests that Hh acts primarily during the earliest stages of limb myogenesis.
What is the relationship between the differentiation-promoting and slow MyHC-promoting actions of Shh on C2 cells? Whereas intracellular signalling and terminal differentiation was triggered rapidly in all C2 cells, slow MyHC up-regulation required longer Shh exposure. Therefore, one can argue that differential cell survival could account for the Shh-dependent increase in slow MyHC, particularly as Shh, or tissues that secrete it, have been shown to promote survival of some myogenic cells [31, 32, 52, 75]. However, we think a purely survival effect is unlikely, for several reasons. First, slow MyHC accumulation in single cells is greater with Shh, suggesting induction rather than simply enhanced survival. Second, assays for apoptosis in our C2 cultures revealed very little cell death, and this was unaffected by Shh exposure (unpublished result). Third, blockade of Hh in the wing bud reduces slow MyHC without a proportional reduction in differentiated muscle (Figs 7,8). Fourth, Hh over-expression in vivo induces ectopic slow while simultaneously reducing total differentiation (Fig. 5). Fifth, in cultured zebrafish blastomeric cells, some of which spontaneously form muscle, Shh induces conversion to a slow fate without affecting cell survival . Conversely, reduction of Hh signalling in zebrafish prevents slow myogenesis without inducing cell death . As altered cell survival does not explain the differentiation promoting activity of Hh, it seems unnecessary to invoke it in regard to slow myogenesis. In C2 cells, blockade of apoptosis appears unlikely to explain the slow promoting activity of Hh, leaving promotion of slow differentiation as the prime explanation. In vivo, the potential combination of direct and indirect effects of Hh, possibly on several myoblast subsets, make attribution of direct effects to Hh action on myoblasts impossible (see below). Nevertheless, our in vitro findings highlight direct induction of slow differentiation by Hh as a mechanism requiring serious consideration.
Could simply enhancing terminal differentiation account for the increase in slow? In vivo manipulations fail to reveal a correlation between increased slow expression and enhanced differentiation (Fig. 5). Nor do the number of nuclei in a cultured myotube (a rough assay of maturity) predict whether slow MyHC is induced (Fig. 3). Indeed, C2/4 cells differentiate well in response to Shh but fail to show the up-regulation of slow MyHC elicited by Shh in C2X cells, which differentiate less extensively with or without Shh. In addition, not all myoblasts in any line respond similarly to Shh exposure. It seems probable, therefore, that both intrinsic and micro-environmental differences between myoblasts regulate their response to Shh and could influence whether the response is simply terminal differentiation, or includes other events, such as slow MyHC accumulation.
Myoblast hetereogeneity of response to Hh
Intrinsic myoblast heterogeneity, possibly based on cell lineage, may also influence Hh response. As with C2 cells, not all cultured chick limb bud myoblasts respond similarly to Shh exposure. Shh efficiently enhanced terminal differentiation from 50% to ~80% of myogenic cells, showing that at least 30% of myogenic cells are likely to be Shh-responsive. However, only a few percent of chick myoblasts acquired slow MyHC in response to Shh (Fig. 1). Early limb myogenic cells have distinct clonally-heritable tendencies to either express slow MyHC or not do so [3, 45, 48, 77]. We suggest that, while most myoblasts may be Shh sensitive, sub-populations may respond differently based on their intrinsic capacity. This view parallels that of Stockdale and colleagues based on experiments showing differences in the myoblast populations forming distinct limb muscles [6, 7]. Fibres in distinct muscles differ in slow MyHC from their inception . So it is possible, by analogy with the situation in Drosophila  that the increase in slow fibres reflects a change in muscle identity of founder myoblasts, rather than a direct induction of slow MyHC. Altered myoblast identity could contribute to the failure of muscle splitting after Shh over-expression in vivo. Thus, by showing differential effects of Shh on distinct clonal myoblast lines that parallel those in primary cultures and in vivo (see below), our findings indicate that cell intrinsic differences determine the response of myogenic cells to Hh.
In the zeugopod, the earliest muscle differentiation is reduced, but not ablated, by introduction of anti-Hh antibody or in Shh-/- mice (Figs 7,8; ). Hh signalling is required for some but not all early somitic myogenesis . This shows that some myoblast populations do not require Hh for differentiation. In later limbs, blocking Hh reduces fibre formation and extra Shh augments differentiation (Figs 5,6,7,8). Similarly, Hh blockade reduces slow MyHC and Shh over-expression augments slow fibres early, but has little or no effect on slow MyHC expression in later muscle. Taken together, these observations indicate that myoblasts generating the earliest fibres in the zeugopod (before about HH28) may respond differently to Hh from those contributing to DMM growth after this stage. Many limb signals other than Hhs undoubtedly influence muscle pattern and likely affect the response to Hh [78, 79]. Resolution of whether myoblast lineage or environmental effects underlie this difference will be important.
Indirect proliferative effects of Hh on myogenic cells
Direct pro-differentiative effects of Hh on some myoblasts do not rule out other direct or indirect effects of Hh. Our results confirm and extend previous reports showing that Shh can induce proliferation of myoblasts [49, 50], both in limb buds and in primary cultures. However, other cell types are affected in limbs because Shh-treated limbs are bigger (Fig. 6). There is good evidence for effects of Hh on non-myogenic limb tissue. Shh over-expression causes limb hypertrophy with up-regulation of ptc1 and gli1 outside muscle masses and increase in non-myogenic tissue area (Figs 5,6). Similarly, Shh causes growth of non-myogenic as well as myogenic cells in our chick primary cultures (Fig. 1) and in somite explants . Conversely, inhibition of Hh in wing with anti-Hh antibody reduces limb growth in addition to reducing muscle mass size: this effect is first noticeable in non-muscle tissue (Figs 7,8). Nobody has reported a proliferative effect of Shh on myoblasts independently of a proliferative effect on other cells. On the contrary, in the C2 cell line we clearly found no mitogenic effect of Shh (Fig. 2). Moreover, in zebrafish, Shh is not a mitogen for slow muscle precursors: muscle differentiation is delayed in the sonic-you mutants that lack Shh  and induced in embryos over-expressing Shh [18, 19]. Therefore, either chick wing cells respond differently to Shh compared with other myoblasts or mitogenic effects of Shh on myoblasts are indirect. It is highly likely that Hh action on non-myogenic cells leads to release of myoblast mitogens. One hypothesis has already proposed that Shh acts through BMPs to amplify the number of myogenic cells  and BMP induction by Ihh causes cartilage proliferation indirectly . So the temporary inhibition of terminal differentiation by Shh over-expression in limbs (Fig. 5; ) may be a consequence of an indirect effect of Hh signalling on myoblast proliferation.
Combining our results with published data, we propose a model in which Hh promotes muscle differentiation directly in myoblasts within the muscle masses. But Hh also promotes myoblast proliferation indirectly by eliciting muscle growth factors from non-myogenic limb cells. This hypothesis explains how Hh may contribute to growth of muscle masses by increasing myoblasts at the edge, where proliferative signals from non-myogenic cells would predominate over the direct differentiative signal. Deeper within the muscle mass, pro-differentiative signals including Hh would be in the ascendant, adding new primary fibres at the periphery of the existing differentiated zone. At early stages distinct levels of Hh signalling may trigger slow myogenesis, possibly in sub-populations of myoblasts. Once muscle splitting commences Hh signalling declines, as indicated by reduced gli1 expression, and other influences probably determine the decision of later myoblasts to divide or differentiate. Muscle-specific ablation of Hh responsiveness will be required to test this hypothesis definitively.
We show that Hh can directly promote myoblast differentiation, at least in vitro. In vivo in chick limb bud, Hh signalling is occurring at the right time and place to affect early slow myogenesis. We introduce a new methodology, Cellagen implants of hybridoma cells secreting functionally-blocking antibody, and show that Hh is required for proper early slow (but not fast) muscle differentiation. Conversely, Hh over-expression induces ectopic early slow muscle in chick limb bud. Neither gain nor loss of later Hh function affects differentiation of later-formed slow muscle. Thus early limb Hh levels promote slow myogenesis, but are unlikely to be solely responsible for the details of slow fibre pattern. The data suggest a simple model of how direct and indirect effects of Hh sculpt early limb myogenesis.
Manipulated chick forelimbs
Chick embryo fibroblasts (CEF) were transfected with Shh/RCAS, a replication-competent retrovirus containing the entire cShh coding sequence. Anti-Hh 5E1 hybridoma cells (~2 × 104) were embedded in a 10 μl Cellagen block. Pellets (~100 μm diameter) or block fragments (2 μl) were implanted into Rhode Island Red chick embryo right wing buds at Hamburger and Hamilton stages (HH) 21-24, avoiding complications due to skeletal pattern alteration by grafting on the dorsal side in the future forewing region, as previously described . Embryos were maintained in a humidifier at 37°C, for 2–4 days and analysed at HH27-29 (E6) pre-splitting, and HH30-32 (E7/8) mid/post-splitting of DMM into its component muscles.
Embryos were fixed at -20°C in methanol, rehydrated in graded PBS, soaked for 2 hrs in 20% sucrose, transferred to a 2:1 mixture of 20% sucrose and Tissue-Tek cryoprotectant (Bright), experimental and contralateral wings aligned and frozen in a single block and cryosectioned. Primary monoclonal antibody supernatants of A4.1025 , BA-D5, A4.840 and N3.36  were diluted 1:10 [61, 80]. EB165 and Na8 ascites, gifts of E. Bandman (University of California, Davis) were used at 1:5000 [57, 82]. To detect ectopic Shh protein, 5E1 supernatant  was dilutied 1:10. MF20 and most other antibodies used in this study are available from Developmental Studies Hybridoma Bank. First antibodies were detected with biotin-conjugated horse-derived anti-mouse IgG, or a biotin-conjugated goat-derived anti-mouse IgM (Vector) and ABC Vectastain kit as described . In situ mRNA hybridisation was after . Identification of chick forewing muscles was according to  and staging based on limb and muscle mass morphology according to .
Following the methods of Stockdale [3, 85], both forelimbs were removed from embryos around HH22 in Dulbecco's modified Eagle's medium (DMEM, Sigma). Limbs were washed with sterile PBS, incubated with trypsin:EDTA (Gibco) for 10 mins, dissociated by trituration and the cells washed, filtered through two 80 μm pore filters (Gibco) and pre-plated on a 90 mm collagen-coated dish for 10 min at 37°C. After this incubation period, 30–40% of cells stuck to the dish but fewer than 1:1000 were myogenic. Non-adherent cells were collected and plated in triplicate at either 2 × 105 (low density) or 4 × 105 (high density) cells per Nunc 35 mm plate in either unconditioned DMEM with 10% horse serum (HS) and 2% chick embryo extract (CEE) or medium that had been conditioned for 24 hours on 90% confluent RCAS/Shh infected QT6 quail fibroblasts (ShhQT6) or the parent QT6 fibroblasts [49, 86]. Fresh medium (conditioned or not) was added after 24 hours, and the cells fixed 50–55 hrs after plating. Prior to conditioning QT6 and ShhQT6 cells were maintained in DMEM 10% foetal bovine serum (FBS -Gibco) and 2% chick serum (Gibco). Cultures were washed in PBS, fixed for 5 mins in -20°C methanol, rehydrated in PBS and stained as for the cryosections. Replicate dishes were singly stained with antibodies A4.840 to detect slow MyHC, A4.1025 to detect pan-MyHC and anti-desmin (Sigma, 1:500 dilution) to detect both myoblasts and myotubes. Dual immunofluorescence showed that all MyHC-reactive cells are strongly desmin-reactive and that immunohistochemistry is more sensitive (data not shown). Total nuclear (cell) numbers were counted on a Zeiss Axioplan 2 microscope in ten separate 10x fields on each dish. All nuclei within immunohistochemically-stained cells were counted on each dish.
Growth and addition of Shh conditioned medium to mouse myoblasts
C2 cells were obtained from three sources i) C2C12 from ATCC, ii) C2/4 from Y. Nabeshima and iii) C2X which arose in late passage cultures of C2C12 from the lab of H. Blau. All were maintained on plastic by standard procedures prior to plating on collagen-coated glass chamber slides (16-well, LAB-TEK, Nalge Nunc International, USA) in DMEM 10% FBS, 2% chick serum with antibiotics and differentiated by switching to DMEM 2% HS. Conditioned medium was created by incubating QT6 or ShhQT6 cells for 36 hours with either growth or differentiation medium. Each QT6 cell culture was used to condition only a single batch of medium. Purified Shh was synthesised in vitro and the biologically active proportion of the protein in each preparation is unknown, low and varies between batches (P. Ingham and T. Jessell personal communication) so no meaningful concentration can be given. C2 cells were fixed with methanol, stained by dual immunofluorescence with A4.840, A4.1025 and/or BA-D5 using class-specific secondary reagents (Jackson) and viewed under epifluorescence on a Zeiss Axiophot. Unless otherwise stated quantitation of differentiation was by scoring the number of nuclei in MyHC-containing cytoplasm (i.e. the number of C2 cells that differentiated into myocytes, whether or not these subsequently fused). Bromodeoxyuridine was added for the last two hours of a 24 hour culture in QT6 or QT6Shh conditioned growth medium. In situ hybridisation was performed on chamber slide cultures fixed with 4% paraformaldehyde followed by methanol and employed digoxigenin-labelled riboprobes essentially as described .
We thank Everett Bandman for antibodies, Tom Jessell for antibodies, 5E1 hybridoma cells and mouse Shh protein, Peter Currie and Philip Ingham for zebrafish Shh protein, Alex Joyner, Andrew Lumsden and Susanne Dietrich for cDNAs, Andy McMahon for Ihh-/- mice, Chin Chiang for Shh-/- mice and Abi Jensen, Graham Dunn, Pete Currie and Phil Ingham for advice. SMH was supported by MRC and EU QLK6-2000-530, XL by an EC Biomed 2 Ageing grant, and DD by the CNRS. CSB held a MRC PhD studentship and part of this work was reported in his PhD thesis (London University, 1999).
- Kardon G: Muscle and tendon morphogenesis in the avian hind limb. Development. 1998, 125: 4019-4032.PubMedGoogle Scholar
- Crow MT, Stockdale FE: Myosin expression and specialization among the earliest muscle fibres of the developing avian limb. Developmental Biology. 1986, 113: 238-254.View ArticlePubMedGoogle Scholar
- Miller JB, Stockdale FE: Developmental origins of skeletal muscle fibres: clonal analysis of myogenic cell lineages based on expression of fast and slow myosin heavy chains. Proceedings of the National Academy of Sciences USA. 1986, 83: 3860-3864.View ArticleGoogle Scholar
- Miller JB, Stockdale FE: Developmental regulation of the multiple myogenic cell lineages of the avian embryo. J Cell Biol. 1986, 103: 2199-2208. 10.1083/jcb.103.6.2197.View ArticleGoogle Scholar
- Van Swearingen J, Lance-Jones C: Slow and fast muscle fibres are preferentially derived from myoblasts migrating into the chick limb bud at different developmental times. Dev Biol. 1995, 170: 321-337. 10.1006/dbio.1995.1218.View ArticlePubMedGoogle Scholar
- DiMario JX, Stockdale FE: Both myoblast lineage and innervation determine fiber type and are required for expression of the slow myosin heavy chain 2 gene. Developmental Biology. 1997, 188: 167-180. 10.1006/dbio.1997.8619.View ArticlePubMedGoogle Scholar
- Nikovits W., Jr., Cann GM, Huang R, Christ B, Stockdale FE: Patterning of fast and slow fibers within embryonic muscles is established independently of signals from the surrounding mesenchyme. Development. 2001, 128: 2537-2544.PubMedGoogle Scholar
- Hughes SM, Salinas PC: Control of muscle fibre and motoneuron diversification. Curr Opin Neurobiol. 1999, 9: 54-64. 10.1016/S0959-4388(99)80007-5.View ArticlePubMedGoogle Scholar
- Stockdale FE, Nikovits W., Jr., Christ B: Molecular and cellular biology of avian somite development. Dev Dyn. 2000, 219: 304-321. 10.1002/1097-0177(2000)9999:9999<::AID-DVDY1057>3.0.CO;2-5.View ArticlePubMedGoogle Scholar
- Borycki AG, Emerson C. P., Jr.: Multiple tissue interactions and signal transduction pathways control somite myogenesis. Curr Top Dev Biol. 2000, 48: 165-224.View ArticlePubMedGoogle Scholar
- Cossu G, De Angelis L, Borello U, Berarducci B, Buffa V, Sonnino C, Coletta M, Vivarelli E, Bouche M, Lattanzi L, Tosoni D, Di Donna S, Berghella L, Salvatori G, Murphy P, Cusella-De Angelis MG, Molinaro M: Determination, diversification and multipotency of mammalian myogenic cells. Int J Dev Biol. 2000, 44: 699-706.PubMedGoogle Scholar
- Pownall ME, Gustafsson MK, Emerson C. P., Jr.: Myogenic regulatory factors and the specification of muscle progenitors in vertebrate embryos. Annu Rev Cell Dev Biol. 2002, 18: 747-783. 10.1146/annurev.cellbio.18.012502.105758.View ArticlePubMedGoogle Scholar
- Borycki AG, Mendham L, Emerson C.P. Jr: Control of somite patterning by Sonic hedgehog and its downstream signal response genes. Development. 1998, 125: 777-790.PubMedGoogle Scholar
- Borycki AG, Brunk B, Tajbakhsh S, Buckingham M, Chiang C, Emerson C. P., Jr.: Sonic hedgehog controls epaxial muscle determination through Myf5 activation. Development. 1999, 126: 4053-4063.PubMedGoogle Scholar
- Zhang XM, Ramalho-Santos M, McMahon AP: Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R asymmetry by the mouse node. Cell. 2001, 105: 781-792. 10.1016/S0092-8674(01)00385-3.View ArticlePubMedGoogle Scholar
- Tajbakhsh S, Rocancourt D, Buckingham M: Muscle progenitor cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice. Nature. 1996, 384: 266-270. 10.1038/384266a0.View ArticlePubMedGoogle Scholar
- Kahane N, Cinnamon Y, Kalcheim C: The origin and fate of pioneer myotomal cells in the avian embryo. Mech Dev. 1998, 74: 59-73. 10.1016/S0925-4773(98)00066-5.View ArticlePubMedGoogle Scholar
- Blagden CS, Currie PD, Ingham PW, Hughes SM: Notochord induction of zebrafish slow muscle mediated by Sonic Hedgehog. Genes Dev. 1997, 11: 2163-2175.PubMed CentralView ArticlePubMedGoogle Scholar
- Du SJ, Devoto SH, Westerfield M, Moon RT: Positive and negative regulation of muscle cell identity by members of the hedgehog and TGF-b gene families. Journal of Cell Biology. 1997, 139: 145-156. 10.1083/jcb.139.1.145.PubMed CentralView ArticlePubMedGoogle Scholar
- Stickney HL, Barresi MJ, Devoto SH: Somite development in zebrafish. Dev Dyn. 2000, 219: 287-303. 10.1002/1097-0177(2000)9999:9999<::AID-DVDY1065>3.3.CO;2-1.View ArticlePubMedGoogle Scholar
- van Raamsdonk W, van't Veer L, Veeken K, Heyting C, Pool CW: Differentiation of muscle fiber types in the teleost Brachydanio rerio, the zebrafish. Posthatching development. Anat Embryol (Berl). 1982, 164: 51-62.View ArticleGoogle Scholar
- Devoto SH, Melancon E, Eisen JS, Westerfield M: Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation. Development. 1996, 122: 3371-3380.PubMedGoogle Scholar
- Kalcheim C, Cinnamon Y, Kahane N: Myotome formation: a multistage process. Cell Tissue Res. 1999, 296: 161-173. 10.1007/s004410051277.View ArticlePubMedGoogle Scholar
- Tajbakhsh S, Buckingham M: The birth of muscle progenitor cells in the mouse: spatiotemporal considerations. Curr Top Dev Biol. 2000, 48: 225-268.View ArticlePubMedGoogle Scholar
- Münsterberg AE, Kitajewski J, Bumcrot DA, McMahon AP, Lassar AB: Combinatorial signalling by sonic hedgehog and Wnt family members induces myogenic bHLH gene expression in the somite. Genes and Development. 1995, 9: 2911-2922.View ArticlePubMedGoogle Scholar
- Fan C-M, Lee CS, Tessier-Lavigne M: A role for WNT proteins in induction of dermomyotome. Developmental Biology. 1997, 191: 160-165. 10.1006/dbio.1997.8713.View ArticlePubMedGoogle Scholar
- Tajbakhsh S, Borello U, Vivarelli E, Kelly R, Papkoff J, Duprez D, Buckingham M, Cossu G: Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development. 1998, 125: 4155-4162.PubMedGoogle Scholar
- Capdevila J, Tabin C, Johnson RL: Control of dorsoventral somite patterning by Wnt-1 and b-catenin. Developmental Biology. 1998, 193: 182-194. 10.1006/dbio.1997.8806.View ArticlePubMedGoogle Scholar
- Ikeya M, Takada S: Wnt signaling from the dorsal neural tube is required for the formation of the medial dermomyotome. Development. 1998, 125: 4969-4976.PubMedGoogle Scholar
- Borycki A, Brown AM, Emerson C. P., Jr.: Shh and Wnt signaling pathways converge to control Gli gene activation in avian somites. Development. 2000, 127: 2075-2087.PubMedGoogle Scholar
- Teillet M, Watanabe Y, Jeffs P, Duprez D, Lapointe F, Le Douarin NM: Sonic hedgehog is required for survival of both myogenic and chondrogenic somitic lineages. Development. 1998, 125: 2019-2030.PubMedGoogle Scholar
- Cann GM, Lee JW, Stockdale FE: Sonic hedgehog enhances somite cell viability and formation of primary slow muscle fibers in avian segmented mesoderm. Anat Embryol (Berl). 1999, 200: 239-252. 10.1007/s004290050276.View ArticleGoogle Scholar
- Sacks LD, Cann GM, Nikovits Jr. W, Conlon S, Espinoza NR, Stockdale FE: Regulation of myosin expression during myotome formation. Development. 2003, 130: 3391-3402. 10.1242/dev.00541.View ArticlePubMedGoogle Scholar
- Chevallier A, Kieny M, Mauger A: Limb-somite relationship: origin of the limb musculature. J Embryol Exp Morphol. 1977, 41: 245-258.PubMedGoogle Scholar
- Christ B, Jacob HJ, Jacob M: Experimental analysis of the origin of the wing musculature in avian embryos. Anat Embryol (Berl). 1977, 150: 171-186.View ArticleGoogle Scholar
- Ordahl CP, Le Douarin NM: Two myogenic lineages within the developing somite. Development. 1992, 114: 339-353.PubMedGoogle Scholar
- Williams BA, Ordahl CP: Pax-3 expression in segmental mesoderm marks early stages in myogenic cell specification. Development. 1994, 120: 785-796.PubMedGoogle Scholar
- Tajbakhsh S, Rocancourt D, Cossu G, Buckingham M: Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD. Cell. 1997, 89: 127-138. 10.1016/S0092-8674(00)80189-0.View ArticlePubMedGoogle Scholar
- Dietrich S, Schubert FR, Healy C, Sharpe PT, Lumsden A: Specification of the hypaxial musculature. Development. 1998, 125: 2235-2249.PubMedGoogle Scholar
- Mankoo BS, Collins NS, Ashby P, Grigorieva E, Pevny LH, Candia A, Wright CV, Rigby PW, V Pachnis: Mox2 is a component of the genetic hierarchy controlling limb muscle development. Nature. 1999, 400: 69-73. 10.1038/21892.View ArticlePubMedGoogle Scholar
- Robson LG, Kara T, Crawley A, Tickle C: Tissue and cellular patterning of the musculature in chick wings. Development. 1994, 120: 1265-1276.PubMedGoogle Scholar
- Duprez D, Lapointe F, Edom-Vovard F, Kostakopoulou K, Robson L: Sonic Hedgehog (SHH) specifies muscle pattern at tissue and cellular chick level, in the chick limb bud. Mechanisms of Development. 1999, 82: 151-163. 10.1016/S0925-4773(99)00040-4.View ArticlePubMedGoogle Scholar
- Hughes SM, Blau HM: Muscle fiber pattern is independent of cell lineage in postnatal rodent development. Cell. 1992, 68: 659-671. 10.1016/0092-8674(92)90142-Y.View ArticlePubMedGoogle Scholar
- Kardon G, Campbell JK, Tabin CJ: Local extrinsic signals determine muscle and endothelial cell fate and patterning in the vertebrate limb. Dev Cell. 2002, 3: 533-545. 10.1016/S1534-5807(02)00291-5.View ArticlePubMedGoogle Scholar
- DiMario JX, Fernyak SE, Stockdale FE: Myoblasts transferred to the limbs of embryos are committed to specific fibre fates. Nature. 1993, 362: 165-167. 10.1038/362165a0.View ArticlePubMedGoogle Scholar
- DiMario JX, Stockdale FE: Differences in the developmental fate of cultured and noncultured myoblasts when transplanted into embryonic limbs. Experimental Cell Research. 1995, 216: 431-442. 10.1006/excr.1995.1054.View ArticlePubMedGoogle Scholar
- Robson LG, Hughes SM: The distal limb environment regulates MyoD accumulation and muscle differentiation in mouse-chick chimaeric limbs. Development. 1996, 122: 3899-3910.PubMedGoogle Scholar
- Robson LG, Hughes SM: Local signals in the chick limb bud can override myoblast lineage commitment: induction of slow myosin heavy chain in fast myoblasts. Mech Dev. 1999, 85: 59-71. 10.1016/S0925-4773(99)00060-X.View ArticlePubMedGoogle Scholar
- Duprez D, Fournier-Thibault C, Le Douarin N: Sonic Hedgehog induces proliferation of committed skeletal muscle cells in the chick limb. Development. 1998, 125: 495-505.PubMedGoogle Scholar
- Amthor H, Christ B, Weil M, Patel K: The importance of timing differentiation during limb muscle development. Curr Biol. 1998, 8: 642-652.View ArticlePubMedGoogle Scholar
- Bren-Mattison Y, Olwin BB: Sonic hedgehog inhibits the terminal differentiation of limb myoblasts committed to the slow muscle lineage. Dev Biol. 2002, 242: 130-148. 10.1006/dbio.2001.0528.View ArticlePubMedGoogle Scholar
- Kruger M, Mennerich D, Fees S, Schafer R, Mundlos S, Braun T: Sonic hedgehog is a survival factor for hypaxial muscles during mouse development. Development. 2001, 128: 743-752.PubMedGoogle Scholar
- Pirskanen A, Kiefer JC, Hauschka SD: IGFs, insulin, Shh, bFGF, and TGF-beta1 interact synergistically to promote somite myogenesis in vitro. Dev Biol. 2000, 224: 189-203. 10.1006/dbio.2000.9784.View ArticlePubMedGoogle Scholar
- Yoshida N, Yoshida S, Koishi K, Masuda K, Nabeshima Y: Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates 'reserve cells'. Journal of Cell Science. 1998, 111: 769-779.PubMedGoogle Scholar
- Bayol S, Loughna PT, Brownson C: Phenotypic expression of IGF binding protein transcripts in muscle, in vitro and in vivo. Biochem Biophys Res Commun. 2000, 273: 282-286. 10.1006/bbrc.2000.2667.View ArticlePubMedGoogle Scholar
- Ingham PW, McMahon AP: Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001, 15: 3059-3087. 10.1101/gad.938601.View ArticlePubMedGoogle Scholar
- Bourke DL, Wylie SR, Theon A, Bandman E: Myosin heavy chain expression following myoblast transfer into regenerating chicken muscle. Basic and Applied Myology. 1995, 1: 43-56.Google Scholar
- Page S, Miller JB, DiMario JX, Hager EJ, Moser A, Stockdale FE: Developmentally regulated expression of three slow isoforms of myosin heavy chain: Diversity among the first fibers to form in avian muscle. Developmental Biology. 1992, 154: 118-128.View ArticlePubMedGoogle Scholar
- Vortkamp A, Pathi S, Peretti GM, Caruso EM, Zaleske DJ, Tabin CJ: Recapitulation of signals regulating embryonic bone formation during postnatal growth and in fracture repair. Mechanisms of Development. 1998, 71: 65-76. 10.1016/S0925-4773(97)00203-7.View ArticlePubMedGoogle Scholar
- Kerst B, Mennerich D, Schuelke M, Stoltenburg-Didinger G, von Moers A, Gossrau R, van Landeghem FK, Speer A, Braun T, Hubner C: Heterozygous myogenic factor 6 mutation associated with myopathy and severe course of Becker muscular dystrophy. Neuromuscul Disord. 2000, 10: 572-577. 10.1016/S0960-8966(00)00150-4.View ArticlePubMedGoogle Scholar
- Maggs AM, Taylor-Harris P, Peckham M, Hughes SM: Evidence for differential post-translational modifications of slow myosin heavy chain during murine skeletal muscle development. J Muscle Res Cell Motil. 2000, 21: 101-113. 10.1023/A:1005639229497.View ArticlePubMedGoogle Scholar
- St-Jacques B, Hammerschmidt M, McMahon AP: Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes and Development. 1999, 13: 2072-2086.PubMed CentralView ArticlePubMedGoogle Scholar
- Condon K, Silberstein L, Blau HM, Thompson WJ: Development of muscle fiber types in the prenatal rat hindlimb. Dev Biol. 1990, 138: 256-274.View ArticlePubMedGoogle Scholar
- Rubinstein NA, Kelly AM: Development of muscle fiber specialization in the rat hindlimb. J Cell Biol. 1981, 90: 128-144. 10.1083/jcb.90.1.128.View ArticlePubMedGoogle Scholar
- Amthor H, Christ B, Patel K: A molecular mechanism enabling continuous embryonic muscle growth - a balance between proliferation and differentiation. Development. 1999, 126: 1041-1053.PubMedGoogle Scholar
- Marigo V, Scott MP, Johnson RL, Goodrich LV, Tabin CJ: Conservation in hedgehog signaling: Induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development. 1996, 122: 1225-1233.PubMedGoogle Scholar
- Watanabe Y, Duprez D, Monsoro-Burq AH, Vincent C, Le Douarin NM: Two domains in vertebral development: antagonistic regulation by SHH and BMP4 proteins. Development. 1998, 125: 2631-2639.PubMedGoogle Scholar
- Wu Q, Zhang Y, Chen Q: Indian hedgehog is an essential component of mechanotransduction complex to stimulate chondrocyte proliferation. J Biol Chem. 2001, 276: 35290-35296. 10.1074/jbc.M101055200.View ArticlePubMedGoogle Scholar
- Coutelle O, Blagden CS, Hampson R, Halai C, Rigby PW, Hughes SM: Hedgehog signalling is required for maintenance of myf5 and myoD expression and timely terminal differentiation in zebrafish adaxial myogenesis. Dev Biol. 2001, 236: 136-150. 10.1006/dbio.2001.0193.View ArticlePubMedGoogle Scholar
- Lewis KE, Currie PD, Roy S, Schauerte H, Haffter P, Ingham PW: Control of muscle cell-type specification in the zebrafish embryo by Hedgehog signalling. Dev Biol. 1999, 216: 469-480. 10.1006/dbio.1999.9519.View ArticlePubMedGoogle Scholar
- Schauerte HE, van Eeden FJ, Fricke C, Odenthal J, Strahle U, Haffter P: Sonic hedgehog is not required for the induction of medial floor plate cells in the zebrafish. Development. 1998, 125: 2983-2993.PubMedGoogle Scholar
- Wolff C, Roy S, Ingham PW: Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo. Curr Biol. 2003, 13: 1169-1181. 10.1016/S0960-9822(03)00461-5.View ArticlePubMedGoogle Scholar
- Barresi MJ, D'Angelo JA, Hernandez LP, Devoto SH: Distinct mechanisms regulate slow-muscle development. Curr Biol. 2001, 11: 1432-1438. 10.1016/S0960-9822(01)00428-6.View ArticlePubMedGoogle Scholar
- Grimaldi A, Tettamanti G, Martin BL, Gaffield W, Pownall ME, Hughes SM: Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis. Development. 2004, 131: 3249-3262. 10.1242/dev.01194.View ArticlePubMedGoogle Scholar
- Asakura A, Tapscott SJ: Apoptosis of epaxial myotome in Danforth's short-tail (Sd) mice in somites that form following notochord degeneration. Dev Biol. 1998, 203: 276-289. 10.1006/dbio.1998.9050.View ArticlePubMedGoogle Scholar
- Norris W, Neyt C, Ingham PW, Currie PD: Slow muscle induction by Hedgehog signalling in vitro. J Cell Sci. 2000, 113: 2695-2703.PubMedGoogle Scholar
- Schafer DA, Miller JB, Stockdale FE: Cell diversification within the myogenic lineage: in vitro generation of two types of myoblasts from a single progenitor cell. Cell. 1987, 48: 659-670. 10.1016/0092-8674(87)90244-3.View ArticlePubMedGoogle Scholar
- Seed J, Hauschka SD: Clonal analysis of vertebrate myogenesis. VIII. Fibroblasts growth factor (FGF)-dependent and FGF-independent muscle colony types during chick wing development. Dev Biol. 1988, 128: 40-49.View ArticlePubMedGoogle Scholar
- Anakwe K, Robson L, Hadley J, Buxton P, Church V, Allen S, Hartmann C, Harfe B, Nohno T, Brown AM, Evans DJ, Francis-West P: Wnt signalling regulates myogenic differentiation in the developing avian wing. Development. 2003, 130: 3503-3514. 10.1242/dev.00538.View ArticlePubMedGoogle Scholar
- Dan-Goor M, Silberstein L, Kessel M, Muhlrad A: Localization of epitopes and functional effects of two novel monoclonal antibodies against skeletal muscle myosin. J Muscle Res Cell Motil. 1990, 11: 216-226.View ArticlePubMedGoogle Scholar
- Hughes SM, Cho M, Karsch-Mizrachi I, Travis M, Silberstein L, Leinwand LA, Blau HM: Three slow myosin heavy chains sequentially expressed in developing mammalian skeletal muscle. Developmental Biology. 1993, 158: 183-199. 10.1006/dbio.1993.1178.View ArticlePubMedGoogle Scholar
- Gardahaut MF, Fontaine-Perus J, Rouaud T, Bandman E, Ferrand R: Developmental modulation of myosin expression by thyroid hormone in avian skeletal muscle. Development. 1992, 115: 1121-1131.PubMedGoogle Scholar
- Ericson J, Morton S, Kawakami A, Roelink H, Jessell TM: Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell. 1996, 87: 661-673. 10.1016/S0092-8674(00)81386-0.View ArticlePubMedGoogle Scholar
- Shellswell GB, Wolpert L: The pattern of muscle and tendon development in the chick wing. Vertebrate limb and somite morphogenesis. Edited by: Ede DA, Hinchcliffe JR and Balls M. 1977, Cambridge, Cambridge University PressGoogle Scholar
- O'Neill MC, Stockdale FE: A kinetic analysis of myogenesis in vitro. Journal of Cell Biology. 1972, 52: 52-65. 10.1083/jcb.52.1.52.PubMed CentralView ArticlePubMedGoogle Scholar
- Riddle RD, Johnson RL, Laufer E, Tabin C: Sonic hedgehog mediates the polarizing activity of the ZPA. Cell. 1993, 75: 1401-1416.View ArticlePubMedGoogle Scholar
- Jensen AM, Wallace VA: Expression of Sonic hedgehog and its putative role as a precursor cell mitogen in the developing mouse retina. Development. 1997, 124: 363-371.PubMedGoogle Scholar
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