Developmental Fate of the Mammalian Myotome

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1 a DEVELOPMENTAL DYNAMICS 239: , 2010 RESEARCH ARTICLE Developmental Fate of the Mammalian Myotome Marianne Deries, 1,4 Ronen Schweitzer, 2,3 and Marilyn J. Duxson 1 * The myotome is a segmented paraxial muscle present in all early vertebrate embryos, which in amniotes disappears in mid-embryogenesis, and is replaced by complex epaxial and hypaxial musculature. Little is known about how this transition occurs. Here, we describe the detailed morphogenesis of the epaxial muscles from the epaxial myotome, in rodent embryos. The results show there is no apoptosis of myotomal fibres during the transition, and that the epaxial muscles arise by translocation, re-orientation, and elongation of the myotomal myocytes followed by cleavage of the myotomal masses. Myotomal myocytes transit from a mononucleated to a multinucleated state just before onset of this transformation. Each newly-formed epaxial muscle anlagen includes populations of Pax3- and Pax7-positive muscle progenitors, with different distributions. Using transgenic mouse embryos bearing a GFP marker for Scleraxis, we show that tendon progenitors are tightly associated with the sides and ends of myotomal myocytes as they re-orient and elongate. 239: , VC 2010 Wiley-Liss, Inc. Key words: myotome; rat; mouse; development; morphogenesis; apoptosis; Pax3; Pax7; scleraxis Accepted 16 August 2010 INTRODUCTION This report examines the manner in which the epaxial muscles of the vertebrate (the deep back muscles) arise from the embryonic myotome. The myotome is a metameric muscle that forms within the somite, lying between the lateral dermomyotome and the medial sclerotome, and is the first muscle to develop in every vertebrate. Its mononucleated myocytes span the whole length of each segment and lie parallel to the axis of the embryo (Denetclaw et al., 1997; Kahane et al., 1998; Venters et al., 1999). In birds and mammals, myotomes are transitory muscles and disappear to be replaced in the epaxial domain by the deep muscles of the back. These attach to the vertebrae, where they act as extensor muscles, and receive their innervation from the dorsal ramus of the mixed spinal nerve. The adult epaxial muscles comprise three main muscle groups, the transversospinalis, longissimus, and iliocostalis muscles, which are identifiable all along the axis (Vallois, 1922). At a thoracic level, the levatores costarum muscles form a fourth group attaching to the ribs and elevating them during respiration (Sato, 1973; Smith and Hollyday, 1983). The transition from the simple unisegmental myotome to the complex morphology of the deep back muscles occurs rapidly during midembryogenesis, but remarkably little is known about how it occurs. A key question is whether the original myocytes of the myotome are retained and contribute to the formation of epaxial muscles. Although it has been proposed that they do (Butcher, 1929; Christ et al., 1983; Christ and Ordahl, 1995), the only direct evidence for retention of myotomal fibres within the definitive axial muscles comes from the work of Cinnamon et al. (1999) where hypaxial myotomal myocytes labelled with DiI during early development were found to persist throughout the segmental intercostal muscles. If the original myocytes are retained within all epaxial and hypaxial derivatives of the myotome, then the further processes that lead to the transformation of the 1 Department of Anatomy and Structural Biology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand 2 Shriners Hospital for Children, Research Division, Portland, Oregon 3 Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon 4 Centro de Biologia Ambiental, Faculdade de Ciencias, Universidade de Lisboa, Lisbon, Portugal *Correspondence to: Marilyn J. Duxson, Department of Anatomy and Structural Biology, Otago School of Medical Sciences, University of Otago, PO Box 913, Dunedin, New Zealand. marilyn.duxson@stonebow.otago.ac.nz DOI /dvdy Published online 23 September 2010 in Wiley Online Library (wileyonlinelibrary.com). VC 2010 Wiley-Liss, Inc.

2 FATE OF MAMMALIAN MYOTOME 2899 simple segmental myotomal scaffold into the complex forms of the adult axial muscles are almost completely unknown. Other possibilities are that the myotomal fibres might undergo programmed death and the axial muscles form de novo from dermomyotomally derived progenitors, or that the myotomal fibres might give rise directly to some muscle groups with simple segmental morphology (e.g., intercostal muscles hypaxially; unisegmental transversospinal muscles epaxially) while muscles with non-segmental forms are formed de novo by migration of precursors from the dorsomedial lip of the dermomyotome. Our current lack of understanding of the fate of the myotome and of the origin of the adult axial muscles is in stark contrast to our detailed knowledge about early myotome formation (Kalcheim et al., 1999; Venters and Ordahl, 2002; Gros et al., 2004; Hollway and Currie, 2005) and the origins of the migratory hypaxial muscles (reviewed in Buckingham, 2001). Transformation of the embryonic myotome into the complex arrays of epaxial and hypaxial muscles of the trunk is unlikely to occur only through the actions of myogenic cells. Muscular, skeletal, and connective tissues develop in close spatial and temporal association and their morphogenesis must be synchronised in order to form functional musculoskeletal units. Stopak and Harris (1982) have shown that connective tissue fibroblasts in vitro can actively organise extracellular matrix so as to exert traction, resulting in movement of dispersed muscle cells into organised muscle masses with functional relations to the forming bones. Similarly, Kieny and Chevallier (1979) and then Chevallier and Kieny (1982) have used chick-quail grafting experiments to demonstrate that the segregation and morphogenesis of limb muscle masses in vivo are dependent on the somatopleural mesenchyme of the limbs. Overall, the consensus from experiments in the limb is that muscle progenitor cells are not obviously committed to any specific muscle or region (Christ et al., 1977; Kardon et al., 2002; Rees et al., 2003); rather, they are instructed by TCF4- expressing mesenchymal cells to differentiate in a specific area and pattern (Kardon, 1998; Kardon et al., 2003). The somitic compartment that gives rise to the connective tissue of axial muscles is the syndetome (Schweitzer et al., 2001; Brent et al., 2003). The progenitor cells of the syndetome specifically express the transcription factor Scx but not MyoD (specific to muscle progenitor cells) or Pax1 (specific to sclerotomal cells) (Brent et al., 2003). At later times, Scx is expressed in a sub-group of developing connective tissues including tendons and aponeuroses. (Schweitzer et al., 2001). Recent lineage studies suggest that internal muscle connective tissues are also derived from Scx-expressing cells (Pryce and Schweitzer, unpublished data), as expression of the ScxGFP transgenic reporter can be detected here, likely due to persistence of the highly expressed GFP transcript and protein. Early interactions between myotome, syndetome, and sclerotome have been studied and induction of Scx found to be dependent on FGF proteins secreted by the myotome (Brent et al., 2003, 2005). However little is known about the later relationship between the Scx-expressing cells and the myotomal muscle fibres over the period when the myotome transforms into the epaxial musculature. A particular question is whether the Scx-expressing cells are appropriately positioned to play a part in the reorganisation of the muscular tissues in the region. After initial formation of the epaxial muscles, their further growth and differentiation requires a continuing source of myogenic progenitors, both to increase fibre number and for addition of nuclei during fibre enlargement. This raises the question of the origin of these progenitors. Epaxial progenitor cells may already be present in the epaxial muscular anlagen as they segregate to form the different muscle groups, as seems likely from the work of Gros et al. (2004, 2005). However, this has never been formally demonstrated. Alternately, progenitors might migrate from the dorsomedial lip of the dermomyotome to colonise the new epaxial muscle masses. This second system would imply a mechanism similar to that of the migration of limb progenitors. The aim of this study was to understand the late development of the mammalian epaxial myotome and exactly how it gives rise to the definitive epaxial muscles. Here we report that there is virtually no death of myotomal myocytes during this transition. Confocal microscopy on wholemount specimens shows that the differentiated fibres of the epaxial myotome cleave into discrete masses, simultaneously altering their orientation and length, to give rise to the anlagen of the epaxial muscles. During this translocation, Scx-expressing connective tissue cells are tightly associated with the ends and sides of the myotomal myocytes, consistent with an active role in shaping the muscles. As the myotomal fibres cleave into the discrete groups that will form each future epaxial muscle, a pool of Pax3- and Pax7-positive myogenic progenitors intermingles with them, providing the source of their future growth and development. RESULTS Epaxial Myotome Undergoes a Progressive Transformation to Form the Epaxial Muscles To understand the morphogenesis of epaxial myotome into epaxial muscles, the morphology of the region was initially studied in rat embryos at the anterior trunk level (just posterior to the forelimb), at half day intervals over the period from E12.5 to E14.5 and at E15.5. This period spans from about a day after the myotome is first established, through until the segmental myotomes have disappeared at forelimb level (E14.5) and the epaxial musculature is well defined (E15.5) (Deries et al., 2008). Myocytes, myofibres, and surrounding connective tissues were visualised in situ and in three dimensions within whole mount preparations dually immunostained for myosin heavy chain (MHC) and tenascin. Tenascin is an extracellular matrix glycoprotein broadly expressed within developing connective tissues including tendons, ligaments, cartilage, and perichondrium (Riou et al., 1992). Images shown are photomontages of multiple (up to 40) confocal Z-stacks. Interpretation of the whole mount preparations was aided by parallel studies of transverse sections and the two are presented together in Figure 1.

3 2900 DERIES ET AL. Fig. 1. Progressive transformation of the myotome into epaxial muscles. Fluorescent images of rat embryos immunostained for MHC (red) and tenascin (green) at anterior trunk level, on transverse sections (A, C, E, I, L, O) or whole mounts viewed in the parasagittal plane (B, D, F H, J, K, M, N, P R). B and D are flattened confocal Z-stacks, all other parasagittal views are 10-mm optical sections. The dotted lines on some transverse sections (E, I, L, O) indicate the approximate plane of the corresponding sagittal sections. A,B: E12.5. C,D: E13.0. E H: E13.5. Arrows in G indicate shortened myocytes in the dorsal region. H is a slightly more advanced specimen, stained only for myosin; the arrow indicates the beginnings of the first cleavage plane. I K: E14.0. Arrows in I and K indicate the first cleavage plane; arrowheads in K indicate the second, more ventral, cleavage plane; asterisks show where fibres from adjacent segments are beginning to blend together. L N: E14.5. The four muscle masses of the thoracic level are almost segregated. Arrows in N indicate some vestiges of segmentation within iliocostalis muscle. O R: E The four epaxial muscle groups are now well defined. MHC, myosin heavy chain, Dm, dermomyotome; Sc, sclerotome; NT, neural tube; DRG, dorsal root ganglia; Ep, epaxial; Hyp, hypaxial; Ve, vertebra; lc, levatores costarum; ts, transversospinalis; long, longissimus; ilio, iliocostalis; HypM, hypaxial muscle; D, dorsal; V, ventral; M, medial; L, lateral; R, rostral; C, caudal.

4 FATE OF MAMMALIAN MYOTOME 2901 At E12.5 and E13.0, the epaxial myotome presents in its classical segmented form with distinct tenascinstained intersegmental boundaries and arrays of axially-oriented parallel myocytes (Fig. 1A D). In cross section, the dermomyotome is visible as an unstained region superficial to the myotome and surrounded by a tenascin-positive extracellular matrix at E12.5 (Fig. 1A), but begins to disperse in its central region by E13.0 (Fig. 1C). All fibres of the myotome span the whole segment, but at E13.0 tenascin expression begins to expand into the myotome in some regions, blurring the intersegmental boundaries (Fig. 1D). E13.5 marks the onset of transformation of the myotome, especially in the dorsal region (Fig. 1E H). While the deeper part of the myotome still has parallel fibres spanning the whole length of the segment (Fig. 1F), the most dorsal and superficial myocytes no longer extend to the segmental boundary (arrows, Fig. 1G). In the most advanced embryos, these dorsalmost myocytes have tilted upwards at the anterior end of each segment, forming a cleavage plane that begins to separate them from their ventral neighbours (arrow, Fig. 1H). Half a day later, the transformation is more apparent (E14.0, Fig. 1I K). In transverse sections, the dorsal cleavage plane starts to become visible as an indentation of the myotomal mass (arrow, Fig. 1I). The deeper muscle masses remain segmented, still recognisably in the pattern of the myotome, but the tenascin-positive intersegmental zone has expanded to form the nascent ribs (Fig. 1J). In contrast, bundles of fibres lying just superficial to the ribs start to elongate and blend together (asterisks, Fig. 1K), marking initiation of the formation of the long back muscles, iliocostalis and longissimus. In this superficial region, two cleavage planes are now visible. Above the first (arrows, Fig. 1K), the dorsalmost fibres of the myotome have continued to tilt dorsally at their rostral ends, segregating a group of fibres that most likely represent the future transversospinalis muscles. A second cleavage plane (arrowheads, Fig. 1K), ventral to the first one, separates the future iliocostalis from longissimus. Within the next 12 hr, further major changes occur, resulting in the clear separation of the four epaxial muscle groups at thoracic level by E14.5. The most medial mass can now be clearly identified as levatores costarum (Fig. 1L, M); the three other adult muscle groups, transversospinalis, longissimus, and iliocostalis, are visible more laterally (Fig. 1L, N). At this stage, iliocostalis muscle still shows the vestiges of its segmental origin (arrows, Fig 1N). The cleavage and re-orientation of the muscles continues over the next 24 hr, so that by E15.5 the four major epaxial muscle masses have largely attained their distinctive features (Fig. 1O R). No aspect of their morphology now resembles the segmented myotome. Myotomal Transformation Does Not Involve Myocyte Apoptosis Transformation of the myotome might occur either by death and replacement of the embryonic myotomal myocytes, or by their morphogenetic re-shaping. To assess whether programmed cell death of myotomal myocytes occurs, we examined the epaxial region in serial frontal sections of the region just behind the forelimb at E12.5, E13.5, and E14.0, using caspase-3 staining to detect the apoptotic cascade (Wolf et al., 1999), TUNEL to detect DNA breaks, and Hoechst nuclear staining to detect nuclear fragmentation and condensation. Detection of apoptosis was combined with immunohistochemistry to detect either muscle precursors and myoblasts (using a combination of Pax3, Pax7 and MyoD monoclonal antibodies) or differentiated muscle masses (MHC antibodies) (Fig. 2). At all three stages, expression of caspase-3, accompanied by nuclear condensation, was seen regularly in the embryos (arrows and arrowheads in Fig. 2A F), but extremely rarely in myogenic cells. After extensive searching (serial sections of three segments just caudal to the forelimb level, examined for 3 embryos at each age), no more than a single caspase-3 positive myogenic cell was seen in any embryo. Detection using TUNEL gave the same result (Fig. 2G L). When apoptosis was found in the myotomal area, it was of non-muscle cells (arrowheads, Fig. 2K, L). As an internal positive control, a dying population of non-myogenic cells was found consistently with both techniques, just lateral to the E12.5 myotome and close to the base of the forelimb (arrows, Fig. 2A, B, G, H). The lack of apoptosis within the transforming myotome is consistent with our morphological observations (in whole mount preparations) that no population of myotomal myocytes or myofibres was ever seen to show fragmentation, nuclear condensation (in Hoechst staining), or other features suggestive of large-scale cell death. Thus, the bulk of the differentiated muscle cells of the myotome survive through the period of myotome-toepaxial transformation, rather than undergoing morphogenetic cell death, as happens in other regions where dramatic re-shaping of tissues takes place. Appearance of Multinucleated Myocytes Myotomal myocytes are mononucleated cells during their early development, whereas the later epaxial musculature is composed of multinucleated muscle fibres. This raises the possibility that addition of nuclei from a different lineage might be the factor that initiates transformation within the myotomal myocytes. We, therefore, examined the timing of the transition from mononucleation to multinucleation in the muscle cells of the epaxial region, using dual immunostaining for MHC and myogenin on parasagittal sections. At the level of the forelimb, occasional binucleate myotomal cells could be found at E13.0 (arrow, Fig. 3A, enlarged in Fig. 3 B), with these becoming common throughout the myotome by E13.5 (arrow, Fig. 3C, enlarged in Fig. 3D). Thus, the transition to multinucleation just precedes initiation of myotomal transformation at the forelimb level. Scleraxis-Positive Cells Are Tightly Associated With Myotomal Myocytes During Mouse Epaxial Morphogenesis The above results suggest mammalian epaxial muscles develop through

5 2902 DERIES ET AL. Fig. 2. Myogenic cells do not undergo apoptosis in the transforming myotome of the rat. Frontal sections of E12.5 (A,B,G,H), E13.5 (C,D,I,J), and E14.0 (E,F,K,L) rat embryos, taken just caudal to the forelimb and immunostained to mark muscle progenitor cells (Pax3, Pax7, and MyoD; red, A,C,E,G,I,K) or myocytes (MHC, red; B,D,F,H,J,L) combined with caspase-3 (green, A F) or TUNEL (green, G L). Nuclear staining is with Hoechst (blue). A dying population is marked by caspase-3 and TUNEL at E12.5 in the region lateral to the myotome (arrows, A,B,G,H). Neither caspase-3 nor TUNEL mark any myogenic cells but a few non-muscle cells are dying within the muscle masses (arrowheads, A E,G,H,K,L). L, lateral; M, medial; R, rostral; C, caudal; MHC, myosin heavy chain. a progressive transformation of the epaxial myotomes. Such a process would require the translocation of differentiated muscle fibres. It is know that tendon primordia form independently of muscles (Chevallier and Kieny, 1982; Kardon, 1998) and that the connective tissues associated with muscle direct myogenic patterning (Kardon et al., 2003). To begin to understand the mechanisms involved in epaxial transformation, morphogenesis of the developing tendon Fig. 3. Fig. 3. Transition from mono- to multi-nucleation in rat epaxial myocytes. Confocal images showing dual immunohistochemistry for MHC (red) and myogenin (green) on 10-mm parasagittal sections at forelimb level. A,B: At E13.0, most myotomal myocytes remain mononucleated, but an occasional binucleated cell can be located (arrow in A, enlarged in B). C,D: E13.5. Binucleated myocytes are now common throughout the epaxial myotome (arrows in C, the lower arrowed cell is enlarged in D). L, lateral; M, medial; R, rostral; C, caudal; MHC, myosin heavy chain.

6 FATE OF MAMMALIAN MYOTOME 2903 primordia and muscle-associated connective tissue was followed alongside that of the muscle fibres, using scleraxis expression to identify these cells (Schweitzer et al., 2001). After failing in many attempts to reliably detect scleraxis mrna or protein in the rat, we used a transgenic ScxGFP mouse line in which Scx gene function is not affected (Pryce et al., 2007) for these experiments. For brevity, we focussed only on development of the most dorsal group of myotomal fibres, which gives rise to the transversospinalis muscle group. First we established that development of epaxial muscles in the mouse occurs similarly to rat, but with all events occurring about 2 days earlier in gestation. We then studied ScxGFP mice at E11.5, E12.5, and E13.5, at both anterior trunk (just behind forelimb) and hindlimb levels. Because myotomal development at hindlimb level lags about half a day behind the forelimb level, this allowed examination of intermediate developmental stages. As described by Brent et al. (2003), Scx-positive cells were first found in a layer covering the medial and ventral sides of the myotome (Fig. 4A, arrow), and in the intersegmental spaces (Fig. 4B, arrows). (Note that scattered yellow staining in Fig. 4B, and to a lesser extent in Fig. 4D, M, is autofluorescence in red blood cells.) As the dorsalmost myotomal myocytes started to translocate (Fig. 4C E; E11.5 forelimb level and Fig. 4F, G; E12.5 hindlimb level), Scx-positive cells invaded the spaces between the myotomal myocytes (arrows, Fig. 4C, D), and were particularly prominent around the dorsally migrating, anterior ends of the myocytes(fig.4d,e,g,arrowheads)and in regions where cleavage planes were forming (arrows, Fig. 4F, G). Over the subsequent stages, dense clusters of Scx-positive cells formed dorsally, marking the positions of the tendon anlagen associated with the spinous process of each vertebra (asterisks in Fig. 4I), and the fibres of the transversospinal group progressively diverged into uni- and multisegmental portions (illustrated by blue highlighted fibres in Fig. 4I, K, M). This latter divergence was first apparent at E12.5, when the anterior end of all fibres in a segment lay anchored within the dorsal Scx-positive cell cluster, but, caudally, the more ventral fibres began to extend so they blended with those of the next posterior segment (longer highlighted fibre in Fig. 4I). A large number of Scx-positive cells were spread widely within all muscle regions at this stage, so it was not possible to say if specific Scx-positive cell clusters were also associated with the caudal ends of individual elongating myocytes. This elongation and divergence into uni- and multi-segmental bundles within the transversospinalis muscle group consolidated over the next two stages, with the longer ventral fibres eventually attaching together onto a continuous lateral tendon expressing Scx (asterisks, Fig. 4K, M). Pax3- and Pax7-Positive Muscle Progenitors Segregate Along With The Epaxial Muscle Masses The above results show that myotomal fibres form the core of the emerging epaxial muscles, but do not define the source of the muscle progenitors that will allow for their growth. A population expressing Pax3 and Pax7, and believed to represent the pool of myogenic progenitors, has been described in the myotome and in limbs, in both chick (Ben-Yair and Kalcheim, 2005; Gros et al., 2005) and mouse (Kassar-Duchossoy et al., 2005; Relaix et al., 2005). We thus examined the distribution of Pax3- and Pax7-expressing cells in relation to the differentiating epaxial muscle masses, as they segregated from the myotome, in rat embryos. Figure 5 shows the extent of differentiation of the myotome and epaxial muscles in the left column, as assessed by expression of MHC (red) and myogenin (green), while the middle- and right-hand columns show Pax3- and Pax7-positive progenitors (red) in relation to myogenin (green). Before the segregation of the epaxial muscles begins (E12.5, Fig. 5A C), the populations of muscle precursors expressing Pax3 and Pax7 are present throughout the myotome and intermingle relatively homogeneously with the differentiated myotomal myocytes expressing myogenin (arrows, Fig. 5B, C). Within the overlying dermomyotome, their distributions are also similar, except that Pax3 is expressed more strongly within the dorsal lip (Dm, Fig. 4B, C). As the myotome starts to cleave into the epaxial masses (E13.5, 14.0 Fig. 4D I), the distributions of Pax3- and Pax7-positive cells within the muscle masses also begin to diverge. Pax3 is now expressed predominantly at the lateral edge of the muscle masses where almost no differentiated cells are present (arrowheads, Fig 5E, H), while Pax7-positive cells lie throughout the cleaving and differentiating epaxial masses (Fig 5F, I). Finally, Pax3 expression becomes restricted to a small ventral region (arrowhead in Fig. 5K) that is largely MHC negative (compare Fig. 5J and K) and so presumed to be the last region to differentiate, and then disappears by E15.5 (Fig. 5N). This leaves the developing epaxial muscle masses populated by progenitors that express solely Pax7 (Fig. 5O). Pax3 and Pax7 Are Associated With Sequential Waves of Epaxial Muscle Growth Given the different distributions of Pax3- and Pax7-positive muscle precursors within the cleaving epaxial muscle masses, it seems likely they are contributing differently to epaxial development. We, therefore, examined the proliferative and differentiative behaviour of the populations through the main period of cleavage of these muscles, from E13.5 to E14.5. Over this period, both populations are in a highly proliferative state, as measured by their ability to incorporate BrDU (Table 1 and Fig. 6E G, L N). Both populations are also transiting into terminal differentiation, as measured by their co-expression of myogenin (Table 1 and Fig. 6A D, H K), but this occurs with distinctively different spatial distributions. As Pax3-positive cells rapidly reduce in number and became restricted to lateral and ventral regions of the epaxial muscle masses (E13.5 E14.5, Fig. 5E, H, K), Pax3/myogenin co-expression is observed in a modest proportion of nuclei within these areas (Fig. 6B D). In contrast, Pax7 and myogenin co-

7 2904 DERIES ET AL. TABLE 1. Proportion of Pax3- or Pax7-Positive Progenitors Co-Expressing BrDU or Myogenin a Pax3/BrDU (%) Pax3/myog (%) Pax7/BrDU (%) Pax7/myog (%) E , 42 (40) 4.4, 2.3 (3.4) 27, 36 (32) 16, 12 (14) E , 35 (40) 15, 12 (14) 36, 31 (34) 18, 16 (17) E , 47 (43) 18, 10 (14) 32, 38 (35) 24, 19 (22) a Numbers are for individual animals, with the average shown in brackets. expression occurs at a similar frequency (Table 1) but throughout the whole of the epaxial muscle masses from E13.5 onwards. (Fig. 6I K). This suggests that the bulk of new myonuclei in the growing muscles at these stages originates from Pax7-positive precursors, while the small number of cells co-expressing Pax3 and myogenin may be involved in completing an earlier stage of myogenesis at the lateral and ventral edges of the masses. One possibility, consistent with the timing and distribution of the two populations, is that Pax3-positive cells are responsible for the initial establishment of new myocytes during expansion of the epaxial masses, while Pax7-positive cells fuse into the initial myocytes to drive their growth and establish them as multinuclear muscle fibres. DISCUSSION Mammalian Epaxial Myotome: A Scaffold for the Epaxial Muscle Masses Our results suggest that the epaxial musculature of the rat and mouse are formed by a process of transformation of the embryonic epaxial myotome, involving translocation, re-orientation, and elongation of existing myotomal myocytes. We found no morphological or other evidence suggesting cell death within the myotome during this transformation process. All three techniques used to detect apoptosis within the myotomal myocytes gave consistent negative results at every stage examined, and the fine morphology similarly showed no myocyte degeneration or fragmentation. Thus, although apoptosis is known to be a feature of some muscles during morphogenesis (e.g., chick biventer cervicis, McClearn et al., 1995; tadpole tail, Watanabe and Sasaki, 1974; rat rectus abdominus, Lynch, 1984), it does not seem to be a feature of myotomal transformation. The myotomal cells instead survive and contribute to epaxial muscle formation. The rat myotome begins as a segmental series of paraxially-positioned, parallel myocytes. However, at E13.5 the dorsal-most myocytes of each segment begin to shorten, and to tilt upwards at their rostral ends, so that they cleave away from the bulk of the myotome. These changes continue at E14.0, by which time the first cleavage is more marked, and a second, more ventral, cleavage plane has opened up. Simultaneously, the myocytes begin to lengthen, particularly in the more ventral regions, so that the intersegmental boundaries of the original myotome are obliterated. A factor in the lengthening of the myotomal fibres may be their transition from a mononucleated to a multinucleated form, which begins at about E13.0 at forelimb level. By E14.5 and E15.5, four muscle masses, corresponding to the transversospinalis, iliocostalis, longissimus, and levatores costarum are distinguishable, divided by cleavage planes. The more ventral epaxial muscles now span multiple segments, with the extension of the muscle mass duebothtofibreelongationandtothe merging of adjacent segments. Thus, the simple myotomal myocytes become transformed into the complex scaffold of the epaxial musculature. This transformation clearly requires movement of differentiated muscle cells, which is in contrast to what we know of limb muscle formation. In the limbs, a pre-pattern of connective tissue influences the orientation of muscle fibres, which is appropriate for each muscle from the time of their first appearance (Kardon, 1998; Kardon et al., 2002, 2003). However, movement of differentiated fibres occurs in other systems too. For example, intercostal muscles, are known to derive from the parallel myocytes of the hypaxial myotome (Cinnamon et al., 1999) yet the adult intercostal muscles are formed of three layers, the internal, the middle, and the external intercostal muscles, all with different orientations. The hypaxial myotome also forms some of the abdominal muscles (Bardeen, 1900; Rizk and Adieb, 1982; Christ et al., 1983). In this case again, differentiated cells end in very different places and with different orientations to those of the myotome. Finally, the translocation of differentiated muscle fibres has been observed in the head during migration of extraocular muscles to their final destination (Noden and Francis-West, 2006) and in the body during the formation of perineal muscles (Evans et al., 2006). Mechanism for Morphogenesis of Epaxial Muscles The movement of differentiated myotomal myocytes might occur by their active migration over the extracellular matrix or passively, when the structures to which they are anchored migrate or change position. Migrating cells, finding their way across a surface, characteristically exhibit branching lamellipodia and filopodia (Bray, 1992). Indeed, newly differentiated myocytes in the rat limb bud briefly exhibit a complex branched form as they search for their correct orientation (personal observation, M.J. Duxson). At the time when the myotomal fibres are altering their position and orientation, however, they do not show any kind of branching, as seen in our whole mount preparations. For example, as the dorsal-most fibres tilt upwards, the only visible change of morphology is a shortening of the myocytes accompanied by an expansion of the intersegmental territory occupied by Scx-positive cells. These observations suggest that active myocyte migration is unlikely to be involved in the morphogenesis of the epaxial muscles. Similarly, during translocation of muscle fibres in the head and despite the presence of Lbx1,

8 FATE OF MAMMALIAN MYOTOME 2905 Fig. 4. Morphogenesis of transversospinal muscles in ScxGFP mouse embryos. Immunohistochemistry on 20-mm transverse sections for tenascin (blue) and MHC (red) combined with intrinsic GFP signal (green) (A, C, F, H, J, L) and on whole mount specimens for MHC (red) and GFP (green) (B, D, E, G, I, K, M). B is a flattened confocal Z-stack, all other sagittal views are single 10-mm optical sections. Dotted lines in the transverse sections indicate the approximate plane of the accompanying sagittal sections. A,B: E11.5, hindlimb level. Scx-positive cells line the medial and ventral myotome and fill the intersegmental spaces (arrows). C E: E11.5, interlimb level. As the dorsalmost myocytes tilt upwards, Scx-positive cells invade between the myocytes (arrows) and cluster around their ends (arrowheads). F,G: E12.5, hindlimb level. Scx-positive cells remain prominent at the ends of the tilting fibres (arrowhead in G) and also fill the forming cleavage planes (arrows in F, G). H,I: E12.5, interlimb level. Dense clusters of Scx-positive cells lie at the rostral ends of the transversospinal muscle bundles (asterisks), overlying the spinous process. Long and short fibre bundles are apparent (blue highlighted fibres). J,K: E13.5, hindlimb level. The fibre bundles are more clearly demarcated. The long bundles attach to a Scx-positive lateral tendon sheath (asterisks). L,M: E13.5, interlimb level. MHC, myosin heavy chain; Scx, scleraxis; Dm, dermomyotome; Sc, sclerotome; Ep, epaxial; Hyp, hypaxial; Ve, vertebra; ts, transversospinalis; long, longissimus; ilio, iliocostalis; D, dorsal; V, ventral; M, medial; L, lateral; R, rostral; C, caudal. Fig. 6. Fig. 6. Differentiation and proliferation within Pax3 and Pax7 precursor populations. Serial sections of the epaxial muscle mass in an E14 rat embryo, at forelimb level, stained to show Pax 3 or Pax7 alongside markers for proliferation or differentiation. A,H: Low-magnification images showing the overall pattern of staining of the myotome with Pax3 and myogenin (A) or with Pax7 and myogenin (H). B G: Highmagnification panels showing details taken from an area indicated approximately by the box in A showing co-expression of Pax3 and myogenin (B D) or Pax3 and BrDU (E G). I N: High-magnification panels showing details of the boxed area in H, showing coexpression of Pax7 and myogenin (I K) or Pax7 and BrDU (L N). Many cells within both Pax3 and Pax7 populations have incorporated BrDU, indicating they are in an active proliferative state. Both populations also show coexpression of myogenin, a marker for terminal myogenic differentiation. Note that nuclei of two distinct cross-sectional sizes stain for myogenin: large nuclei are presumed to be those of myogenic precursors, and some of these co-stain for Pax3 or Pax7; small nuclei are those of differentiated fibres, and these never co-stain for Pax3 or Pax7.

9 2906 DERIES ET AL. Fig. 5. Association of Pax3- and Pax7-expressing muscle precursors with myotome and emerging epaxial muscle masses. Adjacent 10-mm transverse sections of rat embryos at forelimb level at stages from E12.5 to E15.5 to detect MHC and myogenin (left column), Pax3 and myogenin (middle column), or Pax7 and myogenin (right column). A C: E12.5. Arrows in B and C indicate intermingling of Pax3- or Pax7-positive cells with differentiated cells within the myotome. D F: E13.5. The expression of Pax3 has decreased within the myotome but persists laterally, where few differentiated cells are present (arrowhead in E). Pax7-positive cells are scattered throughout the muscle mass (F). G I: E14.0. Pax3-positive cells now lie more towards the lateral edge of the muscles, where differentiation is less advanced (arrowheads in H), whereas Pax7-positive progenitors are widespread. Arrows indicate forming cleavage planes. J O: E14.5 and E15.5. As segregation of the epaxial muscles continues, Pax3 becomes restricted to the least differentiated, ventral areas (arrowhead in K), while Pax7-positive cells are prominent throughout all muscle masses. MHC, myosin heavy chain; NT, neural tube; DRG, dorsal root ganglia; Dm, dermomyotome; M, medial; L, lateral; R, rostral; C, caudal.

10 FATE OF MAMMALIAN MYOTOME 2907 a migratory marker for precursor muscles in the trunk (Mootoosamy and Dietrich, 2002), the main hypothesis remains that the differentiated cells are moved by their environment, rather than by their own active migration (Noden and Trainor, 2005; Noden and Francis-West, 2006). If the muscle fibres are not actively moving, then what is the motor that drives them? The Scx-positive cells, which will form muscle tendons and connective tissues, are one possible candidate. Our observations show that these cells are tightly associated with the myotomal myocytes, and particularly with their rostral and caudal ends (Fig. 4). As the dorsalmost fibres start to move, separate clusters of Scx-positive cells move in association with them, and, at any one boundary, different clusters move in different directions. These observations are consistent with the connective tissues acting as a motor for the movement of differentiated muscle fibres, although they do not prove that this is the mechanism. Alternatively, the Scx-positive cells may simply form the attachment points to cartilaginous structures such as the vertebral processes, which are enlarging by appositional growth and carrying the myocytes along with them. To further investigate the role of Scx expressing connective tissue cells in muscle morphogenesis, it would be informative to disrupt them. Murchison et al. (2007) recently created an Scx null mutant mouse in which the early tendon progenitors seemed normal, but there was failure of differentiation of many tendons at the time when they normally begin to individuate and condense. Failure of tendon formation was particularly severe for the longer tendons that transmit force across joints, such as at the distal ends of many limb muscles and for the long tendons of the tail. Interestingly, function of the back muscles was particularly badly affected in Scx / animals, but this aspect of the phenotype was not investigated in detail. A tantalizing possibility is that normal morphogenesis of the epaxial muscles may have failed in the absence of maturation of the tendon progenitors, resulting in dysfunctional muscular anatomy. Alternately, Scx itself may not be critical for epaxial muscle morphogenesis, in which case it might be necessary to entirely delete the Scx-expressing cells at critical windows of time, perhaps using an inducible Scx-DTA construct, or utilizing some of the TGFbeta mutants in which Scx-expressing cells initially form, but fail to develop into tendons (Pryce et al., 2009). Epaxial Muscle Progenitor Populations Our results show that myogenic precursors expressing Pax3 and Pax7 are intermingled with the differentiated muscle fibres of the late myotome, as expected from previous reports (Gros et al., 2005; Relaix et al., 2005) and segregate along with each epaxial muscle mass as it cleaves. Both populations are actively proliferating throughout this period, as shown here by their incorporation of BrDU (Fig. 6E G, L N). Interestingly, although the patterns of Pax3 and Pax7 distribution are initially similar within the myotome (see Fig. 5B,C), once transformation towards the epaxial musculature begins, their distribution diverges. Pax3-positive cells steadily decline in frequency within the muscle, despite their proliferative activity, and become progressively restricted to the lateral and ventral edges of the differentiated muscle mass (Fig. 5E, H, K, N), where a proportion of cells co-express myogenin (Fig. 6B D). In contrast, from E13.5 onwards, Pax7-expressing progenitors are found in large numbers throughout the muscle mass, also showing coexpression with myogenin. The different spatial domains and dynamics of the two progenitor populations suggest they may be playing different roles in epaxial muscle development, as in the limb. In limb muscles, Pax3-positive muscle progenitors are associated with the embryonic phase of muscle development, when initial formation of primary myotubes occurs, while Pax7-positive progenitors drive later, foetal stages of muscle growth including secondary myogenesis (Biressi et al., 2007; Hutcheson et al., 2009). Our results in the epaxial muscles of the rat are broadly consistent with this pattern. Pax3-positive precursors are widespread throughout the myotome during its initial formation and growth and until E13.0, but then contract towards the edge of the epaxial muscle masses, where perhaps the last primary myotubes/myocytes are forming, just as the masses start to cleave. Loss of the highly proliferative Pax3- positive population at this stage (E ) seems likely to result from a majority of the daughter cells transiting into expression of Pax7 (Hutcheson et al., 2009). Secondary myogenesis in rat epaxial muscles does not start until E16.5 (E14.5 in the mouse, Auda-Boucher et al., 1997), well after the time when we observe that the Pax3-positive population of progenitors has disappeared. Solely Pax7-positive progenitors must, therefore, be responsible for the secondary phase of myogenesis. There is, however, a crucial intermediate phase from E13.5 until E16.5, before secondary myogenesis commences, when many Pax7-positive nuclei co-express myogenin throughout the epaxial masses, in regions where no Pax3 expression remains. In this lag between primary and secondary myogenesis, few new myotubes form, suggesting that differentiating Pax7 nuclei are most likely to be fusing with existing primary myocytes, contributing to the switch from a mononuclear form to a multinuclear one and perhaps also driving changes in phenotype. Despite the parallels with limb muscle development, the origins and formation of the epaxial muscles remain unique in that at least a proportion of their primary myotubes are built upon the transformed scaffold of the larval myotomal fibres, rather than forming de novo from a migratory population of precursors. EXPERIMENTAL PROCEDURES Collection of Dated Embryos Wistar rat couples were mated overnight, with the morning on which a copulation plug was found considered embryonic day 0.5 (E0.5). At the required stages (between E11.5 and E15.5), pregnant females were deeply anaesthetised by intraperitoneal injection of xylazine (20 mg/kg; Wyeth- Ayerst Veterinary Laboratories, New York, NY) and ketamine (100 mg/ kg;

11 2908 DERIES ET AL. Monarch Pharmaceuticals, Bristol, TN). In some cases, the dams were injected intraperitoneally with bromodeoxyuridine (BrDU, 25 mg/kg) 1 hr before administration of the anaesthetic. Embryos were individually extracted from the uterus, then quickly beheaded and eviscerated before processing as below. All procedures were approved by the University of Otago Committee on the Ethical Use of Animals. Homozygous ScxGFP male mice (Pryce et al., 2007) were crossed to Swiss Webster females to produce ScxGFP-positive embryos at three different stages: E11.5, E12.5, and E13.5. Embryos were fixed in 2% PFA/PB overnightat4 C before processing for whole mount immunohistochemistry or frozen section immunohistochemistry as below. Whole Mount Fluorescent Immunohistochemistry All embryos were processed as described in Deries et al, (2008). In brief, tissues were fixed in 4% buffered paraformaldehyde, washed, dehydrated into methanol, then stored in a 1:4 mixture of DMSO:methanol for 3 weeks to permeabilise tissues. Subsequently, they were washed in methanol, rehydrated, and passaged through a standard immunohistochemistry protocol, using an extended (overnight 2 day, 4 C) incubation in the primary antibodies and overnight incubation (4 C) in the secondary antibodies, with 20% DMSO present in the diluent. Embryos were finally washed extensively, dehydrated in methanol, then cleared and mounted in BA:BB (1:2, benzyl alcohol:benzyl benzoate) on concave well slides. Fluorescent Immunohistochemistry on Frozen Sections Embryos for frozen sectioning were prepared exactly as described in Bajanca et al. (2004). Briefly, embryos were fixed overnight in 0.2% paraformaldehyde, then progressively processed into a sucrose/gelatine mixture, which was then used as the cryoembedding medium. Sections were either 10 or 20 mm in thickness and were fixed on the slides with 0.2% paraformaldehyde in phosphate buffer (PFA/PB) before processing through a standard immunohistochemical protocol. For immunodetection of incorporated BrDU, sections were pre-incubated in 4M hydrochloric acid at 37 C for 10 min to denature DNA, then washed in 0.1M sodium tetraborate to neutralise, before immunostaining. TUNEL Combined With Immunohistochemistry TUNEL (In Situ Cell Death Detection Kit, Fluorescein, Roche Applied Science, Nutley, NJ) was used to detect apoptotic cells. It was combined with fluorescent immunohistochemistry to detect muscle cells using a method adapted from Hurst et al (2006). Embryos prepared for immunohistochemistry were cryosectioned at 8 mm and sections dried then fixed on the slide with 0.2% PFA/PB. After incubation with primary antibodies, the TUNEL reaction was performed according to the manufacturer s protocol. The final step was the secondary antibody incubation. Antibodies Differentiated muscle cells were detected with a mix of mouse monoclonal antibodies to embryonic myosin heavy chain (MHC) (F59; 1:20; provided by F. Stockdale) and pan-mhc (MF20; 1:20; provided by D. Fischman). Nuclei of muscle progenitor cells were detected with mouse monoclonal anti-pax3 (Developmental Studies Hybridoma Bank [DSHB]; 1:100) and anti-pax7 (DSHB; 1:20) antibodies; myoblast nuclei were detected with a mouse monoclonal antibody against MyoD (BD Pharmigen TM ; San Jose, CA, 1:100); and nuclei of differentiated muscle cells with a rabbit polyclonal antibody to myogenin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:200). Connective tissues in rat embryos were detected with a rabbit polyclonal antibody to tenascin (antibody 2873 provided by H Erickson; 1:500); while developing tendons and muscle connective tissue in ScxGFP mice were detected with a rabbit polyclonal anti- GFP (Molecular Probes, Eugene, OR; 1:5,000). Caspase-3 detection used a rabbit polyclonal antibody against active forms of caspase-3 (R&D Systems, Minneapolis, MN; 1:200). BrDu detection used a sheep polyclonal antibody (Abcam, Cambridge, MA, ab1893; 1:50). Primary antibodies were detected with goat anti-mouse, goat anti-rabbit, or goat anti-sheep F(ab 0 )2 fragment secondary antibodies conjugated to Alexa fluor VR 568/ 488/ 633 as appropriate (Molecular Probes; all at 1:2,000). Cell Counting Dual labelling of Pax3 or Pax7 with BrDU or myogenin was assessed in at least 4 sections from each of 2 animals, with sections selected in a random systematic fashion from a serial set through the forelimb level of the myotome. A counting grid was placed over the image of each selected section and all Pax-positive cells falling within every fourth grid square (in the red channel) were assessed for dual staining with myogenin or BrDU in the green channel. A minimum of 145 and a maximum of 385 Pax-positive cells were assessed for each animal and staining protocol. Imaging Specimens (sections and whole mount) were viewed with a Zeiss laser scanning confocal microscope and digital image stacks manipulated and flattened using ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, nih.gov/ij/, ). For whole mount specimens, up to 50 separate Z- stacks were taken in register to cover the whole surface of the specimen. Each stack was then split into individual 10-mm optical sections and sections from the same layer montaged to obtain an image of the whole specimen at each level. Montages were then recombined into stacks to produce the final Z-series that were then manipulated with the ImageJ software. Figures were produced with Adobe Photoshop and Adobe Illustrator (VC Adobe Systems Incorporated, San Jose, CA) software packages. ACKNOWLEDGMENTS The authors acknowledge the support of the Department of Anatomy and Structural Biology in providing a PhD bursary to Marianne Deries, and to

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