stereotyped axonal bundle formation and neuromeric...

Stereotyped Axonal Bundle Formationand Neuromeric Patterns in Embryosof a Cyclostome, Lampetra japonica

SHIGERU KURATANI,1* NAOTO HORIGOME,1 TATSUYA UEKI,1 SHINICHI AIZAWA,1

AND SHIGEKI HIRANO2

1Department of Morphogenesis, Institute of Molecular Embryology and Genetics (IMEG),Kumamoto University School of Medicine, Kumamoto 862, Japan

2Department of Anatomy, Niigata University School of Medicine, Niigata 951, Japan

ABSTRACTEarly embryonic development of the nervous system of a lamprey, Lampetra japonica,

was studied by using immunohistochemical techniques and by scanning electron microscopy.The earliest appearance of axons was detected at Tahara’s stage 21-, when dorsolateral andventral longitudinal fasciculi were present in the hindbrain and spinal cord regions. Thebranchiomeric nerve roots began to appear at stage 22; the fibers were joined to thedorsolateral fasciculus proximally and also extended distally into each pharyngeal arch.The anterior neural tube was divided into several neuromeres: the mid-hindbrain sulcusbecame apparent first, then the portion rostral to this sulcus was subdivided into two portionsby the syn-parencephalic boundary. In the hindbrain around stage 23, rhombomeresdeveloped transiently, of which, rhombomere 4 was the most distinctive. Putative crest cellsforming the octavofacial nerve root anlage were selectively adhering to rhombomere 4,whereas no crest cells were found on rhombomere 3. The assignment of the crest-derivednerve anlage to rhombomeres is conserved between gnathostomes and L. japonica. Theneuromerical scheme of the neural tube of L. japonica is also mostly in accordance with that ingnathostomes, sharing the basic developmental patterning of axon bundles at early develop-mental stages. The most distinct difference between these two groups is the topographicalrelationships between the hindbrain neuraxis and pharyngeal arches, as well as the oticplacode. J. Comp. Neurol. 391:99–114, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: rhombomeres; cranial nerves; neural crest; lampreys

The attention of embryologists has been devoted to thesegmentation of the vertebrate neural tube since its discov-ery by von Baer (1828) in a 3-day-old chick embryo. Themetamerism of the nervous system attracts reawakenedinterest from recent developmental biology providing anew background of molecular biology and developmentalneurology, in that the early distribution pattern of pioneerneurons is known to correspond to the neural segmenta-tion, thereby providing an important factor that results indiscrete, precise, and stereotyped axon bundle develop-ment (reviewed by Kimmel, 1993). The segmentation ofthe brain and early axonogenesis is, therefore, regarded asa prepattern of the complicated architecture of the verte-brate brain.

The segmental bulges of the neural tube were first calledneuromeres by Orr (1887), who recognized in lizard em-bryos several common histologic features shared by bulgesin the hindbrain, the rhombomeres. These features are (1)rhombomeres are bounded by internal and external sulci

on the neurepithelium, (2) each rhombomere has neurepi-thelial cells arranged in a fan-shaped pattern, (3) no singlecell spans two successive rhombomeres, (4) each rhombo-mere has its center of proliferation in its middle part, and(5) it develops symmetrically on either side of the hind-brain. It has recently been shown that rhombomeres areestablished through the restriction of cell lineages; oncethe boundary is determined on the neuraxis, cells inneighboring segments will not intermingle over the sulci(Fraser et al., 1990; also see Birgbaur and Fraser, 1994).

Grant sponsor: Ministry of Education, Science, and Culture of Japan;Grant sponsor: Science and Technology Agency of Japan.

*Correspondence to: Shigeru Kuratani, Department of Morphogenesis,Institute of Molecular Embryology and Genetics Kumamoto UniversitySchool of Medicine, 4-24-1 Kuhonji, Kumamoto, Kumamoto 862, Japan.E-mail: [email protected]

Received 19 November 1996; Revised 3 September 1997; Accepted 10September 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 391:99–114 (1998)

r 1998 WILEY-LISS, INC.

Although some authors regarded neuromeres as artifactsor paid little attention to their morphologic importance(Balfour, 1881; Froriep, 1891, 1892; Streeter, 1933), othersconsidered these structures to be a profound segmentalplan of the vertebrate axis (Locy, 1895; Hill, 1899, 1900;also see Neal, 1896).

Of the neuromeres, rhombomeres are especially curiousbecause they are associated with cranial nerve roots whichare metamerical in the sense that the iterative nature ofbranchial arches constitutes the branchiomerism. In manyvertebrate embryos, branchiomeric nerve roots emergefrom even-numbered rhombomeres, thereby being associ-ated with the head metamerism (Remak, 1855; Beranek,1884; Kuhlenbeck, 1935; Lumsden and Keynes, 1989;Kuratani, 1991; Kuratani and Eichele, 1993). Isolation ofhomeobox-containing genes in vertebrates provided theabove scheme for mechanical interpretations of head pat-terning. Of those, Hox genes in amniotes are expressed in anested pattern; each expression domain is limited byrhombomeric sulci (reviewed by McGinnis and Krumlauf,1992, and by Krumlauf, 1993). Not only the hindbrain, butalso the branchiomeric components such as pharyngealectoderm, endoderm and the mesenchyme express thegenes in the same order, thereby establishing the Hox codeof the embryonic head (reviewed by McGinnis and Krum-lauf, 1992, and by Mark et al., 1995).

As Romer (1972) has postulated, the vertebrate bodyplan seems to possess two distinct types of metamerisms;branchiomerism in the head and somitomerism in thetrunk (in this context, the occipital somites are regarded astrunk components; see Kuratani, 1997). It remains to besolved where this unique plan emerged in the evolutionarypath leading to vertebrates. In amphioxus, a sister groupof vertebrates, Hox gene cognates are expressed in asegment-specific fashion along the axis, as in vertebrates(Holland et al., 1992; Holland and Garcia-Fernandez,1996). However, there is no known expression patternrestricted by a cell lineage-based compartment; nor arethere any rhombomeres in this animal.

In the rostral brain, a series of neuromeres are alsoknown to exist in a number of vertebrates. Interestingly,

these bulges share some traits with rhombomeres and alsoexpress a number of regulatory genes in patterns re-stricted by segmental sulci (reviewed by Figdor and Stern,1993; Price, 1993; Boncinelli et al., 1993; Rubenstein et al.,1994; Shimamura et al., 1995). These bulges are alsounique to vertebrates, and it is not known when and howsegmental organization of the central nervous system(CNS) arose in chordate evolution.

Among vertebrates, it is mostly in Gnathostoma speciesthat the CNS development has been examined in detail. Itis a prerequisite in the evolutionary context that a speciesdistantly related to gnathostomes should be examined.The present paper is intended to provide a precise descrip-tion of the early neurogenesis and differentiation of theneural tube in a cyclostome, Lampetra japonica. Theauthors previously described the late embryonic develop-ment of cranial nerves in this animal (Kuratani et al.,1997), but no rhombomere-like structures were seen inthose embryos. In the present study, the morphology of thenervous system in younger lamprey embryos at prehatch-ing stages were examined in an attempt to search forrhombomeres. By observing the distribution of acetylatedtubulin (AT)-like immunoreactivities and three-dimen-sional morphology of the neural tube by scanning electronmicroscopy (SEM), we found that obvious segmentation inthe developing hindbrain, albeit faint, appeared before thecompletion of cranial nerve root patterning.

MATERIALS AND METHODS

Embryos

Adult male and female of Lampetra japonica werecollected in a tributary of the Miomote River, Niigata,Japan, during the breeding season (late May throughJune) in 1995 and 1996. The eggs were artificially fertil-ized and kept in fresh water at 18oC or in 10% Steinberg’ssolution (Steinberg, 1957) below 23oC. Embryos were fixedeither with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PFA/PBS), pH 7.6, for immunostaining orwith 0.01 M phosphate-buffered 2.5% glutaraldehyde forscanning electron microscopy and plastic sectioning. Someembryos were also fixed with Bouin’s fixative for lectinstaining. Because embryonic development was easilychanged by temperature, they were staged morphologi-cally according to the table of Tahara (1988) for L. reiss-neri, a brook species of L. japonica. The animals used inthis study were treated under protocols approved byKumamoto University guidelines for animal experiments.

Whole-mount immunostaining

Whole-mount embryos were prepared as described previ-ously (Kuratani et al., 1997) with slight modifications.After fixation with PFA/PBS at 4oC for 1 day, embryos werewashed in normal saline, dehydrated in a graded series ofmethanol (50, 80, 100%), and stored at -20oC. The samplesto be stained were placed in 20% dimethyl sulfoxide(DMSO)/methanol with 10% (v/v) hydrogen peroxide solu-tion and gently agitated on a shaking platform. Afterwashing in Tris-HCl-buffered saline (TST) (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Triton X-100) containing5% (v/v) DMSO (TST/DMSO), the samples were sequen-tially blocked with 5% dry non-fat milk in TST/DMSO(TSTM/DMSO) overnight.

Abbreviations

DLF dorsolateral fasciculuseo ectopic olfactory sensory cellsep epiphysisISN interstitial nucleusm midbrainMLF medial longitudinal fasciculusolep olfactory epitheliumon olfactory nerveoph ophthalmic nerveos optic stalkot otic placode or otocystph pharynxplat posterior lateral line nerveprm mid-hindbrain boundaryPOT postoptic tractp1-4 pharyngeal pouches 1-4r1-6 rhombomeres 1-6RB Rohon-Beard cellss1- somites 1-sv ventral spinal nervesSOT supraoptic tractVLF ventral longitudinal fasciculusV trigeminal nerveVII octavofacial nerveIX glossopharyngeal nerveX vagus nerve

100 S. KURATANI ET AL.

For whole-mount immunostaining of cranial nerves,monoclonal antibody (Mab) raised against a-acetylatedtubulin (monoclonal anti-acetylated tubulin antibody, No.T-6793, Sigma Chemical Company, St. Louis, MO) wasused. Embryos were incubated in primary antibody (di-luted 1/1,000 in spin-clarified TSTM/DMSO containing0.1% sodium azide) for 2 to 4 days at room temperaturewhile being gently agitated. Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (ZYMEDLaboratories Inc., San Francisco, CA) diluted 1/200 inTSTM/DMSO was used as the secondary antibody. After afinal washing in TST, the embryos were preincubated withperoxidase substrate 3,38-diaminobenzidine (DAB, 100mg/ml) in Tris-HCl-buffered saline (TS) for 1 hour andallowed to react in TS with the same concentration of DABand hydrogen peroxide dissolved at 0.01% for 20 to 40minutes at 0oC. The reaction was stopped and embryoswere dehydrated with a graded series of methanol, thenwere clarified in a mixture of benzyl alcohol and benzylbenzoate (1:2) for observation.

Histology

Some whole-mount immunostained embryos were dehy-drated and embedded in paraffin to be sectioned. Theywere counter stained by hematoxylin (Gill’s hematoxylinV, Muto Pure Chemicals, Ltd., Tokyo, Japan). For Eponsectioning, glutaraldehyde-fixed specimens were washedin 0.01 M phosphate buffer (PB), pH 7.6, and post-fixedwith 1% osmium tetroxide/PB. Embryos were washed withPB several times, dehydrated with a graded series ofethanol, then embedded in Epon 812, and sectioned at 1.5mm. The sections were mounted on glass slides andstained with toluidine blue diluted 0.1% in 0.01 M phos-phate-buffered saline, pH 7.6.

Scanning electron microscopy

To observe the neuromeric bulges on the pial surface ofthe embryonic neural tube, some embryos were preparedfor observation by SEM. Glutaraldehyde-fixed specimenswere washed in 0.01 M phosphate buffer (PB), pH 7.6, andpost-fixed with 4% osmium tetroxide. The embryos wererinsed in PB in which they were skinned with a sharpenedtungsten needle, then were dehydrated with a gradedseries of ethanol, and freeze-dried with t-butyl alcohol.They were mounted on an aluminum stub, sputter-coatedwith gold-palladium alloy, and viewed with the scanningelectron microscope (JSM-5800, JEOL).

RESULTS

Stages 21-–21

The stage 21- embryos were the youngest that exhibitedAT-like immunoreactive axon bundles in the whole-mounts at the level of future hindbrain and spinal cord(Fig. 1A,B). One of the bundles was the dorsolateralfasciculus (DLF), probably formed by axons of the Rohon-Beard (RB) cells distributing along the fasciculus. Therewas another group of cells, commissure neurons, locatedslightly ventral to the RB cells. As in gnathostomes (Shigaand Oppenheim, 1991; Bernhardt, 1994), the axons ex-tended ventrally, and most of these axons turned rostrallynear the ventral aspect of the neural tube (Fig. 1B). At thisstage, the mid-hindbrain boundary is only vaguely de-tected. Immunoreactive cells were more abundantly accu-

mulated in the neurepithelium rostral to the boundary(Fig. 1A).

Slightly later, at stage 21, the mid-hindbrain boundaryis clearly detected in the neural tube, approximately at thelevel dorsal to the first pharyngeal pouch (Fig. 1C). Anaccumulation of AT-like immunoreactive products wasdistributed in a neuromeric pattern, showing the establish-ment of some primitive neuromeres (Fig. 1C). Of those, thecaudal two bulges are separated from each other by arather clear boundary, the mid-hindbrain boundary, thatdelineates the caudal midbrain from the rostral hindbraindorsal to the first pharyngeal pouch (see below). Therostral end of the DLF terminated in the midst of the thirdbulge, which corresponds to the provisional rhombomere112 (r112). At the level of the midbrain, there were a fewneurons whose axons extended caudally (Fig. 1C). Theseneurons correspond to the interstitial nucleus that formsthe anlage of the medial longitudinal fasciculus (MLF).Commissure axons developing in the posterior hindbrainand the spinal cord increased in number and formed theventrolateral fasciculus on the ventral aspect of the neuraltube.

Stage 22

The second pharyngeal pouch and the otic placode hadappeared by stage 22 (Fig. 2A). The number of RB cells andcommissure axons increased in the posterior hindbrainand the spinal cord. The otic placode was located lateral toa new subdivision of the hindbrain stained darkly com-pared with the adjacent boundary stripes in the neuraltube (Fig. 2B). This rhombomere arose at the neuraxiallevel dorsal to the second pharyngeal pouch, and corre-sponds to r4 of gnathostomes because it possesses theoctavofacial nerve root. The bulge was not detected inhistologic sections (not shown). The neurectoderm wasmore heavily stained toward the apical surface, and evenmore at the center of each segment. The most heavilystained portion along the brain axis was the posteriormidbrain (Fig. 2); staining of this portion lasted until stage24.

The octavofacial nerve root developed first. It consistedof a few irregularly oriented axon bundles; they extendedrostroventrally and passed along the medial side of the oticplacode (Fig. 2). The bundle was traced proximally to theDLF, although the cell bodies were not found. In someembryos, there was another axonal bundle arising fromthe DLF rostral to the octavofacial nerve root associatedwith the second pharyngeal arch; it grew caudal to the firstgill pouch (Fig. 2). This bundle represents an accessorialnerve of the octavofacial nerve, probably belonging to theanterior lateral line nerve that eventually merges into theoctavofacial nerve trunk (Kuratani et al., 1997; see below),but the placodal origin of these neurons has not yet beendetermined. Some of the cells in the ectoderm covering theforebrain had differentiated into the AT-positive olfactoryneurons, some sending processes toward the forebrain(Fig. 2B).

Stage 23

The olfactory epithelium became a compact plate ofepithelium beneath the forebrain showing high levels ofAT-like immunoreactivity (Fig. 3). Dorsal to the olfactoryepithelium there were independent accessorial olfactoryneurons whose processes also grew toward the forebraintransiently (Fig. 3B). The staining of their cell bodies was

AXONOGENESIS AND NEUROMERES IN THE LAMPREY 101

Fig. 1. Head region of L. japonica embryos at stages 21- and 21.Immunohistochemically stained with the anti-acetylated tubulin Mab.A: Stage 21- embryo. Arrows indicate the rostral swelling of the neuraltube that shows accumulation of peroxidase-reaction products.B: Higher magnification of the box in A. Arrows indicate the commis-sure neurons growing axons ventrally and then rostrally. The dorsolat-eral fasciculus (DLF) is located dorsal to the commissure neuron cellbodies. C: Stage 21 embryo. The rostral neural tube shows weak

constrictions (an arrow and a broken line) dividing the tube into threeportions. The arrow marks the position of the mid-hindbrain boundary(prm). The DLF has become thicker, and its rostral end is seen withinthe third segment that corresponds to the rostral hindbrain. Arrow-heads indicate the initial development of the medial longitudinalfasciculus. The position of the first pharyngeal pouch (p1) is marked bya broken circle. Scale bars 5 100 µm.

Fig. 2. Photomicrographs of a stage 22 embryo of L. japonica focusedon different depths. A: Focused on the developing neurons. Ventral spinalnerves (sv) are seen at the level of the spinal cord. The medial longitudinalfasciculus (MLF) consists of axon bundles that extend caudally from cellbodies (ISN, interstitial neurons) in the transitional area between therostral two neuromeres. Pharyngeal pouches (p1, p2) are shown by solidlines. Note the development of the octavofacial nerve root (VII) medial tothe otocyst (broken circle). The otocyst develops lateral to r4 (compare with

B). B: Focused on a deeper level to show the neuromeres. The equivalentborders are shown by the same line and arrow as in Figure 1C. The fourthrhombomere (r4, demarcated by two small arrows) is more darkly stainedthan the rest of the adjacent neural tube with the anterior and posteriorsulci that show only low levels of staining. Olfactory neurons are seen inthe ectoderm covering the forebrain (arrowheads) sending axons towardthe forebrain. For abbreviations, see list. Scale bar 5 100 µm (applies toA,B).

Fig. 3. Whole-mount L. japonica embryos at stage 23. The thirdpharyngeal pouch (p3) is developing. A: Early axonogenesis is shown.The trigeminal nerve root (V) is present. Arrowhead indicates anaccessorial nerve associated with the octavofacial nerve (VII). Rohon-Beard cell bodies (RB) are heavily stained. B: Enlargement of therostral-most part of another embryo at the same stage. Arrowheadsshow a cluster of commissure axons in the vicinity of the futuretrigeminal nerve root. The large arrow indicates the mid-hindbrain

boundary, and small arrows indicate rhombomeric sulci. Postoptictract (POT) is seen caudal to the optic stalk (OS). The olfactoryepithelium (olep) is now seen as a solid AT-positive portion of thesurface ectoderm. Dorsal to the definite olfactory epithelium, a fewectopic neurosensory cells (eo) are seen in the ectoderm, and these alsosend processes toward the forebrain. For abbreviations, see list. Scalebars 5 100 µm.

similar to younger olfactory cells (Fig. 2), but the fibers didnot form a fascicular appearance as olfactory axons (Fig.3B). The ectopic neurons disappeared by stage 24. In theforebrain, interstitial neurons had increased in number(Fig. 3B). Caudal to the optic stalk, supra-, and postoptic tractsfirst developed (Fig. 3B). Optically, the axons of the olfactorynerve were closely associated with the supraoptic tract.

The trigeminal nerve root first appeared at this stage(Fig. 3A). As in the case of the octavofacial nerve, the rootarose from the DLF and the nerve bundle grew ventrallyinto the cheek process representing the mandibular arch(Fig. 3A). A few additional axon bundles were associatedwith this definite trigeminal nerve root; they alwaysdeveloped caudal to the latter (Fig. 3A). They representedaccessorial roots of the octavofacial nerve, similar to theones seen at stage 22 (Fig. 2A). These roots were fascicu-

lated as a solid octavofacial nerve root by stage 25 (seeKuratani et al., 1997).

Rhombomeres were most clearly visible at this stage,although the neurepithelial bulges were very faint com-pared with those reported in amniote embryos. Histologicsections showed stronger immunostaining at the luminalside of r4 neurectoderm (Fig. 4A) and boundaries werevisible on the luminal surface (Fig. 4A,B). These bound-aries were restricted to the dorsal portion of the neurecto-derm, and ventrally no indication of rhombomeric pattern-ing was detected. The neurectodermal cells of r4 showed afan-shaped configuration as reported in gnathostomes(Fig. 4A).

As seen in the whole-mounts of a slightly older stagefrom the dorsal view (Fig. 5), the luminal distribution ofAT-like immunoreactive products was the most distinct in

Fig. 4. Histologic observations of the hindbrain of L. japonica atstage 23. A: A horizontally sectioned specimen that had been whole-mount labeled with the anti-AT monoclonal antibody, counterstainedwith hematoxylin. Rhombomeric boundaries on the right side areindicated by arrows on the left. Note, in r4, the fan-shaped configura-tion of the neurepithelial cells and accumulation of the brownish

reaction products in the luminal side of the neurepithelium (asterisk).B: Epon-sectioned specimen of a same stage embryo. Arrows indicatethe rhombomere boundaries weakly protruding on the luminal surfaceof the hindbrain. VII, octavofacial nerve root located medial to theotocyst. For abbreviations, see list. Scale bars 5 100 µm in A, 50 µmin B.


r4 to r6; each segment was separated by AT-negativeboundary regions (Fig. 5A). In some precocious embryos,the ophthalmic nerve developed in the r112 region (Fig.5B). This nerve possessed an independent root at the levelrostral to the trigeminal nerve root. On the ventral aspectof the hindbrain, the commissure axons were present,extending toward the contralateral sides (Fig. 5A). Thetrajectory of these axons showed no sign of the transversescaffold associated with the rhombomeric boundaries. Un-like these boundaries, the mid-hindbrain boundary was a

rather clear indentation on the dorsal surface, whichstayed visible throughout the development.

The rhombomeric sulcus was also vaguely detectable inthe skinned embryo when observed by SEM (Fig. 6). Thesulci detected were only those at levels of mid-hindbrain,r2/3, r3/4, and often r4/5. To be noted was the putativecrest cell population adhering specifically to r4, extendingventrally medial to the otocyst (Fig. 6). This cell populationrepresented the octavofacial nerve anlage. Proximally, thecell covered the entire dorsal surface of r4. Another cell

Fig. 5. Rhombomeres in a stage 23 embryo of L. japonica. Dorsalviews of whole-mount specimens. The broken line indicates thesyn-parencephalic border, and the large arrows indicate the mid-hindbrain border. A: Epiphysis is seen (ep). The photograph is focusedon the ventral aspect of the hindbrain to show commissure axons(arrowheads). Note that the axons do not extend in a transversedirection. B: Enlargement of the same embryo focused on a differentdepth. The contour of the otocyst (ot) is encircled by a solid line. The

earliest anlage of the ophthalmic nerve (oph) is seen in this specimen.The mid-hindbrain boundary is indicated by a large arrow, andrhombomeric sulci are indicated by small arrows. Note in eachrhombomere that AT-like immunoreactive products are accumulatedtoward the luminal side in the neurepithelial cells (asterisks), and theaccumulation is strongest in the posterior end of the midbrain (doubleasterisks). For abbreviations, see list. Scale bars 5 100 µm in A, 50 µmin B.


population representing the anlage of the maxillomandibu-lar nerve root was seen on the caudal part of r112 (Fig. 6).No such cell mass was associated with r3 or r5.

Stages 24 and 25

At these stages, RB cells caudal to the posterior hind-brain stained heavily with the anti-AT antibody (Fig.7A,B). The mid-hindbrain boundary had become moreconspicuous than in previous stages. Some of the embryoshad developed the earliest anlage of the vagus nerve root,and in a few cases, the glossopharyngeal nerve as well. Thevagus nerve root was directed ventrally into the fourthbranchial arch (Fig. 7B). By stage 24, AT-positive commis-sure axons had accumulated in the hindbrain, roughlycorresponding to the sites of cranial nerve roots (Fig. 7B).These neurons were not seen in the rostral-most portion ofthe hindbrain or in the midbrain (Fig. 7A,B).

The definite anlage of the epiphysis appeared at stage24, at the level corresponding to the anterior-most sulcusobserved at stage 21 (Fig. 7C). The posterior commissurewas clearly stained at the site of the dien-mesencephalicborder (Kuratani et al., 1997). All the branchiomericnerves had developed by stage 25 (Kuratani et al., 1997),whereas in the hindbrain, rhombomeric sulci had disap-peared (Figs. 7, 8); they could not be found on the externalsurface by SEM either (not shown). This condition lastedthroughout the developmental stages observed (to stage31).

DISCUSSION

In the present study, the early axonogenesis and segmen-tation of the brain were described in a series of embryos ofan extant jawless craniate, L. japonica. By using whole-

Fig. 6. Stereomicrographs of a stage 23 embryo of L. japonica byscanning electron microscopy. Seen from the right side. Anterior is atthe top. The embryo has been skinned and most of the crest cells havebeen removed to expose the neural tube. Dots indicate neuromericsulci, small arrows rhombomeric sulci, and the large arrow the

mid-hindbrain boundary, respectively. Squared dots indicate the edgeof the removed otocyst. Some presumptive neural crest cells are intactabove the neural tube. Note that cells representing the octavofacialnerve anlage (VII) are adhering specifically to r4 and that no such cellsare found on r3 or r5. For abbreviations, see list. Scale bar 5 100 µm.


mount immunostaining and by SEM, segmental bulgessimilar to amniote rhombomeres were described in lam-prey embryos for the first time. Comparison with the

developmental pattern in gnathostomes poses severalinteresting questions as to the establishment of the verte-brate body plan.

Fig. 7. Stage 24 embryos of L. japonica. Stained with the anti-ATmonoclonal antibody. A: Lateral view of a whole-mount embryo. Thefourth pharyngeal pouch (p4) is beginning to form. Mid-hindbrainboundary (large arrow) and rostral and caudal sulci of r4 (smallarrows) are indicated on the neural tube. Note the extensive accumula-tion of interstitial neurons (ISN) in the syn-mesencephalic regions.Rohon-Beard cells (RB) are heavily stained. Arrowheads show accesso-

rial nerves of the octavofacial nerve. B: Another embryo of the samestage, slightly older than A. The vagus nerve root (X) has developed.Note that commissure neurons are heavily stained (arrowheads) andare lacking in the posterior midbrain. Also note the spatial relationbetween the epiphysis (ep) and the syn-parencephalic border (brokenline). For abbreviations, see list. Scale bar 5 100 µm (applies to A,B).


In the hindbrain of amniotes, cephalic crest cell popula-tions adhere to even-numbered rhombomeres in the preo-tic region (r2 and r4) and to r6 as well, thereby the crestcells develop into cranial nerve anlage (Kuratani, 1991;Kuratani and Eichele, 1993; Niederlander and Lumsden,1996). Such bi-rhombomeric repetition would probably beassociated with the metamerism of the branchial arch,comprising developmental units of the vertebrate head(Kontges and Lumsden, 1996). This morphologic schemeconfirms the mechanism of the segmental specification interms of molecular biology, i.e., a series of homeoticselector genes, the Hox genes, are expressed along theneuraxis. The expression domains of each Hox gene shiftsits anterior border by a bi-rhombomeric length, therebyestablishing the nesting pattern of gene expressions, theHox code (reviewed by McGinnis and Krumlauf, 1992; andby Graham, 1992).

The existence of rhombomeres is not widespread in allchordates. For example, no neuromere-like structureshave been found in amphioxus (Hatschek, 1881; Willey,1891; Franz, 1927; Lacalli et al., 1994). Neal (1918) andNeal and Rand (1936) pointed out that rhombomeresapparently become clearer in embryos of more advancedvertebrates. Although evolutionary grades cannot always

be measured between sister groups, it is true that amnioterhombomeres are much more distinguishable than thosein known anamniotes. Rhombomeres must have evolvedsomewhere in the evolutionary lineage to vertebrates, andit is therefore important to determine whether or not theyhave developed in lampreys that have long been dichoto-mized from the ancestor of gnathostomes (reviewed byJanvier, 1993).

Compared with the hindbrain in amniotes, the rhombo-meric boundaries in L. japonica were very faint and lastedonly for a short period during development. They weremost clearly segmented near the r4 level, and dissociationof r1 and r2 was not determined through the stagesobserved (summarized in Fig. 9). Importantly, semi-bi-rhombomeric patterning seems to exist also in L. japonica,as shown in relation to crest cells and cranial nerve roots:the ophthalmic and trigeminal nerve arise from r112,octavofacial root from r4, and glossopharyngeovagal rootfrom r6 (Figs. 5, 6, 8A). In the chick embryo, the adhesionof crest cells on even-numbered rhombomeres plays animportant role in nerve root formation (Moody and Hea-ton, 1983; Kuratani and Eichele, 1993; Niederlander andLumsden, 1996). In L. japonica, the crest cells represent-ing the anlage of the octavofacial nerve covered the entire

Fig. 8. Embryonic brains of L. japonica at stages 24 and 25.Stained with the anti-AT monoclonal antibody. Dorsal views. A: Stage24 embryo. The photograph is focused on the luminal surface ofrhombomeres. Rhombomeric boundaries are indicated by small ar-rows and the mid-hindbrain border by a large arrow, respectively.B: The ventral aspect of the hindbrain showing the commissure axons

(a few of them are indicated by small arrows). C: Stage 25 embryo. Theluminal plane of the hindbrain is indicated by arrowheads at thefocused level. Note that the rhombomeric bulges have disappeared bythis stage. For abbreviations, see list. Scale bar 5 100 µm (applies toA–C).


length of r4, whereas no crest cells were adhering to r3(Fig. 6). More precise distribution pattern of crest cells willbe described elsewhere together with the development ofcranial ganglia.

So far, there have been no descriptions of agnathanneuromeres clear enough to compare with modern data ingnathostomes. For example, segmental bulges were illus-trated in Bdellostoma by von Kupffer (1900), which arealmost the only data obtained so far in myxinoids. How-ever, the morphology of the brain by their descriptionssuggests that the developmental stage is rather too late toobserve the neurepithelial bulge, and the segments aredubious as real rhombomeres. Nor is there any informa-tion about cranial nerve roots in this embryo (Dean, 1899).In lampreys also, the observed embryos were too old todetect the real rhombomeres (von Kupffer, 1895). Polygo-nal subdivisions or neuromeres were described in ratherold larvae of Petromyzon by Bergquist and Kallen (1953a,1953b). It is unlikely that these segments represent truedevelopmental compartments, because at least in L. ja-

ponica, the real rhombomeres totally disappear long beforethe stage corresponding to the embryos observed (Fig. 8C).In addition to the bi-rhombomeric registration of thecranial nerves to the hindbrain, the lamprey rhombomeresin the present observation share some features in commonwith gnathostomes (Orr, 1887): the dorsal part of rhombo-meres at early stages exhibit a fan-shaped arrangement ofneurepithelial cells, and the apical side of the cells isconstricted in the middle of each rhombomere (Fig. 4). Thisneurepithelial configuration is reported to occur in amni-ote rhombomeres also (Stunkard, 1922; Tuckett and Mor-riss-Kay, 1985; Heyman et al., 1993) and it has beenexplained by the formation of rhombomeres being inducedby cytoskeletal elements in the neurepithelial cells (Tuck-ett and Morriss-Kay, 1985). It remains to be studiedwhether the accumulation pattern of acetylated tubulin-like immunoreactive products has any relation to theneurepithelial compartmentalization.

Bi-rhombomeric repetition appears to be partly depen-dent on alternating cell adhesion for even- and odd-

Fig. 9. Schematic representation of the developing nervous systemof L. japonica. Neurons and neuromeres are shown in the scheme.Neuromerical swellings are shaded. Due to complexity, neuromeres instage 24 embryo are not shaded except for r4. Three rostral bulges atstage 22- correspond to, from rostral to caudal direction, parencephalon1secondary prosencephalon, synencephalon1mesencephalon, and ros-tral hindbrain segment (probably r112), respectively (see insert).Rhombomeres are most clearly seen at stage 23 around the level of r4.No rhombomeres caudal to r6 were found. Note that the topographicalrelationship between neuromeres and pharyngeal pouches is constant

throughout the developmental stages. Circle indicates the optic stalk.Inset: Hypothetical diagram of the neuromerical plan of the brain ofL. japonica. The rhombencephalon probably contains 6 rhombomeres(r1–6), of which the division of r1 and r2 is unclear. The neural tubecaudal to r6 is defined as the spinal cord. The forebrain is to besubdivided into at least three portions. Division of the telencephalon(t) and position of the posterior commissure (pc, dots) are fromKuratani et al. (1997). Cranial nerve roots are indicated by smallcircles. Post, posterior; ant, anterior; paren, parencephalon; mesen,mesencephalon. For other abbreviations, see list.


numbered rhombomeres (Layer and Alber, 1990; Lums-den, 1990; Kuratani, 1991). To detect possible differencebetween the two groups of rhombomeres in L. japonicaembryos, eight kinds of lectins (ConA, Concanavalin A;DBA, Dolichos biflorus agglutinin; LCA, lentil lectin;PHA-E4, phytohemagglutinin-E4; PNA, peanut agglutinin;RCA120, Ricinus communis agglutinin 120; UEA-I, Ulexeuropaeus agglutinin-I; WGA, wheat germ agglutinin)were tried on paraffin-sectioned specimens at stage 23 and24, but no specific binding patterns were obtained on thehindbrain (not shown).

In the hindbrain and pharyngeal systems, one of themost conspicuous differences between the lamprey andgnathostomes is the topographical location of the otocystin relation to cranial nerves. In gnathostomes, there isconsiderable difference in reported locations of the otocyst;it generally develops at the level between r5 and r6 (chick,early stage of rodents) or lateral to r5 (zebrafish, laterstage of rodents), roughly dorsal to the second pharyngealarch. Importantly, the otocyst in these animals alwaysseparates the caudal two crest cell populations in the head.In L. japonica, on the other hand, the otocyst is locatedlateral to r4 (Figs. 3, 6). In relation to this topography, theoctavofacial nerve root passes for a certain distance alongthe medial aspect of the otocyst in this animal (Fig. 7A).Such morphology is in accordance with the earliest configu-ration of the acustico-facialis ganglionic complex describedby Fisk (1954).

Compared with gnathostome pharyngula, the position ofthe whole pharyngeal system of lamprey is also shiftedrostrally in relation to the neuraxis, and the secondpharyngeal pouch arises ventral to the otic placode or r4(Fig. 10). This topographical situation is similar to that inPetromyzon as described by Veit (1939) and Damas (1944),which also resembles some fossil osteostracans, closerelatives of L. japonica (Janvier, 1996). The cranial nerveroots in these animals are thus oriented rostrally afterissuing from the brainstem (Fig. 10; also see Kuratani etal., 1997).

The early phase of axonal growth in the embryonic brainhas been described for a variety of gnathostome species(Windle and Austin, 1936; Windle and Baxter, 1936;Herrick, 1937; Stroer, 1956; Lyser, 1966; Windle, 1970;Keyser, 1972; Wilson et al., 1990; Easter et al., 1993;Hartenstein, 1993; Figdor and Stern, 1993; Burril andEaster, 1994). As is generally accepted, the early axonaltrajectories, especially those along the circumferentialpath, depend on the neurepithelial segmental boundaries(Figdor and Stern, 1993; reviewed by Wilson et al., 1993).In the chick hindbrain, accumulation of neurofilament isseen at the rhombomeric boundary regions (Lumsden andKeynes, 1989; Keynes and Lumsden, 1990; Guthrie andLumsden, 1991). In the lamprey embryos, however, nosuch accumulation was observed at any stage of develop-ment, nor did the commissure axons on the ventral aspectof the hindbrain (Figs. 5, 8) follow any segmental pattern.Likewise, the site of the posterior commissure that firstbecomes apparent at stage 25 (Kuratani et al., 1997) is notpreceded by any visible boundaries on the neurectoderm,which delineate the anterior border of the definite mid-brain.

It has been recognized in the zebrafish development thatreticulospinal neurons are arranged in a metamericalpattern; the same sets of neurons are distributed in eachrhombomere (Kimmel et al., 1982, 1985; Metcalfe et al.,1986; Mendelson, 1986a, 1986b). Such a feature may bewidespread in teleost embryos because a similar phenom-enon is reported in the goldfish (Lee et al., 1993), but hasnever been seen in other vertebrates (Tello, 1923; Mannsand Fritzsch, 1992; Lumsden et al., 1994). The branchialnerve efferent neurons are arranged in a pseudo-metameri-cal pattern in some vertebrates but are not always clearlylimited by rhombomeric boundaries (Bok, 1915; Tello,1923; reviewed by Gilland and Baker, 1993). In larvae oradult lampreys and hagfish, there is also no apparentmetamerical arrangement of particular neurons recog-nized in the hindbrain (Saito, 1928; Schober, 1964; Nieu-wenhuis, 1972; Rovainen et al., 1973; Ronan, 1989; Swainet al., 1993, 1994; Jacobs et al., 1996). At the 1-cm stage ofL. japonica, retrograde labeling of reticulospinal neuronsby 3,000-molecular weight dextran amines showed theaccumulation of labeled neurons at levels of cranial nerveroots (unpublished observation). This pattern was essen-tially similar to the accumulation of commissure neuronsthat starts at stage 24 when rhombomeres begin to disap-pear (Fig. 7), and the accumulation never coincided withrhombomeric boundaries. It remains to be tested by DiI-tracing method whether any homologous set of reticulospi-nal neurons develops in each rhombomere in earlierembryos.

The initial axons developing in the brain and the spinalcord are thought to function in guiding late developingaxons, thereby forming constant anatomical neuronal

Fig. 10. Topographical relationships between branchiomeric nervesand pharyngeal pouches of the lamprey and amniote embryos. Thewhole pharynx of L. japonica embryo is located more rostrally tothe hindbrain than that in the amniote embryos. Concomitantly, thebranchiomeric nerve roots are oriented rostrally after issuing from thehindbrain. The otic placode in the lamprey is also located morerostrally (r4 level) than that in amniotes (r5 to r6). For abbreviations,see list.


tracts (Shiga and Oppenheim, 1991; Chitnis et al., 1992;reviewed by Kimmel, 1993). Such pattern is also depen-dent on the neuromerical configuration of the neurecto-derm, which is expected to be rather constant amongvertebrates (reviewed by Kimmel, 1993). The early brainof the lamprey embryo showed rather a conserved patternof tract formation. For example, the interstitial nucleus orthe precursor of MLF is one of the earliest neurons todevelop in gnathostomes (Windle and Austin, 1936; Windleand Baxter, 1936; Lyser, 1966; Windle, 1970; Easter et al.,1993).

The mesencephalic trigeminal neurons also developquite early in some gnathostome embryos (Kuratani et al.,1988; Covell and Noden, 1989; Easter et al., 1993). Theseneurons are believed to arise from the mesencephalicneural crest (Piatt, 1945; Covell and Noden, 1989) andresemble RB cells in that their cell bodies remain in theneurectoderm. The mesencephalon of the lamprey showedno sign of axons arising from the mid-sagittal line ofthe mesencephalic roof from stages 20 through 30 (untilthe completion of ammocoete larva), which should coverthe period of neurogenesis in the neural crest. Probably,unlike gnathostomes, the lamprey mesencephalic cresthas no potential for Rohon-Beard cell-like neurogenesis.This notion is consistent with the absence of the mesence-phalic trigeminal nucleus in the adult lamprey (reviewedby Sarnat and Netzky, 1981). The dorsal medullar neuronsthat have been suggested to be homologous with thegnathostome mesencephalic trigeminal nucleus (North-cutt, 1979; but see Finger and Rovainen, 1982, and Koyamaet al., 1987) were not found at any developmental stagesobserved by the methods used in the present study.

There have been various opinions as to the commonsegmental plan of the vertebrate brain. The position andnumber of the neuromeres is still an intriguing issue,which is profoundly connected to the position of theneuraxis (or the site of sulcus limitans and/or the floorplate) and the rostral end of the neural tube (reviewed byPuelles et al., 1987; Puelles and Rubenstein, 1993; Ruben-stein et al., 1994; Shimamura et al., 1995). The earlymorphology of the lamprey brain does not show the fullsets of neuromeres that have been suggested in gnathos-tome embryos (Fig. 9). Observation of the brain in thewhole-mounts is a prerequisite, but is made difficult by thesize of the specimen. Still, the site of the epiphysis at latestage 24 (Fig. 7) suggests that the rostral-most faintboundary seen at stage 21 would probably correspond tothe D2/D3 boundary of Figdor and Stern (1993) or theP1/P2 boundary of Puelles and Rubenstein (1993). If this isthe case, the boundary shown by dotted lines throughoutthe figures (Figs. 1C, 2B, 3B, 5A, 7) would be equivalent tothe syn-parencephalic border of Puelles et al. (1987) in thechick embryo, as well as to the posterior diencephalicborder of Coggeshall (1964) in mammals. It remains to beexamined whether clear boundaries develop in the lam-prey in the same pattern as those in amniotes delineatingdevelopmental compartments. Focal injections of dyes andin situ analyses of some regulatory gene expression pat-terns will be necessary to clarify this problem.

ACKNOWLEDGMENTS

We thank Nobutoshi Maeda and Miyuki Yamamoto inthe Institute for Laboratory Animals, Niigata UniversitySchool of Medicine for maintenance of animals. We also

thank Drs. Hiromichi Koyama, Ruth T. Yu, and NorikoOsumi for their helpful discussion. Sincere gratitude isextended to Dr. Syosuke Kawamura for his valuablecomments on the manuscript. This work was supported bygrants-in-aid from the Ministry of Education, Science andCulture of Japan (Specially Promoted Research), and theScience and Technology Agency of Japan.

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stereotyped axonal bundle formation and neuromeric...

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