The evolutionary origins and diversity of the neuromuscular system of paired appendages in batoids

Abstract

Appendage patterning and evolution have been active areas of inquiry for the past two centuries. While most work has centred on the skeleton, particularly that of amniotes, the evolutionary origins and molecular underpinnings of the neuromuscular diversity of fish appendages have remained enigmatic. The fundamental pattern of segmentation in amniotes, for example, is that all muscle precursors and spinal nerves enter either the paired appendages or body wall at the same spinal level. The condition in finned vertebrates is not understood. To address this gap in knowledge, we investigated the development of muscles and nerves in unpaired and paired fins of skates and compared them to those of chain catsharks. During skate and shark embryogenesis, cell populations of muscle precursors and associated spinal nerves at the same axial level contribute to both appendages and body wall, perhaps representing an ancestral condition of gnathostome appendicular neuromuscular systems. Remarkably in skates, this neuromuscular bifurcation as well as colinear Hox expression extend posteriorly to pattern a broad paired fin domain. In addition, we identified migratory muscle precursors (MMPs), which are known to develop into paired appendage muscles with Pax3 and Lbx1 gene expression, in the dorsal fins of skates. Our results suggest that muscles of paired fins have evolved via redeployment of the genetic programme of MMPs that were already involved in dorsal fin development. Appendicular neuromuscular systems most likely have emerged as side branches of body wall neuromusculature and have been modified to adapt to distinct aquatic and terrestrial habitats.

Keywords: Hox; fin; migratory muscle precursor; muscle; nerve; skate.

Figure 1.

Musculature development in paired fins of skate. (a–c) Whole-mount in situ hybridization of Pax3 and Lbx1. (a,b) Dorsal and ventral views of the expression patterns of Pax3 in the pectoral fin. Pax3 is expressed in both muscles of the pectoral fin (arrow) and dermomyotome (arrowhead) (a), or the pectoral fin (arrow) and body wall muscles (arrowhead) (b). White arrow points to the umbilical cord (b). (c) Ventral view of Lbx1 expression pattern in the pectoral fin. Lbx1 is expressed only in the pectoral fin (arrow) and not in the body wall muscles (arrowhead). (d–o) Immunostaining of skate embryos by myosin heavy chain antibody at stage 29 (d–i) or stage 30 (j–o) in lateral (d–f, j–l) or ventral (g–i, m–o) view. At stage 29, the constrictor branchialis, cucullaris and other cephalic muscles develop (d,g). Abductor and adductor muscles start to develop in the pectoral (e,h) and pelvic fins (f,i), yet they do not fully develop distally. Note that the abductor and adductor muscles of the pectoral fin and hypaxial muscles of the body wall develop at the same axial level (e,h). At stage 30, cephalic muscles are more developed compared to stage 29. Cucullaris extends dorsal to branchial arches (j). Interhyoideus and coracomandibularis are clearly identified (m). Abductor and adductor muscles in the pectoral fins (k,n) and pelvic fins (l,o) develop towards the distal direction. abd.m., abductor muscles; add.m., adductor muscles; a.m., adductor mandibulae; c.a. + c.b., a complex of coraco arcualis and coracobranchialis; c.h.d., constrictor hyoideus dorsalis; c.m., coracomandibularis; e.m., epaxial muscles; h.m., hypaxial muscles; i.h. + i.m., a complex of interhyoideus and intermandibularis; l.h., levator hyomandibulae. All scale bars are 0.5 mm. (Online version in colour.)

Figure 2.

Developmental pattern of nerves in chondrichthyan paired fins. Immunostaining of nerves in developing embryos of skate (L. erinacea) and shark (S. retifer) with 3A10 antibody. (a–e) Skate embryos at stage 29. (a) Dorsal view shows the innervation patterns for the pectoral and pelvic fins. The brachial plexus consists of Sp nerves. (b) Dorsal (sensory) and ventral (motor) root of spinal nerves for paired appendages. (c) Lateral view of the branchial arch domain. Occipital nerves do not contribute to the brachial plexus; only spinal nerves form the brachial plexus. The hypoglossal nerve and the pectoral fin nerves were observed. (d) Dorsolateral view of the pectoral fin region. Spinal nerves first branch into the pectoral fin laterally (arrowhead) and the body wall muscle ventrally (arrow) (shown in inset). Then, a second branching event occurred and the pectoral fin nerves innervate the dorsal and ventral muscles of the pectoral fin. (e) Dorsolateral view of the pelvic fin region. Inset shows the branching event where the spinal nerves split and innervate the pelvic fin (arrowhead) and body wall (arrow) muscles. (f–g) Skate embryos at stage 31. (f) Dorsal view of the pectoral fin region. Sp nerves directly innervate the posterior pectoral fin without forming the brachial plexus. (g) Ventral view of the pectoral fin region showing the brachial plexus and a second plexus-like structure (*) innervating the pectoral fin. (h) Three-dimensional reconstruction of neuromuscular systems in skate embryos at stage 29. The ventral roots of Sp nerves (green) exit from the ventral neural tube and innervate into dorsal and ventral pectoral fin (orange) as well as body wall muscles. (i,j) Shark embryos at stage 28. (i) Dorsal view of the pectoral region. Inset shows the branching event as the Sp nerves split and innervate the pectoral fin (arrowhead) and the body wall (arrow) muscles. Distally, the pectoral fin nerves branch dorsoventrally. (j) Ventral view of the pectoral fin. The v.b.s. is observed in the body wall. (k) Reconstruction of neuromuscular systems in shark embryos at stage 29. The ventral roots of Sp nerves (green) innervate into dorsal and ventral pectoral fin (red) as well as body wall muscles (yellow). a.b., abductor muscle of the pectoral fin; a.d., adductor muscle of the pectoral fin; b.m., body wall muscle; b.p., brachial plexus; d.b.p., dorsal branch of the pectoral nerve; d.g., dorsal root ganglion; d.r., dorsal root; h.g.n., hypoglossal nerve; v.b., ventral branch of spinal nerves; n.t., neural tube; p.f.n., pectoral fin nerves; sp., spinal nerves; v.b.p., ventral branch of the pectoral nerve; v.b.s., ventral branch of the spinal nerve; v.r., ventral root. All scale bars are 1 mm. (Online version in colour.)

Figure 3.

Expression pattern of Hox genes during skate gastrulation. (a–p) Whole-mount in situ hybridization of Fgf8, Wnt3, Cyp26a1, Cdx2 and Hox groups A and D in L. erinacea at stage 23. Note that Hox genes show colinear expression along the anteroposterior axis. In inset h, the black arrow, the white arrowhead and the black arrowhead point to the anterior limits of the expression in the neural tube, PAM and LPM, respectively. All scale bars are 1 mm. The photos are scaled except for E, G, N and P, which are scaled with each other. (q) Schematic summary of Hox expression patterns in L. erinacea in the neural tube (red), pharyngeal arches (yellow), PAM (green) and LPM (blue). Expression levels of Hox genes are indicated by colour darkness. The darkest bars show the anterior limit of expression of each Hox gene in S. retifer embryos [38], indicating that expression patterns of Hoxa9, a10, a11 and d8 have shifted posteriorly in L. erinacea. The posterior limits of Hox expressions in S. retifer embryos are not available in previous studies. Anterior limits of each gene in the neural tube (NT), LPM and PAM were determined by extending the anterior border of the adjacent somite (Material and methods). (r) Innervation staining of an RA-L. erinacea embryo. Note the overlap of the pectoral and pelvic fins (arrow). (s,t) Innervation staining of L. erinacea embryos cultured with retinoic acid, resulting in narrower pectoral fin size than RA embryos and creating a thoracic region between the pectoral and pelvic fins (bracket) (t). (u,v) Maximum-intensity projections of sections of innervation staining after RA treatment. At the pectoral fin level, spinal nerves exclusively innervate the pectoral fin in embryos treated by retinoic acid (p.f.n.), whereas they exclusively enter the body wall at the newly created thoracic domain (b.w.n). b.w.n., body wall nerve; p.f.n., pectoral fin nerve; v.r., ventral nerve. All scale bars are 1 mm. (Online version in colour.)

Figure 4.

MMP cells in dorsal fins. (a) Pax3 expression in the first and second dorsal fins at stage 29. The expression is confirmed at the base of the dorsal fins (arrows). (b,c) Lbx1 expression in the dorsal fins at stage 29 (b) and 30 (c). Lbx1 is highly expressed in the muscle precursor cells in the first and second dorsal fins at stage 29 and the expression extends in the distal direction at stage 30. (d) Section of the embryo stained by whole-mount in situ hybridization of Lbx1. Arrowheads indicate expression of Lbx1. (e,f) Section staining of a dorsal fin by phalloidin (actin; green) and DAPI (nucleus; blue). MMP cells are observed right under the epidermal tissues. f is a magnified image of e without overlay of DAPI. All scale bars are 1 mm. (Online version in colour.)

Figure 5.

Summary and hypothesis of neuromuscular evolution in appendages. Summary of neuromuscular evolution in appendages. In skates, sharks, zebrafish and lungfish, muscles and nerves branch into the body wall and paired appendages at the same axial level (blue in the tree represents animals that possess the dual contribution of neuromuscular systems). Amniotes lost this branching pattern, and neuromuscular components exclusively contribute to either the body wall or the paired appendages during embryonic development. In skates, the dual contribution of neuromuscular components is extended posteriorly to support undulatory swimming. Note that skate dorsal fins also show Lbx1 expression in their MMP cells. Lbx1 expression in unpaired fins raises the possibility that genetic networks of MMPs may have been deployed from unpaired to paired fins during evolution. Phylogenetic tests of Lbx1 expression in other species are critical to conclude the evolutionary history of Lbx1. (Online version in colour.)