developmental morphology of the head mesoderm and ...developmental morphology of the head mesoderm...

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Developmental Biology 210, 381–400 (1999)Article ID dbio.1999.9266, available online at http://www.idealibrary.com on

Developmental Morphology of the Head Mesodermand Reevaluation of Segmental Theories of theVertebrate Head: Evidence from Embryos of anAgnathan Vertebrate, Lampetra japonica

Shigeru Kuratani,*,1 Naoto Horigome,* and Shigeki Hirano†*Department of Biology, Okayama University, Faculty of Science, Okayama, Japan; and†Department of Anatomy, Niigata University School of Medicine, Niigata, Japan

Due to the peculiar morphology of its preotic head, lampreys have long been treated as an intermediate animal which linksamphioxus and gnathostomes. To reevaluate the segmental theory of classical comparative embryology, mesodermaldevelopment was observed in embryos of a lamprey, Lampetra japonica, by scanning electron microscopy and immuno-

istochemistry. Signs of segmentation are visible in future postotic somites at an early neurula stage, whereas the rostralesoderm is unsegmented and rostromedially confluent with the prechordal plate. The premandibular and mandibularesoderm develop from the prechordal plate in a caudal to rostral direction and can be called the preaxial mesoderm as

pposed to the caudally developing gastral mesoderm. With the exception of the premandibular mesoderm, the headesodermal sheet is secondarily regionalized by the otocyst and pharyngeal pouches into the mandibular mesoderm, hyoidesoderm, and somite 0. The head mesodermal components never develop into cephalic myotomes, but the latter develop

nly from postotic somites. These results show that the lamprey embryo shows a typical vertebrate phylotype and that theasic mesodermal configuration of vertebrates already existed prior to the split of agnatha–gnathostomata; lamprey does notepresent an intermediate state between amphioxus and gnathostomes. Unlike interpretations of theories of headegmentation that the mesodermal segments are primarily equivalent along the axis, there is no evidence in vertebratembryos for the presence of preotic myotomes. We conclude that mesomere-based theories of head metamerism arenappropriate and that the formulated vertebrate head should possess the distinction between primarily unsegmented head

esoderm which includes preaxial components at least in part and somites in the trunk which are shared in all the knownertebrate embryos as the vertebrate phylotype. © 1999 Academic Press

Key Words: lamprey; head mesoderm; neural crest; somites; segmentation; pharyngeal arch.

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INTRODUCTION

Whether the vertebrate head is segmented as is the trunkhas long been an intriguing issue of vertebrate morphologyand embryology. Goethe (1820) and Oken (1807) first putforth the “vertebral theory” of the skull, in which the skullwas proposed to be composed of several transformed verte-brae. Supported by great anatomists (Owen, 1848; Rathke,1839; Reichert, 1838), the theory became temporarily themainstream of the head problem (Kopffrage) and was then

1 To whom correspondence should be addressed at Departmentof Biology, Okayama University Faculty of Science, 3-1-1 Tsushi-

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manaka, Okayama, Okayama 700-8530, Japan. Fax: 181-86-251-7876. E-mail: [email protected].

0012-1606/99 $30.00Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

efined through embryological studies by Gegenbaur (1872),alfour (1878), van Wijhe (1882), and others (reviewed byoodrich, 1930; de Beer, 1937; Starck, 1978; Jarvik, 1980;

nd Jefferies, 1986). The segment assumed in this theoryas a primordial metameric unit comparable to a preverte-ra or a somite in the trunk.The theory of head mesoderm segmentation was at one

ime forgotten but resurrected by new methods of embryo-ogical observation. For example, in a series of descriptiveorks based on scanning electron microscopy, the presencef primordial segmentation in the mesoderm was detectednd these incomplete segmental units were named cephalicomitomeres (reviewed by Jacobson, 1993). The concept of
he segmented body plan has also gained molecular bases ofetamerism as can typically be seen in some homeobox-
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382 Kuratani, Horigome, and Hirano

containing genes (reviewed by Krumlauf, 1993; and Ruben-stein and Puelles, 1994) as well as a number of regulatorygenes involved in the segmentation of paraxial mesodermin the trunk (reviewed by Tajbakhsh and Sporle, 1998),again increasing its importance on a new paradigm ofdevelopmental biology.

Until now, the segmentation theory has been biased byidiosyncrasy established by elasmobranch embryology aswell as morphology of the amphioxus. This is partly be-cause the elasmobranch embryos exhibit tandemly ar-ranged mesodermal segments or “head cavities,” which isreminiscent of the amphioxus condition. Lampreys, thesister group of gnathostomes, have been less extensivelystudied than elasmobranchs and have been regarded as anintermediate group linking amphioxus and gnathostomes.They are also one of the animal groups whose mesodermalmorphology remains unexplored by scanning electron mi-croscopy. The present paper is thus intended to give aprecise description of the developing mesoderm in Lam-petra japonica, to evaluate its developmental pattern on thephylogenetic tree, and also to reevaluate the problem ofvertebrate head segmentation in search of the most appro-priate formulation of the vertebrate head as a component ofvertebrate phylotype, the shared specification of embryonicbody plan (reviewed by Hall, 1998). By use of scanningelectron microscopy (SEM), we confirmed in this animalthat there is no complete segmentation in the head meso-derm and that myotomes are restricted to postotic meso-derm as seen in gnathostome embryos.

MATERIALS AND METHODS

Embryos and scanning electron microscopy. Lamprey em-bryos were obtained and maintained in the laboratory as previouslydescribed (Horigome et al., 1999). Developmental stages weredefined according to the description by Tahara (1988) and moredetailed stages of 19.5, 20.5, 21.5, and 22.5 are as described(Horigome et al., 1999). Embryos were fixed in 2.5% glutaraldehydein 0.01 M phosphate buffer (pH 7.4; PB) and processed for SEMobservation as described previously (Horigome et al., 1999).

Immunohistochemistry. Embryos were fixed with 4% parafor-aldehyde in 0.1 M phosphate-buffered saline (PFA/PBS), washed

n 0.9% NaCl/distilled water, dehydrated in a graded series ofethanol (50, 80, 100%), and stored at 220°C. The immunostain-

ng procedure has been described in Kuratani et al. (1997a). Briefly,fter washing in TST (TST is Tris–HCl-buffered saline: 20 mMris–HCl, pH 8.0, 150 mM NaCl, 0.1% Triton X-100), the samplesere blocked with aqueous 1% periodic acid and with 5% nonfatry milk in TST (TSTM) and incubated in the primary antibody,H-1 (purchased from the Developmental Studies Hybridomaank, Iowa City, IA; diluted 1/1000 in spin-clarified TSTM con-aining 0.1% sodium azide) which recognizes tropomyosin. Theecondary antibody used was horseradish peroxidase (HRP)-onjugated goat anti-mouse IgG (ZYMED Lab. Inc., San Francisco,A) diluted 1/200 in TSTM. After washing in TST, the embryos
ere allowed to react in TS with the peroxidase substrate 3,39-iaminobenzidine (DAB, 150 mg/ml) and 0.01% hydrogen peroxide.
Copyright © 1999 by Academic Press. All right

Histochemistry. Embryos were fixed with PFA/PBS for 1 h atoom temperature and briefly washed with 0.9% NaCl. They weret once treated with acetylcholinesterase (AChE) according to therocedure of Karnovsky and Roots (1964). The reaction was stoppedy postfixation with PFA/PBS and the embryos were stored in PBS.or observation, they were dehydrated with a graded series ofethanol, clarified with BABB, and mounted on depression slide

lass.

RESULTS AND DISCUSSION

Development of the head mesoderm in L. japonica.One of the reasons for the misconception that lampreysrepresent an intermediate state between the amphioxus andgnathostomes was the morphology of the preotic mesodermwhich was illustrated to be segmented as myotomes inamphioxus. The most precise and influential description inthis respect has been that by Damas (1944) in L. fluviatilis,

hich has been cited in major literatures (Grasse, 1954;arvik, 1980; Jefferies, 1986). Unlike the report by Damas1944), however, preotic mesoderm is never segmented in L.japonica.

The first overt mesoderm of the L. japonica embryo isseen as the separation of a layer from the endoderm at stage19 (Fig. 1); a faint boundary is being formed lateral to thedorsal midline, delineating the mesoderm laterally which ismore conspicuous at caudal levels. Rostrally, the mesodermgradually merges into the endoderm with no clear demar-cation (Figs. 1A and 1B). At the rostral end, the chorda andparaxial mesoderms as well as the endoderm are exten-sively continuous with the foregut endoderm, forming asingle medial plate, or the prechordal plate (Figs. 1B and1C).

Segmental boundaries in the paraxial mesoderm are re-stricted in the future trunk region, appearing in a rostral tocaudal direction, the rostralmost one representing thesomite 0/1 boundary (Figs. 1B and 2). Bilateral asymmetry iscommonly observed in the segmental pattern (Fig. 1). Simi-lar observations were made by Veit (1939), who noticed inPetromyzon planeri that the left mesoderm develops fasterthan the right. However, in L. japonica, examination of anumber of specimens and comparison with older embryosindicated that there is no clear preference as to which sideof the embryo develops larger mesoderm rostral to theboundary (Fig. 2). Bilateral asymmetry is somehow compen-sated at later stages, presumably due to differential growthof the mesoderm.

Through stages 19 and 20, the rostralmost mesoderm iscontinuous with the prechordal plate medially (Figs. 1B–1E), but ventrolaterally the mesoderm is gradually sepa-rated from the endoderm (Figs. 1F and 3A). The process ofseparation proceeds from a caudal to rostral direction (Figs.1E and 1F). This mesoderm shows slight lateral expansion(Fig. 3B), delineated caudally by an endodermal swellingrepresenting pharyngeal pouch 1 (Fig. 3B). This mandibular
mesoderm develops as a typical enterocoel as has alreadybeen reported in lamprey embryos (Hatta, 1891; de Selys-
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383Head Mesoderm in the Lamprey

FIG. 1. Development of the mesoderm in L. japonica at stages 19 and 19.5. (A) The lateral view of an embryo whose surface ectoderm haseen removed. The lateral border of the mesoderm is indicated by dots. Rostrally the border disappears, indicating that the mesoderm inhis region is not completely dissociated from the endoderm. (B) A slightly older embryo of the same stage as seen from the dorsal view.he surface ectoderm as well as the neural rod has been removed. Note the continuity between the notochord (nt) and the prechordal plate

pc). The prechordal plate also continues into the endoderm (en) rostrally and the mesoderm (mes) laterally. Formation of the segmentaloundaries in the paraxial mesoderm (arrows), representing somite 0/1 boundaries, shows an asymmetrical developmental pattern. Theateral notch of the rostral mesoderm is indicated by an asterisk. (C) The mesoderm on the left side has been removed from this embryo.ote that the rostral portion of the notochord is still continuous with the endoderm laterally (arrows). (D) Anterior portion of the embryo

een from the dorsal view. Surface ectoderm, neural rod (nr), as well as the notochord (nt) have been partially removed. Early mesodermmes) is seen paraxially on the left side (top of the figure), consisting of cells smaller than the underlying endodermal cells (en) that arexposed on the right side of the embryo. Arrows indicate the cut edge of the ectoderm. Ventral to the notochord (nt), the endodermal roofs exposed which rostrally merges to the prechordal plate (pc). The endodermal roof is partially removed (*) and the cut edge (singlerrowhead) extends to the prechordal plate. Double arrowhead corresponds to the somite 0/1 boundary. (E) The rostral mesoderm has grownaterally to form the mandibular mesoderm (mm). Arrows indicate segmental boundaries of somites and dots the lateral limit of the

andibular mesoderm. (F) A slightly older embryo seen from the right lateral view. Only the surface ectoderm has been removed. Theandibular mesoderm (mm) is a distinctive component whose rostral end is not yet dissociated from the endoderm (arrowhead). Caudal

o the mandibular mesoderm, a part of endoderm (*) is exposed, representing the future pharyngeal pouch 1. Arrows indicate the cut edgef the mesoderm. en, endoderm; mes, mesoderm; nr, neural rod; nt, notochord; pc, prechordal plate; s1–s2, somites. Bar: 100 mm.

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.

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Longchamps, 1910; Damas, 1944). Through stages 20 to 21,some distinct regions can be distinguished in the rostralmesoderm (Figs. 3A–3D); the mesoderm caudal to the firstpharyngeal pouch is further regionalized into two portionsrostrocaudally by the otocyst and second pharyngeal pouch(Figs. 3E–3G and 4). The rostral subdivision is incorporatedinto the second pharyngeal arch and is called the hyoidmesoderm. The caudal subdivision, or somite 0, resemblesa somite in both size and shape, but lacks a clear rostralboundary (Figs. 3C and 3E).

By stage 21, the mandibular mesoderm is mostly delin-eated from the prechordal plate (Fig. 5E), which has clearlydiminished in size (see Figs. 1 and 3). At this stage theanterior tip of the notochord can be separated from theprechordal plate: the prechordal plate is now an indepen-dent entity (Fig. 5B), which by stage 22 has grown laterallyto form a pair of mesoderm in front of the mandibularmesoderm (Fig. 5C). From the location (caudal to the eye,

FIG. 3. Mesodermal development in L. japonica embryos betweemesoderm is regionalized into the mandibular mesoderm (mm) an(pp1). The mandibular mesoderm has separated from the endoderm.is to the right. The surface ectoderm, neural rod, as well as the notobroken portion (*) shows the fusion of the rostral notochord, the plateral view of a stage 20.5 embryo. An indentation (arrowhead) fo

FIG. 2. Diagrams of the segmental boundary pattern in several emboundary represents the somite 0/1 boundary. There is no clear ri

0 (s0). (D) Anterior view of another stage 20.5 embryo whose surface ecnotochord (nt) and the prechordal plate (pc) which laterally continues int


ostral to the mandibular mesoderm) and morphologypaired configuration connected at the midline) (Fig. 5D),his mesoderm is identified as the premandibular meso-erm.Thus, in L. japonica embryos, the head mesoderm con-

ists of the mandibular, hyoid, and premandibular meso-erm as well as somite 0. Of those, only the last components located in a postotic position. Unlike the postoticomites that are segmented by clear dorsomedial bound-ries, these cephalic components are confluent with eachther through the stages observed, with the exception of theremandibular mesoderm whose appearance is rather late.Development of myotomes in L. japonica reveals the

hared phylotype of vertebrates. The second reason forhe misconception of the lamprey as an intermediate crea-ure was the belief that ammocoete larvae possess myo-omes apparently in preotic levels (Fig. 6A; Hatschek, 1892;oltzoff, 1901; Neal, 1914, 1918a,b; Neal and Rand, 1942;

ges 20 and 21. (A) Left lateral view of a stage 20 embryo. The headid mesoderm (hm) 1 somite 0 at the level of pharyngeal pouch 1andibular mesoderm as seen from the dorsoanterior view. Anteriord have been removed. The endodermal roof (er) is exposed and theordal plate (pc), and the endodermal roof at this position. (C) Leftby the otocyst regionalizes the hyoid mesoderm (hm) and somite

s stages 19.5 through 20.5. In every case, the rostralmost segmentaleft preference in the number and rate of boundary formation.

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toderm and neural rod have been removed. Note the fusion of theo the mandibular mesoderm (mm). Asterisk indicates an artifactual



hole in the mandibular mesoderm. (E) Lateral view of a stage 21 embryo. The hyoid mesoderm (hm) and somite 0 (s0) are still continuous.(F and D) In slightly older embryos, pharyngeal pouch 2 has appeared through a slit on the mesoderm, pharyngeal slit 2 (ps2). en, endoderm;er, endodermal roof; hm, hyoid mesoderm; lm, lateral mesoderm; mes, mesoderm; mm, mandibular mesoderm; nr, neural rod; nt,
notochord; pc, prechordal plate; pp1, pharyngeal pouch 1; ps2, pharyngeal slit 2; se, surface ectoderm; s0–s2, somites; TC, trigeminal crestcells. Bars: 100 mm (A, C, E–G) and 50 mm (B, D).

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also see Jollie, 1973). These myotomes were often treated asdirect derivatives of preotic segments (Neal, 1918a,b),which is not actually the case as shown below.

By staining with CH-1 monoclonal antibody that recog-nizes tropomyosin, the initial myotome development isdetected at stage 21 of L. japonica (Fig. 6B). All immunore-active myotomes are postotic in position: comparison witha SEM-observed embryo (Fig. 2E) revealed that the rostral-most myotome (m1) arises from s1, the first completesomite developing postotically. In the following develop-

FIG. 5. Developmental sequence of the premandibular mesodermthe rostral portion of the neural tube have been removed. (A) At stand the mandibular mesoderm (mm). The rostral notochord is still chas now attained a clear rostral tip (arrow). (C) By stage 22, the pre(pm). (D) Stage 24 embryo from which all the mesenchyme has be

FIG. 4. Sequential regionalization of the cephalic mesoderm in L.japonica. At stage 19.5, the rostralmost clear segmental boundaryrepresents the somite 0/1 boundary. Caudal to this boundarydevelop a series of metamerical somites by stage 20. On the ventralaspect, a notch is formed at the level of pharyngeal pouch 1 (pp1).At this point, the rostral mesoderm is regionalized into twoportions: the mandibular mesoderm (mm) and the caudal portion.By stage 20.5, the otocyst and first pharyngeal pouch regionalizethe caudal mesodermal portion into the hyoid mesoderm (hm) andsomite 0 (s0).

of the premandibular mesoderm (arrow) is the remnant of the prechordaoptic vesicle (ev). (E) Stage 21 embryo. Trigeminal crest cells (TC) are


ental stages, the rostral myotomes shift rostrally, withhe first myotome sliding ventral to the otocyst (Figs.C–6E). The second myotome (m2) is always characteristi-ally regionalized into dorsoventral halves. Detailed obser-ation of the intermediate stages between stages 24 and 25evealed no sign of additional myotome development ros-ral to m1 (Fig. 6D). Thus, rostral myotomes are all postoti-ally derived: m1 develops into the rostral half of thenfraoptic myotome and dorsal and ventral halves of m2nto the supraoptic and caudal half of the infraoptic myo-omes, respectively (Figs. 6A and 7). The basic myotomeevelopmental pattern is thus very similar between lam-reys and gnathostomes.In gnathostome embryogenesis, myotomes always de-

elop only from trunk (or postotic) somites (Froriep, 1883,902a, 1905, 1917; Rabl, 1892b; Goodrich, 1911; de Beer,922; reviewed by Starck, 1963). The rostralmost somiteften fails to form a myotome since it disintegrates early inevelopment (Hinsch and Hamilton, 1956). Myotomes areistinct embryonic structures that form epaxial myomeresn amniote embryos (Christ et al., 1983, 1986; Ordahl ande Douarin, 1991), and their initial differentiation is exem-lified by the early expressions of myogenic marker geneselonging to the MyoD family and Pax3 genes (see Sporlend Schughart, 1997; Hacker and Guthrie, 1998) or earlymmunoreactivity to CH-1 and MF-20 monoclonal antibod-es as well as AChE reactivity. In this connection, all thearly somites in amphioxus express the alkali myosin lighthain gene (AmphiMLC-alk), a marker of myotome (Hol-and et al., 1995), indicating that rostral mesoderm inmphioxus is more like somites than the head mesoderm ofertebrates.In the head mesoderm, the above-listed markers appear

ubstantially late compared to myotomes (Hacker anduthrie, 1998, and references therein), which is also the

ase with branchial muscles in the lamprey (Fig. 6A). In thisegard, Myf-5 expression has been known to involve aeparate control that is only activated in the head meso-erm but not in somites (Patapoutian et al., 1993). More-ver, regulatory genes that are associated with initiation ofhe somitic segmentation (Sek: Nieto et al., 1992; paraxis:

Burgess et al., 1995; Notch: Conlon et al., 1995; hairy:Muller et al., 1996; Mesp: Saga et al., 1996, 1997; Buch-berger et al., 1998) are not expressed in the preotic regionwith any metamerical patterns; it is these segmental traitsthat make somites distinct from the head mesoderm. Al-

. japonica. In all the embryos except D, the surface ectoderm and0.5, a boundary is being formed between the prechordal plate (pc)uous with the prechordal plate (arrow). (B) Stage 21. The notochordal plate has grown laterally to form the premandibular mesoderm

moved. Seen from the left lateral view. Note that the middle part

in Lage 2ontigchorden re

l plate, located rostral to the notochordal tip (nt) and caudal to thecolored green and the mandibular mesoderm (mm) yellow. The



anterior aspect of the mandibular mesoderm is covered by a subpopulation of TC cells (asterisk). The mandibular mesoderm as well as thecrest cells is removed on the right side of the embryo. Dots on the left side of the photograph represent the connection between theprechordal plate (pc) and the mandibular mesoderm. Anteriormost portion of the TC cell mass has been broken (dots). (F) Stage 24 embryo.Similarly colored as in E. The premandibular mesoderm (pm) has grown from the prechordal plate (pc) into a rostral subpopulation of TCcells. A distinct cell strand (arrows) delineates the premandibular mesoderm from the mandibular mesoderm (mm). ev, position of the optic
vesicle; hm, hyoid mesoderm; mm, mandibular mesoderm; ne, neural tube; nt, notochord; pc, prechordal plate; pf, pharyngeal floor; pm,premandibular mesoderm; pp1, pharyngeal pouch 1; TC, trigeminal crest cells. Bars: 50 mm (A–C, E, F) and 100 mm (D).

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though the above-listed segmental genes have not yet beencloned in the lamprey, it seems most likely that thedifference between the myotome-producing somites androstral mesoderm is distinct in all the vertebrates, and there

FIG. 6. Development of myotomes in L. japonica. In all the figurehistochemically by AChE activity (A and D) or immunochemicallya stage 281 larva (A), rostral myotomes are apparently located prestages 21 (B), 24 (C), 24.5 (D), and 25 (E). Pharyngeal pouches are ind(B and C). The rostralmost myotome (m1) is of the first somite origtwo halves dorsoventrally after stage 24.5 (D). Note that rostralmyotome appears in front of m1. m1 to m8, myotomes; ot, otocys

is no positive evidence at present, in the evolutionarylineage of craniates, to show that preotic myotome or a

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esodermal component of an intermediate trait may havence existed in any hypothetical ancestors.Morphology of the head mesoderm in vertebrates: Topo-

raphical conservation. The classical concept of verte-

erior to the left, except in D. Muscle precursors were either shownned with CH-1 antibody that recognizes tropomyosin (B, C, E). Inlly. (B–E) Early development of myotomes is shown in embryos at

by broken lines. All the myotomes are at first located postoticallyee Fig. 3D). The second myotome is characteristically divided intotomes move anteriorly through development and no additional1 to pp5, pharyngeal pouches. Bars: 100 mm.

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(Kopfhole) are present during the late pharyngular period;there are three pairs of cavities in the preotic region: fromrostral to caudal, premandibular, mandibular, and hyoidcavities (Balfour, 1878; van Wijhe, 1882; reviewed by Goo-drich, 1930; Jarvik, 1980; Jefferies, 1986). Each of theseepithelial structures later gives rise to extrinsic ocularmuscle subsets, which are innervated by each ocular cranialnerve in the same order (Marshall, 1881; reviewed byGoodrich, 1930; de Beer, 1937; Neal and Rand, 1942; Jarvik,1980; Jefferies, 1986). Similar epithelial cavities have alsobeen observed in a vestigial form to give rise to extrinsicocular muscles in bony fishes and amniotes (reviewed byBrachet, 1935; Wedin, 1949; Jacob et al., 1984). In additiono the three pairs of cavities, some elasmobranch speciesave another pair of mesoderm in front of the premandibu-ar mesoderm which has been termed Platt’s vesicle (Platt,891). It is now generally believed that this vesicle only

FIG. 7. Reinterpretation of mesodermal morphology of lampreyindicated by a dotted line on the scheme originally drawn by Neapp2, first and second pharyngeal pouches. (B) Fully grown ammocindicated by broken lines and numbered according to the observatdorsal half of the second myotome (m2), while the infraoptic myotofrom s1) and the ventral half of m2. hbm, hypobranchial muscles;

epresents a portion of the premandibular cavity (Goodrich,918; Jefferies, 1986; but see Jarvik, 1980).

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The three preotic mesodermal components found in theamprey embryo show a striking similarity to the sharkead mesoderm especially before head cavities becomeistinct (Fig. 8A); the premandibular mesoderm in the sharks found caudal to the optic vesicle, medial to the mandib-lar element, and both counterparts are fused medially inontact with the rostral tip of the notochord, exhibiting theame topographical relationships as the lamprey preman-ibular mesoderm (Figs. 8A; Bjerring, 1977). The earliestorphology of Squalus mesoderm in which mandibular

nd hyoid mesoderm form a contiguous mesenchymalheet (Scammon, 1911; reviewed in Jefferies, 1986) roughlyorresponds to the stage 19 embryo of L. japonica (Fig. 1);he mesoderm caudal to the mandibular mesoderm inqualus also appears to be regionalized by the otocystBjerring, 1977).

Regionalization of the preotic mesoderm is less complete

) Pharyngular state of the lamprey. Mesodermal distribution is4). ev, optic vesicle; nhp, nasohypophysial plate; ot, otocyst; pp1,larva of the lamprey. Redrawn from Neal (1897). Myotomes are

n the present study. The supraoptic myotome is derived from thes brought about by fusion of the entire first myotome (m1, derivedhypoglossal nerve.

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n amphibia than in the shark and lamprey; the newtmbryo develops an unsegmented mesoderm in the preotic



region which is contiguous with somite 0 (Jacobson andMeier, 1984). Although a clear premandibular mesodermhas not been described, there is a possibility that the ventralhalf of the mandibular mesoderm represents the preman-dibular mesoderm; this mesodermal portion may only besecondarily incorporated into the mandibular arch (Fig. 8A).

As in the amphibia, amniote embryos do not show clearlyregionalized preotic mesoderm. In early stages of amnioteembryos, however, six or seven incomplete mesenchymalsegments have been recognized by SEM observation, collec-tively called cephalic somitomeres by Meier and his col-leagues (chick: Meier, 1979; quail: Meier, 1982; mouse:Tam and Meier, 1982; Tam et al., 1982; snapping turtle:Meier and Packard, 1984), although the existence of somi-tomeres has recently been doubted by Freunt and others(1996). Since similar pseudosegments have also been de-scribed in teleost embryos (Martindale et al., 1987), itappears that mesodermal reorganization took place inde-pendently more than once in the vertebrate evolution.

In terms of early arrangement of the mesoderm, thelamprey–gnathostome difference is not so great as has beenexpected. Rather, a line can be drawn between amniotesand anamniotes (excluding teleosts). In the latter, apparentdevelopmental factors that seem to yield similarities ofmesodermal regionalization are more or less epigenetic innature, being brought about by the secondarily establishedtopographical relationships with growing otocyst and pha-ryngeal pouches (Fig. 4). The above difference may partly bedue to heterochrony; in amniotes, the deposition of headmesoderm seems to be accelerated compared to sharks andlampreys, as judged from otocyst and pharyngeal archdevelopment. The same is true for teleosts in which pha-ryngeal arch formation is delayed (Richardson, 1995; Rich-ardson et al., 1997).

Aside from the origin and developmental mechanism ofcephalic somitomeres, the question is how we can assignthe somitomeres in amniotes to four pairs of head mesoder-mal components. Simply, the head mesodermal componentof the shark may correspond to two successive somitomeres

FIG. 8. (A) Comparative morphology of the head mesoderm.elasmobranch embryos (shark); three pairs of mesodermal comrecognized. In amphibia (newt), definitive premandibular mesodermm?). In every case, the rostralmost postotic mesoderm, s0, lacksnumbered. (B) Relationships between neural crest cells and hedevelopment in L. japonica. Crest cells are shaded. Cephalic crest care canalized during migration into three major cell populations (gracrest (BC) cells (based on Horigome et al., 1999). Each cell populamesoderm through development and by stage 22 the TC cell popula(MC) cells through growth of premandibular mesoderm. (Right) Cojaponica; based on the present study) and amniotes (chick; based o(1–7). The topographical correlation of the head mesoderm is indicacell populations. BC, branchial crest cells; ev, optic vesicle; HC, hy
mandibular mesoderm; nt, notochord; ot, otocyst; pc, prechordal platecrest cells.

in amniotes. This assumption is not supported in terms ofeither developmental fate (origins of extrinsic ocularmuscles) exemplified in the chick (Jacob et al., 1984;Noden, 1988; Couly et al., 1992) or inconstancy amongamniote embryos (Trainor et al., 1994). Somitomeres, ifthey are really present at all, do not seem to possess anymorphological nature which corresponds to mesodermalregionalization seen in shark or lamprey. The developmen-tal fate of the lamprey head mesoderm is still unknown, butmay possibly be similar to shark mesodermal components,though a peculiar innervation pattern has been reported inPetromyzon (Cords, 1928).

Head mesoderm and cephalic crest cells. As in gnathos-tomes, there are three major crest cell populations in L.japonica, each ventrally filling the pharyngeal arch(Horigome et al., 1999). The rostralmost cephalic crest cellpopulation, or TC cells (trigeminal crest cells; Horigome etal., 1999), surrounds the mandibular mesoderm mainlylaterally (Figs. 5E and 5F). The rostral part of TC cells coversthe anterior aspect of the mandibular mesoderm. It is intothis cell mass that the laterally growing premandibularmesoderm penetrates (Figs. 5E, 5F, and 8B), forming adistinct ectomesenchyme surrounding the premandibularmesoderm, although not entirely separated from the rest ofthe TC cells.

In the context of head morphology, the PM cells exhibitan interesting distribution pattern since they occupy apremandibular region (reviewed by Kuratani et al., 1997b).The cranial elements developing in this region are calledtrabecular cartilages that have been shown to be of crestorigin in avian embryos by construction of chick/quailchimeras (Le Lievre, 1978; Couly et al., 1993) and also in alamprey, Petromyzon marinus, by an extirpation experi-ment (Langille and Hall, 1988). In gnathostome chondrocra-nia, the trabecular cartilages are initially a pair of rod-likecartilages beneath the optic chiasm and are situated on bothsides of the hypophysis and forebrain floor (de Beer, 1937), atopography which is again conserved in lampreys (reviewedby Janvier, 1993). The PM cells are also located in front of

similarity is striking between L. japonica (lamprey) and earlynts, the premandibular, mandibular, and hyoid mesoderm, aremissing, probably incorporated into the mandibular arch (pm 1

ostral boundary against the hyoid mesoderm. Pharyngeal slits areesoderm. On the left is the sequence of crest cell–mesodermave a ubiquitous origin on the neuraxis (invisible in the figure) andows), namely trigeminal crest (TC), hyoid crest (HC), and branchialmaintains stereotyped topographical relationships with the head

is subdivided into premandibular crest (PMC) and mandibular crestison of crest cell–mesoderm relationships between the lamprey (L.derson and Meier, 1981). Somitomeres in the chick are numberedased on spatial relationships between mesoderm and cephalic crestrest cells; hm, hyoid mesoderm; MC, mandibular crest cells; mm,

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the notochord and adhere to the forebrain where “pre-chordal neurocranium” (definition by Couly et al., 1993) isexpected to develop. The crest cell distribution pattern isthus conserved not only in the pharyngeal (Horigome et al.,1999), but also in the premandibular region of the lampreyembryo.

Somitomeres have been found to maintain a constanttopographical relationship with brain segments and ce-phalic crest cell populations (Jacobson, 1993; Tam andTrainor, 1994; Trainor et al., 1994). The somitomere–crestell relationship has been described in the chick embryo bynderson and Meier (1981) and in the snapping turtle byeier and Packard (1984), both patterns which are substan-

ially identical: the hyoid crest cells (rostral otic crest inhick by Anderson and Meier, 1981; HC cells in L. japonicay Horigome et al., 1999) pass in the chick embryo, be-

tween somitomeres 4 and 5. If the crest–mesoderm topo-graphical relationships are conserved as the vertebrate phy-lotype, the somitomere 5/6 boundary would correspond tohyoid mesoderm/s0 indentation of L. japonica (Fig. 5B; seeHorigome et al., 1999, for crest cell distribution). Since the

andibular mesoderm is covered by TC cells, the hyoidesoderm in L. japonica would correspond to somitomereof the chick embryo, an assignment which is consistentith Noden (1988). As noted above, such an assignmentoes not imply the conserved fate map in terms of tissueifferentiation (see Couly et al., 1992). This would partly beue to an extensive mixture of mesodermal cells outsidehe pharyngeal arches (Trainor et al., 1994). Probably, thenvironmental signals may play fundamental roles in tissueifferentiation of head mesoderm. In this respect, the headavities found in some vertebrates are peculiar for eachifferentiates into a particular set of extrinsic eye muscles.lso, the homologies of head mesodermal components maye able to establish by means of their regionalization, butot by their developmental fates.Mesoderm derived from the prechordal plate—Gastral

nd preaxial origins of the vertebrate mesoderm. Therechordal plate is commonly observed in early embryos ofertebrates. It is in essence the roof of the foregut, caudallyontinuous with the developing notochord (Jacob et al.,984). The prechordal plate in the lamprey is a single cellayer located beneath the neural tube rostral to the noto-hord (Hatta, 1891; de Selys-Longchamps, 1910). In itsarliest phase of development, the prechordal plate forms aarge part of the embryonic axis and gradually diminishes inize. It is the production of not only the mandibular and

FIG. 9. Segmental theory of the vertebrate head. (A, top) The sGoodrich (1930). (A, bottom) By removing the peripheral nervous son mesomeric formulation, in which head cavities of elasmobranchhm, hyoid mesoderm (hyoid somite); mm, mandibular mesode(premandibular somite); Pv, Platt’s vesicle. (B) Comparison between
genetic code (localized pattern of homeobox gene expressions) is recognneural subdivisions between the two animals (based on Holland and Ho

remandibular mesoderm, but also the rostral portion of theotochord that uses up the material of the prechordal plate;he prechordal plate is not a definitive structure but aiminishing mass of undifferentiated cells whose cell lin-ages are ever separating continuously through the produc-ion of the mesoderm as well as the notochord. Suchehavior is similar to the posterior end of the axial cellass; for example, the notochordal plate in mouse also

hares common morphological properties with the pre-hordal plate (Sulik et al., 1994).Although the cell-labeling experiment is still unsuccess-

ul in our laboratory, the relationships between the pre-hordal plate and mandibular mesoderm as well as theremandibular mesoderm are rather obvious in L. japonicaFigs. 1, 3, 5). In L. fluviatilis also, labeling experiments atastrular stages revealed close relationships between therechordal plate and a part of the head mesoderm (Weissen-erg, 1934, 1935). Similar relationships have also beenbserved in various vertebrates (Platt, 1891; Veit, 1924;ohrn, 1904a,b; Scammon, 1911; Goodrich, 1918; Adel-ann, 1922, 1932; Holmgren, 1940; Bjerring, 1977; Jacob et

l., 1984; Jacobson and Meier, 1984; Jacob and Jacob, 1993;. Gilland, personal communication). The preaxial meso-erm in primitive vertebrates arises as an outer pouch of theostral gut, and in this respect it resembles the anterior gutiverticulum in amphioxus (Hatschek, 1881) or rostraldhesive organs in some primitive fishes (reviewed by Nealnd Rand, 1942). Especially in a shark, Squalus, it has beeneen that the mandibular mesoderm precedes the preman-ibular, as in the lamprey (Scammon, 1911; reviewed inefferies, 1986; E. Gilland, personal communication), i.e.,he appearance of mesodermal elements here proceeds fromcaudal to rostral direction.Probably prechordal plate-derived mesoderm is also

resent in the chick, where labeling of the early prechordallate results in labeling of some extrinsic ocular musclesJacob et al., 1984; Couly et al., 1992; but see below). Aimilar assumption has been made by Adelmann (1922); theuthor called the anteriorly produced mesoderm the pre-xial mesoderm as opposed to the peristomal and gastralesoderm of Rabl (1889, 1892a; Rabl’s classification of

eristomal and gastral mesoderm appears to be an artificialne, and the term “gastral” will be used below to indicatehe mesoderm that comes from either the primitive streakr the blastopore lateral lip). In the lamprey, the gastralaraxial mesoderm is mostly segmented into somitesWeissenberg, 1934, 1935).

ntal scheme of the vertebrate head by Goodrich. Redrawn fromand chondrocranial elements, this scheme is shown to be based

ryos are equated with myotomes. hb, hypobranchial musculature;mandibular somite); ot, otocyst; pm, premandibular mesodermphioxus (top) and vertebrates (bottom). In both animals, the shared

egmeystememb

rm (am

ized in the CNS, possibly inferring the presence of homologies inlland, 1998). The configuration of the mesoderm shows profound



difference between the two; the amphioxus only possesses a series of gastral myotomes, whereas vertebrate embryos show the unsegmented
head mesoderm that is secondarily regionalized and typical myotomes arising only postotically. In possession of the whole array ofmyotomes, Goodrich’s scheme rather fits the amphioxus better than vertebrates. agd, anterior gut diverticulum; m, mouth.


A problem remains as to the boundary between thepreaxial and gastral mesoderm in the paraxial mesoderm;the hyoid mesoderm and somite 0 appear to be the earliestparts of the mesoderm to be produced in the lamprey andshark (Adelmann, 1922); these mesodermal componentsmay originate from an unseparated common mesodermalsource. Still, this does not explain the origins of amniotehead mesoderm, since a large portion of the latter definitelyoriginates from the primitive streak (Lawson et al., 1991;Schoenwolf et al., 1992; Smith et al., 1994; Psychoyos andStern, 1996; Faust et al., 1998; reviewed by Tam andBehringer, 1997). Similarly, the prechordal plate does notdifferentiate into jaw muscles in the chick (Couly et al.,1992). The above may imply that the origins (preaxial orgastral) of the mesoderm are not constant among vertebratespecies either. It appears that only some primitive verte-brates possess an extensive preaxial mesoderm. In am-niotes, on the other hand, experimental studies made so farappear to show that preaxial mesoderm occupies the ros-tromedial portion of the head mesoderm, only differentiat-ing into extrinsic ocular muscles (see Fig. 10). Comparativecell-labeling experiments will be needed among variouschordate embryos to further clarify evolutionary changes ofmesodermal production.

Reevaluation of segmental theories of vertebrate head.The problem of head segments started as a question aboutthe number of skeletal segments, and the conclusion ofGoodrich (1918, 1930; Fig. 9A) marked the end point in thishistory. Based on selachian embryos, his scheme has beenone of the most widely cited theories until today. It is truethat the scheme of Goodrich, at least at certain points indevelopment, seems to hold, not only for the shark, but alsofor the lamprey embryo if the head mesoderm is reallysegmented as it appears. Therefore, as stated by Jefferies(1986), this theory could either be the truth or all thesegmental theories based on mesodermal segments (me-someres) including Goodrich’s as well as recent ones wouldcollapse (Bertmar, 1959; Jollie, 1977; Bjerring, 1977; Jarvik,1980; Gilland and Baker, 1993); Goodrich’s scheme is asuitable starting point from which to deal with the headproblem, especially as a new step into evolutionary andmolecular developmental biology (see Northcutt, 1993;Holland, 1999).

The theories of head segments are actually referring tohead metamerism, implying the existence of simple andrepeating segmental mechanisms involved in head morpho-genesis similar to trunk patterning. Therefore, in themechanistic understanding of the head problem, the devel-opmental burden brought about by the prepattern should bedealt with as the primary cause of the metamerism. Thequestion of head segmentation can be summarized intoseveral points as follows: (1) what can be a primary elementof head segment? (source of the burden); (2) if it is asomite-equivalent unit, does it repeat with the same inter-val as branchial arches? (somitomerism versus branchiom-
erism); and (3) how many head segments are there in thepreotic region of the craniate head? (number of units); and

finally, are the units equivalent to rostral somites in am-phioxus? (homology of units). Of those, dissociation be-tween branchiomerism and somitomerism has already beendiscussed (Marshall, 1881; Froriep, 1902a, 1917; Rabl,1892b; Starck, 1963; Kuratani, 1997, and referencestherein), and the question of the variable number of postoticsegments is answered in part by involvement of the Hoxcode in the patterning of occipital bones (Burke et al., 1995).

In the trunk region, postotic somites pattern the periph-eral nervous system as a morphogenetic burden; the seg-mentation involves primarily the mesoderm. Themetameric pattern of spinal nerves has been shown to beimposed by metamerism of the paraxial mesoderm (De-twiler, 1934; Keynes and Stern, 1984; Rickmann et al.,1985; Tosney, 1987; Bronner-Fraser and Stern, 1991). Inamphioxus, muscle blocks are completely segmented alongthe body axis to the rostral tip of the head, and the PNS isentirely somitomeric, each nerve arising between two suc-cessive myotomes (Dogiel, 1903; Franz, 1927). Therefore, itwas reasonable that cephalic mesomeres or the neuromereswere extensively sought in comparative vertebrate embry-ology (Locy, 1895; Hill, 1900; Neal, 1896, 1918a; Johnston,1905; reviewed by Neal and Rand, 1942). Goodrich’sscheme was on the same line (Fig. 9A).

Typical cephalic mesomeres were believed to be presentas three or four pairs of head cavities in elasmobranchembryos (Balfour, 1878; van Wihje, 1882; Goodrich, 1918;de Beer, 1922, 1937; reviewed by Goodrich, 1930; Jarvik,1980; Jefferies, 1986). More recently, cephalic somitomereswere recognized in amniotes, amphibians, and teleosts bySEM observation. These two streams share the same mor-phological formulation as the background (theories of seg-mentalists), although they do not reconcile to each other interms of number of segments (reviewed by Northcutt,1993). In either case, the most serious problem with thesegmental theory is that the scheme is more suitable foramphioxus than for vertebrates (Fig. 9). As judged from theillustrations by Hatschek (1881; also see Presley et al.,1996; and the cover photograph of the May 1997 issue ofDevelopment by Holland et al., 1997), all the myotomes areequivalent and they arise as typical gastral mesodermwhose cell lineage is separated early from the rostral noto-chord and foregut, sequentially developing in an anterior toposterior direction, just like the trunk mesoderm of verte-brates. Moreover, the vertebrate head mesoderm does notimpose metameric pattern onto the PNS as a developmen-tal burden, and there is no intermediate animal that showsthe transition from the segmental amphioxus-like animalto the vertebrate pattern. The problem of head segmenta-tion is thus linked to the formulation of the chordatephylotype and homology of mesodermal components.

As shown in the present work as well as our previousstudies (Kuratani et al., 1997a, 1998b; Horigome et al.,1999), the lamprey pharyngula shares a series of featuresand clearly belongs to the phylotype of vertebrates; pro-
someres in the forebrain, clearly demarcated midbrain,postotic myotomes, odd–even pattern of rhombomeres and

n

esodered


cranial nerve roots, pharyngeal arches of which the first two

FIG. 10. Evolution of the head mesoderm. Different patterns of mesPhylotypes that represent the shared embryonic patterns of given montree, tricoelomate larvae of deuterostome animals are presented as ahave arisen with several different sources (reviewed by Starck, 197paraxially in the tail. In the amphioxus (b), cell lineages of notochordarise as gastral components, possessing myotomal traits (horizontallvertebrates (c and d), myotomes are all gastrally originated (horizontpreaxial (gray) and gastral (white) origins. The head mesoderm is primwhile in some derived groups like amniotes (d) it fails to be regionalimesoderm of the latter group appears less extensive than in the prototyin amniotes, giving rise to extrinsic ocular muscles (based on Couly etto resemble that of amphioxus; the body plan of the latter may have bmesodermal components. agd, anterior gut diverticulum; hm, hyoid mpm, premandibular mesoderm; s0, somite 0; 1–7, somitomeres (numb

are highly modified, three populations of dorsally migratingcephalic crest cells, and unsegmented head mesoderm:

tn


one of these characteristics is found in amphioxus. Unlike

al organization are indicated on the hypothetical phylogenetic tree.letic groups are also placed on the tree. At the top of the phylogeneticble ancestral type from which paired mesodermal components maydpole larvae of tunicates (a) possess only unsegmented mesodermrimitive gut are separated early, and all of the mesodermal segmentsched). The anterior gut diverticulum originates from the foregut. Inatched), and unsegmented head mesoderm and the notochord haveregionalized by surrounding structures into several components (c),ut may be segmented incompletely into somitomeres. The preaxiald may possibly be localized in the medial part of the head mesoderm

992). Note that the phylotype of the vertebrate ancestor does not havestablished through the loss of head mesoderm including the preaxialerm; mm, mandibular mesoderm; nt, notochord; pc, prechordal plate;).

odermophypossi8). Taand py hatally harilyzed bpe an

al., 1een e

hese vertebrate-specific features, the morphology of theeural tube may have a common ground plan among


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chordates. For example, a number of homologous ho-meobox genes are expressed along the neuraxis, showing atripartite configuration of the neural tube as the chordateprototype (Fig. 9B; Holland et al., 1992; Wada et al., 1996a,997, 1998; Wada et al., 1995, 1996b; reviewed by Hollandnd Graham, 1995; Holland, 1996; Holland and Holland,998). Moreover, anatomical similarities have been recog-ized in the fore-midbrains between amphioxus and verte-rates (reviewed by Lacalli, 1996). Therefore, the chordatehylotype, which the segmental schemes of the vertebrateead were supposed to infer, is only able to include antero-osteriorly patterned neuraxis and the shared genetic codehat may indicate homologous subdivisions of the centralervous system among chordates, but nothing can be de-cribed as to the paraxial mesoderm at present, except thatt may or may not be segmented partially; if it is to refer tohe shared pattern of the head mesoderm, the scheme muste the vertebrate phylotype and it cannot contain theegmented head mesoderm that never existed within theroup Vertebrata (Fig. 9B). The idea fits the “New Head”heory of Gans and Northcutt (1983) as well as the distinc-ion of branchiomerism and somitomerism (reviewed byuratani, 1997). Both refer to the anteroposteriorly differ-ntiated mesenchymal embryonic environment, throughhich cephalic crest cells contribute to head formationnique to vertebrates. Simultaneously, it is suggested thathe reorganization of paraxial mesodermal and preaxialtructures may have played a fundamental role in theertebrate and amphioxus evolution.Evolution of chordate mesoderm. Where could we find

he origin of the vertebrate phylotype? In vertebrate em-ryos, a part of the head mesoderm arises preaxially sharingommon origins with the preoral gut endoderm and theotochord. The notochord in amphioxus always possesses alear rostral end during development implying early disso-iation of cell lineages from the foregut, leaving no space forhe prechordal plate to show itself (Fig. 10); the amphioxusotochord elongates secondarily during development, andts rostral tip in the adult state never reflects its originalxtension (Hatschek, 1881). If we are to locate a structurehat is best equivalent to the prechordal plate in am-hioxus, it might be sought in the rostral portion of therimitive gut that gives rise to the anterior gut diverticu-um, although the diverticulum never differentiates into

uscles nor does it maintain a connection with the noto-hord; there is no exact prechordal plate homologue inmphioxus.Whatever the ancestor may have looked like, the discus-

ion so far implies that evolution of vertebrates and am-hioxus should primarily involve a profound alteration ofesodermal configuration. As one possible scenario based

n an amphioxus-like ancestor, the unsegmented headesoderm is a newly formed structure that has been gained

ndependently in the lineage of craniates (including myx-nes) and no equivalent tissue has ever been present in the
craniate lineage. In that case, the head mesoderm is uniqueo vertebrate lineages (Froriep, 1902b; Gans and Northcutt,

983). As a second scenario, some of the rostral somites inhe ancestor have lost segmental nature (Fig. 10); thus,omologies can exist between these somites and vertebrateead mesoderm components as stated by several authors

Gilland and Baker, 1993; Holland, 1999). The latter shouldnclude also the creation of the prechordal plate and itserivatives, i.e., the rostral notochord and some of the headesoderm. It is equivalently possible that amphioxus rep-

esents a secondary condition through an extensive reduc-ion of rostral structures leaving no homologies betweennterior mesodermal components (unsegmented head me-oderm and prechordal derivatives) (Fig. 10).The idea about the possible common ancestor between

he two, from which the mesoderm of tunicates might alsoave evolved, would possibly be obtained in the study ofemichordate larvae, the sister group of chordates. It maye interesting, in this context, to mention that tripartiteoeloms are typical in larvae of deuterostomes, to whichhordates and hemichordates belong (Fig. 10; Remane, citedn Starck, 1978); the larval form explains the multipleources of mesoderm and the presence of the rostralmostoelom, or protocoel, that is more or less unpaired in natures premandibular mesoderm in vertebrates.At present, it seems most likely that the possession of the

reaxial mesodermal lineage is plesiomorphic in chordates,hich should have been secondarily lost in lineages ofrochordates and cephalochordates (Fig. 10). This loss willot support the monophyly of the latter two animal groupsince the morphology of the rostral body shares few simi-arities. It is premature to conclude that they are second-rily acquired in the lineage of vertebrates when we have nodea how rostral mesoderm might have looked like in theommon ancestor of chordates, although fossil evidences ofambrian protochordates include some forms that seemed

o have possessed an unsegmented region in the headreviewed by Insom et al., 1995). Importantly, we still doot have much information about the head mesoderm ofagfish, the sister group of vertebrates, which might bringp the remote possibility that the craniate preotic meso-erm was once epithelially segmented in a somitomericashion. Will it force us to recognize another phylotype forhe group Craniata?

ACKNOWLEDGMENTS

We are grateful to Niigata Prefectural Inland Water FisheriesExperiment Station, Niigata, Japan, for the use of the facility and toDrs. Tatsuya Ueki and Shinichi Aizawa for their technical adviceand discussion. We also thank Dr. Ruth T. Yu for her criticalreading of the manuscript. Sincere gratitude is extended to Drs.Kathryn W. Tosney, Drew M. Noden, Hiroshi Wada, and EdwinGilland for their comments and valuable discussion. The CH-1antibody developed by Dr. J. Lin and the MF-20 antibody developedby Dr. D. A. Fischman were obtained from the DevelopmentalStudies Hybridoma Bank (DSHB) maintained by the Department of
Pharmacology and Molecular Sciences, Johns Hopkins UniversitySchool of Medicine (Baltimore, MD), and the Department of


Biological Sciences, University of Iowa (Iowa City, IA), underContract N01-HD-2-3144 from the NICHD. This work was sup-ported by grants-in-aid from the Ministry of Education, Science,and Culture of Japan (Specially Promoted Research).

REFERENCES

Adelmann, H. B. (1922). The significance of the prechordal plate:An interpretative study. Am. J. Anat. 31, 55–101.

Adelmann, H. B. (1932). The development of the prechordal plateand mesoderm of Amblystoma punctatum. J. Morphol. 54, 1–54.

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

Balfour, F. M. (1878). “A Monograph on the Development ofElasmobranch Fishes.” Macmillan, London.

de Beer, G. R. (1922). The segmentation of the head in Squalusacanthias. Q. J. Microsc. Sci. 66, 457–474.

de Beer, G. R. (1937). “The Development of the Vertebrate Skull.”Oxford Univ. Press, London.

Bertmar, G. (1959). On the ontogeny of the chordal skull inCharacidae, with a discussion on the chondrocranial base and thevisceral chondrocranium in fishes. Acta Zool. Stockholm 40,203–364.

Bjerring, H. C. (1971). The nerve supply to the second metamerebasicranial muscle in osteolepiform vertebrates, with some re-marks on the basic composition of the endocranium. Acta Zool.,Stockholm 52, 189–225.

Bjerring, H. C. (1977). A contribution to structural analysis of thehead of craniate animals. Zool. Script 6, 127–183.

Brachet, A. (1935). “Traie d’Embryologie des Vertebres.” Masson &Cie, Etiteurs, Paris.

Bronner-Fraser, M., and Stern, C. (1991). Effects of mesodermaltissues on avian neural crest cell migration. Dev. Biol. 143,213–217.

Buchberger, A., Seidl, K., Klein, C., Eberhardt, H., and Arnold,H.-H. (1998). cMeso-1, a novel bHLH transcription factor, isinvolved in somite formation in chick embryos. Dev. Biol. 199,201–215.

Burgess, R., Cserjesi, P., Ligon, K. L., and Olson, E. N. (1995).Paraxis: A basic helix-loop-helix protein expressed in paraxialmesoderm and developing somites. Dev. Biol. 168, 296–306.

Burke, A. C., Nelson, C. E., Morgan, B. A., and Tabin, C. (1995).Hox genes and the evolution of vertebrate axial morphology.Development 121, 333–346.

Christ, B., Jacob, M., and Jacob, H. J. (1983). On the origin anddevelopment of the ventro-lateral trunk musculature in theavian embryo. An experimental and ultrastructural study. Anat.Embryol. 166, 87–101.

Christ, B., Jacob, M., Jacob, H. J., Brand, B., and Wachtler, F. (1986).Myogenesis: A problem of cell distribution and cell interactions.In “Somites in Developing Embryos,” NATO ASI Series (R.Bellairs, D. A. Ede, and J. W. Lash, Eds.), pp. 261–276. PlenumPress, New York.

Conlon, R. A., Reaume, A. G., and Rossant, J. (1995). Notch1 isrequired for the coordinate segmentation of somites. Develop-ment 121, 1533–1545.

Cords, E. (1928). Uber die Nervenversorgung der Augenmuskelnvon Petromyzon marinus. Anat. Anz. 66, 293–295.


Couly, G. F., Colty, P. M., and Le Douarin, N. M. (1992). Thedevelopmental fate of the cephalic mesoderm in quail–chickchimeras. Development 114, 1–15.

Couly, F. C., Coltey, P. M., and Le Douarin, N. M. (1993). The tripleorigin of skull in higher vertebrates: A study in quail–chickchimeras. Development 117, 409–429.

Damas, H. (1944). Recherches sur le developpement de Lampetrafluviatilis L.—Contribution a l’etude de la cephalogenese desvertebres. Arch. Biol. Paris 55, 1–289.

Detwiler, S. R. (1934). An experimental study of spinal nervesegmentation in Amblystoma with reference to the plurisegmen-tal contribution to the brachial plexus. J. Exp. Zool. 67, 395–441.

Dogiel, A. S. (1903). Das periphere Nervensystem des Amphioxus.Anat. Heft. 21, 143–213.

Dohrn, A. (1904a). Studien zur Urgeschichte des Wirbeltierkorpers.23. Die Mandibularhohle der Selachier. Mitt. Zool. Stat. Neapel.17, 1–116.

Dohrn, A. (1904b). Studien zur Urgeschichte des Wirbeltierkorpers.24. Die Pramandibularhohle der Selachier. Mitt. Zool. Stat.Neapel. 17, 116–294.

Faust, C., Lawson, K. A., Schork, N. J., Thiel, B., and Magnuson, T.(1998). The Polycomb-group gene eed is required for normalmorphogenetic movements during gastrulation in the mouseembryo. Development 125, 4495–4506.

Franz, V. (1927). Morphologie der Akranier. Z. Ges. Anat. 27,465–692.

Freund, R., Dorfler, D., Popp, W., and Wachtler, F. (1996). Themetameric pattern of the head mesoderm—Does it exist? Anat.Embryol. 193, 73–80.

Froriep, A. (1883). Zur Entwickelungsgeschichte der Wirbelsaule,insbesondere des Atlas und Epistropheus und der OccipitalRegion. I. Beobachtung an Huhnerembryonen. Arch. Anat.Physiol. 1883, 177–234.

Froriep, A. (1885). Uber Anlagen von Sinnesorganen am Facialis,Glossopharyngeus und Vagus, uber die genetische Stellung desVagus zum Hypoglossus, und uber die Herkunft der Zungenmus-culatur. Arch. Anat. Physiol. 1885, 1–55.

Froriep, A. (1902a). Zur Entwicklungsgeschichte des Wirbeltierko-pfes. Verh. Anat. Ges. 1902, 34–46.

Froriep, A. (1902b). Einige Bemerkungen zur Kopffrage. Anat. Anz.21, 545–553.

Froriep, A. (1905). Die occipitalen Urwirbel der Amnioten imVergleich mit denen der Selachier. Verh. Anat. Ges. 1905,111–120.

Froriep, A. (1917). Die Kraniovertebralgrenze bei den Amphibien(Salamandra atra). Beitrag zur Entwicklungsgeschichte des Wir-beltierkopfes. Arch. Anat. Physiol. 191, 61–102.

Gans, C., and Northcutt, R. G. (1983). Neural crest and the originof vertebrates: A hew head. Science 220, 268–274.

Gegenbaur, C. (1872). “Untersuchungen zur vergleichenden Anato-mie der Wirbeltiere. III. Das Kopfskelett der Selachier.” WilhelmEngelmann, Leipzig.

Gilland, E., and Baker, R. (1993). Conservation of neuroepithelialand mesodermal segments in the embryonic vertebrate head.Acta Anat. 148, 110–123.

Goethe, J. W. (1820). “Zur Naturwissenschaft uberhaupt, besonderszur Morphologie. I.” Cotta, Stuttgart & Tubingen.

Goodrich, E. S. (1911). On the segmentation of the occipital regionof the head in the batrachia urodela. Proc. Zool. Soc. London1910, 101–120.

Goodrich, E. S. (1918). On the development of the segments of thehead in Scyllium. Q. J. Microsc. Sci. 63, 1–30.



Goodrich, E. S. (1930). “Structure and Development of Verte-brates.” Macmillan, London.

Grasse, P. P. (1954). “Traite de Zoologie (Anatomie, Systematique,Biologie) Tome XII Vertebres.” Masson, Paris.

Hacker, A., and Guthrie, S. (1998). A distinct developmentalprogramme for the cranial paraxial mesoderm in the chickembryo. Development 125, 3461–3472.

Hall, B. K. (1998). “Evolutionary Developmental Biology,” 2nd ed.Chapman & Hall, London.

Hatschek, B. (1881). Studien uber die Entwicklung des Amphioxus.Arb. Zool. Inst. Univ. Wien 4, 1–88.

Hatschek, B. (1892). Die Metamerie des Amphioxus und desAmmocoetes. Verh. Anat. Ges. 6, 136–162.

Hatta, S. (1891). On the formation of the germinal layers inPetromyzon. J. Coll. Sci. Imp. Univ. Tokyo Jpn. 5, 127–147.

Hill, C. (1900). Developmental history of primary segments of thevertebrate head. Zool. Jahrb. 13, 393–446.

Hinsch, G. W., and Hamilton, H. L. (1956). The developmental fateof the first somite of the chick. Anat. Rec. 125, 225–245.

Holland, N. D. (1996). Homology, homeobox genes, and the earlyevolution of the vertebrates. Memoirs Calif. Acad. Sci. 20, 63–70.

Holland, P. W. H. (1999). Embryonic development of heads, skel-etons and amphioxus: E. S. Goodrich revisited. Int. J. Dev. Biol.,in press.

Holland, P. W. H., and Graham, A. (1995). Evolution of regionalidentity in the vertebrate nervous system. Perspect. Dev. Neu-robiol. 3, 17–27.

Holland, L. Z., and Holland, N. D. (1998). Developmental geneexpression in amphioxus: New insights into the evolutionaryorigin of vertebrate brain regions, neural crest, and rostrocaudalsegmentation. Am. Zool. 38, 647–658.

Holland, P. W. H., Holland, L. Z., Williams, N. A., and Holland,N. D. (1992). An amphioxus homeobox gene: Sequence conser-vation, spatial expression during development and insights intovertebrate evolution. Development 116, 653–661.

Holland, L. Z., Pace, D. A., Blink, M. L., Kene, M., and Holland,N. D. (1995). Sequence and expression of amphioxus alkalimyosin light chain (AmphiMLC-alk) throughout development:Implications for vertebrate myogenesis. Dev. Biol. 171, 665–676.

Holland, L. Z., Kene, M., Williams, N. A., and Holland, N. D.(1997). Sequence and embryonic expression of the amphioxusengrailed gene (AmphiEn): The metameric pattern of transcrip-tion resembles that of its segment-polarity homolog in Drosoph-ila. Development 124, 1723–1732.

Holmgren, N. (1940). Studies on the head in fishes. Embryological,morphological, and phylogenetical researches. 1. Developmentof the skull in sharks and rays. Acta Zool. Stockholm 21, 51–267.

Horigome, N., Myojin, M., Ueki, T., Hirano, S., Aizawa, S., andKuratani, S. (1999). Development of cephalic neural crest cells inembryos of a cyclostome, Lampetra japonica, with special refer-ence to the evolution of the jaw. Dev. Biol. 206, 287–308.

Insom, E., Pucci, A., and Simonetta, A. M. (1995). Cambrianprotochordata, their origin and significance. Boll. Zool. 62,243–252.

Jacob, R. S. M., and Jacob, H. J. (1993). The avian prechordal headregion: A morphological study. J. Anat. 183, 75–89.

Jacob, M., Jacob, H. J., Wachtler, F., and Christ, B. (1984). Ontogenyof avian extrinsic ocular muscles. I. A light- and electron-microscopic study. Cell Tiss. Res. 237, 549–557.

Jacobson, A. G. (1993). Somitomeres: Mesodermal segments in the
head and trunk. In “The Vertebrate Skull” (J. Hanken and B. K.Hall, Eds.), Vol. 1, pp. 42–76. Univ. Chicago Press, Chicago.

Jacobson, A. G., and Meier, S. (1984). Morphogenesis of the head ofthe newt: Mesodermal segments, neuromeres, and distribution ofneural crest. Dev. Biol. 106, 181–193.

Janvier, P. (1993). Patterns of diversity in the skull of jawless fishes.In “The Skull” (J. Hanken and B. K. Hall, Eds.), Vol. 2, pp.131–188. Univ. Chicago Press, Chicago and London.

Jarvik, E. (1980). “Basic Structure and Evolution of Vertebrates.”Academic Press, London.

Jefferies, R. P. S. (1986). “The Ancestry of the Vertebrates.” BritishMuseum (Natural History), London.

Johnston, J. B. (1905). The morphology of the vertebrate head fromthe viewpoint of the functional divisions of the nervous system.J. Comp. Neurol. 15, 175–275.

Jollie, M. T. (1973). On the origin of the chordates. Acta Zool.Stockholm 54, 81–100.

Jollie, M. T. (1977). Segmentation of the vertebrate head. Am. Zool.17, 323–333.

Karnovsky, M. J., and Roots, L. (1964). A direct coloring thiocholinemethod for cholinesterases. J. Histochem. Cytochem. 12, 219–221.

Keynes, R. J., and Stern, C. D. (1984). Segmentation in the verte-brate nervous system. Nature 310, 786–789.

Koltzoff, N. K. (1901). Entwicklungsgeschichte des Kopfes vonPetromyzon planeri. Bull. Soc. Nat. Moscou 15, 259–289.

Krumlauf, R. (1993). Hox genes and pattern formation in thebranchial region of the vertebrate head. Trends Genet. 9, 106–112.

Kuratani, S. (1997). Distribution of postotic crest cells in the chickembryo defines the trunk/head interface: Embryological inter-pretation of crest cell distribution and evolution of the vertebratehead. Anat. Embryol. 195, 1–13.

Kuratani, S., Ueki, T., Aizawa, S., and Hirano, S. (1997a). Peripheraldevelopment of cranial nerves in a cyclostome, Lampetra ja-ponica: Morphological distribution of nerve branches and thevertebrate body plan. J. Comp. Neurol. 384, 483–500.

Kuratani, S., Matsuo, I., and Aizawa, S. (1997b). Developmentalpatterning and evolution of the mammalian viscerocranium:Genetic insights into comparative morphology. Dev. Dyn. 209,139–155.

Lacalli, T. C. (1996). Landmarks and subdomains in the larval brainof Branchiostoma: Vertebrate homologs and invertebrate ante-cedents. Isr. J. Zool. 42, 131–146.

Langille, R. M., and Hall, B. K. (1988). Role of the neural crest indevelopment of the trabeculae and branchial arches in embryonicsea lamprey, Petromyzon marinus (L). Development 102, 301–310.

Lawson, K. A., Meneses, J. J., and Pedersen, R. A. (1991). Clonalanalysis of epiblast fate during germ layer formation in themouse embryo. Development 113, 891–911.

Le Lievre, C. S. (1978). Participation of neural crest-derived cells inthe genesis of the skull in birds. J. Embryol. Exp. Morphol. 47,17–37.

Locy, W. A. (1895). Contributions to the structure and develop-ment of the vertebrate head. J. Morphol. 11, 497–594.

Marshall, A. M. (1881). On the head cavities and associated nervesof elasmobranchs. Q. J. Microsc. Sci. 21, 72 (cited in Goodrich,1918).

Martindale, M. Q., Meier, S. P., and Jacobson, A. G. (1987).
Mesodermal metamerism in the teleost, Oryzias latipes. J.Morphol. 193, 241–252.


Meier, S. (1979). Development of the chick mesoblast. Formation ofthe embryonic axis and establishment of the metameric pattern.Dev. Biol. 73, 25–45.

Meier, S. (1982). The development of segmentation in the cranialregion of vertebrate embryos. Scan. Electron Microsc. 3, 1269–1282.

Meier, S., and Packard, D. S., Jr. (1984). Morphogenesis of thecranial segments and distribution of neural crest in embryos ofthe snapping turtle, Chelydra serpentina. Dev. Biol. 102, 309–323.

Muller, M., Weizsacker, E. V., and Campos-Ortega, J. A. (1996).Expression domains of a zebrafish homologue of the Drosophilapair-rule gene hairy correspond to primordia of alternatingsomites. Development 122, 2071–2078.

Neal, H. V. (1896). A summary of studies on the segmentation ofthe nervous system in Squalus acanthias. Anat. Anz. 12, 377–391.

Neal, H. V. (1914). Morphology of the eye muscle nerves. J.Morphol. 25, 1–186.

Neal, H. V. (1918a). Neuromeres and metameres. J. Morphol. 31,293–315.

Neal, H. V. (1918b). History of eye muscles. J. Morphol. 30,433–453.

Neal, H. V., and Rand, H. W. (1942). “Comparative Anatomy.”Blakiston Co., Philadelphia.

Nieto, M. A., Gilardi-Hebenstreit, P., Charnay, P., and Wilkinson,D. G. (1992). A receptor protein tyrosine kinase implicated in thesegmental patterning of the hindbrain and mesoderm. Develop-ment 116, 1137–1150.

Noden, D. M. (1988). Interactions and fates of avian craniofacialmesenchyme. Development 103(Suppl.), 121–140.

Northcutt, R. G. (1993). A reassessment of Goodrich’s model ofcranial nerve phylogeny. Acta Anat. 148, 71–80.

Oken, L. (1807). “Uber die Bedeutung der Schadelknochen.” Gob-hardt, Bamberg.

Ordahl, C. P., and Le Douarin, N. M. (1991). Two myogeniclineages within the developing somite. Development 114, 339–353.

Owen, R. (1848). “On the Archetype and Homology of the Verte-brate Skeleton.” Voorst, London.

Patapoutian, A., Miner, J. H., Lyons, G. E., and Wold, B. (1993).Isolated sequences from the linked Myf-5 and MRF4 genes drivedistinct patterns of muscle-specific expression in transgenicmice. Development 118, 61–69.

Platt, J. B. (1891). A contribution to the morphology of the verte-brate head, based on a study of Acanthias wulgaris. J. Morphol. 5,79–106.

Presley, R., Horder, T. J., and Slipka, J. (1996). Lancelet develop-ment as evidence of ancestral chordate structure. Isr. J. Zool. 42,97–116.

Psychoyos, D., and Stern, C. D. (1996). Fates and migratory routesof primitive streak cells in the chick embryo. Development 122,1523–1534.

Rabl, C. (1889). Theorie des Mesoderms. Morphol. Jahrb. 15,113–252.

Rabl, C. (1892a). Theorie des Mesoderms. Fortsetzung. Morphol.Jahrb. 19, 65–144.

Rabl, C. (1892b). Uber die Metamerie des Wirbeltierkopfes. Ver.Anat. Ges. 6, 104–136.

Rathke, H. (1839). “Bemerkungen uber Entwicklung des Schadelsder Wirbelthiere.” Konigberg.


Reichert, C. (1838). “Vergleichende Entwicklungsgeschichte desKopfes der Amphibien.” Konigberg,.

Richardson, M. K. (1995). Heterochrony and the phylotypic period.Dev. Biol. 172, 412–421.

Richardson, M. K., Hanken, J., Gooneratne, M. L., Pieau, C.,Raynaud, A., Selwood, L., and Wright, G. M. (1997). There is nohighly conserved embryonic stage in the vertebrates: Implica-tions for current theories of evolution and development. Anat.Embryol. 196, 91–106.

Rickmann, M., Fawcett, J., and Keynes, R. J. (1985). The migrationof neural crest cells and the growth of motor axons through therostral half of the chick somite. J. Embryol. Exp. Morphol. 90,437–455.

Rubenstein, J. L. R., and Puelles, L. (1994). Homeobox gene expres-sion during development of the vertebrate brain. Curr. Top. Dev.Biol. 29, 1–63.

Saga, Y., Hata, N., Kobayashi, S., Magnuson, T., Seldin, M. F., andTaketo, M. M. (1996). MesP1: A novel basic helix-loop-helixprotein expressed in the nascent mesodermal cells during mousegastrulation. Development 122, 2769–2778.

Saga, Y., Hata, N., Koseki, H., and Taketo, M. M. (1997). MesP2: Anovel mouse gene expressed in the presegmented mesoderm andessential for segmentation initiation. Genes Dev. 11, 1827–1839.

Scammon, R. E. (1911). Normal plates of the development ofSqualus acanthias. Keibel’s Normaltafeln Entwicklungsge-schichte Wirbeltiere 12, 1–140.

Schoenwolf, G. C., Garcia-Martinez, V., and Dias, M. S. (1992).Mesoderm movement and fate during avian gastrulation andneurulation. Dev. Dyn. 193, 235–248.

de Selys-Longchamps, M. (1910). Gastrulation et formation desfeuillets chez Petromyzon planeri. Arch. Biol. 25, 1–75.

Smith, J. L., Gesteland, K. M., and Schoenwolf, G. C. (1994).Prospective fate map of the mouse primitive streak at 7.5 days ofgestation. Dev. Dyn. 201, 279–289.

Sporle, R., and Schughart, K. (1997). System to identify individualsomites and their derivatives in the developing mouse embryo.Dev. Dyn. 210, 216–226.

Starck, D. (1963). Die Metamerie des Kopfes der Wirbeltiere. Zool.Anz. 170, 393–428.

Starck, D. (1978). “Vergleichende Anatomie der Wirbeltiere aufevolutionsbiologicher Grundlage.” Band 1: Theoretische Grund-lagen. Stammesgeschichte und Systematik unter Berucksichtungder niedern Chordata. Springer Verlag, Berlin.

Sulik, K., Dehart, D. B., Inagaki, T., Carlson, J. L., Vrablic, T.,Gesteland, K., and Schoenwolf, G. C. (1994). Morphogenesis ofthe murine node and notochordal plate. Dev. Dyn. 201, 260–278.

Tahara, Y. (1988). Normal stages of development in the lamprey,Lampetra reissneri (Dybowski). Zool. Sci. 5, 109–118.

Tajbakhsh, S., and Sporle, R. (1998). Somite development: Con-structing the vertebrate body. Cell 92, 9–16.

Tam, P. P., and Behringer, R. R. (1997). Mouse gastrulation: Theformation of the mammalian body plan. Mech. Dev. 68, 3–25.

Tam, P. P. L., and Meier, S. (1982). The establishment of thesomitomeric pattern in the mesoderm of the gastrulating mouseembryo. Am. J. Anat. 164, 209–225.

Tam, P. P. L., and Trainor, P. A. (1994). Segmentation and specifi-cation of the paraxial mesoderm. Anat. Embryol. 189, 275–307.

Tam, P. P. L., Meier, S., and Jacobson, A. G. (1982). Differentiationof the metameric pattern in the embryonic axis of the mouse. II.
Somitomeric organization of the presomitic mesoderm. Differ-entiation 21, 109–122.


Tosney, K. W. (1987). Proximal tissues and patterned neuriteoutgrowth at the lumbosacral level of the chick embryo: Dele-tion of the dermamyotome. Dev. Biol. 122, 540–558.

Trainor, P. A., Tan, S.-S., and Tam, P. P. L. (1994). Cranial paraxialmesoderm: Regionalization of cell fate and impact on craniofacialdevelopment in mouse embryos. Development 120, 2397–2408.

Veit, O. (1924). Beitrage zur Kenntnis des Kopfes der Wirbeltiere II.Fruhstadien der Entwicklung des Kopfes von Lepidosteus osseusund ihre prinzipielle Bedeutung fur die Cephalogenese der Wir-beltiere. Morphol. Jahrb. 53, 320–390.

Veit, O. (1939). Beitrage zur Kenntnis des Kopfes der Wirbeltiere III.Beobachtungen zur Fruhentwicklung des Kopfes von Petromy-zon planeri. Morph. Jahrb. 84, 86–107.

Wada, H., Holland, P. W. H., and Satoh, N. (1996a). Origin ofpatterning in neural tubes. Nature 384, 123.

Wada, H., Holland, P. W. H., Sato, S., Yamamoto, H., and Satoh, N.(1997). Neural tube is partially dorsalized by overexpression ofHrPax-37: The ascidian homologue of Pax-3 and Pax-7. Dev.Biol. 187, 240–252.

Wada, H., Saiga, H., Satoh, N., and Holland, P. W. H. (1998).Tripartite organization of the ancestral chordate brain and theantiquity of placodes: Insights from ascidian Pax-2/5/8, Hox andOtx genes. Development 125, 1113–1122.

Wada, S., Katsuyama, Y., Yasugi, S., and Saiga, H. (1995).Spatially and temporally regulated expression of LIM class


homeobox gene Hrlim suggests multiple distinct functions indevelopment of the ascidian, Halocynthia roretzi. Mech. Dev.51, 115–126.

Wada, S., Katsuyama, Y., Sato, Y., Itoh, C., and Saiga, H. (1996b).Hroth, an orthodenticle-related homeobox gene of the ascidian,Halocynthia roretzi: Its expression and putative roles in the axisformation during embryogenesis. Mech. Dev. 60, 59–71.

Wedin, B. (1949). The development of the head cavities in Alligatormississippiensis Daud. Lunds Univ. Arssikr. N. F. Avs. 2 45,1–32 (cited in Starck, 1963).

Weissenberg, R. (1934). Untersuchungen uber den Anlageplan beimNeunaugenkeim: Mesoderm, Rumpfdarmbildung und Ubersichtder zentralen Anlagezonen. Anat. Anz. 79, 177–199.

Weissenberg, R. (1935). Untersuchungen uber den Anlageplan beimNeunaugenkeim. II. Das genauere Verhalten der Gliederung derzentralen Anlagezonen. Anat. Anz. 82, 20–33.

van Wijhe, J. W. (1882). Uber die Mesodermsegmente und dieEntwicklung der Nerven des Selachierkopfes. Ver. Akad. Wiss.22, 1–50.

Received for publication December 3, 1998
Revised March 4, 1999
Accepted March 4, 1999


developmental morphology of the head mesoderm and ...developmental morphology of the head mesoderm...

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