Developmental Morphology of the Head Mesoderm and Reevaluation

Home , Pharyngeal slit

Developmental Biology 210, 381–400 (1999) Article ID dbio.1999.9266, available online at http://www.idealibrary.com on Developmental Morphology of the Head Mesoderm and Reevaluation of Segmental Theories of the Vertebrate Head: Evidence from Embryos of an Agnathan 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 links amphioxus and gnathostomes. To reevaluate the segmental theory of classical comparative embryology, mesodermal development was observed in embryos of a lamprey, Lampetra japonica, by scanning electron microscopy and immunohistochemistry. Signs of segmentation are visible in future postotic somites at an early neurula stage, whereas the rostral mesoderm is unsegmented and rostromedially conﬂuent with the prechordal plate. The premandibular and mandibular mesoderm develop from the prechordal plate in a caudal to rostral direction and can be called the preaxial mesoderm as opposed to the caudally developing gastral mesoderm. With the exception of the premandibular mesoderm, the head mesodermal sheet is secondarily regionalized by the otocyst and pharyngeal pouches into the mandibular mesoderm, hyoid mesoderm, and somite 0. The head mesodermal components never develop into cephalic myotomes, but the latter develop only from postotic somites. These results show that the lamprey embryo shows a typical vertebrate phylotype and that the basic mesodermal conﬁguration of vertebrates already existed prior to the split of agnatha–gnathostomata; lamprey does not represent an intermediate state between amphioxus and gnathostomes. Unlike interpretations of theories of head segmentation that the mesodermal segments are primarily equivalent along the axis, there is no evidence in vertebrate embryos for the presence of preotic myotomes. We conclude that mesomere-based theories of head metamerism are inappropriate and that the formulated vertebrate head should possess the distinction between primarily unsegmented head mesoderm which includes preaxial components at least in part and somites in the trunk which are shared in all the known vertebrate embryos as the vertebrate phylotype. © 1999 Academic Press Key Words: lamprey; head mesoderm; neural crest; somites; segmentation; pharyngeal arch.

INTRODUCTION reﬁned through embryological studies by Gegenbaur (1872), Balfour (1878), van Wijhe (1882), and others (reviewed by Whether the vertebrate head is segmented as is the trunk Goodrich, 1930; de Beer, 1937; Starck, 1978; Jarvik, 1980; has long been an intriguing issue of vertebrate morphology and Jefferies, 1986). The segment assumed in this theory and embryology. Goethe (1820) and Oken (1807) ﬁrst put was a primordial metameric unit comparable to a preverte- forth the “vertebral theory” of the skull, in which the skull bra or a somite in the trunk. was proposed to be composed of several transformed verte- The theory of head mesoderm segmentation was at one brae. Supported by great anatomists (Owen, 1848; Rathke, time forgotten but resurrected by new methods of embryo- 1839; Reichert, 1838), the theory became temporarily the logical observation. For example, in a series of descriptive mainstream of the head problem (Kopffrage) and was then works based on scanning electron microscopy, the presence of primordial segmentation in the mesoderm was detected 1 To whom correspondence should be addressed at Department and these incomplete segmental units were named cephalic of Biology, Okayama University Faculty of Science, 3-1-1 Tsushi- somitomeres (reviewed by Jacobson, 1993). The concept of manaka, Okayama, Okayama 700-8530, Japan. Fax: ϩ81-86-251- the segmented body plan has also gained molecular bases of 7876. E-mail: [email protected]. metamerism as can typically be seen in some homeobox-

0012-1606/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 381 382 Kuratani, Horigome, and Hirano containing genes (reviewed by Krumlauf, 1993; and Ruben- Histochemistry. Embryos were fixed with PFA/PBS for1hat stein and Puelles, 1994) as well as a number of regulatory room temperature and briefly washed with 0.9% NaCl. They were genes involved in the segmentation of paraxial mesoderm at once treated with acetylcholinesterase (AChE) according to the in the trunk (reviewed by Tajbakhsh and Spo¨rle, 1998), procedure of Karnovsky and Roots (1964). The reaction was stopped again increasing its importance on a new paradigm of by postfixation with PFA/PBS and the embryos were stored in PBS. For observation, they were dehydrated with a graded series of developmental biology. methanol, clarified with BABB, and mounted on depression slide Until now, the segmentation theory has been biased by glass. idiosyncrasy established by elasmobranch embryology as well as morphology of the amphioxus. This is partly be- cause the elasmobranch embryos exhibit tandemly ar- RESULTS AND DISCUSSION ranged mesodermal segments or “head cavities,” which is reminiscent of the amphioxus condition. Lampreys, the Development of the head mesoderm in L. japonica. sister group of gnathostomes, have been less extensively One of the reasons for the misconception that lampreys studied than elasmobranchs and have been regarded as an represent an intermediate state between the amphioxus and intermediate group linking amphioxus and gnathostomes. gnathostomes was the morphology of the preotic mesoderm They are also one of the animal groups whose mesodermal which was illustrated to be segmented as myotomes in morphology remains unexplored by scanning electron mi- amphioxus. The most precise and influential description in croscopy. The present paper is thus intended to give a this respect has been that by Damas (1944) in L. fluviatilis, precise description of the developing mesoderm in Lam- which has been cited in major literatures (Grasse´, 1954; petra japonica, to evaluate its developmental pattern on the Jarvik, 1980; Jefferies, 1986). Unlike the report by Damas phylogenetic tree, and also to reevaluate the problem of (1944), however, preotic mesoderm is never segmented in L. vertebrate head segmentation in search of the most appro- japonica. priate formulation of the vertebrate head as a component of The first overt mesoderm of the L. japonica embryo is vertebrate phylotype, the shared specification of embryonic seen as the separation of a layer from the endoderm at stage body plan (reviewed by Hall, 1998). By use of scanning 19 (Fig. 1); a faint boundary is being formed lateral to the electron microscopy (SEM), we confirmed in this animal dorsal midline, delineating the mesoderm laterally which is that there is no complete segmentation in the head meso- more conspicuous at caudal levels. Rostrally, the mesoderm derm and that myotomes are restricted to postotic meso- gradually merges into the endoderm with no clear demar- derm as seen in gnathostome embryos. cation (Figs. 1A and 1B). At the rostral end, the chorda and paraxial mesoderms as well as the endoderm are extensively continuous with the foregut endoderm, forming a single medial plate, or the prechordal plate (Figs. 1B and MATERIALS AND METHODS 1C). Segmental boundaries in the paraxial mesoderm are re- Embryos and scanning electron microscopy. Lamprey em- stricted in the future trunk region, appearing in a rostral to bryos were obtained and maintained in the laboratory as previously caudal direction, the rostralmost one representing the described (Horigome et al., 1999). Developmental stages were somite 0/1 boundary (Figs. 1B and 2). Bilateral asymmetry is defined according to the description by Tahara (1988) and more commonly observed in the segmental pattern (Fig. 1). Simi- detailed stages of 19.5, 20.5, 21.5, and 22.5 are as described lar observations were made by Veit (1939), who noticed in (Horigome et al., 1999). Embryos were fixed in 2.5% glutaraldehyde Petromyzon planeri that the left mesoderm develops faster in 0.01 M phosphate buffer (pH 7.4; PB) and processed for SEM than the right. However, in L. japonica, examination of a observation as described previously (Horigome et al., 1999). number of specimens and comparison with older embryos Immunohistochemistry. Embryos were fixed with 4% parafor- maldehyde in 0.1 M phosphate-buffered saline (PFA/PBS), washed indicated that there is no clear preference as to which side in 0.9% NaCl/distilled water, dehydrated in a graded series of of the embryo develops larger mesoderm rostral to the methanol (50, 80, 100%), and stored at Ϫ20°C. The immunostain- boundary (Fig. 2). Bilateral asymmetry is somehow compen- ing procedure has been described in Kuratani et al. (1997a). Briefly, sated at later stages, presumably due to differential growth after washing in TST (TST is Tris–HCl-buffered saline: 20 mM of the mesoderm. Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% Triton X-100), the samples Through stages 19 and 20, the rostralmost mesoderm is were blocked with aqueous 1% periodic acid and with 5% nonfat continuous with the prechordal plate medially (Figs. 1B– dry milk in TST (TSTM) and incubated in the primary antibody, 1E), but ventrolaterally the mesoderm is gradually sepa- CH-1 (purchased from the Developmental Studies Hybridoma rated from the endoderm (Figs. 1F and 3A). The process of Bank, Iowa City, IA; diluted 1/1000 in spin-clarified TSTM con- separation proceeds from a caudal to rostral direction (Figs. taining 0.1% sodium azide) which recognizes tropomyosin. The secondary antibody used was horseradish peroxidase (HRP)- 1E and 1F). This mesoderm shows slight lateral expansion conjugated goat anti-mouse IgG (ZYMED Lab. Inc., San Francisco, (Fig. 3B), delineated caudally by an endodermal swelling CA) diluted 1/200 in TSTM. After washing in TST, the embryos representing pharyngeal pouch 1 (Fig. 3B). This mandibular were allowed to react in TS with the peroxidase substrate 3,3Ј- mesoderm develops as a typical enterocoel as has already diaminobenzidine (DAB, 150 ␮g/ml) and 0.01% hydrogen peroxide. been reported in lamprey embryos (Hatta, 1891; de Selys-

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 has been removed. The lateral border of the mesoderm is indicated by dots. Rostrally the border disappears, indicating that the mesoderm in this region is not completely dissociated from the endoderm. (B) A slightly older embryo of the same stage as seen from the dorsal view. The 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 segmental boundaries in the paraxial mesoderm (arrows), representing somite 0/1 boundaries, shows an asymmetrical developmental pattern. The lateral notch of the rostral mesoderm is indicated by an asterisk. (C) The mesoderm on the left side has been removed from this embryo. Note that the rostral portion of the notochord is still continuous with the endoderm laterally (arrows). (D) Anterior portion of the embryo seen from the dorsal view. Surface ectoderm, neural rod (nr), as well as the notochord (nt) have been partially removed. Early mesoderm (mes) is seen paraxially on the left side (top of the ﬁgure), consisting of cells smaller than the underlying endodermal cells (en) that are exposed on the right side of the embryo. Arrows indicate the cut edge of the ectoderm. Ventral to the notochord (nt), the endodermal roof is exposed which rostrally merges to the prechordal plate (pc). The endodermal roof is partially removed (*) and the cut edge (single arrowhead) extends to the prechordal plate. Double arrowhead corresponds to the somite 0/1 boundary. (E) The rostral mesoderm has grown laterally to form the mandibular mesoderm (mm). Arrows indicate segmental boundaries of somites and dots the lateral limit of the mandibular mesoderm. (F) A slightly older embryo seen from the right lateral view. Only the surface ectoderm has been removed. The mandibular mesoderm (mm) is a distinctive component whose rostral end is not yet dissociated from the endoderm (arrowhead). Caudal to the mandibular mesoderm, a part of endoderm (*) is exposed, representing the future pharyngeal pouch 1. Arrows indicate the cut edge of the mesoderm. en, endoderm; mes, mesoderm; nr, neural rod; nt, notochord; pc, prechordal plate; s1–s2, somites. Bar: 100 ␮m.

FIG. 2. Diagrams of the segmental boundary pattern in several embryos stages 19.5 through 20.5. In every case, the rostralmost segmental boundary represents the somite 0/1 boundary. There is no clear right–left preference in the number and rate of boundary formation.

Longchamps, 1910; Damas, 1944). Through stages 20 to 21, rostral to the mandibular mesoderm) and morphology some distinct regions can be distinguished in the rostral (paired configuration connected at the midline) (Fig. 5D), mesoderm (Figs. 3A–3D); the mesoderm caudal to the first this mesoderm is identified as the premandibular meso- pharyngeal pouch is further regionalized into two portions derm. rostrocaudally by the otocyst and second pharyngeal pouch Thus, in L. japonica embryos, the head mesoderm con- (Figs. 3E–3G and 4). The rostral subdivision is incorporated sists of the mandibular, hyoid, and premandibular meso- into the second pharyngeal arch and is called the hyoid derm as well as somite 0. Of those, only the last component mesoderm. The caudal subdivision, or somite 0, resembles is located in a postotic position. Unlike the postotic a somite in both size and shape, but lacks a clear rostral somites that are segmented by clear dorsomedial bound- boundary (Figs. 3C and 3E). aries, these cephalic components are confluent with each By stage 21, the mandibular mesoderm is mostly delin- other through the stages observed, with the exception of the eated from the prechordal plate (Fig. 5E), which has clearly premandibular mesoderm whose appearance is rather late. diminished in size (see Figs. 1 and 3). At this stage the Development of myotomes in L. japonica reveals the anterior tip of the notochord can be separated from the shared phylotype of vertebrates. The second reason for prechordal plate: the prechordal plate is now an indepen- the misconception of the lamprey as an intermediate crea- dent entity (Fig. 5B), which by stage 22 has grown laterally ture was the belief that ammocoete larvae possess myo- to form a pair of mesoderm in front of the mandibular tomes apparently in preotic levels (Fig. 6A; Hatschek, 1892; mesoderm (Fig. 5C). From the location (caudal to the eye, Koltzoff, 1901; Neal, 1914, 1918a,b; Neal and Rand, 1942;

FIG. 3. Mesodermal development in L. japonica embryos between stages 20 and 21. (A) Left lateral view of a stage 20 embryo. The head mesoderm is regionalized into the mandibular mesoderm (mm) and hyoid mesoderm (hm) ϩ somite 0 at the level of pharyngeal pouch 1 (pp1). The mandibular mesoderm has separated from the endoderm. (B) Mandibular mesoderm as seen from the dorsoanterior view. Anterior is to the right. The surface ectoderm, neural rod, as well as the notochord have been removed. The endodermal roof (er) is exposed and the broken portion (*) shows the fusion of the rostral notochord, the prechordal plate (pc), and the endodermal roof at this position. (C) Left lateral view of a stage 20.5 embryo. An indentation (arrowhead) formed by the otocyst regionalizes the hyoid mesoderm (hm) and somite 0 (s0). (D) Anterior view of another stage 20.5 embryo whose surface ectoderm and neural rod have been removed. Note the fusion of the notochord (nt) and the prechordal plate (pc) which laterally continues into 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 crest cells. Bars: 100 ␮m (A, C, E–G) and 50 ␮m (B, D).

mental stages, the rostral myotomes shift rostrally, with the first myotome sliding ventral to the otocyst (Figs. 6C–6E). The second myotome (m2) is always characteristically regionalized into dorsoventral halves. Detailed observation of the intermediate stages between stages 24 and 25 revealed no sign of additional myotome development rostral to m1 (Fig. 6D). Thus, rostral myotomes are all postotically derived: m1 develops into the rostral half of the infraoptic myotome and dorsal and ventral halves of m2 into the supraoptic and caudal half of the infraoptic myotomes, respectively (Figs. 6A and 7). The basic myotome developmental pattern is thus very similar between lampreys and gnathostomes. In gnathostome embryogenesis, myotomes always develop only from trunk (or postotic) somites (Froriep, 1883, 1902a, 1905, 1917; Rabl, 1892b; Goodrich, 1911; de Beer, 1922; reviewed by Starck, 1963). The rostralmost somite often fails to form a myotome since it disintegrates early in development (Hinsch and Hamilton, 1956). Myotomes are distinct embryonic structures that form epaxial myomeres in amniote embryos (Christ et al., 1983, 1986; Ordahl and Le Douarin, 1991), and their initial differentiation is exemplified by the early expressions of myogenic marker genes belonging to the MyoD family and Pax3 genes (see Spo¨rle FIG. 4. Sequential regionalization of the cephalic mesoderm in L. and Schughart, 1997; Hacker and Guthrie, 1998) or early japonica. At stage 19.5, the rostralmost clear segmental boundary immunoreactivity to CH-1 and MF-20 monoclonal antibod- represents the somite 0/1 boundary. Caudal to this boundary ies as well as AChE reactivity. In this connection, all the develop a series of metamerical somites by stage 20. On the ventral aspect, a notch is formed at the level of pharyngeal pouch 1 (pp1). early somites in amphioxus express the alkali myosin light At this point, the rostral mesoderm is regionalized into two chain gene (AmphiMLC-alk), a marker of myotome (Hol- portions: the mandibular mesoderm (mm) and the caudal portion. land et al., 1995), indicating that rostral mesoderm in By stage 20.5, the otocyst and first pharyngeal pouch regionalize amphioxus is more like somites than the head mesoderm of the caudal mesodermal portion into the hyoid mesoderm (hm) and vertebrates. somite 0 (s0). In the head mesoderm, the above-listed markers appear substantially late compared to myotomes (Hacker and Guthrie, 1998, and references therein), which is also the case with branchial muscles in the lamprey (Fig. 6A). In this also see Jollie, 1973). These myotomes were often treated as regard, Myf-5 expression has been known to involve a direct derivatives of preotic segments (Neal, 1918a,b), separate control that is only activated in the head meso- which is not actually the case as shown below. derm but not in somites (Patapoutian et al., 1993). More- By staining with CH-1 monoclonal antibody that recog- over, regulatory genes that are associated with initiation of nizes tropomyosin, the initial myotome development is the somitic segmentation (Sek: Nieto et al., 1992; paraxis: detected at stage 21 of L. japonica (Fig. 6B). All immunore- Burgess et al., 1995; Notch: Conlon et al., 1995; hairy: active myotomes are postotic in position: comparison with Mu¨ller et al., 1996; Mesp: Saga et al., 1996, 1997; Buch- a SEM-observed embryo (Fig. 2E) revealed that the rostral- berger et al., 1998) are not expressed in the preotic region most myotome (m1) arises from s1, the first complete with any metamerical patterns; it is these segmental traits somite developing postotically. In the following develop- that make somites distinct from the head mesoderm. Al-

FIG. 5. Developmental sequence of the premandibular mesoderm in L. japonica. In all the embryos except D, the surface ectoderm and the rostral portion of the neural tube have been removed. (A) At stage 20.5, a boundary is being formed between the prechordal plate (pc) and the mandibular mesoderm (mm). The rostral notochord is still contiguous with the prechordal plate (arrow). (B) Stage 21. The notochord has now attained a clear rostral tip (arrow). (C) By stage 22, the prechordal plate has grown laterally to form the premandibular mesoderm (pm). (D) Stage 24 embryo from which all the mesenchyme has been removed. Seen from the left lateral view. Note that the middle part of the premandibular mesoderm (arrow) is the remnant of the prechordal plate, located rostral to the notochordal tip (nt) and caudal to the optic vesicle (ev). (E) Stage 21 embryo. Trigeminal crest cells (TC) are colored 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 the crest cells is removed on the right side of the embryo. Dots on the left side of the photograph represent the connection between the prechordal 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 TC cells. 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 ␮m (A–C, E, F) and 100 ␮m (D).

FIG. 6. Development of myotomes in L. japonica. In all the figures, anterior to the left, except in D. Muscle precursors were either shown histochemically by AChE activity (A and D) or immunochemically stained with CH-1 antibody that recognizes tropomyosin (B, C, E). In a stage 28ϩ larva (A), rostral myotomes are apparently located preotically. (B–E) Early development of myotomes is shown in embryos at stages 21 (B), 24 (C), 24.5 (D), and 25 (E). Pharyngeal pouches are indicated by broken lines. All the myotomes are at first located postotically (B and C). The rostralmost myotome (m1) is of the first somite origin (see Fig. 3D). The second myotome is characteristically divided into two halves dorsoventrally after stage 24.5 (D). Note that rostral myotomes move anteriorly through development and no additional myotome appears in front of m1. m1 to m8, myotomes; ot, otocyst; pp1 to pp5, pharyngeal pouches. Bars: 100 ␮m.

though the above-listed segmental genes have not yet been mesodermal component of an intermediate trait may have cloned in the lamprey, it seems most likely that the once existed in any hypothetical ancestors. difference between the myotome-producing somites and Morphology of the head mesoderm in vertebrates: Topo- rostral mesoderm is distinct in all the vertebrates, and there graphical conservation. The classical concept of verte- is no positive evidence at present, in the evolutionary brate head metamerism was based on the morphology of lineage of craniates, to show that preotic myotome or a shark embryos where clearly segmented head cavities

FIG. 7. Reinterpretation of mesodermal morphology of lampreys. (A) Pharyngular state of the lamprey. Mesodermal distribution is indicated by a dotted line on the scheme originally drawn by Neal (1914). ev, optic vesicle; nhp, nasohypophysial plate; ot, otocyst; pp1, pp2, ﬁrst and second pharyngeal pouches. (B) Fully grown ammocoete larva of the lamprey. Redrawn from Neal (1897). Myotomes are indicated by broken lines and numbered according to the observation in the present study. The supraoptic myotome is derived from the dorsal half of the second myotome (m2), while the infraoptic myotome is brought about by fusion of the entire ﬁrst myotome (m1, derived from s1) and the ventral half of m2. hbm, hypobranchial muscles; XII, hypoglossal nerve.

(Kopfho¨le) are present during the late pharyngular period; The three preotic mesodermal components found in the there are three pairs of cavities in the preotic region: from lamprey embryo show a striking similarity to the shark rostral to caudal, premandibular, mandibular, and hyoid head mesoderm especially before head cavities become cavities (Balfour, 1878; van Wijhe, 1882; reviewed by Goo- distinct (Fig. 8A); the premandibular mesoderm in the shark drich, 1930; Jarvik, 1980; Jefferies, 1986). Each of these is found caudal to the optic vesicle, medial to the mandib- epithelial structures later gives rise to extrinsic ocular ular element, and both counterparts are fused medially in muscle subsets, which are innervated by each ocular cranial contact with the rostral tip of the notochord, exhibiting the nerve in the same order (Marshall, 1881; reviewed by same topographical relationships as the lamprey preman- Goodrich, 1930; de Beer, 1937; Neal and Rand, 1942; Jarvik, dibular mesoderm (Figs. 8A; Bjerring, 1977). The earliest 1980; Jefferies, 1986). Similar epithelial cavities have also morphology of Squalus mesoderm in which mandibular been observed in a vestigial form to give rise to extrinsic and hyoid mesoderm form a contiguous mesenchymal ocular muscles in bony ﬁshes and amniotes (reviewed by sheet (Scammon, 1911; reviewed in Jefferies, 1986) roughly Brachet, 1935; Wedin, 1949; Jacob et al., 1984). In addition corresponds to the stage 19 embryo of L. japonica (Fig. 1); to the three pairs of cavities, some elasmobranch species the mesoderm caudal to the mandibular mesoderm in have another pair of mesoderm in front of the premandibu- Squalus also appears to be regionalized by the otocyst lar mesoderm which has been termed Platt’s vesicle (Platt, (Bjerring, 1977). 1891). It is now generally believed that this vesicle only Regionalization of the preotic mesoderm is less complete represents a portion of the premandibular cavity (Goodrich, in amphibia than in the shark and lamprey; the newt 1918; Jefferies, 1986; but see Jarvik, 1980). embryo develops an unsegmented mesoderm in the preotic

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. 390 Kuratani, Horigome, and Hirano region which is contiguous with somite 0 (Jacobson and in amniotes. This assumption is not supported in terms of Meier, 1984). Although a clear premandibular mesoderm either developmental fate (origins of extrinsic ocular has not been described, there is a possibility that the ventral muscles) exemplified in the chick (Jacob et al., 1984; half of the mandibular mesoderm represents the preman- Noden, 1988; Couly et al., 1992) or inconstancy among dibular mesoderm; this mesodermal portion may only be amniote embryos (Trainor et al., 1994). Somitomeres, if secondarily incorporated into the mandibular arch (Fig. 8A). they are really present at all, do not seem to possess any As in the amphibia, amniote embryos do not show clearly morphological nature which corresponds to mesodermal regionalized preotic mesoderm. In early stages of amniote regionalization seen in shark or lamprey. The developmen- embryos, however, six or seven incomplete mesenchymal tal fate of the lamprey head mesoderm is still unknown, but segments have been recognized by SEM observation, collec- may possibly be similar to shark mesodermal components, tively called cephalic somitomeres by Meier and his col- though a peculiar innervation pattern has been reported in leagues (chick: Meier, 1979; quail: Meier, 1982; mouse: Petromyzon (Cords, 1928). Tam and Meier, 1982; Tam et al., 1982; snapping turtle: Head mesoderm and cephalic crest cells. As in gnathos- Meier and Packard, 1984), although the existence of somi- tomes, there are three major crest cell populations in L. tomeres has recently been doubted by Freunt and others japonica, each ventrally filling the pharyngeal arch (1996). Since similar pseudosegments have also been de- (Horigome et al., 1999). The rostralmost cephalic crest cell scribed in teleost embryos (Martindale et al., 1987), it population, or TC cells (trigeminal crest cells; Horigome et appears that mesodermal reorganization took place inde- al., 1999), surrounds the mandibular mesoderm mainly pendently more than once in the vertebrate evolution. laterally (Figs. 5E and 5F). The rostral part of TC cells covers In terms of early arrangement of the mesoderm, the the anterior aspect of the mandibular mesoderm. It is into lamprey–gnathostome difference is not so great as has been this cell mass that the laterally growing premandibular expected. Rather, a line can be drawn between amniotes mesoderm penetrates (Figs. 5E, 5F, and 8B), forming a and anamniotes (excluding teleosts). In the latter, apparent distinct ectomesenchyme surrounding the premandibular developmental factors that seem to yield similarities of mesoderm, although not entirely separated from the rest of mesodermal regionalization are more or less epigenetic in the TC cells. nature, being brought about by the secondarily established In the context of head morphology, the PM cells exhibit topographical relationships with growing otocyst and pha- an interesting distribution pattern since they occupy a ryngeal pouches (Fig. 4). The above difference may partly be premandibular region (reviewed by Kuratani et al., 1997b). due to heterochrony; in amniotes, the deposition of head The cranial elements developing in this region are called mesoderm seems to be accelerated compared to sharks and trabecular cartilages that have been shown to be of crest lampreys, as judged from otocyst and pharyngeal arch origin in avian embryos by construction of chick/quail development. The same is true for teleosts in which pha- chimeras (Le Lievre, 1978; Couly et al., 1993) and also in a ryngeal arch formation is delayed (Richardson, 1995; Rich- lamprey, Petromyzon marinus, by an extirpation experi- ardson et al., 1997). ment (Langille and Hall, 1988). In gnathostome chondrocra- Aside from the origin and developmental mechanism of nia, the trabecular cartilages are initially a pair of rod-like cephalic somitomeres, the question is how we can assign cartilages beneath the optic chiasm and are situated on both the somitomeres in amniotes to four pairs of head mesoder- sides of the hypophysis and forebrain floor (de Beer, 1937), a mal components. Simply, the head mesodermal component topography which is again conserved in lampreys (reviewed of the shark may correspond to two successive somitomeres by Janvier, 1993). The PM cells are also located in front of

FIG. 8. (A) Comparative morphology of the head mesoderm. The similarity is striking between L. japonica (lamprey) and early elasmobranch embryos (shark); three pairs of mesodermal components, the premandibular, mandibular, and hyoid mesoderm, are recognized. In amphibia (newt), deﬁnitive premandibular mesoderm is missing, probably incorporated into the mandibular arch (pm ϩ mm?). In every case, the rostralmost postotic mesoderm, s0, lacks its rostral boundary against the hyoid mesoderm. Pharyngeal slits are numbered. (B) Relationships between neural crest cells and head mesoderm. On the left is the sequence of crest cell–mesoderm development in L. japonica. Crest cells are shaded. Cephalic crest cells have a ubiquitous origin on the neuraxis (invisible in the ﬁgure) and are canalized during migration into three major cell populations (gray arrows), namely trigeminal crest (TC), hyoid crest (HC), and branchial crest (BC) cells (based on Horigome et al., 1999). Each cell population maintains stereotyped topographical relationships with the head mesoderm through development and by stage 22 the TC cell population is subdivided into premandibular crest (PMC) and mandibular crest (MC) cells through growth of premandibular mesoderm. (Right) Comparison of crest cell–mesoderm relationships between the lamprey (L. japonica; based on the present study) and amniotes (chick; based on Anderson and Meier, 1981). Somitomeres in the chick are numbered (1–7). The topographical correlation of the head mesoderm is indicated based on spatial relationships between mesoderm and cephalic crest cell populations. BC, branchial crest cells; ev, optic vesicle; HC, hyoid crest cells; hm, hyoid mesoderm; MC, mandibular crest cells; mm, mandibular mesoderm; nt, notochord; ot, otocyst; pc, prechordal plate; PMC, premandibular crest cells; s0–s7, somites; TC, trigeminal crest cells.

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. Head Mesoderm in the Lamprey 391 392 Kuratani, Horigome, and Hirano the notochord and adhere to the forebrain where “pre- premandibular mesoderm, but also the rostral portion of the chordal neurocranium” (definition by Couly et al., 1993) is notochord that uses up the material of the prechordal plate; expected to develop. The crest cell distribution pattern is the prechordal plate is not a definitive structure but a thus conserved not only in the pharyngeal (Horigome et al., diminishing mass of undifferentiated cells whose cell lin- 1999), but also in the premandibular region of the lamprey eages are ever separating continuously through the produc- embryo. tion of the mesoderm as well as the notochord. Such Somitomeres have been found to maintain a constant behavior is similar to the posterior end of the axial cell topographical relationship with brain segments and ce- mass; for example, the notochordal plate in mouse also phalic crest cell populations (Jacobson, 1993; Tam and shares common morphological properties with the pre- Trainor, 1994; Trainor et al., 1994). The somitomere–crest chordal plate (Sulik et al., 1994). cell relationship has been described in the chick embryo by Although the cell-labeling experiment is still unsuccess- Anderson and Meier (1981) and in the snapping turtle by ful in our laboratory, the relationships between the pre- Meier and Packard (1984), both patterns which are substan- chordal plate and mandibular mesoderm as well as the tially identical: the hyoid crest cells (rostral otic crest in premandibular mesoderm are rather obvious in L. japonica chick by Anderson and Meier, 1981; HC cells in L. japonica (Figs. 1, 3, 5). In L. fluviatilis also, labeling experiments at by Horigome et al., 1999) pass in the chick embryo, be- gastrular stages revealed close relationships between the tween somitomeres 4 and 5. If the crest–mesoderm topo- prechordal plate and a part of the head mesoderm (Weissen- graphical relationships are conserved as the vertebrate phy- berg, 1934, 1935). Similar relationships have also been lotype, the somitomere 5/6 boundary would correspond to observed in various vertebrates (Platt, 1891; Veit, 1924; hyoid mesoderm/s0 indentation of L. japonica (Fig. 5B; see Dohrn, 1904a,b; Scammon, 1911; Goodrich, 1918; Adel- Horigome et al., 1999, for crest cell distribution). Since the mann, 1922, 1932; Holmgren, 1940; Bjerring, 1977; Jacob et mandibular mesoderm is covered by TC cells, the hyoid al., 1984; Jacobson and Meier, 1984; Jacob and Jacob, 1993; mesoderm in L. japonica would correspond to somitomere E. Gilland, personal communication). The preaxial meso- 5 of the chick embryo, an assignment which is consistent derm in primitive vertebrates arises as an outer pouch of the with Noden (1988). As noted above, such an assignment rostral gut, and in this respect it resembles the anterior gut does not imply the conserved fate map in terms of tissue diverticulum in amphioxus (Hatschek, 1881) or rostral differentiation (see Couly et al., 1992). This would partly be adhesive organs in some primitive fishes (reviewed by Neal due to an extensive mixture of mesodermal cells outside and Rand, 1942). Especially in a shark, Squalus, it has been the pharyngeal arches (Trainor et al., 1994). Probably, the seen that the mandibular mesoderm precedes the preman- environmental signals may play fundamental roles in tissue dibular, as in the lamprey (Scammon, 1911; reviewed in differentiation of head mesoderm. In this respect, the head Jefferies, 1986; E. Gilland, personal communication), i.e., cavities found in some vertebrates are peculiar for each the appearance of mesodermal elements here proceeds from differentiates into a particular set of extrinsic eye muscles. a caudal to rostral direction. Also, the homologies of head mesodermal components may Probably prechordal plate-derived mesoderm is also be able to establish by means of their regionalization, but present in the chick, where labeling of the early prechordal not by their developmental fates. plate results in labeling of some extrinsic ocular muscles Mesoderm derived from the prechordal plate—Gastral (Jacob et al., 1984; Couly et al., 1992; but see below). A and preaxial origins of the vertebrate mesoderm. The similar assumption has been made by Adelmann (1922); the prechordal plate is commonly observed in early embryos of author called the anteriorly produced mesoderm the pre- vertebrates. It is in essence the roof of the foregut, caudally axial mesoderm as opposed to the peristomal and gastral continuous with the developing notochord (Jacob et al., mesoderm of Rabl (1889, 1892a; Rabl’s classification of 1984). The prechordal plate in the lamprey is a single cell peristomal and gastral mesoderm appears to be an artificial layer located beneath the neural tube rostral to the noto- one, and the term “gastral” will be used below to indicate chord (Hatta, 1891; de Selys-Longchamps, 1910). In its the mesoderm that comes from either the primitive streak earliest phase of development, the prechordal plate forms a or the blastopore lateral lip). In the lamprey, the gastral large part of the embryonic axis and gradually diminishes in paraxial mesoderm is mostly segmented into somites size. It is the production of not only the mandibular and (Weissenberg, 1934, 1935).

FIG. 9. Segmental theory of the vertebrate head. (A, top) The segmental scheme of the vertebrate head by Goodrich. Redrawn from Goodrich (1930). (A, bottom) By removing the peripheral nervous system and chondrocranial elements, this scheme is shown to be based on mesomeric formulation, in which head cavities of elasmobranch embryos are equated with myotomes. hb, hypobranchial musculature; hm, hyoid mesoderm (hyoid somite); mm, mandibular mesoderm (mandibular somite); ot, otocyst; pm, premandibular mesoderm (premandibular somite); Pv, Platt’s vesicle. (B) Comparison between amphioxus (top) and vertebrates (bottom). In both animals, the shared genetic code (localized pattern of homeobox gene expressions) is recognized in the CNS, possibly inferring the presence of homologies in neural subdivisions between the two animals (based on Holland and Holland, 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 of myotomes, Goodrich’s scheme rather ﬁts the amphioxus better than vertebrates. agd, anterior gut diverticulum; m, mouth.

A problem remains as to the boundary between the ﬁnally, are the units equivalent to rostral somites in am- preaxial and gastral mesoderm in the paraxial mesoderm; phioxus? (homology of units). Of those, dissociation be- the hyoid mesoderm and somite 0 appear to be the earliest tween branchiomerism and somitomerism has already been parts of the mesoderm to be produced in the lamprey and discussed (Marshall, 1881; Froriep, 1902a, 1917; Rabl, shark (Adelmann, 1922); these mesodermal components 1892b; Starck, 1963; Kuratani, 1997, and references may originate from an unseparated common mesodermal therein), and the question of the variable number of postotic source. Still, this does not explain the origins of amniote segments is answered in part by involvement of the Hox head mesoderm, since a large portion of the latter deﬁnitely code in the patterning of occipital bones (Burke et al., 1995). originates from the primitive streak (Lawson et al., 1991; In the trunk region, postotic somites pattern the periph- Schoenwolf et al., 1992; Smith et al., 1994; Psychoyos and eral nervous system as a morphogenetic burden; the seg- Stern, 1996; Faust et al., 1998; reviewed by Tam and mentation involves primarily the mesoderm. The Behringer, 1997). Similarly, the prechordal plate does not metameric pattern of spinal nerves has been shown to be differentiate into jaw muscles in the chick (Couly et al., imposed by metamerism of the paraxial mesoderm (De- 1992). The above may imply that the origins (preaxial or twiler, 1934; Keynes and Stern, 1984; Rickmann et al., gastral) of the mesoderm are not constant among vertebrate 1985; Tosney, 1987; Bronner-Fraser and Stern, 1991). In species either. It appears that only some primitive verte- amphioxus, muscle blocks are completely segmented along brates possess an extensive preaxial mesoderm. In am- the body axis to the rostral tip of the head, and the PNS is niotes, on the other hand, experimental studies made so far entirely somitomeric, each nerve arising between two suc- appear to show that preaxial mesoderm occupies the ros- cessive myotomes (Dogiel, 1903; Franz, 1927). Therefore, it tromedial portion of the head mesoderm, only differentiat- was reasonable that cephalic mesomeres or the neuromeres ing into extrinsic ocular muscles (see Fig. 10). Comparative were extensively sought in comparative vertebrate embry- cell-labeling experiments will be needed among various ology (Locy, 1895; Hill, 1900; Neal, 1896, 1918a; Johnston, chordate embryos to further clarify evolutionary changes of 1905; reviewed by Neal and Rand, 1942). Goodrich’s mesodermal production. scheme was on the same line (Fig. 9A). Reevaluation of segmental theories of vertebrate head. Typical cephalic mesomeres were believed to be present The problem of head segments started as a question about as three or four pairs of head cavities in elasmobranch the number of skeletal segments, and the conclusion of embryos (Balfour, 1878; van Wihje, 1882; Goodrich, 1918; Goodrich (1918, 1930; Fig. 9A) marked the end point in this de Beer, 1922, 1937; reviewed by Goodrich, 1930; Jarvik, history. Based on selachian embryos, his scheme has been 1980; Jefferies, 1986). More recently, cephalic somitomeres one of the most widely cited theories until today. It is true were recognized in amniotes, amphibians, and teleosts by that the scheme of Goodrich, at least at certain points in SEM observation. These two streams share the same mor- development, seems to hold, not only for the shark, but also phological formulation as the background (theories of seg- for the lamprey embryo if the head mesoderm is really mentalists), although they do not reconcile to each other in segmented as it appears. Therefore, as stated by Jefferies terms of number of segments (reviewed by Northcutt, (1986), this theory could either be the truth or all the 1993). In either case, the most serious problem with the segmental theories based on mesodermal segments (me- segmental theory is that the scheme is more suitable for someres) including Goodrich’s as well as recent ones would amphioxus than for vertebrates (Fig. 9). As judged from the collapse (Bertmar, 1959; Jollie, 1977; Bjerring, 1977; Jarvik, illustrations by Hatschek (1881; also see Presley et al., 1980; Gilland and Baker, 1993); Goodrich’s scheme is a 1996; and the cover photograph of the May 1997 issue of suitable starting point from which to deal with the head Development by Holland et al., 1997), all the myotomes are problem, especially as a new step into evolutionary and equivalent and they arise as typical gastral mesoderm molecular developmental biology (see Northcutt, 1993; whose cell lineage is separated early from the rostral noto- Holland, 1999). chord and foregut, sequentially developing in an anterior to The theories of head segments are actually referring to posterior direction, just like the trunk mesoderm of verte- head metamerism, implying the existence of simple and brates. Moreover, the vertebrate head mesoderm does not repeating segmental mechanisms involved in head morpho- impose metameric pattern onto the PNS as a developmen- genesis similar to trunk patterning. Therefore, in the tal burden, and there is no intermediate animal that shows mechanistic understanding of the head problem, the devel- the transition from the segmental amphioxus-like animal opmental burden brought about by the prepattern should be to the vertebrate pattern. The problem of head segmenta- dealt with as the primary cause of the metamerism. The tion is thus linked to the formulation of the chordate question of head segmentation can be summarized into phylotype and homology of mesodermal components. several points as follows: (1) what can be a primary element As shown in the present work as well as our previous of head segment? (source of the burden); (2) if it is a studies (Kuratani et al., 1997a, 1998b; Horigome et al., somite-equivalent unit, does it repeat with the same inter- 1999), the lamprey pharyngula shares a series of features val as branchial arches? (somitomerism versus branchiom- and clearly belongs to the phylotype of vertebrates; pro- erism); and (3) how many head segments are there in the someres in the forebrain, clearly demarcated midbrain, preotic region of the craniate head? (number of units); and postotic myotomes, odd–even pattern of rhombomeres and

FIG. 10. Evolution of the head mesoderm. Different patterns of mesodermal organization are indicated on the hypothetical phylogenetic tree. Phylotypes that represent the shared embryonic patterns of given monophyletic groups are also placed on the tree. At the top of the phylogenetic tree, tricoelomate larvae of deuterostome animals are presented as a possible ancestral type from which paired mesodermal components may have arisen with several different sources (reviewed by Starck, 1978). Tadpole larvae of tunicates (a) possess only unsegmented mesoderm paraxially in the tail. In the amphioxus (b), cell lineages of notochord and primitive gut are separated early, and all of the mesodermal segments arise as gastral components, possessing myotomal traits (horizontally hatched). The anterior gut diverticulum originates from the foregut. In vertebrates (c and d), myotomes are all gastrally originated (horizontally hatched), and unsegmented head mesoderm and the notochord have preaxial (gray) and gastral (white) origins. The head mesoderm is primarily regionalized by surrounding structures into several components (c), while in some derived groups like amniotes (d) it fails to be regionalized but may be segmented incompletely into somitomeres. The preaxial mesoderm of the latter group appears less extensive than in the prototype and may possibly be localized in the medial part of the head mesoderm in amniotes, giving rise to extrinsic ocular muscles (based on Couly et al., 1992). Note that the phylotype of the vertebrate ancestor does not have to resemble that of amphioxus; the body plan of the latter may have been established through the loss of head mesoderm including the preaxial mesodermal components. agd, anterior gut diverticulum; hm, hyoid mesoderm; mm, mandibular mesoderm; nt, notochord; pc, prechordal plate; pm, premandibular mesoderm; s0, somite 0; 1–7, somitomeres (numbered).

cranial nerve roots, pharyngeal arches of which the first two none of these characteristics is found in amphioxus. Unlike are highly modified, three populations of dorsally migrating these vertebrate-specific features, the morphology of the cephalic crest cells, and unsegmented head mesoderm: neural tube may have a common ground plan among

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. 396 Kuratani, Horigome, and Hirano chordates. For example, a number of homologous ho- 1983). As a second scenario, some of the rostral somites in meobox genes are expressed along the neuraxis, showing a the ancestor have lost segmental nature (Fig. 10); thus, tripartite configuration of the neural tube as the chordate homologies can exist between these somites and vertebrate prototype (Fig. 9B; Holland et al., 1992; Wada et al., 1996a, head mesoderm components as stated by several authors 1997, 1998; Wada et al., 1995, 1996b; reviewed by Holland (Gilland and Baker, 1993; Holland, 1999). The latter should and Graham, 1995; Holland, 1996; Holland and Holland, include also the creation of the prechordal plate and its 1998). Moreover, anatomical similarities have been recog- derivatives, i.e., the rostral notochord and some of the head nized in the fore-midbrains between amphioxus and verte- mesoderm. It is equivalently possible that amphioxus rep- brates (reviewed by Lacalli, 1996). Therefore, the chordate resents a secondary condition through an extensive reduc- phylotype, which the segmental schemes of the vertebrate tion of rostral structures leaving no homologies between head were supposed to infer, is only able to include antero- anterior mesodermal components (unsegmented head me- posteriorly patterned neuraxis and the shared genetic code soderm and prechordal derivatives) (Fig. 10). that may indicate homologous subdivisions of the central The idea about the possible common ancestor between nervous system among chordates, but nothing can be de- the two, from which the mesoderm of tunicates might also scribed as to the paraxial mesoderm at present, except that have evolved, would possibly be obtained in the study of it may or may not be segmented partially; if it is to refer to hemichordate larvae, the sister group of chordates. It may the shared pattern of the head mesoderm, the scheme must be interesting, in this context, to mention that tripartite be the vertebrate phylotype and it cannot contain the coeloms are typical in larvae of deuterostomes, to which segmented head mesoderm that never existed within the chordates and hemichordates belong (Fig. 10; Remane, cited group Vertebrata (Fig. 9B). The idea fits the “New Head” in Starck, 1978); the larval form explains the multiple theory of Gans and Northcutt (1983) as well as the distinc- sources of mesoderm and the presence of the rostralmost tion of branchiomerism and somitomerism (reviewed by coelom, or protocoel, that is more or less unpaired in nature Kuratani, 1997). Both refer to the anteroposteriorly differ- as premandibular mesoderm in vertebrates. entiated mesenchymal embryonic environment, through At present, it seems most likely that the possession of the which cephalic crest cells contribute to head formation preaxial mesodermal lineage is plesiomorphic in chordates, unique to vertebrates. Simultaneously, it is suggested that which should have been secondarily lost in lineages of the reorganization of paraxial mesodermal and preaxial urochordates and cephalochordates (Fig. 10). This loss will structures may have played a fundamental role in the not support the monophyly of the latter two animal groups vertebrate and amphioxus evolution. since the morphology of the rostral body shares few simi- Evolution of chordate mesoderm. Where could we find larities. It is premature to conclude that they are second- the origin of the vertebrate phylotype? In vertebrate em- arily acquired in the lineage of vertebrates when we have no bryos, a part of the head mesoderm arises preaxially sharing idea how rostral mesoderm might have looked like in the common origins with the preoral gut endoderm and the common ancestor of chordates, although fossil evidences of notochord. The notochord in amphioxus always possesses a Cambrian protochordates include some forms that seemed clear rostral end during development implying early disso- to have possessed an unsegmented region in the head ciation of cell lineages from the foregut, leaving no space for (reviewed by Insom et al., 1995). Importantly, we still do the prechordal plate to show itself (Fig. 10); the amphioxus not have much information about the head mesoderm of notochord elongates secondarily during development, and hagfish, the sister group of vertebrates, which might bring its rostral tip in the adult state never reflects its original up the remote possibility that the craniate preotic meso- extension (Hatschek, 1881). If we are to locate a structure derm was once epithelially segmented in a somitomeric that is best equivalent to the prechordal plate in am- fashion. Will it force us to recognize another phylotype for phioxus, it might be sought in the rostral portion of the the group Craniata? primitive gut that gives rise to the anterior gut diverticulum, although the diverticulum never differentiates into muscles nor does it maintain a connection with the noto- ACKNOWLEDGMENTS chord; there is no exact prechordal plate homologue in amphioxus. We are grateful to Niigata Prefectural Inland Water Fisheries Whatever the ancestor may have looked like, the discus- Experiment Station, Niigata, Japan, for the use of the facility and to sion so far implies that evolution of vertebrates and am- Drs. Tatsuya Ueki and Shinichi Aizawa for their technical advice phioxus should primarily involve a profound alteration of and discussion. We also thank Dr. Ruth T. Yu for her critical mesodermal configuration. As one possible scenario based reading of the manuscript. Sincere gratitude is extended to Drs. Kathryn W. Tosney, Drew M. Noden, Hiroshi Wada, and Edwin on an amphioxus-like ancestor, the unsegmented head Gilland for their comments and valuable discussion. The CH-1 mesoderm is a newly formed structure that has been gained antibody developed by Dr. J. Lin and the MF-20 antibody developed independently in the lineage of craniates (including myx- by Dr. D. A. Fischman were obtained from the Developmental ines) and no equivalent tissue has ever been present in the Studies Hybridoma Bank (DSHB) maintained by the Department of acraniate lineage. In that case, the head mesoderm is unique Pharmacology and Molecular Sciences, Johns Hopkins University to vertebrate lineages (Froriep, 1902b; Gans and Northcutt, School of Medicine (Baltimore, MD), and the Department of

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