Novel developmental bases for the evolution of hypobranchial muscles in vertebrates

Abstract

Background: Vertebrates are characterized by possession of hypobranchial muscles (HBMs). Cyclostomes, or modern jawless vertebrates, possess a rudimentary and superficial HBM lateral to the pharynx, whereas the HBM in jawed vertebrates is internalized and anteroposteriorly specified. Precursor cells of the HBM, marked by expression of Lbx1, originate from somites and undergo extensive migration before becoming innervated by the hypoglossal nerve. How the complex form of HBM arose in evolution is relevant to the establishment of the vertebrate body plan, but despite having long been assumed to be similar to that of limb muscles, modification of developmental mechanisms of HBM remains enigmatic.

Results: Here we characterize the expression of Lbx genes in lamprey and hagfish (cyclostomes) and catshark (gnathostome; jawed vertebrates). We show that the expression patterns of the single cyclostome Lbx homologue, Lbx-A, do not resemble the somitic expression of mammalian Lbx1. Disruption of Lbx-A revealed that LjLbx-A is required for the formation of both HBM and body wall muscles, likely due to the insufficient extension of precursor cells rather than to hindered muscle differentiation. Both homologues of Lbx in the catshark were expressed in the somitic muscle primordia, unlike in amniotes. During catshark embryogenesis, Lbx2 is expressed in the caudal HBM as well as in the abdominal rectus muscle, similar to lamprey Lbx-A, whereas Lbx1 marks the rostral HBM and pectoral fin muscle.

Conclusions: We conclude that the vertebrate HBM primarily emerged as a specialized somatic muscle to cover the pharynx, and the anterior internalized HBM of the gnathostomes is likely a novelty added rostral to the cyclostome-like HBM, for which duplication and functionalization of Lbx genes would have been a prerequisite.

Keywords: Development; Evolution; Hagfish; Hypobranchial muscle; Lamprey; Lbx genes; Shark; Skeletal muscle; Vertebrates.

Fig. 1

Lbx-A gene in expression in lamprey and hagfish embryos. a A pre-ammocoete larva doubly stained with phalloidin (green; skeletal muscles) and acetylated tubulin antibody (green; neurons). HBM, hypobranchial muscle; m, mouth; XII, hypoglossal nerve. b–e Expression of LjLbx-A gene in the rostral part of lamprey embryos. Arrowheads indicate the rostral end of hypobranchial muscle (HBM) primordia. Arrows indicate the ventral edge of the extending body wall muscle primordia. b, d Left lateral views. c Ventral view. e Left-ventral view. f Differentiated somitic muscles visualized by myosin heavy chain antibody MF20. g–l Anterior portion of the stage 30 embryos stained with MyHC antibody MF20, viewed from the left (g, i, k) and right (h, j, l) sides. Compared to the embryos injected with Cas9 only (g, h), LjLbx-A-disrupted embryos have thinner and shorter HBM (i–l). m 3D reconstruction of an HE-stained stage 53 hagfish embryo showing location of the hypobranchial-equivalent muscle anlage (dark green; Oisi et al. [17]). Dorsolateral segmented muscles (m. par) are shown by the repeated pattern of light greens. m. obl/rect, anterior oblique muscles and the rectus muscles; m. par, parietal muscles. n, o Expression of EbLbx-A in the trunk of stage 53 hagfish embryo. Arrowheads show the expression at the ventral edge of the somites. Transverse sections at pharyngeal (n) and cloacal (o) levels. no, notochord; nt, neural tube. Scale bars, 200 mm

Fig. 2

Expression of Lbx1 and Lbx2 in S. torazame. Expression of Lbx1 (a–d) and Lbx2 (e–g) detected by whole-mount in situ hybridization. Arrowhead and arrows indicate the patch of Lbx1 expression in CMD and paired fin muscle primordia, respectively. Scale bars, 0.5 mm (a, b, e, f), 1 mm (c, g), and 2 mm (d)

Fig. 3

Shark Lbx1 and Lbx2 are expressed in distinct muscle primordium in the trunk. Transverse sections of S. torazame embryos at a–c stage 27, d–f stage 28, and g–i stage 29. a Expression of Lbx1 detected in the pectoral muscle precursor cells (arrows). b A serial section to a, Lbx2 is expressed in the ventral somite in the body wall. c At the pectoral fin level, Lbx2 is expressed in rectus abdominus muscle primordia (dotted bracket), which is not yet differentiated, as shown by the absence of MyHC staining (c). d, e Lbx1 is expressed in the pectoral fin muscle primordia (arrow) which is also stained with ZO-1 tight junction antibody. g–i At stage 29, Lbx1 is expressed in the pectral fin muscle primordia which are still not differentiated. Expression of Lbx2 is no longer detectable. Muscle differentiation is observed only in the rectus abdominus (dotted bracket) but not yet in the fin (i). j, k At stage 32, muscle differentiation is observed in the appendicular (arrow in j) and median (arrow in k) fins. l At stage 29, Lbx1 is expressed in the muscle precursor cells in the dorsal median fin (arrow). Scale bars, a, d, g 0.1 mm; j, k 0.5 mm; l 0.05 mm

Fig. 4

Anterior part of the shark hypobranchial muscles is marked by the expression of Lbx1. a–d Whole-mount in situ hybridization of S. torazame embryos at stage 27.5. a, b Expression of Lbx2 in ventral (a) and left lateral (b) views. c, d Expression of Lbx1 in ventral (c) and left lateral (d) views. Arrows show the expression in the anterior extremity of HBM primordium. e–g” Transverse sections of a stage 29 embryo at the levels indicated in h (ventral view stained with MyHC antibody). e’–g’ The enlarged views of the areas marked in e–g. f” and g” are the adjacent sections to f’ and g’, respectively, hybridized with different probes. e’ Lbx1 is expressed in the medial HBM precursor cells (arrowhead) located dorsal to the hyoid arch-derived muscles (double arrowheads). f’, f” Lbx1 is expressed in the medial HBM precursor cells, whereas Lbx2 expression is restricted in the bilateral HBM primordium (f”). g’, g” Earliest differentiation of HBM occurs in the cells in which Lbx2 is downregulated. i At stage 29.5, the anterior medial part of HBM differentiates as coracomandibularis (cmd) muscle. j At stage 30, the anterior tip of early m. coracoarcualis (cac, originally Lbx2-positive) extended anteriorly, forming coracohyoideus (chy) muscle connecting cac muscle and basihyal cartilage, overlapping laterally with m. coracomandibularis. Coracomandibularis now forms a single medial bundle of muscle fibers that connects the coracoarcualis muscle and the lower jaw. k Meckel’s cartilage expressing Sox9 (asterisk). l, m The cac muscle has four segments corresponding to the adjacent pharyngeal arches (pa3-6). l Ventral view. m Left ventrolateral view. ht, heart; m, mouth. Scale bars, a–l 0.5 mm; m 0.2 mm

Fig. 5

Evolutionary scheme of vertebrate skeletal muscles with reference to the embryonic expression of Lbx genes. In the hagfish (a), rectus muscle (m. rect) and anterior oblique muscle (m. decussatus) are the hypobranchial muscle counterpart and derived from Lbx-A-positive ventral cells of the somites. In the lamprey (b), Lbx-A is required in the formation of body wall muscles (bwm, light green) and hypobranchial muscle (HBM, dark green). In gnathostomes (c), Lbx1 (blue) and Lbx2 (green) regulate distinct domains of hypobranchial muscles. Lbx1 functions in the tongue (cmd, m. coracomandibularis in the shark) and appendicular muscles (pfm, pectoral fin muscles) both of which are lacking in cyclostomes. Pharyngeal muscles derived from head mesoderm are shown in orange