Development of the EOMs and their innervation
To clarify the disposition and innervation pattern of EOMs in lamprey larvae, we first performed 3D reconstruction of a ca. 100 mm ammocoete larva (Fig. 2a–d). At this stage, six EOMs were already differentiated as distinct muscle primordia attached to the surface of the eye (Fig. 2a). They consisted of four rectus muscles (ar, dr, cr, and vr) and two oblique muscles (ao and co). This muscle organization was the same as that known in the adult lamprey (Fig. 1b, [30–32]). To confirm the muscle identities, we also analyzed the innervation patterns of these EOMs. As reported for adult specimens (Fig. 1b, [30–32]), the oculomotor nerve (III) innervated the ‘ar’, ‘dr’, and ‘ao’ muscles (Fig. 2b), the trochlear nerve (IV) the ‘co’ muscle (Fig. 2c), and the abducens nerve (VI) the ‘vr’ and ‘cr’ muscles (Fig. 2d). Notably, the pathways of the trochlear and abducens nerves partially overlapped those of the trigeminal nerve (V), and the trochlear nerve ramifies into sub-bundles and become fasciculated again near its terminus (Fig. 2c). We confirmed this observation by immunofluorescence analysis using an anti-acetylated tubulin antibody in early larvae, as described below. Furthermore, the attachment site of the ‘ao’ muscle to the cartilaginous orbital wall was relatively more ventral (Fig. 2b) than that in the adult, in which the ‘ao’ muscle crossed over the ‘ar’ muscle (Fig. 1).
For the comparison, we constructed 3D images of a pre-hatching stage (72 mm long) embryonic shark (Scyliorhinus torazame) to represent gnathostomes (Fig. 3a–d). Consistent with the adult anatomy (Fig. 1b) as well as previous descriptions [30–32], the oculomotor nerve (III) innervated the ‘mr’, ‘sr’, ‘ir’, and ‘io’ muscles (Fig. 3b), the trochlear nerve (IV) the ‘so’ muscles (Fig. 3c), and the abducens nerve (VI) the ‘lr’ muscles (Fig. 3d). The ciliary ganglion was observed in the orbit (inset in Fig. 3b1), but no similar ganglion was found in the lamprey (Fig. 2).
To determine whether the disposition of the lamprey EOMs changes during development, we performed a histological analysis by hematoxylin and eosin (HE) staining in stage (st.) 30 prolarvae (Fig. 4), 35 mm (about half a year old, Fig. 5a),100 mm larvae (Fig. 5b), metamorphic (Fig. 5c) and adult lampreys (Fig. 5d). In st.30 prolarvae, we found no muscle fibers suggestive of EOM differentiation, but only mesenchymal cell masses surrounding the eyeball (Fig. 4).
In the 35 mm larvae, EOMs were found as fibrous, distinguishable six-cell clusters (Fig. 5a), identities of which were obvious from their disposition. Rectus muscles were located at the anterior, dorsal, ventral, and caudal parts in the orbit; thus, we named them the anterior, dorsal, ventral, and caudal rectus muscles, respectively. As for two oblique muscles, one primordium originated slightly ventral to ‘ar’, and was directed caudally; it was therefore identified as the ‘ao’ muscle. The other, identified as the ‘co’, originated from the dorsocaudal region in the orbit, was directed ventrally.
In the 100 mm larvae, the EOMs become more clearly compartmentalized and discriminable (Fig. 5b), suggesting the growth of the EOMs of this animal during the larval period, ranging 4–5 years. The topological disposition of the EOMs was the same as that in the 35 mm larvae. In the metamorphic stage, the external part of the EOMs became thinner and wider (Fig. 5c1), suggesting rigid attachment to the eyeball to exert its functional movement. The relatively immature state of larval EOMs may be due to the larval life style of this animal, in which the eyes do not possess image-forming vision [44–47]. Through all of the stages examined, the positions of the EOMs did not show radical changes, and it seemed likely that the EOM morphological pattern is established during the pre-metamorphic stages. However, the little change in the relationship between the ‘ar’ and ‘ao’ muscles was notable. During the larval period, these muscles at first run in parallel to each other (Fig 2a4, Bb), and cross each other in the adults (Figs. 1b and 5d4). This change is likely to occur during metamorphosis.
Developmental mechanism of EOMs and patterning of head mesoderm
To trace further the developmental origin of the lamprey EOMs, immunofluorescence analysis was performed using an anti-tropomyosin antibody in younger lamprey larvae (Fig. 6a–c). We did not detect any EOMs in st. 28 or st. 30 prolarvae, but did detect other muscles, including somatic/branchial muscles; supraocularis, subocularis, elevator labialis ventralis (elv), velocranialis, and constrictor buccalis (Fig. 6a, b, see also [48]).
Next, we traced the developmental origin of EOMs by analyzing more upstream regulatory genes for EOMs. In gnathostomes, the genetic cascade involved in the development of EOMs has already been reported; genes encoding muscle-related factors (MRFs) act as determination and differentiation genes, Pitx2 acts upstream of MRFs in cranial muscle progenitor cells, and Pitx2-null embryos lack EOMs [49]. This cascade is also conserved in sharks, in which Pitx2 and Myf5 (a member of the MRF family) are expressed in developing head mesoderm/cavities [50]. In a st. 26 prolarva, although MrfA (a member of the MRF family) and MA2 (a muscle differentiation marker) were not expressed [41], we detected Pitx2 transcripts in the head mesoderm (Fig. 7a). In contrast, in the 90 mm ammocoete larvae, MrfA and MA2 were expressed in EOM prmordia, while the Pitx2 expression ceased (Fig. 8).
Furthermore, we found that there was distinct genetic regionalization in the dorsal head mesoderm. In gnathostomes, Gsc is expressed in the prechordal plate [51], from which the premandibular mesoderm (pm) is thought to arise (lampreys: [28]; sharks: [36]). Gsc plays a dominant role as an organizer in head formation, including head muscle differentiation [52]. We found that Gsc was expressed in the anteriormost head mesoderm in the st. 26 lamprey prolarvae (Fig. 7b), and expression corresponded to that in the premandibular mesoderm. Simultaneously, TbxA transcripts were detected in the paraxial head mesoderm located anterior to the otic vesicle (Fig. 7c). In sharks, an equivalent expression has been observed in the hyoid cavity [50]. In the mouse, Tbx1 (homolog of the lamprey TbxA) regulates craniofacial myogenesis [53]. Thus, TbxA expression in the lamprey is expected to represent that in the paraxial portion of the hyoid mesoderm (hm). These results suggest that the dorsal head mesoderm of lamprey, characterized by PitxA expression along the anteroposterior axis, is further specified through expression of Gsc and TbxA, i.e., pm: Gsc+, TbxA-; mm: Gsc-, TbxA-; hm: Gsc-, TbxA+ (Fig. 7d).
Developmental lineage of the head mesoderm: origin of the differentiated EOMs
On examination of expression of Pitx, Gsc, and Tbx, three distinct domains were identifiable in the lamprey dorsal head mesoderm, in a pattern similar to those in gnathostomes. Thus, via immunofluorescence analysis using an anti-acetylated tubulin antibody, we examined differentiation of the three lamprey head mesodermal portions into the specific EOM groups innervated by the respective cranial motor nerves as seen in shark head cavities [5]. In the st. 28 prolarva, although the head mesoderm was not differentiated into the EOMs (Fig. 6a), PitxA was expressed in the three components of the dorsal head mesoderm (Fig. 9a). At this stage, the EOM-innervating nerves were already extending their fibers, and the distribution pattern corresponded to each portion of the dorsal head mesoderm: the oculomotor nerve (III) reached the premandibular mesoderm, the trochlear nerve (IV) the mandibular mesoderm, and the abducens nerve (VI) the hyoid mesoderm (Fig. 9b, c). These fibers approached the orbit in the 15 mm larvae (Fig. 9d), and their distribution pattern was maintained through the larval period, by which time the EOMs had already been formed (35 mm; Fig. 9e, see also Fig. 6c). These results indicate that the three components of the dorsal head mesoderm are assigned morphologically to respective nerves in a modern gnathostome pattern, and that nerve innervation is maintained through differentiation into EOMs, supporting differentiation of the specific paraxial head mesodermal portion into specific subsets of EOMs.
In lamprey development, the premandibular mesoderm is derived from the prechordal plate, and the mandibular and hyoid mesoderm are regionalized rostrocaudally from each other by the growth of the first pharyngeal pouch [28]. Based on our results (Fig. 7), each of these subdivisions appears to correspond to a genetically-specified subdivision, as described above. However, there is another possibility that the mesenchymal cells are mixed and then become re-specified. Thus, we performed cell-labeling experiments to determine whether each head mesodermal portion retained its cohesion from its origin or became mixed. First, only DiO was injected into the prechordal plate region in st. 21 embryos (Fig. 10a) and incubated until st. 27. At st. 27, a DiO signal was observed around the eyeball, although the eyeball itself was also labeled as an artifact (Fig. 10b, c). Subsequently, triple dye injections were performed; DiO was injected into the prechordal plate, DiI into the mandibular mesoderm, and DiD into the hyoid mesoderm in st. 21 embryos (Fig. 10d). The mesodermal portions retained their cohesion and did not mix with each other in almost all of the larvae at st. 27 (n = 43/48; no fluorescent signal was detected in the remaining 5 samples), (Fig. 10e). The positions of these mesodermal portions also corresponded to the expression patterns of Gsc and TbxA as described above (Fig. 7d, e). These results indicate that the above noted dorsal head mesoderm is regionally specified early in development as well, with respect to their developmental fates.