Muscle development in the shark Scyliorhinus canicula: implications for the evolution of the gnathostome head and paired appendage musculature

Abstract

Background: The origin of jawed vertebrates was marked by profound reconfigurations of the skeleton and muscles of the head and by the acquisition of two sets of paired appendages. Extant cartilaginous fish retained numerous plesiomorphic characters of jawed vertebrates, which include several aspects of their musculature. Therefore, myogenic studies on sharks are essential in yielding clues on the developmental processes involved in the origin of the muscular anatomy.

Results: Here we provide a detailed description of the development of specific muscular units integrating the cephalic and appendicular musculature of the shark model, Scyliorhinus canicula. In addition, we analyze the muscle development across gnathostomes by comparing the developmental onset of muscle groups in distinct taxa. Our data reveal that appendicular myogenesis occurs earlier in the pectoral than in the pelvic appendages. Additionally, the pectoral musculature includes muscles that have their primordial developmental origin in the head. This culminates in a tight muscular connection between the pectoral girdle and the cranium, which founds no parallel in the pelvic fins. Moreover, we identified a lateral to ventral pattern of formation of the cephalic muscles, that has been equally documented in osteichthyans but, in contrast with these gnathostomes, the hyoid muscles develop earlier than mandibular muscle in S. canicula.

Conclusion: Our analyses reveal considerable differences in the formation of the pectoral and pelvic musculatures in S. canicula, reinforcing the idea that head tissues have contributed to the formation of the pectoral appendages in the common ancestor of extant gnathostomes. In addition, temporal differences in the formation of some cranial muscles between chondrichthyans and osteichthyans might support the hypothesis that the similarity between the musculature of the mandibular arch and of the other pharyngeal arches represents a derived feature of jawed vertebrates.

Keywords: Cranial; Cucullaris; Fin; Head; Limb; Muscles; Pectoral; Pelvic; Shark.

Fig. 1

Pectoral fin development in Scyliorhinus canicula. Developmental stages (St.) at the top. a Scan electron microscopy (SEM) showing initial outgrowth of the lateral plate mesoderm (arrowheads), in the ventrolateral region between somites 6 and 16 (S6; S16), which will give rise to the pectoral fins. b-i SEM in upper panels (anterior to top) and Mallory’s trichrome stains of histological transversal sections in lower panels (dorsal to top). Dashed lines in upper panels indicate approximate plane of section shown in lower panels. b Initial pectoral fin buds (Pec) prior to the formation of the apical ectodermal ridge (AER). c Formation of the AER in the most distal ectoderm. d-e Formation of the apical ectodermal fold (AEF) by convergence of dorsal and ventral ectodermal cells at the distal fin tip. Note dermomyotome projections (Myo) starting to enter the pectoral fin buds. f Expansion of the abductor superficialis (Abd) and adductor superficialis (Add) muscles, ventrally and dorsally, respectively. g Pectoral fin with two identifiable domains: a proximal domain in which the endoskeleton elements differentiate (End) and a distal finfold (Ff), filled with mesenchymal cells and still capped with an AEF at this stage. Note: first chondrogenic condensations in the anterior part of the fins marked in blue (arrow) and abductor and adductor muscles in all presumptive End region. h Chondrogenic condensations in the End region, prominent finfold and AEF undetectable. Prominent abductor superficialis and adductor superficialis muscles covering the radials (Rad). i Shoulder girdle (Grd) forms parallel to the pharyngeal arches (Pha) that sustain the gills (Gl). Chondrogenesis of the radials (Rad) is close to completion as is development of the abductor superficialis and adductor superficialis muscles. j-p MyHC antibody stains throughout pectoral fin development. Dorsal views in J, K, L, M and P and ventral views in N and O. Note muscle projection expanding ventrally towards the fin field at stage 27 and entering the fin field by stage 28 (arrows). Adductor muscles are detected earlier (stage 29) than abductor muscles (stage 30). Adductor superficialis muscles are detected with MyHC stain in the entire endoskeleton domain between stages 31 and 32

Fig. 2

Pelvic fin development in Scyliorhinus canicula. Developmental stages (St.) at the top. a Scan electron microscopy (SEM) and Mallory’s trichrome stains of histological transversal sections showing initial pelvic fin outgrowth (Pel), laterally to the prospective cloaca region (Cl). b-f SEMs showing the progression of pelvic fin development (ventral views). Note formation of the apical ectodermal fold (AEF) at stage 26 and separation of a proximal domain in which the endoskeleton differentiates (End) and a distal domain, the finfold (Ff) by stage 32. g-k Muscle development between stages 28 and 33 shown with Mallory’s trichrome stains (g, i, k) and MyHC antibody stain (H,J). g Muscle projections entering the fin territory (arrows). h Abductor muscles observed ventrally (Abd). i Abductor and adductor superficialis (Add) muscles detected ventrally and dorsally, respectively. j Dorsal view showing adductor muscles detected from the proximal part of the fin to the distal border of the endoskeleton domain (End), but not in the finfold (Ff). k Chondrogenesis is close to completion, with the pelvic girdle (Grd) clearly detected proximally and prominent abductor and adductor muscles

Fig. 3

MyHC expression during muscle differentiation between stages 23 and 27 in Scyliorhinus canicula. In all figures anterior (or cranial) is towards the left. a-d left lateral views; e ventral view. a Stage 23; six pharyngeal arches (1–6) and the first three pharyngeal clefts (C1–3, opening of the pouches 1–3) are visible. The first pharyngeal arch is the mandibular arch, the second is the hyoid arch and the following four are branchial arches I-IV. b Stage 24; first cephalic muscle anlagen are indicated by faint staining laterally in the hyoid arch (constrictor hyoideus dorsalis) and the first three branchial arches (constrictores branchiales I-III); the opening of the fourth pharyngeal pouch (C4) is visible; first external gill buds appear. c Stage 26; the pectoral fin bud is clearly developed. d-e Stage 27. d Muscle development is clear in the hyoid arch (constrictor hyoideus dorsalis) and branchial arches I to III (constrictores branchiales I-III). e First muscle staining appears ventrally in the mandibular arch. Scale bar = 1 mm

Fig. 4

MyHC expression during muscle differentiation between stages 28 and 29 in Scyliorhinus canicula. Cranial to the left. a-c Stage 28; a While the dorsal constrictor of the mandibular arch is just now appearing, the dorsal constrictor of the hyoid arch is already diving into the adductor arcus palatini and the constrictor hyoideus dorsalis. b The ventral muscles of the mandibular and hyoid arch are all present, while the more posterior ventral branchial muscles are still not developed. c Ventral view of the pectoral fin with the first fin muscle appearing. d-f Stage 29; d The dorsal constrictor of the mandibular arch divides into the spiracularis and the levator arcus palatini. e Enlarged window to show the faint cucullaris staining. f Ventral branchial and hypobranchial muscles further differentiated. Scale bar = 1 mm

Fig. 5

MyHC expression during muscle differentiation between stages 30 and 31 in Scyliorhinus canicula. a, Cranial to the right; b-e, Cranial to the left. a-b Stage 30; a Spiracularis and levator arcus palatini start to separate, while the dorsal muscles of the hyoid arch are widely separated. The cucullaris extends, but does not attach to branchial arches. b Branchial and hypobranchial muscles further differentiated. c-e Stage 31; c Mandibular and hyoid arch muscles become better distinguishable. d Ventrally the intermandibularis and interhyoideus come into contact and four coracobranchiales are now visible. e In the pectoral fin, the adductor superficialis and abductor superficialis are clearly visible. Scale bar = 1 mm

Fig. 6

MyHC expression during muscle differentiation at stage 32 in Scyliorhinus canicula; cranial to the left. a The constrictores branchiales I-IV completely cover the branchial arches; deep branchial muscles cannot be identified. b The cucullaris muscle stretches dorsally to the branchial arches with dorsally attaching to the most caudal one. c The ventral aspect is almost completely covered by muscles. Deep branchial muscles, such as the coracobranchiales, are no longer visible. d Ventral view of pectoral region. The abductor and the adductor superficialis are expanded. Scale bar = 1 mm