Myogenesis in Xenopus laevis

neutmphil elastase and cathepsin G. J Biol Chem 265:6092-6097. Pratt CW, Whinna HC, Church FC: 1992. A comparison of three heparin-binding serine proteinase inhibitors. J Biol Chem 267:87958801. Roberts HR, Lozier JN: 1992. New perspectives on the coagulation cascade. Hosp Pratt 27:97-l 11. Senior RM, Skogen WF, Griffin GL, Wilner GD: 1986. Effects of fibrinogen derivatives upon the inflammatory response: studies with fibrinopeptide B. J Clin Invest 77: 10 141019. Stubbs MT, Bode W: 1993. A player of many parts: the spotlight falls on thrombin’s structure. Thromb Res 69: l-58. Van Deerlin VMD, Tollefsen DM: 1991. The N-terminal acidic domain of heparin cofactor II mediates the inhibition of a-thrombin

Myogenesis Tim Mohun,

in the presence of glycosaminoglycans. Biol Chem 266:20,223-20,23 1.

J

Vu TH, Hung DT, Wheaton VI, Coughlin SR: 1991. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057-1068. Wachtfogel YT, Kucich U, James HL, et al.: 1983. Human plasma kallikrein releases neutrophil elastase during blood coagulation. J Clin Invest 72: 1672-l 677. Whinna HC, Blinder MA, Szewczyk M, Tollefsen DM, Church FC: 1991. Role of lysine 173 in heparin binding to heparin cofactor II. J Biol Chem 266:8129-8135. Whinna HC, Choi HU, Rosenberg LC, Church FC: 1993. Interaction of heparin cofactor II with biglycan and decorin. J Biol Chem 26813920-3924.

TCM

in Xenopus

laevis

Robert Wilson, Elisa Gionti, and Malcolm

Logan

The amphibian embryo provides a convenient experimental system with which to study myogenesis. The earliest steps in the formation of axial and cardiac muscle are accessible for investigation using both embryological and molecular approaches. We review the origins of skeletal and cardiac muscle in the Xenopus embryo, the molecular markers available to detect muscle differentiation, and the use of embryo explants to investigate the regulation of myogenesis. (Trends Cardiovasc

l

Origins

Med

1994;4:146-15

of Embryonic

1)

Muscle

The amphibian embryo has long been a favored subject of developmental biologists. Taking advantage of its size and robustness, experimental embryologists have traced the origins of many tissues in the early embryo and identified cellular interactions that are decisive in establishing the body plan [reviewed in Slack (1991)]. As in other vertebrates, muscle Tim Mohun, Robert Wilson, Elisa Gionti, and Malcolm Logan are at the Laboratory of Developmental Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 lAA, England.

146

tissue in Xenopus is derived from the mesodermal cell layer formed during gastntlation. At the 32-cell stage, the progeny of almost half of the cells in the Xenopus embryo will eventually contribute to muscle. Lineage-labeling studies show that these lie in the central two tiers of the embryo (Figure 1). During the subsequent period of rapid cell division, no cell movement occurs and, in the blastula embryo, the precursors of somitic muscle are localized in a broad equatorial band extending around much of the embryo circumference. By the gastrula stage, tissue fated to form somitic muscle is localized to the newly forming

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mesoderm, on either side of the dorsal lip (Keller 1976). As a result of morphogenetic movements during gastrulation, these regions form the dorsal mesoderm that lies on either side of the notochord and extends along the anteroposterior axis of the embryo. This tissue progressively segments from the anterior end of the embryo into discrete somites that comprise blocks of about nine cells each (Hamilton 1969). The newly formed somite rotates 90°, with the result that each somite cell extends along the anteroposterior axis of the embryo and spans the entire length of the somite. Unlike in other vertebrates, in Xenopus the majority of cells within the somite form muscle tissue of the myotome, and few apparently contribute to the dermatome and sclerotome. Unusually also, the myotomal cells remain unfused even after they have differentiated and contractile function is acquired prior to any innervation of the myotome blocks. Amphibian embryos also differ from those of mammals or birds in the relative timing of cardiac and skeletal myogenesis. In Xenopus, cardiac muscle differentiation is a late event in embryogenesis, commencing many hours after the myotomes have formed. Fate maps of the gastrula identify the precursors of cardiac muscle as paired regions of dorsal mesoderm, distinct from somitic precursors and restricted to the anterior end of the embryo (Figure 1). By the end of gastrulation, the cardiac mesoderm lies at the anterior edge of the neural plate, on either side of the embryo. As neurulation progresses, these regions move to the ventral midline, where they fuse immediately behind the cement gland. Many hours elapse before a heart tube is formed, and circulation only commences in the free-swimming tadpole (Nieuwkoop and Faber 1956).

l

Muscle Embryo

Formation Explants

in

A useful feature of amphibian embryos is the ability of isolated tissue fragments to continue differentiation for many days, relying entirely on their intracellular yolk store for nutrients. This has been exploited to study the origins of embryonic mesoderm and the capacity of mesodermal cells to differentiate. TCM

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Mesoderm originates from an inductive interaction between animal and vegetal halves of the early embryo that can be reproduced with explant combinations [reviewed in Slack (199 l)]. Animal pole explants are induced by signals from the vegetal tissue to form axial structures that contain large amounts of muscle tissue. Similar results can be obtained with animal pole explants alone, by exposure to members of the transforming growth factor l3 (TGFB) and basic fibroblast growth factor (bFGF) families (Figure 2). The range and types of mesodermal derivatives obtained depend on the dose and identity of the growth factor added to the culture medium (Smith et al. 1993). Thus, the TGFl3 family member activin A readily induces dorsal mesodermal derivatives such as myotomal muscle, and at higher doses will induce cardiac muscle (Logan and Mohun 1993). In contrast, bFGF is incapable of inducing the most dorsal tissues (notochord and cardiac muscle). Although the identity of the natural inducers within the embryo remains uncertain, it seems likely that patterning of the mcsoderm along the anteroposterior and dorsoventral axes results from localization or concentration gradients of mesoderminducing signals within the embryo. Such signals must trigger the first steps in muscle formation.

l

Molecular Markers Muscle Differentiation

of Skeletal

cDNAs encoding muscle-specific structural proteins provide useful molecular markers for terminal differentiation and the most widely used of these is the cardiac actin gene transcript (Mohun et al. 1984). As in other vertebrates, the cardiac actin gene is expressed in the developing axial musculature of amphibian embryos and is restricted to cardiac muscle only after metamorphosis (Mohun et al. 1988). Transcripts can be detected by RNase protection assay at the midgastrula stage and are localized within the presomitic mesoderm. Indeed, by reverse transcription-polymerase chain reaction, cardiac actin mRNA accumulation can be detected almost immediately after the onset of gastrulation (Figure 3). Terminal differentiation of skeletal muscle therefore commences even before segmentation of the dorsal mesoderm into somites. In this respect, TCM

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32 cell (stage 6)

gastrula (stage 10)

early neurula (stage 14)

SOMITE,.,,

@

3 hours

late neurula (stage 20)

tadpole (stage 33)

0D

44 hours (heartbeat)

22 hours

1. Muscle formation in Xenopus early development. At the 32-cell stage, ceils fated to form much of the embryo musculature lie in the central two tiers (Dale and Slack 1987). In the early gastrula, somitic muscle precursors (light shading) arise from the dorsal and lateral marginal zone (Keller 1976). Prospective heart mesoderm (durk shading) lies in the deep layer, adjacent to the blastopore lip. During neurulation, this moves from the anterior edge of the neural plate on either side of the embryo to the ventral midline, where the heart finally differentiates. Figure

the timetable differs from mammals.

l

Expression

of myogenesis in Xenoptls that found in birds and

of the MyoD

Family

Activation of the cardiac actin gene has been used as a model to identify transcription factors regulating the differentiation of myotomal cells, By monitoring the expression of the cloned gene microinjected into fertilized eggs, regulatory regions within the cardiac actin gene promoter have been identified (Figure 4). Muscle-specific expression of this gene in either normal embryos or growthfactor-induced animal pole explants requires several promoter elements, including a binding site for the ubiquitous serum response factor (SRF) and a site recognized by the MyoD family of musclespecific transcription factors (Mohun et al. 1989, Taylor et al. 1991). Three members of the MyoD gene family are expressed in Xenoptls embryos [reviewed in Mohun (1992)]. Some

Figure

XMyoD transcripts are inherited from the unfertilized egg and distributed throughout the cleaving embryo. In the blastula embryo, the XMyoD gene is briefly activated, but the transcripts appear to be unstable, and no XMyoD protein has been detected. In the early gastrula, renewed expression results in rapid accumulation of both transcripts and protein in the newly forming mesoderm. XMyf5 expression also commences in the presomitic mesoderm of the early gastrula, but subsequently accumulates largely in the posterior region. A third MyoD family member, XMRF4, can be detected in the myotomes of the tailbud embryo after much of the embryo musculature has differentiated. Interestingly, no transcripts of the Xenopus myogenin gene have been detected in embryonic or adult tissues. This is a striking illustration of the complex functional redundancy among members of the MyoD family since, in mice, gene-knockout experiments have shown that the myo-

2. Blastula animal pole explants can be induced to form muscle by exposure to growth

factors.

Activin A [high]

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Skeletal Cardiac Muscle Muscle

147

Embryo stage /o.& 2, + +@ + I’s

Femoral

(a31

Skeletal (a.3

l

Figure 3. Sarcomeric actin genes are activated during gastrulation. Transcripts of the cardiac (ccl) actin gene can be detected in the early gastrula (stage 10.5) with the reverse transcription-polymerase chain reaction and are the most prevalent sarcomeric actin transcripts in the early embryo. The two genes encoding skeletal muscle-specific actin isofonns are activated later during gasttulation; femoral (a2) actin transcripts accumulate from stage II while skeletal (a3) actin mRNA is first detected at stage f2.

genin gene is essential for the formation of skeletal muscle (Hasty et al. 1993, Nabeshima et al. 1993).

l

Regulation

of Skeletal

Myogenesis

It is tempting to attribute the spatial restriction of myotomal muscle differentiation to localized transcription of the MyoD gene family in the somitic mesoderm. However, several observations challenge this view. First, in the early gastrula, XMyoD mRNA is initially found throughout the mesoderm (Frank and Harland 1991), and the XMyoD protein can be detected in the nuclei of all but the most dorsal mesodermal cells that subsequently form the notochord (Hopwood et al. 1992). Staining for the protein is most intense in dorsal mesodermal cell nuclei and becomes restricted to the premyotomal cells as gastrulation proceeds. This suggests that both transcripts and protein may be transiently expressed in cells other than those that ultimately form muscle. Consistent with this view, “ventralized” embryos produced by exposure to UV light accumulate transcripts of the XMyoD gene despite forming little or no myotomal muscle tissue (Frank and Harland 1991). Second, ectopic expression of XMyf5 or XMyoD in embryos results in transactivation of endogenous targets, such as the cardiac

148

actin gene, but does not trigger the full muscle phenotype (Hopwood and Gurdon 1990, Hopwood et al. 1991). Taken together, these results suggest that differentiation of somitic mesoderm involves mechanisms that restrict the accumulation and activity of the myogenic proteins to the dorsal mesoderm and perhaps requires regulatory factors distinct from the MyoD family. Muscle Formation and Mesoderm Patterning

The importance of regional signals in restricting muscle formation within the mesoderm is well established from embryologic studies. At the blastula stage, explants comprising the entire equatorial region (from which the mesoderm is formed) differentiate into reasonably complete embryos containing axially arranged blocks of myotomal muscle. Explants from the dorsal side of the embryo also form muscle tissue, but lateral explants fail to do so, despite the fact that much of the myotomal muscle in normal development is formed from this region of the embryo. The lateral cells therefore require-additional signals before they will differentiate according to their normal fate. Patterning of mesoderm along the dorsoventral axis is believed to result from a “dorsalizing” signal provided by a small region above the dorsal lip of the gastrula (“the organizer”), first identified many decades ago by its capacity to induce axis duplication in grafting experiments (Slack 1991). A number of genes have now been identified that are either expressed in the organizer region or can promote the formation of dorsal mesoderm in UV-ventralized embryos (Sive 1993, Smith et al. 1993). Their products may therefore be components of the “dorsalizing” signal. How might this signal regulate muscle formation? One possibility is that it triggers a regional difference in the level or activity of XMyoD/XMyR proteins. Positive autoregulation of the XMyoD and XMyR genes by their products could amplify such differences, resulting in the accumulation of the proteins above a threshold necessary to initiate myogenic differentiation. Little is known about the stability of these transcripts, but several mechanisms have been identified that may regulate the activity of the proteins

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directly. These include phosphorylation (Olson et al. 1991), interaction with members of the jun family of cellular proto-oncogenes (Bengal et al. 1992, Li et al. 1992), availability of E-protein partners, and the distribution of Id-like negative regulators that compete for binding to E proteins. Neither the phosphorylation status of XMyoD or XMyf5 during early development nor their interaction with jun family proteins has been documented, but the availability of a high-affinity anti-XMyoD monoclonal antibody may allow these questions to be investigated. The Xenopus E2A gene is ubiquitously expressed in the early embryo, and functional protein can be detected in regions of the embryo other than the somitic mesoderm (Rashbass et al. 1992). Members of the Id family of negative regulators are present in the early embryo (T.M. and R.W. unpublished data) but they too are widely distributed, and their role in regulating the differentiation of somitic mesoderm is unclear.

l

The RSRF/MEFZ

Family

A second family of transcription factors that play an important role in skeletal myogenesis are the RSRF or MEF2 proteins. These are believed to act as regulators for both MyoD-dependent and MyoDindependent branches of the myogenic program (Cserjesi and Olson 1991) and may also regulate expression of the MyoD gene family (Edmondson et al. 1992, Yee and Rigby 1993). Four RSRF/MEFZ genes have been identified in mammals, and each gene produces several distinct proteins as a result of alternative splicing (Pollock and Treisman 1991, Breitbart et al. 1993). The proteins can bind their target sequences as either homo- or heterodimers (Pollock and Treisman 1991), and a large number of different binding activities may well be produced from this small gene family. RSRF/MEFZ-like binding activities are present in many cell lines and tissue types. In cultured muscle cells, an RSRF-containing DNA-binding activity (termed MEFZ) is upregulated during differentiation, and the same activity is detected during myogenic conversion of fibroblasts by members of the MyoD family (Cserjesi and Olson 1991, Lassar et al. 1991). In Xenopus, two RSRF/MEFZ genes (SL-1 and SL-2) have been identified TCM Vol. 4, No. 3, 1994

298

a b (Chambers et al. 1992), and at least until the neurula stage these appear to be under the control of the MyoD family. Transcripts of both genes are present in the unfertilized egg, but zygotic transcription commences during gastrulation. The SL- 1 gene is activated in dorsal, presomitic mesoderm shortly after onset of XMyoD accumulation, while the SL-2 gene is expressed only during terminal differentiation of muscle cells. In the tailbud embryo, transcription of both genes switches from being somite specific to ubiquitous, and RSRF/MEF2binding activities are subsequently detected in many tadpole and adult tissues. This suggests that the RSRFIMEFZ gene products have other functions in addition to their role in myotomal muscle differentiation.

l

Differentiation

of Cardiac

Muscle

Cardiac mesoderm, like its somitic counterpart, becomes specified during gastrulation as a result of a dorsalizing signal from the “organizer” region of the embryo. Removal of the dorsal lip from early gastrulae prevents heart formation, while removal of the precardiac mesoderm at this stage results in the repatterning of more lateral mesoderm to form heart tissue (Sater and Jacobson 1990b). These results indicate that an essential first step in normal cardiogenesis is the patterning of dorsal mesoderm by signals from the organizer region. The restriction of cardiac mesoderm to discrete regions of anterior, dorsal mesoderm presumably reflects the character TCM

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Figure 4. A total 580 nucleotides of cardiac actin promoter sequence drive somite-specific expression of reporter genes in Xenopus embryos. (a) Expression of the human f3-globin gene linked to the Xenoptls cardiac actin promoter is localized to the dorsal half of the neurula embryo (assayed by an RNase protection). In cultured blastula explants, the injected gene is activated in the equatorial region (Eq). Transcripts are detected only in animal pole explants that have been cultured as conjugates with vegetal tissue (Conj) or induced by exposure to the growth factor, activin A. M, approximate size markers (Hinfl-digested pBR322 DNA): P, undigested RNA probe; and t, tRNA control hybridization. (b) Expression of a cardiac actin/LacZ chimeric gene is restricted to the dorsal, paraxial mesoderm in the neurula embryo. A whole, neurula (stage 18) embryo is shown from the dorsal side (ant&or end upper right) after staining for 8-galactosidase activity. Expression is mosaic (as with all injected genes) but localized within the developing somites.

of the signaling rather than an inherent property of these cells. The dosedependent induction of cardiac muscle differentiation in blastula animal pole explants by activin A is consistent with this interpretation. In urodele amphibians, a second cellular interaction between the cardiac mesoderm and the pharyngeal endoderm is necessary for heart formation. Explants of cardiac mesoderm from late neurulae will differentiate into complete beating hearts in isolation while those from early neurulae will do so only if maintained in contact with the underlying endoderm (Smith and Armstrong 1990). In Xenopus (an anuran amphibian), it has proved impossible to determine whether a similar endodermal signal is necessary for heart differentiation. Removal of superficial, pharyngeal endoderm from early gastrulae has little effect on heart formation, and explants of cardiac mesoderm from late gastrulae will form beating heart in culture. A second region of anterior endoderm is in contact with the prospective heart mesoderm from the late blastula stage onward

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and could be important for cardiac mesoderm specification. This tissue lies within the embryo, however, and cannot be separated from the mesoderm with which it is in contact (Sater and Jacobson 1989). In normal development, the two regions of cardiac mesoderm fuse at the ventral midline long before heart tube differentiation. Each region can give rise to a complete heart, however, if the other is removed, and explants of cardiac mesoderm from either side of the embryo will differentiate independently into a complete heart. This indicates that further cellular interactions regulate formation of a single heart tube from specified mesodermal tissue. The total region of heart-forming potency (or heart “morphogenetic field”) in the neurula is much larger than the area destined to become heart tissue, encompassing both anteroventral and adjacent lateral mesoderm (Sater and Jacobson 1990a). By tailbud stage 28, the lateral mesoderm loses its capacity to form heart tissue. Nothing is known about the molecular signals that restrict cardiac muscle dif-

149

ferentiation to the most ventral cardiac mesodetm and trigger regulative heart formation in ablated embryos. Molecular markers exclusive to embryonic cardiac muscle have proved difficult to identify, since much of the myogenic program in vertebrate embryos is common to both cardiac and skeletal muscle. However, mRNAs encoding the Xenopus cardiac myosin heavy chain (Logan and Mohun 1993), myosin light chain II (T.M. and M.L. unpublished data), and troponin I (P. Krieg personal communication) proteins are all heart specific during premetamorphic development. These transcripts are first detected in the tailbud embryo, localized to the anterior ventral mesoderm from which the heart is formed. Their appearance coincides with that of cardiac actin RNA in this region, suggesting that, in contrast to skeletal muscle markers, the cardiac-specific structural genes may be coordinately regulated. In the tadpole heart, these markers are expressed in the myocardium. Regulation

l

of Cardiac

Myogenesis

To date, no members of the MyoD family have been detected in the embryonic heart, although a very low level of XMyoD transcripts has been reported in adult Xenopus heart tissue (Jennings 1992). Differentiation of skeletal and cardiac muscle therefore seems to be regulated by distinct mechanisms. The absence of convenient experimental models for cardiac myogenesis has hampered the identification of myogenic regulators, but recent studies have now implicated two gene products. In mouse embryos, a homeobox gene related to Drosophila tinman is expressed in the precardiac regions of the mesoderm and subsequently restricted largely to the developing heart tube (Komuro and Izumo 1993, Lints et al. 1993). Its counterpart in Xenopus is expressed in the cardiac mesoderm from the early neurula stage and accumulates in an anterior ventral region of the embryo that encompasses the heart primordium (Tonissen et al. 1994). In flies, the tinman gene is essential for heart formation (Bodmer 1993), and the expression pattern of its vertebrate homologues points toward some conservation in function. RSRFlMEF2 proteins may also be important in cardiac myogenesis. A bind-

1.50

ing site recognized by these proteins is essential for cardiac-specific expression of the rat myosin light chain II (Braun et al. 1989, Zhu et al. 1991) and human phosphoglycerate mutase (Nakatsuji et al. 1992) genes. In Xenopus embryos, the RSRF/MEFZ gene, SL- 1, accumulates in the anteroventral region containing the cardiac mesoderm several hours before any terminal differentiation of heart muscle can be detected (TM. unpublished data). Expression of its mammalian counterpart has also been detected in primary cardiocyte cultures, including within cells that have not apparently undergone terminal differentiation (Breitbart et al. 1993).

l

Future

Questions

In myotomal muscle, products of the MyoD and RSRF/MEFZ gene families activate terminal differentiation, but the mechanisms that restrict their activity to the somitic mesoderm are unclear. In cardiac muscle, the equivalent regulatory proteins have yet to be identified, as do the tissue interactions that limit heart formation to the ventral midline. With the identification of candidate signals responsible for mesoderm patterning in Xenopus, embryological and molecular approaches can be combined to investigate these questions.

l

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