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Dynamic modification of oral innervation during metamorphosis in Branchiostoma belcheri, the oriental lancelet.


Contrary to its similarity to vertebrates in basic body structures, the lancelet (amphioxus) develops a nervous system with many unique features. All vertebrates send somatic motor elements from the central nervous system (CNS) through ventral roots and receive sensory elements through dorsal roots. Their cranial nerves show a very stable innervation pattern across the vertebrate members, with some special exceptions, despite the fact that this taxon exhibits great variation in the head form (see Fig. 3 in Discussion for comparison with the innervation pattern in lancelet head region). On the other hand, it is well known that in lancelets motor neurons supplying myomeres and chordal cells, the latter of which have myofibrils, do not exit the CNS; instead they make synapses with cell processes that extend from muscle or chordal cells at the basement membrane surrounding the CNS (Platt, 1892; Flood, 1966; Yasui et al., 1998). Axons from neurons within the CNS or in the periphery thus pass only through the dorsal roots. In the head region of common lancelets Branchiostoma, the peripheral nerves show a metameric pattern similar to that of the cranial nerves in vertebrates; in addition, they show specific asymmetric innervation patterns (Franz, 1923; Kaji et al., 2001). The velum is a median structure, but it is innervated only by the left fourth and fifth (and sometimes sixth) nerves. In the oral hood, there are two nerve plexuses surrounding its distal margin, the inner and outer oral hood nerve plexuses. These two nerve plexuses look like symmetric structures, but the inner oral hood nerve plexus on the right side receives only the left third and fourth nerves (Franz, 1923; Kaji et al., 2001).

Despite the classical observations on the asymmetric innervation, difficulty in obtaining research materials has, until recently, prevented progress in understanding how the asymmetric pattern is established. The first elucidation on the developing oral nerves was the discovery of a circum-oral nervous system called the oral nerve plexus (referred to here as the oral nerve ring, or ONR) in 8- to 14-day larvae (Lacalli et al., 1999). The mouth opening in common lance-lets Branchiostoma appears first on the left side of the pharyngeal region on the second day after fertilization (Conklin. 1932; Hirakow and Kajita. 1994; Stokes and Holland. 1995). Clusters of monociliate cells called oral spine cells appear along the margin of the mouth, and each cluster develops an oral spine that consists of long cilia tightly uniting together (Willey, 1891; Andersson and Olsson, 1989; Lacalli et al., 1999). By 8 days after fertilization, Florida lancelets Branchiostoma floridae have developed the oral nerve plexus with about a dozen fibers around the mouth opening (Lacalli et al., 1999). The nerve plexus at that stage receives neurites from a few extrinsic neurons, intrinsic neurons located on the nerve plexus, and efferent fibers from the CNS. The intrinsic neurons synapse with oral spine cells and seem to increase in number during the expansion of the mouth. From these observations, it was first recognized that the lancelet larval mouth is probably controlled by its own local neurons and some centrally derived elements (Lacalli et al., 1999).

Application of immunostaining to developing lancelet embryos, larvae just before metamorphosis, and juveniles has demonstrated the dynamic change of innervation pattern in the oral region (Yasui et al., 1998; Kaji et al., 2001). The mouth at the initial stage is supplied by the left second and third nerves (sometimes fourth as well) from the CNS. In accordance with the expansion of the larval mouth and caudal shift as a whole, however, the left second and third nerves are separated from the oral region or the ONR, whereas posterior nerves join the ONR successively. A developmental study also depicted nerve fibers that extend contralaterally and seem to produce the asymmetric innervation in the oral region (Kaji et al., 2001).

The characterization of the peripheral nervous system in lancelets, therefore, suggests a specific innervation pattern in the oral region, though the segmental property seems to be comparable with that of vertebrates. Modern lancelets are believed to retain ancestral body form, a belief that seems to be strengthened by the analysis of the Florida lancelet genome (Putnum et al., 2008). However, considering the deep divergence of the lancelet ancestor from vertebrates, as evidenced by the earliest vertebrate fossils, Myllokummingia and Haikouichthys from the Early Cambrian (Shu et al., 1999, 2003), their body plan is expected to be a mixture of primitive and derived features (Northcutt, 2001). To avoid ambiguity in the character state of the lancelet nervous system, a precise understanding on the developmental process based on critical developmental stages is required.

We have established a long-term mass culture system of laboratory offspring (Yasui et al., 2007), making it possible to examine enough samples during metamorphosis. Furthermore, to observe a higher resolution of nerve networks, the fluorescent immunostaining method was applied and larvae were observed during neural development three-dimension-ally in whole-mount with a confocal laser microscope. In the present study, we show the dynamic modification of nerve networks in the oral region from the mouth opening and characterize precisely how the asymmetric innervation pattern in the oral region is established.

Materials and Methods


Animals in the present study were obtained from two populations of progenies bred in the laboratory (Urata et al., 2007; Yasui et al., 2007). The parental animals were collected from a natural habitat of Branchiostoma belcheri (or B. japonicum: see Zhang et al., 2006) in the Ariake Sea, Kumamoto, Japan. Sampling was carried out after the first observation of metamorphosing individuals in the culturing tank. Since individual variation in later larval growth is great (Yasui et al., 2007), we were able to get suitable stages for 1 to 2 months. We collected more than 20 individuals at each stage before metamorphosis, early and later stages of metamorphosis, and a juvenile stage just after metamorphosis. Early larval stages before and after the opening of the mouth were also collected in the breeding season in 2007. They were fixed with 4% paraformaldehyde in 0.22-mm Millipore filtered seawater (FSW) overnight at 4 [degrees]C. The fixed samples were washed with FSW, dehydrated through a graded methanol series, and stored in absolute methanol at -20 [degrees]C until use.


Immunostaining of the peripheral nervous system was performed as in the previous study (Kaji et al., 2001). In brief, fixed materials were macerated in 2.5 N HC1 for 5 days before the immunostaining procedure. The antibody used was anti-acetylated alpha tubulin monoclonal antibody (T-6793, Sigma). A second antibody labeled with Alexa Fluor 488 (Molecular Probes Invitrogen, Carlsbad, CA) was used to get three-dimensional images using a laser confocal system (CISi, Nikon, Tokyo).

Retrograde labeling

Individuals ranging from those just developing an oral hood but before the appearance of oral cirri on the left side to those developing young oral cirri were fixed with 4% paraformaldehyde in FSW overnight at 4 [degrees]C, and then washed with FSW. Fluorescent dye labeling to the distal margin of the oral hood and developing oral cirri were carried out with Dil (1,l'-dioctadecyl-3, 3, 3', 3'-tetramethyl-lindocarbocyanine perchlorate)-saturated dimethyIformamide solution by using a glass needle. The labeled specimens were stored in the dark for a week in FSW supplemented with several drops of 4% seawater paraformaldehyde and then observed under the laser confocal system.


Oral nerve ring (plexus) and asymmetric innervation to velum

The oral nerve ring (ONR) is formed soon after the opening of the larval mouth around 36 h post-fertilization. The process of ONR formation varied between individuals (n = 48). However, most larvae first form the ventral half of the ONR with two extrinsic neurons, one of which is located near the preoral pit and the other at the ventral surface of the body, and soon newly formed intrinsic neurons extend neurites, making a circular ONR by 48 h post-fertilization (Fig. 1 A, B). Centrally derived nerve fibers join slightly later than the local formation of the ONR. Although several neurons appear around the mouth, some contribute to make the ONR, whereas others extend their axons toward the CNS. The left second nerve has its branch near the mouth opening, which is an axon from a peripherally located neuron but does not connect with the ONR. Neurons located near the preoral pit extend axons toward the oral papilla, suggesting a functional relationship between the latter and control of mouth opening and closing.

The ONR is still found in larvae just prior to metamorphosis, in which the mouth is most expanded, and in contact with the left fourth to tenth nerves from the CNS (Fig. 1C). Although we cannot conclude whether all of the centrally derived nerves send fibers to the ONR, metamerically distributing nerves ventral to the mouth seemingly correspond to the posterior nerves and suggest that at least the eighth to tenth nerves do not supply their fibers to the ONR (Fig. 1C).

During the phase of mouth shrinkage, the ONR follows the mouth but delays its shrinkage (Fig. ID). Behind the shrinking mouth, the ONR remains a hairpin loop. In accordance with the development of the integumental fold above the larval mouth, the distal part of the left fourth nerve changes its route, turning dorsally and then posteriorly and retaining the anastomosis with the ONR (Fig. ID). The posterior nerves such as eighth to tenth are drawn toward the posterior tip of the hairpin loop (Fig. ID). When the secondary series of gill pores (intrinsic right-side gills) becomes heart-shaped owing to growing tongue bars, the mouth is rotated inward at its anterior margin and wholly located between the fifth and sixth nerves. The distal part of the fourth nerve is drawn by the rotating ONR and elongates as the contralateral branch to the velum. The fifth nerve has two or three branches that project to the ONR, which turn in the integumental fold like the fourth (Fig. 1E). The other branches of the fifth nerve, when they exist, do not anastomose with the ONR. The main branch of the sixth nerve seems to anastomose with the ONR; however, it becomes freed from the connection during the rotation of the larval mouth. As a result, only the left fourth and fifth nerves are involved during the transformation from the larval mouth into the velar mouth. When observed dorsally, the branch from the fourth to innerve the velum passes above the Hatschek's pit and enters the velum dorsally on the right side, whereas the fifth nerve innervates it on the left side (Fig. 1G, H).

Although the velar tentacles are well grown soon after the completion of the mouth rotation (now the mouth can be called the velum), fibers from primary sensory neurons on their surface are not observed. The incomplete nerve ring in the velum after metamorphosis suggests that the ONR has been degraded during metamorphosis (Fig. 1I), and the velar nerve ring is formed secondarily from the left fourth and fifth nerves, as well as afferent axons from primary neurons.

Inner oral hood nerve plexus

During the expansion of the larval mouth before the onset of metamorphosis, the left third nerve extends a branch to the ventral margin of the preoral cavity, passing along the anterior limit of the cavity (Kaji et al., 2001). The left third nerve also sends a very thin fiber backward into the integumental fold. The former branch is a precursor of the right half of the inner oral hood nerve plexus (IOHNP), and the latter becomes that of the left half. When the secondary gill row appears, the left fourth nerve issues a thick branch forward, and then a thin fiber extends ventrally at the anterior end of the integument fold and runs along the ventral margin of the preoral cavity along with the fiber from the third nerve (Fig. 1E). This fiber also contributes to the formation of the right IOHNP. The contralateral branches from the left third and fourth nerves that contribute to form the right IOHNP are thus formed via the anterior margin of the oral region when the entire oral apparatus is still asymmetrically located on the left side. The right half of the IOHNP is discernible as such when the mouth has been considerably shrunk but still opens on the left side. In contrast, the left half of the IOHNP delays in formation and is identified as a horizontal branch in the integumental fold (Fig. 1E), which comprises fibers from the third and fourth nerves. This branch was previously termed the prebuccal branch (Kaji et al., 2001).

When left and right oral hoods are located symmetrically, 9 to 10 oral cirri grow from the distal margin of each hood, and the IOHNP is easily identifiable on both sides (Fig. 2A, B). The anterior half of the IOHNP is mainly composed of the left third nerve and the posterior half of the left fourth nerve on both sides. Contrary to the initial stage of the formation, the right half of the IOHNP is delayed in forming compared with the left half, and it is easily identifiable in the third and fourth compositions (compare Fig. 2A and C with B and D). The IOHNP establishes a complete loop connecting both sides with a contralateral branch of the left third nerve anteriorly and communicating fibers posteriorly.


Outer oral hood nerve plexus

The outer oral hood nerve plexus (OOHNP) is first recognized as several fibers from the left seventh and eighth nerves, which spread toward the posterior distal margin of the primordial oral hood (Fig. 2E, E'). When the oral hoods take on a symmetric appearance, the fifth to eighth nerves issue branches passing over the inner oral hood nerve plexus and reaching the intercirral margin of the oral hood. At the distal end of these nerves, these branches extend fibers both anteriorly and posteriorly and form a plexus (Fig. 2A, C). In some of the oral cirri, longitudinal axons extend and anastomose with the newly forming OOHNP (Fig. 2C). Although the initial extension of descending branches from the left seventh and eighth nerves in the posterior region of the primordial oral hood is asymmetric, the nerve network pattern in the OOHNP on both sides appears symmetrical. The formation of the OOHNP progresses forward.


Afferent fibers and outer oral hood nerve plexus

Since the earliest nerve fibers toward the distal margin of the left primordial oral hood (integumental fold) seem to extend prior to the appearance of oral cirri (Fig. 2E, E'), we performed retrograde labeling at the distal margin of the primordial oral hood or growing cirri with DiI to examine whether the initial fibers are efferent or afferent from primary sensory neurons at the periphery (Fig. 2F', G'). At the stage just prior to the budding of cirral primordia on the margin of the primordial oral hood, no fibers were labeled with DiI (Fig. 2F, F'). Fibers were labeled when small protrusions of cirral primordia appeared (Fig. 2G, G'). These observations suggest that the formation of the outer oral nerve hood plexus is initiated by efferent fibers from the CNS.


Re-interpretation of oral nerve ring

In the previous study (Kaji et al., 2001), the oral nerve ring (ONR) in larvae was hypothesized to be the precursor of both the inner oral hood nerve plexus (IOHNP) and the velar nerve ring. In that interpretation, the prebuccal nerve branch extending from the ONR was the important structure that demonstrated the division of the ONR into the IOHNP and the velar nerve ring. The three-dimensional analysis of fluorescent immunostained specimens in the present study has clarified that the prebuccal branch is a composite structure that contains posterior fibers from the left third nerve, anterior fibers from the left fourth nerve, and the branch of the fourth nerve to the ONR. From the former two branches, the IOHNP will develop, and the branch to the ONR is the future contralateral branch to the velar nerve ring.

The precise observation on a sufficient number of metamorphosing specimens in the present study confirms that the larval ONR is a transient structure that appears on the second day after fertilization and is retained until the transformation of the larval mouth into the velar mouth during metamorphosis. The ONR is mainly composed of neurites from peripheral neurons that appear ventral to the preoral pit and around the larval mouth opening (Lacalli et al., 1999). Numerous synapses between neurites and oral spine cells suggest peripheral integrative activities in the rejection response by sensing with the oral spines (Lacalli et al., 1999). Since oral spines are a larval adaptation to prevent harmful intake through a large mouth before oral cirri and velar tentacles develop, the degradation of the ONR coincident with that of the oral spines suggests that the ONR, which has its own neurons, is specific to the larval life.

It would be intriguing to know whether the ONR has a counterpart in any other animals. Ciliate bands developed around the mouth in dipleurula-type larvae were referred to by Lacalli et al, (1999). This idea is interesting because the ONR in lancelets is also a transient structure retained only during larval life. Unlike the ciliate bands in dipleurula larvae, however, the ONR develops later than the persistent CNS and centrally derived peripheral nerves. One of the neurotransmitters in the nerve networks in the ciliate bands, serotonin, is absent in the lancelet oral region (Holland and Holland, 1993). In the present study, we have shown that the epidermis has numerous immunopositive cells that extend their axons toward nearby centrally derived peripheral nerves (see dorsal regions in Fig. 1D, E), suggesting an explanation for why the innervation pattern of peripheral nerves in lancelets is easily changeable (Kaji et al., 2001). The potential to differentiate diffuse neurons or primary sensory neurons in the epidermis, which might be derived from an ancestral diffuse nervous system, suggests that lancelet larvae could develop peripheral nerve plexuses in situ based on the local neurogenic potential.

Segmental pattern in lancelet head region

The oral expansion beyond mesodermal segments in lancelet larvae provides a unique model system to contrast the relationship between peripheral nerve patterning and neural crest cell/mesodermal cell populations in vertebrates (Fig. 3). In vertebrates, the cranial nerves display a branchiomeric pattern that depends in part on the segmental nature of the rhombencephalon and neural crest cells that derive from this region of the brain (Hunt et al., 1991), whereas spinal nerves are in general metamerically arranged with somites. In the postotic pharyngeal region in vertebrates, dorsal somites called epibranchial somites and ventral pharyngeal arches coexist and produce a specific interface between head and trunk (Kuratani, 1997).


The branchial region in vertebrates, including agnathans, is occupied by cephalic neural-crest-derived mesenchymes divided into preotic and postotic cell populations by the otic vesicle. In the preotic region, neural crest cells make the prepattern of trigeminal and facial nerves according to the segmental character of the rhombencephalon (hindbrain), whereas the postotic crest cells have no rhombencephalic restriction but are limited in their migration by the anterior boundary of the trunk region, and thus make the prepattern of glossopharyngeal and vagus nerves (Shigetani et al., 1995; Ferguson and Graham, 2004). The patterning of peripheral nerves in the trunk region is affected by somites. However, those passing through the posterior occipital somites are affected by the caudal most cephalic crest mesenchymal population at the posterior pharyngeal region and thus give rise to the hypoglossal nerve prepattern (Mackenzie et al., 1998). Since this principle seems to be true in many vertebrate species (Kuratani, 1997), it had been expected that some precursor to this pattern might exist in lancelets.

Development of the peripheral nerves in the lancelet oral region is characterized by two specificities. The first is that the ONR seemingly controlling the larval mouth develops locally, and the connection with the CNS occurs slightly later. Although primary gills open almost at the same time, no local nerve network is developed. This may be related to the inhalant function of the mouth as in many other suspension feeders. In vertebrates, however, the mouth in ammocoete larvae, which are also suspension feeders, is exclusively innervated by efferent fibers (maxillomandibular nerve) from the CNS (Kuratani et al., 1998). Although feeding strategy is comparable between lancelets and ammocoetes, there are substantial differences in their patterns of oral innervation.

Recent molecular phylogenetic studies have indicaed that cephalochordates diverged first from the chordate stem (Delsuc et al., 2006; Bourlat et al., 2006; Putnam et al., 2008), but it is believed that they retain ancestral characters more than the other chordate groups (Putnam et al., 2008). On the other hand, molecular phylogenetic analysis within cephalochordates has suggested that the lateral asymmetry of lancelets is a primitive character (Kon et al., 2007)--that is, the cephalochordate lineage has evolved from a laterally asymmetric body to a symmetric body. This phylogenetic estimation of the lateral asymmetry is consistent with the developmental changes of common lancelets, in which the asymmetric body develops prior to metamorphosis and then changes into a symmetric appearance. The oral innervation pattern in lancelets seems to be related to the asymmetric body plan of a possible ancestor, and it is rather difficult to find a prepattern of vertebrate cranial nerves.

The second specificity is related to mesoderm-nerve interrelationships. The body of lancelets is characterized by myomeres that are present from the anterior end to the posterior end, except in the case of asymmetron lancelets, which have a urostyloid process. Each centrally derived peripheral nerve issues between myomeres. The spatial interrelationships between nerves and myomeres in lancelets are thus comparable to those in vertebrates, although alignment of myomeres on one side is out of register by half a block from the other side in lancelets. The larval mouth has been thought to develop in the first mesodermal domain (van Wijhe, 1906; Franz, 1927) and expands up to the region of the eighth myomere, though it does not cross over the myomere domain. If the innervation pattern were controlled as in vertebrates, it could be expected that the mouth would be innervated by the originally positioned nerves, the second and third, and sometimes the fourth, during the expansion. However, centrally derived peripheral nerves to the mouth detach anteriorly and join posteriorly during development (Fig. 4). Finally, the fourth and fifth nerves retain connection with the ONR, which is out of register from the original position. The sixth and seventh nerves seem to anastomose with the ONR but become freed from it.


What are the differences between lancelets and vertebrates in the development of peripheral nerves? One is the absence of neural-crest-cell-like mesenchyme that will play a role in axonal guidance and the lack of branchial musculature in lancelets, though larvae before metamorphosis transiently develop muscle fibers around the primary gills (Bone, 1959). Second is the absence of somatic muscles from the body wall ventrally to the metapleural origin; the somatic muscles extend into the metapleural folds but not into the ventral half of the original body wall. Although all young gills are colocalized with myomeres, the mouth and gill pores develop in the unsegmented ventral wall--that is, ventral to the left oral hood or the metapleural fold. Since, unlike the vertebrate body, the lancelet body has no mesenchymal populations or placodal development in the branchial region (Meulemans and Broner-Fraser, 2007) to mediate interactions between the myomeric dorsal half and branchial ventral half, the branchiomerism does not affect the innervation pattern. All of the centrally derived peripheral nerves in the branchial region on the right side extend toward the metapleural fold and terminate to form an intercommunicating bundle at the base of the fold (not shown; Bone, 1959). On the oral side, in the absence of a motile cell population such as the neural crest cells that play a role in axonal guidance, the ONR, which accompanies the expanding and shrinking larval mouth, is the only structure that is involved in axonal guidance.

In lancelets, we can find somitomeric and branchiomeric patterns similar to those in vertebrates. Despite this similarity, however, the metameric framework guiding peripheral nerve outgrowth is very different in lancelets than in vertebrates. In lancelets, neurite attraction to existing nerve bundles may be the most important factor in the patterning of the peripheral nervous system.


We thank Tokiko Ishii, Makoto Urata, Toshio Mitani, and Nobuo Yamaguchi of Hiroshima University for their help in culturing lancelets in the laboratory.

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(1) Department of Lifestyle Medicine, Biomedical Engineering Center, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan; (2) Department of Neurosurgery, Kochi Medical School, Kochi University, Oko-cho Kohasu, Nankoku, Kochi 783-8505, Japan; (3) Department of Craniofacial Biology, University of Colorado Denver, Denver, Colorado 80010; and (4) Marine Biological Laboratory, Graduate School of Science, Hiroshima University, 2445 Mukaishima-cho, Onomichi, Hiroshima 722-0073, Japan

Received 16 December 2008; accepted 2 June 2009.

* To whom correspondence should be addressed. E-mail:

Abbreviations: CNS, central nervous system; FSW, filtered seawater; IOHNP, inner oral hood nerve plexus; ONR, oral nerve ring; OOHNP, outer oral hood nerve plexus.
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Author:Kaji, Takao; Shimizu, Kelji; Artinger, Kristin Bruk; Yasui, Kinya
Publication:The Biological Bulletin
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Geographic Code:1USA
Date:Oct 1, 2009
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