Development of Cephalic Neural Crest Cells in Embryos of Lampetra japonica, with Special Reference to the Evolution of the Jaw

Developmental Biology 207, 287–308 (1999) Article ID dbio.1998.9175, available online at http://www.idealibrary.com on

Development of Cephalic Neural Crest Cells in Embryos of Lampetra japonica, with Special Reference to the Evolution of the Jaw Naoto Horigome,* Miyoko Myojin,* Tatsuya Ueki,† Shigeki Hirano,‡ Shinichi Aizawa,§ and Shigeru Kuratani* ,1 *Department of Biology, Okayama University Faculty of Science, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan; †Mukaishima Marine Biological Laboratory, Faculty of Science and Laboratory of Marine Molecular Biology, Graduate School of Science, Hiroshima University, 2445 Mukaishima, Hiroshima 722-0000, Japan; ‡Department of Anatomy, Niigata University School of Medicine, 1 Asahimachi, Niigata 951-8510, Japan; and §Department of Morphogenesis, IMEG, Kumamoto University School of Medicine, 2-1-1 Honjo, Kumamoto 860, Japan

Neural crest cells contribute extensively to vertebrate head morphogenesis and their origin is an important question to address in understanding the evolution of the craniate head. The distribution pattern of cephalic crest cells was examined in embryos of one of the living agnathan vertebrates, Lampetra japonica. The initial appearance of putative crest cells was observed on the dorsal aspect of the neural rod at stage 20.5 and ventral expansion of these cells was first seen at the level of rostral somites. As in gnathostomes, cephalic crest cells migrate beneath the surface ectoderm and form three major cell populations, each being separated at the levels of rhombomeres (r) 3 and r5. The neural crest seems initially to be produced at all neuraxial levels except for the rostral-most area, and cephalic crest cells are secondarily excluded from levels r3 and r5. Such a pattern of crest cell distribution prefigures the morphology of the cranial nerve anlage. The second or middle crest cell population passes medial to the otocyst, implying that the otocyst does not serve as a barrier to separate the crest cell populations. The three cephalic crest cell populations fill the pharyngeal arch ventrally, covering the pharyngeal mesoderm laterally with the rostral-most population covering the premandibular region and mandibular arch. The third cell population is equivalent to the circumpharyngeal crest cells in the chick, and its influx into the pharyngeal region precedes the formation of postotic pharyngeal arches. Focal injection of DiI revealed the existence of an anteroposterior organization in the neural crest at the neurular stage, destined for each pharyngeal region. The crest cells derived from the posterior midbrain that express the LjOtxA gene, the Otx2 cognate, were shown to migrate into the mandibular arch, a pattern which is identical to gnathostome embryos. It was concluded that the head region of the lamprey embryo shares a common set of morphological characters with gnathostome embryos and that the morphological deviation of the mandibular arch between the gnathostomes and the lamprey is not based on the early embryonic patterning. © 1999 Academic Press Key Words: lamprey; evolution; mandibular arch; pharyngeal arches; neural crest; Otx gene.

INTRODUCTION In vertebrate development, neural crest cells play fundamental and extensive roles in organogenesis, with a wide repertoire of cell differentiation. Series of experimental 1 To whom correspondence should be addressed. Fax: 181-86251-7876. E-mail: [email protected].

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studies based on chick embryos have suggested that crest cells of the cephalic and trunk regions behave differently, in a manner which is highly associated with the embryonic environment and crest migration pathways (Le Douarin, 1982). In the head of the chick embryo, crest cells pass along the dorsolateral pathway beneath the surface ectoderm, migrating ventrally to fill the pharyngeal arch and form the pharyngeal ectomesenchyme (reviewed by Noden, 1988). In

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amniote embryos, the skeletogenic property of the crest cells is specific to the head region (see Smith and Hall, 1990, for skeletogenic property of trunk crest in anamniote vertebrates), and these cephalic crest cells give rise to the entire viscerocranium (reviewed by Noden, 1988; and by Kuratani et al., 1997a). The neural crest, therefore, is profoundly associated with the establishment of the vertebrate head itself (Le Douarin, 1982; Gans and Northcutt, 1983; Northcutt and Gans, 1983; Noden, 1984, 1991; Hall and Ho¨rstadius, 1988; Couly et al., 1993). Diversity of cranial morphology in vertebrates is largely due to the functional modification of the pharyngeal arch skeletons including the crest-derived premandibular skeleton (trabecular or ethmoid cartilages). One of the most striking examples can be found in the evolution of the jaw in gnathostomes by modification of an anterior pharyngeal arch skeleton (reviewed by Goodrich, 1930; de Beer, 1937; Romer and Parsons, 1977; Jarvik, 1980; Carroll, 1993; Janvier, 1996). Agnathans do not possess such a structure; they have instead developed the velum, a distinct apparatus for suction and pumping of water. Although it has been suggested that the agnathan velum has evolved into the gnathostome jaws (reviewed by Carroll, 1993; Janvier, 1996), the homology of mandibular arch derivatives remains unsettled (see Holland et al., 1993; and see Kuratani et al., 1997a). Other pharyngeal skeletal elements of vertebrates have also been modified in each animal group depending on their segmental positions. It was in this context that molecular genetics has shed light on the understanding of mechanism of cranial patterning, i.e., a series of homeobox-containing genes, Hox genes, are expressed in a nested fashion along the embryonic pharyngeal arches, thus establishing the cranial Hox code (reviewed by Hunt et al., 1991; McGinnis and Krumlauf, 1992; and by Krumlauf, 1993; Mark et al., 1995). The function of these genes as homeotic selectors has been experimentally shown in the disruption of Hoxa-2 in mouse where the second arch skeleton is transformed into the identity of the first (Rijli et al., 1993; GendronMaguire et al., 1993). The cephalic Hox code is also seen in hindbrain segmentation, where two rhombomeres roughly correspond to a single pharyngeal arch by means of cephalic crest cell populations that connect the two structures. This genetic code seems to be widespread among chordates (Holland et al., 1992; reviewed by Holland and GarciaFernandez, 1996). The embryonic distribution pattern of crest cells thus appears to be the basis of not only cranial nerve development, but also the patterning mechanism of the pharyngeal system and its evolution. To investigate the evolutionary history of the craniate head, it is necessary to observe an animal that may reflect an ancestral state of neural crest contribution to embryogenesis. One suitable animal for such analysis is the lamprey, a member of a sister group of gnathostomes. Within the vertebrates, including fossil forms, lampreys belong to

the group Osteostraci that appears closest to gnathostomes (Forey and Janvier, 1993; reviewed by Janvier, 1993, 1996). The lamprey pharyngeal skeleton has been shown to be of neural crest origin (Langille and Hall, 1988; reviewed by Smith and Hall, 1990) and furthermore the branchial skeletons are morphologically comparable between gnathostomes and lamprey (Mallat, 1984). Still, questions remain as to the homology of cranial skeletal elements between these animals. For example, the identity of the trabecular cartilage is enigmatic, as is the origin of the gnathostome jaw (reviewed by de Beer, 1937; Janvier, 1993; and by Kuratani et al., 1998). One realistic comparison would be to start from identification of the embryonic stages in which similar topographical and cell distribution patterns are established between the two groups. Among osteostracans, Lampetra japonica is easily accessible in the main island of Japan. A large quantity of eggs can be artificially fertilized at one time in the laboratory and embryos are easily maintained. In this species, we have so far found that the neuromeric configuration and early peripheral nerve morphology closely resemble gnathostomes (Kuratani et al., 1997b, 1998). However, rhombomere– crest cell relationships were only briefly described at stage 23, merely in terms of rhombomere development (Kuratani et al., 1998). In later development, melanocyte distribution pattern prefigures the morphology of the peripheral nervous system (Kuratani et al., 1997b). In the present study, observation of crest cell migration and distribution by scanning electron microscopy (SEM) revealed well-known characteristics of the vertebrate crest cell behavior as well as unique features of the lamprey crest cells, both of which would be relevant to the origin of the craniate head. By comparing the results with those studies made in gnathostome embryos, this paper is intended to consider the early evolutionary processes that took place in the evolution of the vertebrate head.

MATERIALS AND METHODS Embryos. Adult male and female lampreys (L. japonica) were collected in a tributary of the Miomote River, Niigata, Japan, during the breeding season (late May through June) from 1995 through 1997. The eggs were artificially fertilized and kept in fresh water at 18°C or in 10% Steinberg’s solution (Steinberg, 1957) below 23°C. Embryos were fixed either with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PFA/PBS) for immunostaining or with 0.01 M phosphate-buffered 2.5% glutaraldehyde for SEM observation. Some embryos were also fixed with Bouin’s fixative for paraffin sectioning. Since embryological development is easily affected by temperature, they were staged morphologically according to the table of Tahara (1988) for L. reissneri, a brook species of L. japonica. For neural crest cell development, however, we often needed more minute stages than those described in Tahara’s time table. To make a precisely timed sequence of crest cell development, we have set intermediate stages between each successive stage as stage 21, 21.5, 22, 22.5, and so forth.

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Immunohistochemistry. For whole-mount immunostaining of the cranial nerves, embryos were prepared as described (Kuratani et al., 1997b) with minor modifications. After fixation with PFA/PBS at 4°C for 1 day, embryos were washed in 0.9% NaCl/distilled water, dehydrated in a graded series of methanol (50, 80, and 100%), and stored at 220°C. The samples to be stained were placed on ice in 2 ml DMSO (dimethyl sulfoxide)/methanol (1:1) until they sank. Five-tenths milliliter of 10% Triton X-100/distilled water was added and the embryos were further incubated for 30 min at room temperature. After washing in TST (Tris–HCl-buffered saline: 20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% Triton X-100), the samples were sequentially blocked with aqueous 1% periodic acid and with 5% nonfat dry milk in TST (TSTM). The embryos were incubated in the primary antibody (monoclonal anti-acetylated tubulin, No. T-6793, Sigma Chemical Co., St. Louis, MO; diluted 1/1000 in spin-clarified TSTM containing 0.1% sodium azide) for 2 to 4 days at room temperature while being gently agitated on a shaking platform. The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZYMED Lab. Inc., San Francisco, CA) diluted 1/200 in TSTM. After final washing in TST, the embryos were preincubated with peroxidase substrate 3,39-diaminobenzidine (DAB, 100 mg/ml) in TS for 1 h and allowed to react in TS with the same concentration of DAB with 0.01% hydrogen peroxide for 20 to 40 min at 0°C. The reaction was stopped and embryos were clarified with 30% glycerol in 0.5% KOH. The stained embryos were transferred to a 60% glycerol/water mixture and mounted on depression slide glasses for observation. Scanning electron microscopy. Glutaraldehyde-fixed specimens were washed in 0.01 M phosphate buffer (PB) and postfixed with 4% osmium tetraoxide. The embryos were rinsed in PB in which they were skinned with a sharpened tungsten needle. They were then dehydrated with a graded series of ethanol and freezedried with t-butyl alcohol. The embryos were placed on an aluminum stub, sputter coated with gold–palladium alloy, and viewed with the scanning electron microscope (JSM-5800, JEOL). DiI labeling of the neural crest. Embryos at stages 19 and 19.5 were employed for the injection, since crest cells have not yet emigrated from the dorsal aspect of the neural rod by these stages. Embryos were injected with 0.05% solution of 1,1-dioctadecyl3,3,39,39-tetramethylindocarbocyanine perchlorate (DiI, Molecular Probes), prepared according to the method by Serbedzija et al. (1992). The embryos were excised from the egg membranes in the full-strength Steinberg’s solution (Steinberg, 1957) and placed in wells made in a solidified agar in a plastic dish. Injections were performed with a fine glass pipet into the neural ridge at various levels along the anteroposterior axis. The embryos were kept alive for 2 days until they reached approximately stage 21 and were fixed in 2% paraformaldehyde and 1.25% glutaraldehyde in PBS. They were then rinsed with PBS, transferred to 30% glycerol/distilled water to obtain transparency, and mounted on a depression slide glass with glycerol. Observation was performed with fluorescence microscope. The neuraxial level of the injection was retrospectively determined at the time of observation, by the labeling site on the neural rod or neural tube of the embryo. For the identification of neural tube regions, our previous description of lamprey neuromere development (Kuratani et al., 1998) was consulted. In situ hybridization. L. japonica embryos were fixed in PFA/ PBS overnight at 4°C, dehydrated, and stored in 80% ethanol/H 2O at 220°C. Specimens were transferred to PBS containing 0.1%

Tween 20 (PBT) and digested by 10 mg/ml proteinase K (Sigma) in PBT for 25 min at RT. They were then acetylated in 0.1 M triethanolamine (pH 8) containing 0.25% acetic anhydride for 5 min at RT, followed by addition of acetic anhydride to a final concentration of 0.5% and incubation for 5 min. The specimens were postfixed with 4% paraformaldehyde in PBT for 20 min at RT, washed five times in PBT, and incubated in a hybridization buffer [50% formamide, 53 sodium sodium citrate (SSC), 13 Denhardt’s solution, 5 mM ethylenediaminetetraacetic acid (EDTA)–Na 2, 0.5 mg/ml yeast RNA, 0.1% Tween 20, 0.1% Chaps, 100 mg/ml heparin sulfate] for at least 2 h at 65°C. They were transferred to a fresh hybridization buffer with 0.1 mg/ml digoxigenin-labeled RNA probe and incubated for at least 16 h at 15°C. After hybridization, the specimens were washed in 50% formamide, 53 SSC, 0.3% Chaps for 10 min at 60°C and the solution was substituted gradually with 23 SSC containing 0.3% Chaps. RNaseA was added at a final concentration of 20 mg/ml and incubated for 10 min at RT. The samples were washed once in 23 SSC containing 0.3% Chaps for 30 min at 60°C, once in PBT containing 0.3% Chaps for 10 min, and once in PBT for 10 min. For immunological detection of digoxigenin epitopes, the embryos were blocked with PBT containing 0.5% blocking reagent (Boehringer-Mannheim) for 60 min before incubation with 1:2000-diluted anti-digoxigenin–AP Fab fragments (Boehringer-Mannheim), which had been absorbed by the lamprey embryonic powder. Incubation was done at 4°C overnight, and specimens were washed five times for 60 min in PBT containing 0.5% blocking reagent at RT. Alkaline phosphatase activity was detected with the BM purple chromogenic substrate (Boehringer-Mannheim). Stained specimens were fixed by PFA/ PBT and cleared in graded series of glycerol/H 2O.

RESULTS The observations in this study primarily rely on scanning electron micrographs of L. japonica embryos whose surface ectoderms were carefully removed manually. Although caution was paid not to remove any other tissues, it cannot be ruled out that some of the migrating crest cells might have fallen out. This is of particular concern for cephalic crest cells that are expected to migrate along the dorsal pathway (beneath the surface ectoderm) as in gnathostomes. We therefore prepared several specimens for each developmental stage, in order to avoid inaccuracy resulting from artifacts associated with individual preparations. By doing so, accidental loss of cells and tissues could be easily distinguished. The results we obtained were consistent and enabled us to observe all the changes in the distribution of putative crest cells as a continuous sequence throughout the developmental stages. We also tested several monoclonal antibodies including HNK-1 that might possibly recognize migrating crest cells, but none showed unambiguous labeling of crest cells; HNK-1 stained surface ectoderm of embryos around stage 21 and cranial nerve ganglia derived from the ectodermal placode and possibly containing crest cells (not shown).

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Early Development of Putative Crest Cells in L. japonica The first sign of neural crest cell formation was observed at stage 20, after completion of neural rod formation along the axis (Fig. 1A). he neurulation of lamprey involves formation of a rod-like neurepithelial structure, the neural rod, which hollows out secondarily to form the central canal (Hatta, 1891). The hollowed neural tube can be seen from stage 22. At stage 20, rather distinct populations of flattened cells appeared on the dorsal aspect of the neural rod (Fig. 1A). These putative deepithelialized crest cells were much larger than the rest of the neurepithelial cells, which were more compact and visible only on their apical surfaces in the SEM image (Fig. 1C). On the dorsolateral aspect of the rostral neural rod, the putative crest cells formed a sheet of cells along the neuraxis with their leading edge forming a ridge (Fig. 1B). By this stage, some cells had already begun migrating ventrally at the levels of somites 1 through 3 (Fig. 1B). These earliest crest cells constituted the caudal-most population of cephalic crest cells to migrate beneath the ectoderm. By stage 20.5, the lateral ridge of the crest cells had extended caudally (Figs. 1D and 1E), such that the morphology strikingly resembled the “dorsale Hirnplatte” described in Petromyzon by von Kupffer (1891). Rostrally, the ridge extended almost up to the anterior tip of the neural rod, possibly including the level of prospective forebrain, although the very rostral end of the neural rod was devoid of crest cells. The anterior limit of the crest-producing region was difficult to determine due to the absence of landmarks in the early neural rod (Fig. 1E). Caudally the crest cell ridge extended to the neuraxial level approximately corresponding to somite 5, whereas no indication of crest cell migration was apparent in the more caudal levels (not shown). In slightly older embryos at the same stage, putative mesencephalic crest cells had already made contact with the mesoderm within the mandibular arch (Fig. 2A). This was the rostral-most crest cell population in the head region.

Stages 21 and 21.5 By stage 21, the last or middle cephalic crest cell population appeared (Fig. 2B); these cells were seen as an independent population at the level dorsal to somite 0. By stage 21.5, this population had grown ventrally medial to the otocyst, with its leading edge arriving at the indentation on the cephalic mesoderm, i.e., between the hyoid mesoderm and somite 0 (Fig. 2C). At this point, the cells changed their direction of migration slightly anteriorly on the way to the hyoid arch. The acusticofacial nerve ganglion develops in later development at this position (Kuratani et al., 1997). Its destination, developmental fate, and relative position indicated that this crest cell population corresponded to the preotic crest cells of gnathostomes. Thus, three distinct cephalic crest cell populations had appeared (Fig. A). From the distribution pattern of each crest cell population, they were provisionally termed, from rostral to caudal, premandibular/mandibular crest, hyoid crest (HC), and branchial crest (BC) cells, respectively. The premandibular/ mandibular crest could also be named trigeminal crest (TC) since distribution of these cells corresponded to the future trigeminal nerve innervation area (the premandibular region innervated by the first branch). The rostral-most neural rod was devoid of TC cells (Fig. 3A; also see Fig. 1E), suggesting that this part of the neurectoderm does not give rise to crest cells, a pattern which resembles that in the Xenopus embryo (Sadaghiani and Thiebaud, 1987). Although the rostral neural rod was now regionally differentiated, showing signs of the earliest development of some neuromeric bulges, rhombomeres were not yet clear (Fig. 2C). Compared to stage 22 embryos, it was found that the HC cells were specifically associated with the lateral aspect of rhombomere 4 (r4) (Figs. 2C and 3A, also see Figs. 2D to 2F). Crest cells were excluded from the lateral aspects of future r3 and 5, but were still contiguous along the dorsal midline (Figs. 2C and 3A). Thus, these three crest cell populations were ventrally separated from each other by a couple of crest-free regions anterior and posterior to the HC cells. Comparison with acetylated tubulin-immunostained embryos (not shown) revealed that TC cells covered the rostral

FIG. 1. Early development of crest cells in embryos of L. japonica. The surface ectoderm was removed from specimens before observation. (A) Stage 20 embryo seen from the dorsal view. Mesodermal segmentation is clearly visible on both sides of the neural rod. Note the large flattened cells (arrows) that are developing along the dorsal aspect of the neural rod. (B) Another stage 20 embryo viewed from the left lateral side. The earliest emigrating crest cells are seen at levels of somites 1 through 3 (arrowheads), which correspond to branchial crest cells at later stages. Arrows indicate the lateral ridge of crest cells. (C) Stage 20 embryo, dorsal view of the rostral neural rod. Dots indicate the portion which has been broken to show the inside of the neural rod. Note the difference in size and shape of neural crest cells (ncc) and neurepithelial cells (ne). (D) Stage 20.5 embryo, dorsal view. Dots indicate the lateral ridge of crest cells (ncc). (E) Stage 20.5 embryo seen from the dorsolateral view of the right side of the embryo. The first pharyngeal pouch (pp1) is protruding between the first two mesodermal segments. Putative crest cells (ncc) are adhering on the dorsal to lateral aspect of the neural rod, forming a ridge laterally as indicated by arrowheads. hm, hyoid mesoderm; lm, lateral mesoderm; mm, mandibular mesoderm; ncc, neural crest cells; ne, neurectodermal cells; pp1, pharyngeal pouch 1; s0 –s4, somites. Bars, 100 mm (A, B, C, E) and 50 mm (D).

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FIG. 2. Establishment of cephalic crest cell populations in L. japonica. In all the specimens, the surface ectoderm and the otic pit were removed before observation. (A) Stage 20.5 embryo seen from the lateral view. Putative crest cells from rostral brain region (TC, arrowhead) are beginning to cover the mandibular mesoderm (mm). (B) Stage 21 embryo. Note that another crest cell population is arising on the lateral aspect of the hindbrain (arrows) at the level of somite 0 (s0). This population corresponds to future hyoid crest cells. (C) Enlarged lateral view Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.

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hindbrain, midbrain, and prospective posterior forebrain, though it was not determined in the present study whether they had originated in the forebrain or had migrated from the more posterior neural rod (Fig. 3A). The mandibular arch mesoderm was covered laterally by a ventral portion of the same crest cell population. Some of the TC cells were observed to migrate between the neural rod and the mandibular mesoderm, which will be described elsewhere. The influx of crest cells into the mandibular arch was observed at stages 21 through 21.5, during which period the leading edge of the crest cell subpopulation expanded over the lateral surface of the mesoderm (Fig. 4). These invading crest cells were scattered, but mostly resembled mesodermal cells (Fig. 4C). Toward completion of the influx, it became easier to distinguish the two types of mesenchymes by cell shapes; crest cells were often elongated dorsoventrally, probably reflecting their migratory activity, while the underlying mesodermal cells remained spherical (Fig. 4D). The BC cells appeared to have been derived from the posterior or postotic hindbrain as well as the anterior spinal cord corresponding to the level of somite 0 to somite 4 or 5 as judged from the SEM microphotograph (Fig. 3A). They corresponded to the earliest migrating crest cells observed at stage 20 (Fig. 1B) and were now located rather proximally on the lateral aspect of somites, being more sparse than the other two cell populations.

Stage 22 and Older Embryos By stage 22 when protrusion of the eye vesicle had become conspicuous, the leading edge of HC cells extended rostrally to reach the dorsal part of the hyoid arch and had changed the migratory direction ventrally to fill the hyoid arch (Fig. 2D). This was close to the stage when rhombomeric boundaries appeared very conspicuous (Fig. 2E). Rhombomeres 3 and 5 were totally devoid of crest cells and three cephalic crest cell populations were now completely separated from each other (Fig. 2D). Simultaneously, the adhesion of HC cells onto r4 had become focal, prefiguring the acusticofacial nerve root (Fig. 2D). Though TC and BC cells were not associated with single rhombomeres, they

had limits of hindbrain association at r2/3 and r5/6 boundaries, respectively (Fig. 2F). Such a crest cell distribution pattern corresponded to the cranial nerve morphology as observed by acetylated tubulin immunostaining at stage 24 (Fig. 3B; Kuratani et al., 1997b); each cranial nerve root basically developed on even-numbered rhombomeres. By stage 22.5 when pharyngeal pouch 2 had completely penetrated the mesodermal sheet and the pharyngeal mesoderm was separated ventrally (Figs. 5B and 5C), the HC cells had almost colonized the entire hyoid arch (Fig. 5B). Thus, both the mandibular and hyoid arches received crest cells after completion of pharyngeal arch formation. Crest cell emigration was still apparent on the dorsal aspect of the neural tube at the level of somite 2 and posterior (Fig. 5A); no such cells were found more rostrally. In the postotic region, dorsoventrally elongated cells were seen between somites (Fig. 5B). These intersomitic cells appeared to correspond to the above-noted late-emigrating crest cells (Fig. 5A). Ventrally, the cells that had migrated earlier proceeded ventrally to the future branchial arch region that was yet unsegmented (Fig. 5B). Therefore, BC cells fill the ventral body wall before the formation of postotic branchial arches. At stages 23 and 24, the postotic pharyngeal pouches were not seen to penetrate the branchial wall yet, but the BC cells had completely filled the branchial wall, forming a continuous mesenchymal sheet (Figs. 6A and 6B). Elongated superficial crest cells were still found in the intersomitic space or on the lateral aspect of somites (Fig. 6).

DiI Labeling of the Neural Crest in the L. japonica Embryo To detect the migratory behavior and neural crest origin of the pharyngeal ectomesenchyme, focal injection of a lipophilic dye, DiI, was performed (Figs. 7 and 8). Due to the small size and absence of positional landmarks on the neurectoderm, it was not possible to make a clear mapping of crest cell origins along the neuraxis at stage 19 to 19.5 (Fig. 7A). By observation at stage 21 or later (Fig. 8), however, we could roughly map retrospectively the origin of crest cells along the neuraxis and the destination of the

of the stage 21.5 embryo shown in Fig. 3A. Three putative crest cell populations (TC, HC, and BC) are established in the head region. Hyoid crest cells (HC) reach to the dorsal edge of the head mesoderm. Crest cells are excluded from the lateral aspects of r3 and r5, but they are contiguous longitudinally along the dorsal midline of the hindbrain neural rod. (D) Stage 22 embryo. HC cells are migrating into the hyoid arch (arrow). This cell population is now completely isolated from the other two crest cell populations. This is the stage when the earliest optic vesicle (dots) appears. (E) A stage 22 embryo from which most of the HC cells had been removed during preparation. Anterior is to the left. Note that adhesion of HC cells is restricted on r4. Also note the development of clear borders of rhombomeres on the neural tube (arrows). (F) Enlarged lateral view of another stage 22 embryo, anterior to the left. Crest cells are excluded from dorsal aspects of r3 and r5 and the three crest cell populations (TC, HC, and BC) are now completely separated from each other. Rostral limits of BC cells are shown by arrowheads. Arrows indicate positions of rhombomeric boundaries. BC, branchial crest cells; HC, hyoid crest cells; hm, hyoid mesoderm; hy, hyoid arch; TC, trigeminal crest cells; mm, mandibular mesoderm; pp1, pharyngeal pouch 1; pp2, pharyngeal pouch 2; s0 –s2, somites. Bars, 100 mm (A, B, D, E) and 50 mm (C, F).

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FIG. 4. Development of the trigeminal crest cells. Stage 21 (A, C) and 21.5 (B, D) embryos. C and D are enlargements of boxes in A and B, respectively. TC cells are colored in C and D. At stage 21, only the dorsal portion of the mandibular mesoderm (mm) is covered by spherical crest cells. By stage 21.5, most of the mandibular mesoderm is covered by dorsolaterally elongated cells except for the ventral tip. Note growth of the first pharyngeal pouch. Bars, 100 mm (A, B) and 50 mm (C, D).

FIG. 3. Cephalic crest cell distribution, rhombomeres, and cranial nerves. (A) Stage 21.5 embryo seen from the right lateral view. The same embryo as shown in Fig. 2C. The surface ectoderm has been removed together with the otic pit [ventral border of the otic pit is seen as a mesodermal depression indicated by dots below the labeling (ot)]. Putative crest cells are colored green, mesoderm yellow, and the endoderm salmon pink. There are three major populations of putative crest cells at this stage, termed trigeminal crest cells (TC), hyoid crest cells (HC), and branchial crest cells (BC). The TC cells fill the mandibular arch as well as the more rostral region of the head. The dots in the mandibular arch represent the boundary of the leading edge of immigrating crest cells which have fallen out during preparation within this arch. The second pharyngeal pouch is penetrating the mesoderm (arrow, pp2). (B) Rhombomeres and cranial nerve roots at stage 24. The embryo has been immunochemically stained with anti-acetylated tubulin antibody. The two photos are focused at different depths, showing rhombomeres (top) and cranial nerve roots (below). Arrowheads indicate positions of cranial nerve roots. ot, otic vesicle (inner surface indicated by dots); IX, glossopharyngeal nerve root; V, trigeminal nerve root; VII, acusticofacialis nerve root; X, vagus nerve root. Bars, 100 mm.

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crest cells (Fig. 7B). It was apparent that the neural crest was already organized anteroposteriorly at the stage of neurula, and the origins of the three cephalic crest cells did not seem to overlap extensively (Fig. 7B). In one case of the injections (#n091), labeled cells were found in both mandibular and hyoid arches. However, it was not determined, in the present experiment, if the labeled neurectoderm covered the region corresponding to r3 which is known to give rise to crest cells to both arches in amniote embryos (Serbedzija et al., 1992; Sechrist et al., 1993). When DiI-labeled cells were observed to have migrated into the mandibular arch or the cheek process as well as the forebrain region, neural tube labeling was found in the midbrain (Fig. 8A). The cells were superficially located beneath the skin, indicating that they were on the dorsolateral pathway. The labeled cells within the hyoid arch were associated with labeling of the neural tube at the level corresponding to the middle part of the hindbrain (Fig. 8B). Some of the BC cells became labeled when we labeled the postotic neural tube (somite level) (Fig. 8C). In the last case, the labeled putative crest cells seemed to have migrated rostrocaudally lateral to the ventral edge of somites.

LjOtxA Expression in Mandibular Crest Cells We have previously described the L. japonica cognate of the murine Otx2 gene, LjOtxA (Ueki et al., 1998). By in situ hybridization, LjOtxA expression was seen from stage 21 onward, in the midbrain and more rostral portion of the neural tube (Fig. 9). The most conspicuous expression in the first pharyngeal arch mesenchyme was observed at stage 23 (Figs. 9C–9F). To observe the mesenchymal expression, embryos were histologically prepared after hybridization and high levels of LjOtxA message were detected in stage 23 embryos and onward. High levels of LjOtxA were seen in the rostral neural tube and a weak signal was also observed in the outer portion of the mandibular arch mesenchyme (Fig. 9). By comparing with Fig. 4, it was apparent that the neural crest-derived ectomesenchyme expressed the gene within the arch.

DISCUSSION Neural Crest Cells of L. japonica In the present study, the morphological distribution pattern of crest cells in L. japonica has been described primarily based on SEM observation. Although there have been several histological (von Kupffer, 1896; Damas, 1944; Johnels, 1948; Nakao and Ishizawa, 1987) and experimental works (Newth, 1956; Langille and Hall, 1988) on the lamprey crest cells, this is the first to provide threedimensional data on crest cell distribution in this group of animals. The DiI injection also revealed that our speculation concerning crest cell origin along the neuraxis and their distribution was consistent with the labeling pattern, as well as with previous mapping performed by Langille and Hall (1988). Our main findings are (1) in L. japonica embryos, there are three major crest cell populations, termed TC, HC, and BC cells, which are located in superficial positions of the embryo; (2) the neural crest-derived cells seem to be the source of the major mesenchymal component of the pharyngeal arch; (3) each of the three populations is proximally associated with metamerical rhombencephalic bulges, the rhombomeres; the HC cell population is restricted to r4, the TC cell population is associated with r2 and anterior, and BC cells are associated with r6 and caudally; (4) origins of crest cells are organized anteroposteriorly along the neuraxis of the neurula; and especially (5) midbrain-derived crest cells express the LjOtxA gene, homologue of murine Otx2, and migrate into the mandibular arch of this animal. Among the previous descriptions of closely related animals by other authors, minor differences are noted. For example, von Kupffer (1891) illustrated, in Petromyzon, a stream of putative crest cells (Zwischenstrang) connecting the dorsal aspect of the hindbrain and either the mesoderm or the ganglionic primordia, roughly at a stage corresponding to stage 23 of L. japonica. Von Kupffer (1891) also showed some small populations of crest cells which are not in direct contact with the ectoderm. In L. japonica, we could find neither of these cells. We may have failed to observe these cells freely migrating in the space between the ectoderm, mandibular mesoderm, and the midbrain: prior to our SEM observation, the skin ectoderm was removed and therefore it was not certain whether all these

FIG. 5. Late distribution pattern of the crest cells in L. japonica. Surface ectoderm has been removed from all embryos. (A) Stage 22.5 embryo seen from the dorsal view. The rostral region of the embryo has been broken. Putative crest cells are scattered on the dorsal aspect of the neural tube. They elongated in shape with their longer axes oriented transverse to the neuraxis (arrows). They seem to represent late-emigrating crest cells. (B) Stage 22.5 embryo seen from the lateral view. Ectodermal otic pit (ot) remains in this embryo, covering the dorsal portion of the HC cell population. In the space between adjacent somites, putative crest cells of elongated shape are seen (arrowheads). The BC cells are covering the entire postotic pharyngeal arch region. The ventral limit of the BC cells is indicated by arrows. (C) Another stage 22.5 embryo specimen. Enlarged view of the hyoid and branchial regions. The hyoid arch (hy) is now mostly covered by HC cells. The mesoderm of this arch is seen in the ventral portion which is almost separated ventrally from the branchial arch mesoderm (arrowheads) by the growth of the second pharyngeal pouch (pp2). Bars, 100 mm (A, B) and 50 mm (C).

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FIG. 6. Late distribution pattern of crest cells in L. japonica-2. The surface ectoderm has been removed from all embryos which are seen from the lateral views. Otic vesicles were not removed. (A) Stage 23 embryo. The left side of the embryo is shown. Elongated crest cells are not restricted in the intersomitic space but are also seen on the lateral surface of the somite (arrowheads). The third pharyngeal or the first branchial arch is protruding but is not yet separated by the third pharyngeal pouch which is still invisible. (B) Stage 24 embryo. The crest cells within the branchial arch region have become a compact cell population by this stage, contiguous with similar flattened cells over the somites. At these stages, the r4 region (arrows in A and B) becomes distinctly depressed on the dorsal aspect of the neural tube. Bars, 100 mm.

crest cells formed a compact cell mass or presented as isolated cells. This possibility cannot be ruled out especially since the buffer for the fixative was prepared (low

concentration of phosphate buffer, free of NaCl) to wash away extracellular matrices, the substrate for crest cell migration and adhesion. Extensive labeling and utilization

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FIG. 7. Diagram illustrating the DiI labeling and neural crest organization. (A) Lateral and dorsal views of stage 19 to 19.5 lamprey neurula are shown as line drawings. On the dorsal view, sites of focal DiI injection are shown and destinations of labeled cells are indicated. Injection sites on the neuraxis are indicated by ratio of neural rod length measured from the rostral end of the neural ectoderm (rn). Due to the absence of topographical landmarks and spherical morphology of neurula, accurate mapping of the crest was not expected. One of the injection sites resulted in labeled cells in the hyoid arch in one embryo (#s095) and in the branchial arch in the other embryo (#s092). In one injection (#n091), both TC and HC cells were labeled. Numbers indicate individual injections. (B) Hypothetical rostrocaudal organization of the neural crest on the neurula. bp, blastopore; rn, rostral end of the neurectoderm.

of a molecular marker of the crest cells would be necessary to resolve this problem. The embryological description by Damas (1944) in L. fluviatilis, on the other hand, is consistent with our observations; by performing the graphic reconstruction, he illustrated the distribution of crest cells in three cell populations in the vicinity of the neural tube as in Fig. 1E of the present study.

The three major populations of cephalic crest cells in L. japonica are separate, each forming a stream of cells ventrally to fill the pharyngeal arch (Figs. 4 and 5). Typically, each of these cell populations fills, from rostral to caudal, pharyngeal arch 1 as well as the premandibular region (reviewed by Kuratani et al., 1997a), arch 2, and arch 3 and caudal ones (postotic pharyngeal arch region). Such a distri-

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bution pattern has been described in various vertebrate embryos at an early pharyngular state (elasmobranchs: Froriep, 1891, 1902; Goodrich, 1918; de Beer, 1922; teleosts: Sadaghiani and Vielkind, 1990; amphibians: Froriep, 1917; Stone, 1926; Starck, 1963; Zackson and Steinberg, 1986; Hall and Ho¨rstadius, 1988; Mayor et al., 1995; Olsson and Hanken, 1996; reptiles: Meier and Packard, 1984; birds: Anderson and Meier, 1981; Kuratani and Kirby, 1991; Sechrist et al., 1993; mammals: Halley, 1955; Mu¨ller and O’Rahilly, 1980; Tan and Morriss-Kay, 1985; Maden et al., 1992; Osumi-Yamashita et al., 1997). Of the three crest cell populations of vertebrate embryos, the BC cells tend to remain undivided into the branchial ectomesenchyme. Of this cell population, the third arch ectomesenchyme becomes isolated rather early in many species (Neal, 1896; Olsson and Hanken, 1996; Kuratani and Kirby, 1991; Shigetani et al., 1995). Also in L. japonica, isolation of arch 3 (br1) ectomesenchyme was suggestive in some embryos after stage 23 (Fig. 5B). The BC cell population corresponds to circumpharyngeal crest cells (CP cells) in the chick embryo (Kuratani and Kirby, 1991); in both, the pharyngeal arches have not been formed by the time of crest cell influx. It is in the lateral body wall that the CP and BC cells first populate and the segmentation of the pharyngeal wall takes place secondarily in both species. Furthermore, the DiI labeling of the postotic crest of the lamprey resulted in the presence of labeled cells along the dorsal edge of the branchial arch complex (Fig. 8B). This is a site called “Kopfnickerwulst” by Froriep (1885), corresponding to the trunk/pharyngeal wall interface (Kuratani, 1997). Similar rostrocaudal migration of crest cells at this site was also suggestive in the CP cells (Shigetani et al., 1995). Overall, the morphological pattern of crest cell distribution suggests that the similarity between various vertebrate groups is based on a simple embryonic architecture and shared relative timing with which embryonic components are laid down. In relation to the skeletogenic property of the trunk crest in anamniote embryos (reviewed by Langille and Hall, 1988; and by Smith and Hall, 1990), the distribution pattern of the lamprey BC cells seems worth consideration, especially since the branchial region is expanded caudally in lampreys. As with TC cells (Fig. 4), lamprey BC cells are found superficially to the arch mesoderm, indicating that they had migrated along the dorsolateral pathway (Figs. 5B and 5C).

This is also implied by the early distribution pattern of the cells as seen in stage 21.5 embryos (Fig. 3A). It seems most likely, therefore, that the branchial cartilage of the lamprey is also of cephalic crest origin as in the case of gnathostome viscerocranium. Whether there is any contribution of medially migrating crest cells to the branchial skeleton belongs to future study.

Rhombomeres and Crest Cells In vertebrate embryos, cranial sensory ganglia are also partly derived from the cephalic crest (Narayanan and Narayanan, 1977; D’Amico-Martel and Noden, 1983). The dorsal part of each population adheres to the hindbrain at the levels of even-numbered rhombomeres, the neurectodermal segments (reviewed by Orr, 1887; Kuhlenbeck, 1935; Lumsden and Keynes, 1989). Alternatively, it appears to be the odd-numbered rhombomeres that isolate crest cell populations. The otic placode that is located between the second and the third cell populations would also be the barrier, in gnathostome embryos, to inhibit crest cell migration. This, however, is not the case in L. japonica, since the HC cells migrate medial to the developing otocyst. Thus, rhombomere– crest cell relationships are more extensively conserved among vertebrates than the topography of the rhombomeres and otocyst. In the present mapping, rhombomere levels of origins for each crest cell population have not been obtained, although the overall morphological distribution seems to indicate that the similar rhombomeric origins as found in amniote embryos (TC cells from r3 and anterior, HC cells from r3 to r5, and BC cells from r5 and caudal) will be found by future labeling study. Of interest, at the moment, was that the segmental relationships of the rhombomeres and crest cells do not appear to be simply due to the segmented origin of the crest cells in L. japonica. By stage 21.5, for example, the cephalic crest cells are distributed on the dorsal aspect of the whole hindbrain region, leaving no gaps at r3 and r5; on the dorsolateral aspect of the neural tube, the separation is then only clear in distal portions of the cell populations (Figs. 2C and 3A). Complete separation is attained at stage 22 (Fig. 2F); adhesion of the cells seemed to be gradually limited to the r4 region and crest cells are completely excluded from r3 and r5 when the rhombomeres are most conspicuous (Figs. 2D–2F). Such a sequence of crest cell

FIG. 8. DiI labeling of the neural crest cells in L. japonica embryos. Three embryos in which labeled cells were distributed in three different sites are presented. In all embryos, focal microinjection of DiI was performed at stage 19.5 and embryos were grown to stage 21.5 forty-eight hours after injection. (A) Labeled putative crest cells are seen in the mandibular arch and forebrain region. The site of injection is indicated at the level of the midbrain (out of focus, asterisk). (B) Drawing of a same stage embryo to show the neural tube morphology. The site of injection (arrowhead) corresponds to the rostral midbrain region as judged from the mid-/hindbrain boundary (mhb) and synen-/parencephalon boundary (spb). (C) DiI-labeled cells (arrowheads) are found in the second pharyngeal arch region. Neural rod labeling is seen roughly in the middle of the hindbrain (asterisk). (D) Labeled cells are in the posterior hindbrain and suprabranchial regions (arrowheads). The labeling pattern indicates that crest cells are migrating caudally along the suprabranchial area. Bars, 50 mm (A) and 100 mm (C, D).

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separations may explain occasional labeling of crest cells in two successive arches by focal injection of DiI around stage 19 (Fig. 7). The selective adhesion of the cephalic crest cells on even-numbered rhombomeres has been reported to play important roles in establishment of cranial nerve roots in gnathostome embryos (Moody and Heaton, 1983; Kuratani and Eichele, 1993; Niederla¨nder and Lumsden, 1996). In L. japonica too, the rhombomeres with which crest cells are associated are the sites of cranial nerve development (Fig. 3; Kuratani et al., 1998). The secondary separation of crest cell populations holds true for the developmental pattern in the chick embryo; it has been shown that r3 and r5 give rise to crest cells as others do, but these cells merge into either rostral or caudal crest cell streams thus forming two crestcell-free spaces (Sechrist et al., 1993, but also see Lumsden et al., 1991). An alternative, but not necessarily contradicting explanation to this is that cell death takes place specifically in r3 and r5, thus producing crest-cell-free areas in the hindbrain (Graham et al., 1993). Although vital staining with methylene blue was tried on L. japonica embryos, no indication of selective cell death has been found in these rhombomeres (not shown). In some gnathostome embryos and larvae, cell– cell contact-dependent signaling has been suspected to function in crest cell segregation (reviewed by Friedman et al., 1996; Orioli and Klein, 1997; and by Smith et al., 1997), which may also be involved in crest cell distribution in the lamprey.

Crest Cells and Vertebrate Evolution The origin of the craniate head is enigmatic and so is that of the neural crest. The amphioxus (Branchiostoma) seems to have split from the lineage toward craniates after tunicates did (Janvier, 1996). Crest cells are not apparent in this animal (see Holland et al., 1997) nor is there a morphologically clear head; there is no preotic unsegmented mesoderm, although the rostral-most neural tube has been reported to be differentiated at least as a partial forebrain similar to that in craniates (reviewed by Lacalli et al., 1994). Most of the amphioxus neuraxis is regarded to be equivalent to the hindbrain of craniates, but the configuration of the neural tube displays simultaneously rhombencephalic and spinal traits (Fritzsch and Northcutt, 1993) and patterning of the peripheral nervous system (PNS) is entirely somitomeric (Franz, 1927). This supports the idea that the

head–trunk differentiation is not yet completed in this animal (Kuratani, 1997). The neural crest in hagfish embryos is much less understood than any other chordate species. It has been suggested that the neural crest of these animals develops a so called “ganglionic vesicle” that does not seem to deepithelialize before gangliogenesis (von Kupffer, 1896; Cornel, 1942; reviewed by Wicht and Tusch, 1998). Moreover, it is also suggested from the embryonic morphology that the optic vesicle is serially homologous with the neural crest (reviewed by Wicht and Tusch, 1998). The neural crest cell developmental pattern in the lamprey as revealed by the present study as well as by previous descriptions and experiments indicates that the neural crest of lampreys is much closer to that of gnathostome embryos than to hagfish. The lamprey crest does not in any respect represent an intermediate stage leading to gnathostome embryonic pattern. The idea is consistent with the theory that myxinoids are regarded as a sister group of vertebrates (Forey and Janvier, 1993; reviewed by Janvier, 1993, 1996) and also raises a question if the embryonic pattern of the myxinoids shares all the traits of the vertebrate pharyngula described in the present study. The conserved pattern of early crest cell distribution among vertebrate species seems to imply the early establishment of crest cell–pharyngeal arch association in the course of vertebrate evolution. Similar anteroposterior organization of neural crest and branchial arches along the embryonic axis is shown between chick and Petromyzon marinus (Langille and Hall, 1988). Of special interest in this respect is the expression pattern of the lamprey cognate of the Otx2 gene (Ueki et al., 1998); LjOtxA is strongly expressed in the rostral neural tube, and the caudal boundary of expression has been found to be close to the mid-/ hindbrain boundary as that of Otx2 in gnathostome embryos (Simeone et al., 1992; Boncinelli et al., 1993). As shown in Figs. 8 and 9, the cells arising from the LjOtxAexpressing neural crest migrate into the rostral portion of the cheek process that corresponds to the mandibular arch of gnathostome embryos, and these crest cells maintain the gene expression in the periphery of the mandibular mesenchyme (Fig. 9). The same crest–mandibular arch assignment is repeated in amniotes; midbrain-derived crest cells always migrate into the mandibular arch (Le Lievre, 1978; OsumiYamashita et al., 1992; Ko¨ntges and Lumsden, 1996). In connection with this, haploinsufficiency of the Otx2 gene

FIG. 9. LjOtxA expression in the pharyngula of L. japonica. LjOtxA mRNA was detected by in situ hybridization. (A) Stage 22 embryo. LjOtxA expression is restricted to the rostral portion of the neuraxis. The caudal limit of the expression (arrowhead) is close to the mid-/hindbrain boundary. (B) Stage 24 embryo. The caudal limit of LjOtxA expression is indicated by an arrowhead. Asterisk indicates nonspecific staining. (C and D) Stage 23 embryo. Expression is seen in the rostral neural tube and also in the cheek process (cp) that contains the first pharyngeal arch as well as the first pharyngeal pouch (pp1). Arrows indicate the plane of sections shown in E and F. (E) Transverse section of a stage 23 embryo. Expression of LjOtxA is seen as blue staining. Note the LjOtxA expression in the forebrain as well as in the peripheral mesenchymal portion of the cheek process (arrows). cp, cheek process; fb, forebrain; hb, hindbrain; mb, midbrain; pp1 and pp2, first and second pharyngeal pouches. Bars, 100 mm (A, B, D, E, F) and 200 mm (C).

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in the mouse results in selective loss of the mandible (Matsuo et al., 1995). The first pharyngeal arch of the lamprey goes through a distinctly different pathway from gnathostome embryos; the lamprey first arch forms the upper lip and velum of this animal. Possible heterogeneity of the mandibular arch ectomesenchyme on the lamprey would reveal the homology of skeletal elements within this arch. Both the upper lip and velum contain muscles that are innervated by trigeminal nerves (Kuratani et al., 1997b and citations therein). Both the upper lip and velar muscles are derived from an en-expressing common anlage located in the cheek process of young pharyngula (Holland et al., 1993). Secondarily, expression is lost in the upper lip mesoderm and becomes restricted to the velothyroideus muscle anlage in the velum (Holland et al., 1993). A similar expression of the en gene is again seen in the mandibular process of gnathostome embryos; the en-expressing cells are actually the myoblasts in this pharyngeal arch (Hatta et al., 1990, 1991; Hemmati-Brivanlou et al., 1991; Ekker et al., 1992; Gardner and Barald, 1992; Logan et al., 1993). Therefore, in the first pharyngeal arch, the mesodermal component, crest-derived ectomesenchyme, origin of the ectomesenchyme and genetic code of the neurectoderm of the crest are all shared between the lamprey and gnathostomes (Fig. 10). The above-described conservation implies an importance related to evolution of the vertebrate head; regardless of the enormous metamorphoses of the first pharyngeal arch, the embryonic and cellular components, as well as the early genetic program behind the morphogenesis, seem to have already been established prior to the splitting of lampreys and gnathostomes. The conservative and epigenetic relatedness of morphogenetic elements like tissues, cell populations, pharyngeal arches, rhombomeres, and spatial expression of regulatory genes would constitute the so-called phylotypic stage or the embryonic Bauplan of the vertebrates that is seen in what we call the early pharyngula. We should not expect that this would reflect the adult of the shared ancestor, but it may probably have appeared in the ontogeny of the ancestor. Importantly, it still remains unknown which of the above-listed traits that constitute

the vertebrate phylotype are shared in the pharyngula of the hagfish, the sister group of vertebrates, as phylotypes of craniates. The vertebrate phylotype has already been recognized through gnathostome embryology but now it is not only for gnathostomes. Alternatively, such an embryonic design, or the primordial body plan assumed in the gnathostome embryo, is not meant automatically to create the upper and lower jaws. Rather, the morphogenetic basis of the actual shape of the jaw would have been secondarily built upon, or would be downstream of, such a basic embryonic developmental plan. It will be through molecular means that we will eventually elucidate the unsolved homology of mandibular arch-derived structures between lampreys and gnathostomes. The question is with no doubt related to developmental and molecular mechanisms involved in the morphological patterning of this arch; expression patterns of yet undiscovered genes functioning in the dorsoventral division of the gnathostome mandibular arch, for example, would be most crucial to examine in lamprey embryogenesis.

ACKNOWLEDGMENTS We are grateful to Masakuni Hoshino, Masao Iwahashi, Hideo Murayama, and Masakuni Hoshino in Niigata Prefectural Inland Water Fisheries Experiment Station, Niigata, Japan, for their kind help with our experiments and for the use of the facility. We also thank Drs. Ruth Yu and Drew Noden for their critical reading of the manuscript and helpful discussion. Sincere gratitude is extended to Dr. Kathryn W. Tosney for her technical comments and discussion on SEM observation. This work was supported by grants-in-aid from the Ministry of Education, Science, and Culture of Japan (Specially Promoted Research).

REFERENCES Anderson, C. B., and Meier, S. (1981). The influence of the metameric pattern in the mesoderm on migration of cranial neural crest cells in the chick embryo. Dev. Biol. 85, 385– 402.

FIG. 10. Crest cell–pharyngeal arch association and vertebrate evolution. In both gnathostomes and lampreys, the neural crest is organized anteroposteriorly at the neurula (top), from which three cephalic crest cell populations arise during pharyngular stages of development (middle). These cell populations have conserved topographical relationships with hindbrain bulges, the rhombomeres; the three cell populations are separated from each other by r3 and r5, thereby forming independent cell streams called TC, HC, and BC cells that are destined to become the mandibular arch and premandibular region, the hyoid arch, and branchial arches, respectively. This common basic pattern can differentiate into both gnathostome and lamprey pharyngeal systems which are extremely different from each other (bottom). In gnathostomes, the mandibular arch develops into upper and lower jaws and masticatory muscles, while in the lamprey the velum and the upper lip develop from the same arch. It is not yet known if this mandibular arch subdivision is homologous between these animals. In both, midbrain-derived crest cells populate the mandibular arch. The same pattern of gene expression is again shared by both groups, namely that Otx2 cognates are expressed in the midbrain and more rostral brain and en expression is seen within the mandibular arch myoblasts (shaded). These strongly suggest that metamorphosis of the mandibular arch is based on conserved embryonic topography and gene expression patterns that are likely to have been established before splitting of gnathostomes and agnathans.

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