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First published online 16 June 2004
doi: 10.1242/dev.01194

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Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis

Annalisa Grimaldi^1,*,, Gianluca Tettamanti^1,*,, Benjamin L. Martin², William Gaffield³, Mary E. Pownall⁴ and Simon M. Hughes^1,

¹ Randall Centre, New Hunt's House, Guy's Campus, King's College London, London SE1 1UL, UK
² Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204 USA
³ Western Regional Research Center, Albany CA 94710 USA
⁴ Department of Biology, University of York, York YO10 5YW, UK

View larger version (82K): [in a new window]   Fig. 1. Monoclonal antibodies distinguish two populations of muscle cells in Xenopus. Transverse cryosections of stage 35 (A-E), stage 48 trunk (F,G) and adult hindlimb muscle (H-J) stained with monoclonal antibodies to all skeletal muscle MyHC isoforms (A), MyHC of the myotomal superficial muscle fibre monolayer (B-D,F,I,J), MyHC of the deep muscle layers (D,F, green) and NADH-TR histochemistry of adjacent serial sections showing fibres with high mitochondrial enzyme content (E,G,H). (A-G) Two antibodies (BA-F8 and EB165) preferentially label the outermost muscle fibre layer (arrowheads, A-D,F). Note that the superficial layer develops oxidative metabolism (arrowheads E,G). (H-J) In adult muscle, oxidative fibres weakly express MyHC immunologically, similar to the superficial muscle layer in the larvae (asterisks), whereas glycolytic fibres show only background staining (dots). (K,L) Whole-mount immunohistochemistry reveals that a subset of all muscle fibres marked by the 12/101 muscle marker (K) express the EB165 epitope (L). Note the EB165 expression at the leading edge of the abdominal fibre layer (arrowhead) and its lack in more dorsal fibres (asterisk) at stage 48. A subset of head fibres express slow (EB165). NT neural tube; not, notochord; e, eye; i, intestine; j, jaw; s, somite.

View larger version (100K): [in a new window]   Fig. 2. Slow muscle fibres mature in a posterior to anterior wave. Embryos at successive developmental stages were serially sectioned and stained for slow (EB165, red) and all (BA-D5, green) fibres. Approximate somite number in each row of sections is indicated on the right, counting from anterior. Thus, temporal development of a somite can be followed left to right. (A,B) Only fast fibres (green) are present in the 10 somites formed at stage 22. (C) Summary scale diagram showing the stages described with anteroposterior extent of expression of MyHC markers highlighted [modified, with permission, from Nieuwkoop and Faber (Nieuwkoop and Faber, 1967)]. (D-F) Fast expression continues anteriorly in stage 28 embryos, but in the most posterior region, the fast marker is less apparent. A few cells reactive with slow muscle marker (red) are detected medially only in the most posterior region of this 20 somite embryo (F, inset, brackets). (G-J) By stage 35, cells in post-anal tail express slow markers in a monolayer of superficial cells (I, arrows), which is not apparent in trunk somites (G,H). In the most posterior somites of these 36-somite embryos, slow markers appear to span the somite (J, brackets). (K-N) Fast fibres fill the somite at stage 48, as occurs earlier, and slow fibres form a monolayer at the dorsal and ventral extremes of the lateral myotome (arrows). Note that several antibodies show weak and variable crossreactivity to epidermis. not, notochord. Scale bars: in J, 75 µm for A,B,D-J; in K, 75 µm for K-N; in insets, 86 µm.

View larger version (59K): [in a new window]   Fig. 5. Cyclopamine blocks early slow muscle formation. Xenopus embryos were de-vitellinized, treated with cyclopamine (100 µg/ml), or ethanol vehicle control, fixed at various stages and stained in whole mount for muscle (12/101) or slow (EB165) fibres. (A) Treatment at stage 22 leads to bent embryos with loss of posterior muscle, and severe loss of slow fibres by stage 36 (arrows). A separate group of ventral fast fibres is visible in posterior somites of cyclopamine-treated embryos (arrowheads). Posterior tissue is formed but fails to make muscle (brackets). Insets show the posterior somites at higher magnification. Note poor chevron formation. Slow muscle is greatly reduced or absent. (B) Embryos allowed to develop to stage 41 showing slow myogenesis in anterior somites of both control and cyclopamine-treated embryos, but continued posterior defects in treated embryo (arrowheads). Slow muscle initiates at dorsal and ventral extremities of the somite (arrows). (C,D) Cyclopamine treatment from stage 12 until stage 48 yields similar results. (C) Tail somites 15-30 of untreated embryos (left panels) are extensive and chevron shaped, with a ventral layer of slow fibres (asterisk), separated from a small group of slow fibres at the dorsomedial lip (arrows). Cyclopamine-treated embryos (right panels) have reduced differentiation, dorsoventral extent and less marked chevron shape. Note the initiation of fast myogenesis at dorsoventral extremity of somites in the absence of slow fibres (arrowheads) and lack of a separate row of dorsal slow fibres in the treated embryo (arrow). Slow myogenesis is reduced and commences more anteriorly than in controls (asterisk). (D) In trunk somites, cyclopamine causes reduction in dorsoventral somite extent (brackets), disorganised ventral body wall fast fibres (dot) and reduced somitic slow fibres (asterisk). Note the absence of a separate group of slow fibres at the dorsomedial lip (arrows).

View larger version (90K): [in a new window]   Fig. 3. Overexpression of sonic hedgehog induces ectopic slow muscle fibres. Control lacZ RNA, with (right panels) or without (left panels) RNA encoding zebrafish shh, was injected into one side of four-cell Xenopus embryos and the animals allowed to develop for 2 days until stage 35. Embryos were fixed, serially sectioned and stained for slow (red, EB165) and all sarcomeric (green, A4.1025) MyHCs to identify muscle fibre populations. Whereas lacZ-injected embryos never showed alteration in superficial slow muscle fibre number or position, either close to ß-galactosidase activity or elsewhere, Shh-injected embryos frequently contained ectopic slow fibres in regions showing overexpression of ß-galactosidase. Somite is outlined on X-gal panels. Despite injected RNA frequently being highest in anterior regions, ectopic slow muscle was detected posteriorly within embryos. This suggested that induction of ectopic slow fibres was more readily achieved in regions that normally contain slow superficial cells at this stage.

View larger version (68K): [in a new window]   Fig. 4. Notochord and Hedgehog signalling are required for normal MRF expression. (A) Notochord was ablated at stage 13 and embryos analysed by in situ hybridisation 2 hours later for XMyf5, XMyoD and actin mRNA. Arrowheads indicate adaxial tissue with high XMyf5 expression that is absent after notochord ablation. (B) Xptc2 expression in stage 28 embryos is ablated by cyclopamine treatment, both in the first wave slow muscle-forming region (arrows) and elsewhere (arrowheads). Solanidine has no effect (inset). (C) Embryos treated with cyclopamine, or ethanol vehicle control, at stage 9 and fixed at stage 28 or 32. XMyf5 in PSM is reduced creating a `gap' in tail expression (arrows). However, dorsal and ventral somite borders retain XMyf5 expression (arrowheads). XMyoD is reduced in tail tip (arrows), but unaffected in somitic stripes anteriorly. The dorsal and ventral somite borders fail to upregulate XMyoD in presence of cyclopamine (white arrows). Note reduced chevron form and dorsoventral extent of anterior XMyoD signal.

View larger version (110K): [in a new window]   Fig. 7. XMyoD and XMyf5 expression distinguish several myogenic cell populations in Xenopus somites. XMyf5 (A,C,D,G-I) and XMyoD (B,E,F,J) mRNA was detected in whole-mount in situ hybridisation of stage 35 embryos. Sections of stained embryos at the approximate positions shown in A and B were mounted without further treatment (H,J) or after immunohistochemical staining for MyHC (C-G) or PCNA (I). (A,B) Whole-mount embryos showing the distinct expression of XMyf5 and XMyoD, with section positions marked. (C-F) Trunk level sections showing that the superficial (dermomyotome, brackets) layer of the somite has distinct morphology, lacks MyHC expression and expresses XMyf5 in dorsomedial (shown magnified in D) and ventrolateral lips, and in rare cells away from the lips (C, arrows). XMyoD, by contrast, is expressed within the superficial myotome (E,F, arrowheads) and in the most dorsal dermomyotomal cells (E, arrow). (G-I) Tail sections showing that XMyf5 transcript is located medially in undifferentiated posterior tailbud (G). The outer layer of mesoderm lacks XMyf5 (brackets). Cells with less signal appear orientated perpendicular to the notochord in slightly more anterior regions and are most obvious at dorsal and ventral somite extremes (H). The nuclei of some of these XMyf5-expressing cells contain PCNA (I). (J) XMyoD expression is primarily superficial within the somite in tail tip (bracket). (K-M) Col1a1 is expressed in trunk regions at stage 25 (K) and more widely at stage 37 (L), and vibratome sections reveal expression in epidermis and more weakly in underlying dermomyotome (M, arrowheads).

View larger version (86K): [in a new window]   Fig. 6. Morphologically and molecularly distinct cell monolayer coats, first, trunk then tail somites. Embryos at stage 22(A), stage 28 (B,C) and stage 35 (D-F) were plastic embedded, transversally sectioned and stained with violet fuchsin. NT, neural tube; Not, notochord; Epi, epidermal bilayer. Yolk droplets appear yellow. A distinct superficial layer of cells covers trunk somites prior to superficial slow fibre differentiation (A,B,D, arrows) and anterior tail somites after slow fibre formation is initiated (E, arrows, compare with Fig. 1D). Insets show superficial layer (arrows) in middle of somite (D) and at dorsomedial lip (E) at stage 35. Note the transient lack of this layer in the nascent posterior somites present at stage 28 (C) and stage 35 (F), when single cells can be observed elongated across the somite (arrowheads). (G,H) Electron micrographs show a distinct dermomyotome (arrows) in somite 8 (G) but only spindly cells in somite 18 (H) above a layer of well-differentiated muscle with basal myofibrils (arrowheads). Pax3 mRNA was detected by whole-mount in situ hybridisation of stage 29-38 embryos (I-K, upper panels) and En1 mRNA marked a subset of medial cells in the superficial somite level with the notochord (L, arrowheads). Serial transverse 100 µm vibratome sections revealed that signal is superficial within the somite (I-K, lower panels, arrowheads) and non-overlapping with 12/101, a marker of differentiated muscle. Dorsal and ventral groups of cells in the tailbud express highly (K, inset, arrows). Expression persists in a complex pattern in all somites, but is consistently stronger in trunk somites anterior to about somite 12 at stage 29 (G) and stage 33/34 (H). Subsequently, Pax3 increases in tail somites (I).

View larger version (39K): [in a new window]   Fig. 8. Model of phases of myogenesis in trunk and tail. (A) Slow muscle is first formed in posterior somites (lower series, tail mode). Hh signalling from ventral midline acts on medial somitic cells to promote XMyf5 expression (blue) and early slow myogenesis. These cells rapidly differentiate, express XMyoD (purple) and move to the superficial somite surface (orange arrows) where they elongate anteroposteriorly to make superficial slow fibres (pink). Simultaneously, most somitic cells differentiate into fast fibres, also elongating anteroposteriorly to form the bulk of somitic muscle (yellow). Undifferentiated cells form a dermomyotome (blue arrows). At later stages, a second population of slow muscle fibres (orange) is generated from dermomyotome, probably at dorsomedial and ventrolateral lips, independent of Hh signalling. In anterior somites (upper series, trunk mode), despite early notochord-dependent XMyf5 expression (red arrow), a block on slow muscle formation prevents appearance of the first wave of slow fibres. Fast fibre formation is abundant, and precocious compared with zebrafish. However, some cells remain undifferentiated to form the superficial dermomyotome. Dorsal and ventral dermomyotomal lips continue to express XMyf5 and XMyoD, reflecting their continued role as myogenic centres. Slow fibre formation is initiated from dermomyotome independently of Hh signalling. Extra fast fibres (green) probably also arise from dermomyotome at all anteroposterior levels. At even later stages Hh signalling is again required for XMyoD expression, somite growth and third wave slow fibre formation (dark red) at dermomyotomal lips throughout the axis. (B) How first wave slow fibre migration accompanied by terminal differentiation of fast fibres can appear like somite rotation.

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