Characterization and functional analysis of the cyclostome Lbx-A genes
In the present study, we first examined the functional importance of lamprey Lbx-A gene during development. Since LjLbx-A sequence reported previously had lacked the 5′ part of the coding region [10], we isolated a full-length cDNA clone of LjLbx-A utilizing the updated genomic data and the gene modeling (Additional file 1: Fig. S2). Newly identified N-terminus of deduced amino acid sequence of LjLbx-A contained a 6-amino acid lady bird domain which is shared with Drosophila lady bird and mammalian Lbx2 genes, but not with mammalian Lbx1 genes, implicating a possible functional correlation of LjLbx-A to Lbx2 genes of jawed vertebrates [24].
We subsequently carried out the detailed analyses of the expression of LjLbx-A. In addition to the expression in HBM (Fig. 1b–d), LjLbx-A was also expressed in the ventral edge of the postotic trunk somites across the anteroposterior axis, in addition to the HBM precursor cells (Fig. 1e). These LjLbx-A-expressing cells in the trunk extend ventrally to form the body wall muscle (stage 29; Additional file 1: Fig. S1j), differentiating much later than the dorsal part of the trunk muscle (Fig. 1f, Additional file 1: Fig. S1g-j). In the later ammcoete larval stage (~ 50 mm body length), LjLbx-A was expressed in a cell layer at the dorsal edge of the myotomes, which would later give rise to the muscles in the dorsal median fin (Additional file 1: Fig. S4) [25].
The broad expression of LjLbx-A in the ventral side of the trunk does not resemble the somitic expression of mammalian Lbx1, which is expressed only in the occipital and limb levels, but not in the flank [4, 26]. To clarify which of the lamprey larval muscles require the function of LjLbx-A, we disrupted the LjLbx-A locus during embryogenesis, using CRISPR/Cas9-mediated gene editing (Fig. 1g-l, Additional file 1: Fig. S5 to S8). After injection of the Cas9 protein and gRNA complementary to LjLbx-A, the dorsal somitic muscles of the larvae appeared unaffected, whereas HBM was lost partly or entirely from the ventral floor of the pharynx (Fig. 1g-l, Additional file 1: Fig. S6a-c). These embryos also lost body wall muscles, suggesting that LjLbx-A is required for the formation of both HBM and body wall muscles (Additional file 1: Fig. S6d-f). We also found that LjLbx-A-depleted embryos lost the expression of LjPax3/7-A in the pharyngeal region, which marks the extending HBM primordium (Additional file 1: Fig. S7) [10, 23], suggesting that the loss of HBM and body wall muscles is attributable to the insufficient extension of precursor cells rather than to hindered muscle differentiation.
We also characterized the Lbx gene of the hagfish Eptatretus burgeri, EbLbx-A (Additional file 1: Fig. S3). In hagfish embryos, EbLbx-A was expressed in the ventral edge of the somites (Fig. 1n, o) [17]. This region has been reported to give rise to the anterior oblique muscles (m. decussatus) and the rectus muscles in the ventrolateral aspect of the pharyngeal wall (m. obl/rect; Fig. 1m) [17]. M. obl/rect have been proposed to characterize the HBM of the hagfish, despite the fact that these muscles are innervated by the occipitospinal nerves that pass ventrally as individual segmental nerves, not by a bundled hypoglossal nerve which is the case in the lamprey HBM (Fig. 1a). The morphology of hagfish m. obl/rect also differs significantly from that of the lamprey HBM; the oblique muscles broadly cover the surface of the ventral body from caudal to the mouth to the cloaca, and the rectus muscles lie longitudinally close to the ventral midline. Nevertheless, m. obl/rect have been reported to differentiate at a later stage of embryogenesis [17], which is also the case in the development of the lamprey HBM and body wall muscles (Additional file 1: Fig. S1). The fact that the expression of EbLbx-A is restricted to the ventral somites supports the HBM nature of the m. obl/rect, suggesting that the Lbx-dependent regulation of differentiation would be conserved in the cyclostomes.
Catshark Lbx1 and Lbx2 are differentially expressed during myogenesis
We chose the elasmobranch cloudy catshark Scyliorhinus torazame as a model to investigate how the wide variety of somite-derived hypaxial muscles, such as those in the limbs, appeared in the vertebrate species diverged early in evolution. It has been reported that, similarly to those of Osteichthyan animals, the Lbx1 gene of shark and skates are expressed in precursor cells in muscles [27, 28]. However, involvement of Lbx2 in myogenesis in these animals had remained unexplored, and no insights had been obtained with respect to the differential expression of Lbx1 and Lbx2, which emerged possibly due to the two rounds of vertebrate whole genome duplication (Additional file 1: Fig. S3) [27, 29]. In this study, we examined the expression of Lbx1 and Lbx2 during embryogenesis of S. torazame, using probes specific to each of the two genes. Unlike in mammals, both Lbx1 and Lbx2 were expressed in the somitic muscle primordia, although in a non-overlapping fashion, as detailed below (Fig. 2).
Expression of catshark Lbx1 commenced at the epithelial VLL of postotic somites and became restricted to the pectoral and pelvic fin levels (Fig. 2a–c). Near the fin buds, these Lbx1-positive cells detached from the VLL in bulk, in a similar configuration to the “muscle bud” described by Goodrich (Fig. 3a) [30, 31], to invade the fin bud, where they differentiated into adductor and abductor muscles of the paired fins (Figs. 2a–d and 3d, g, j). Remarkably, these cell aggregates maintained their initial somitomeric pattern and were positive for ZO-1 (zona occuludens-1) antibody, a marker for tight junctions, suggesting that the dermomyotome persisting in the fin muscle primordia is epithelial in nature (Fig. 3e) [32, 33]. This observation is consistent with the classical view that the chondrichthyan fin muscles form from epithelial muscle primordium [30]. Lbx1 was also expressed in the muscle primordia in median fins (Figs. 2d and 3k, l), similarly to the case in LjLbx-A (Additional file 1: Fig. S4).
Catshark Lbx1 was not expressed in the abdominal muscle primordia (Fig. 3a, g), unlike lamprey/hagfish Lbx-A. Remarkably, in contrast to the Lbx1, catshark Lbx2 continued to be expressed in the VLL throughout the trunk (Figs. 2e–g and 3b). Lbx2-positive cells did not enter the fin buds; Lbx2-positive VLL cells extended ventromedially, passing through the medial aspect of the fin buds (Fig. 3b, f), marking the future abdominal rectus muscle (Fig. 3i). Thus, catshark Lbx2, but not Lbx1, marks the developing abdominal muscle, a feature similar to that of lamprey Lbx-A described above (Fig. 1e).
Catshark HBM exhibits differential expression of Lbx1 and Lbx2 during HBM development
Our observations also suggested the differential functions of Lbx1 and Lbx2 in HBM development in S. torazame (Fig. 4). In early embryos, Lbx2 is expressed in a projection of the cells originating from the anterior somites (Fig. 4a, b, Additional file 1: Fig. S9a). The epithelial VLL of the 3rd and 4th somites released the Lbx2-positive cells ventrally along the posterior edge of the pharynx (Additional file 1: Fig. S9a-c). Later, the Lbx2-positive cells accumulated adjacent to the heart, then further proceeded anteriorly within the body wall ventral to the pharynx (Fig. 4a, b; Additional file 1: Fig. S9d-f). In contrast, the expression of Lbx1 was not consecutive along the posterior circumference of the pharynx. At stage 28, a single patch of Lbx1 expression became evident at the level of the 3rd pharyngeal arch (arrowhead in Figs. 2c and 4c, d, e’). This expression of Lbx1 in the anteriormost part of HBM primordium did not overlap with the Lbx2 expression in the midline, whereas dorsally the leading edges of the bilateral portion of HBM transiently express both Lbx1 and Lbx2 (Fig. 4f’ and f”). During the extension of HBM primordium from the somites, the anteriormost portion of Lbx2-positive domain seems to provide Lbx1-positive precursor cells to give rise to the anterior HBM that fuses in the midline.
In late embryos, rows of Lbx2-positive cells have differentiated into coracoarcualis (CAC) muscle, the posterior paired domain of HBM (Fig. 4g, g’, h) [34]. CAC myofibers consist of 4 segments each of which corresponding to the posterior pharyngeal arches (Fig. 4l, m; pa3-6), a remarkable similarity to the lamprey HBM (Additional file 1: Fig. S1l). Anatomically, CAC originates at the scapulocoracoid cartilage and inserts to the anterior, medially located HBM (coracomandibularis muscle, CMD; Fig. 4i, j), a morphological orientation consistent with that of the amniote rectus cervicus (sternohyoideus) muscle [11, 34].
Lbx1-positive cells, on the other hand, differentiated later than CAC, giving rise to CMD muscle in the midline, located anterior to the CAC muscle (Fig. 4i, j). Unlike the CAC muscle, the CMD muscle was composed of a long single segment of myofibers and inserted to the Meckel’s cartilage (Fig. 4k, l), reminiscent of mammalian geniohyoideus muscle [34].
It is also noteworthy that the entire HBM primordia, including both Lbx1- and Lbx2-positive cell populations, were stained with the ZO-1 antibody (Additional file 1: Fig. S10). This observation suggests that, in the shark, both the HBM precursor cells and the fin muscle primordia are epithelial in nature, and both remain as a coherent aggregate during the process of extension into the distal parts of the body.
Duplicated gnathostome Lbx genes and complexity of skeletal muscles
These results provide a new scheme for the developmental homology of HBM elements and appendicular muscles of the vertebrates (Fig. 5). The catshark CAC muscle (associated with Lbx2 expression) and the lamprey HBM (associated with Lbx-A expression) are both attached with the ventral ends of pharyngeal muscles and do not fuse in the midline (Fig. 5b, c) [11]. Moreover, catshark CAC and lamprey HBM are also similar with respect to the pattern of myofiber segmentation which corresponds to the adjacent pharyngeal arches (Additional file 1: Fig. S1k and l; Fig. 4m; Fig. 5b, c), suggesting their muscle differentiation is under the influence of pharyngeal embryonic components such as cephalic neural crest cells and the cranial mesoderm cells. On the other hand, Lbx1-positive CMD of the shark, which is homologous to the tetrapod geniohyoideus and genioglossus muscles, seems to be a novel muscular component acquired in gnathostomes (Fig. 5c). CMD-equivalent muscles, as well as the appendicular muscles, are lacking in the cyclostomes, whose HBM is entirely bilateral and segmented in accordance with the pharyngeal arches.
Considering expression patterns of Lbx1/Lbx2 genes in the wide variety of Osteichthyans, however, the evolutionary pathway seems more complex. In amphibians, only the Lbx1 gene has been identified in genomic sequences, exhibiting a loss of Lbx2 locus in the amphibian lineage (Additional file 1: Fig. S3) [8, 29]. In Xenopus laevis and direct-developing frog Eleutherodactylus coqui, Lbx1 is expressed in the ventral side of the trunk somites [35, 36]. In Xenopus, these Lbx1-positive somitic cells give rise to the “rectus abdominus” muscle that extends from the ventral edge of the somites and eventually surrounds the abdomen of tadpoles, similarly to the body wall muscles of other vertebrates. Xenopus Lbx1 is also expressed in the limb muscle precursors that appear during the metamorphosis [35]. In zebrafish, both Lbx1b and Lbx2 genes were shown to be involved in hypaxial muscles including pectoral fin muscles [8, 37, 38]. It has been suggested that the primary role of Lbx transcription factors is to control the switch of proliferation/differentiation of the muscle precursor cells [8, 39]. Lbx1 and Lbx2 have been suggested to possess overlapping regulatory functions, as the forced expression of Lbx2 could rescue Lbx1 deficiency [8]. Although only limited information about downstream factors of Lbx is currently available [40], the comparative insights shown here suggest that skeletal muscles of vertebrate clades have deployed different combinations of Lbx1 and Lbx2 to ensure differentiation of complex musculature at variable timing of development.