Download Peripheral Development of Cranial Nerves in a Cyclostome

THE JOURNAL OF COMPARATIVE NEUROLOGY 384:483–500 (1997)
Peripheral Development of Cranial
Nerves in a Cyclostome, Lampetra
japonica: Morphological Distribution
of Nerve Branches and the Vertebrate
Body Plan
SHIGERU KURATANI,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 860, Japan
2Department of Anatomy, Niigata University School of Medicine, Niigata 951, Japan
ABSTRACT
The development of peripheral nerves was studied in a Japanese marine lamprey,
Lampetra japonica, in whole-mount and sectioned embryos from hatching until the earliest
ammocoete. Nerve fibers were immunohistochemically stained with a monoclonal antibody
against acetylated tubulin. Branchiomeric nerves first developed in a simple metamerical
pattern, each associated with a single pharyngeal arch. Of those, the ophthalmicus profundus,
maxillomandibular, and facial nerves later developed a highly modified branching pattern,
whereas postotic nerves were less specialized and showed the stereotypical branching pattern
of post-trematic nerves. The early distribution of melanocytes in myotome-free space largely
overlapped with the morphology of the cranial nerve and ganglion anlage, and resembled the
cephalic crest cell distribution pattern in the early chick embryo. It was suggested that the
cephalic crest cell distribution, which is also inhibited by myotomes in the lamprey, would be
the common basis for branchiomeric nerve patterning. In later development of the lamprey
embryo, myotomes 1 through 3, which had originated in the postotic region, grew rostrally
into the preotic region, laterally covering all of the branchiomeric nerves. This results in a
deep position of the cranial nerves, which is not observed in gnathostomes. J. Comp. Neurol.
384:483–500, 1997. r 1997 Wiley-Liss, Inc.
Indexing terms: branchial region; neural crest; lampreys; embryo; mesoderm
The gnathostome embryonic head is characterized by
possession of two metamerisms, branchiomerism and somitomerism. The evolutionary origin of this body plan is still
enigmatic because these two repetitions are not clearly
separated in the nervous system of the amphioxus (e.g.,
Bone, 1959). To obtain insight into the primitive patterns
of head metamerism, it seemed valuable as a first step to
examine the developmental sequence of the pharyngeal
regions in a sister group of gnathostomes. As a candidate
for such animals, we chose Lampetra japonica, a marine
lamprey available during their mating season in northern
rivers of Japan.
Lampreys used to be regarded as primitive representatives of a state between acraniate and gnathostome vertebrates (Neal, 1918a; de Beer, 1931; reviewed by Janvier,
1996. The presence of myotomes in the head region and the
presence of the endostyle in the larval state were viewed
r 1997 WILEY-LISS, INC.
especially as plesiomorphic traits. The pre-ammocoete
developmental process of the peripheral nervous system
(PNS) should give us a clue to the plan of the vertebrate
head morphology, but no detailed embryological studies
have been performed. The description by Johnston (1905)
of the peripheral morphology of the cranial nerve in
Petromyzon was based on analysis of a feeding stage larva.
Grant sponsor: NICHD; Grant number: N01-HD-2-3144; 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 (IMEG), Kumamoto
University School of Medicine, 4-24-1 Kuhonji, Kumamoto, Kumamoto 860,
Japan. E-mail: [email protected]
Received 17 September 1996; Revised 22 January 1997; Accepted 12
February 1997
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S. KURATANI ET AL.
Although spatiotemporal development of the cranial ganglia in Lampetra was also reported (Damas, 1944), the
initial nerve branching pattern, which would be correlated
with the primary branchiomere, has not been reported.
Among the recent advances in embryology, whole-mount
immunostaining has made it possible to observe the
three-dimensional morphology of the nervous system (Ishikawa et al., 1986). Especially in early embryonic stages,
this technique can be used to visualize the PNS, which
cannot be accurately reconstructed from histological sections. Here we describe cranial nerve development in
embryos of L. japonica based on whole-mount and sectioned specimens stained with several monoclonal antibodies. This is the first description of the peripheral morphology of developing cyclostome cranial nerves. At the same
time, it is intended to evaluate the relevance of cyclostome
morphology to understanding vertebrate head development.
MATERIALS AND METHODS
Embryos
Adult male and female lampreys (Lampetra japonica)
were collected in the Miomote River, Niigata, Japan,
during the breeding season (late May through June) in
1995 and 1996. They were brought into the laboratory,
where eggs were artificially fertilized and kept in fresh
water at 18–23°C. Embryos were fixed twice a day with
two kinds of fixatives, each for a different purpose (see
below): 4% paraformaldehyde in 0.1 M phosphate-buffered
TABLE 1. Comparison of Developmental Stages
L. reissneri
by Tahara (1988)
Stage 21
Stage 22
Stage 23
Stage 24
Stage 25
Stage 26
Stage 27
Stage 28
Stage 29
Body length
–3mm
3–3.5mm
4mm
5mm
6–7mm
7.5–8mm
L. fluviatilis
by Damas (1944)
P. marinus
by Piavis (1971)
III–IV
V
VI
VII–VIII
IX–X
XI
XII–XIII
XIV
XV
12
13
13
13–14
14
15
15
16
18
saline (PFA/PBS) and Bouin’s fixative. Because development of the embryos was significantly affected by temperature, they were staged morphologically. The developmental sequence of the L. japonica embryos is similar to that of
the brook species, L. reissneri, so that staging reported by
Tahara (1988) for the latter was used in the present
description. The comparison of developmental sequences
between L. reissneri, L. fluviatilis (Damas, 1944), and
Petromyzon marinus (Piavis, 1971) is listed in Table 1. For
nervous elements, the terminology of Alcock (1898), Johnston (1905), Lindström (1949), Ronan and Northcutt (1987),
and Koyama et al. (1990) was used.
Whole-mount immunostaining
For whole-mount immunostaining, embryos were prepared as described (Kuratani and Eichele, 1993) with
minor modifications. After fixation with PFA/PBS at 4°C
Abbreviations
a3
bc
bv
Cc
dlt
dsr
eb
ebm
ebt
ep
es
eso
fb
fg
gldix
gldx1
glf
glv2,3
hb
hbm
hm
hy
int
io
llp
m
ma
mb
mbc
mc
me
mp
mvl
n
olep
ot
p1–p8
a6 pharyngeal arches
branchial arch cartilage
branchial arch vein
circumpharyngeal crest cells in the chick embryo
dorsolateral fasciculus
dorsal spinal nerves
eyeball
epibranchial muscles
epibranchial tract of the vagus nerve
epiphysis
endostyle
esophagus
forebrain
foregut
epibranchial ganglion of the glossopharyngeal nerve
epibranchial ganglion of the first vagus nerve
facial ganglion
maxillomandibular nerve ganglion
hindbrain
hypobranchial muscles
hyomandibular nerve
hyoid arch
intestinal ramus of the vagus nerve
infraoptic nerve
lower lip
myotomes
adductor muscle of the branchial arch
midbrain
buccal muscle
constrictor muscle
external mandibular ramus
muscle plate of the branchial arch
ventral lip muscle
notochord
olfactory epithelium
otocyst
pharyngeal pouches
pc
ph
pllg
plln
plsp
pnd
prtx1
ps4
rb
rbc
rdix
rc
rm
rmp
rpvii
rrec
rthyr
rv
rvel
sc
so
s1–s8
tb
ulp
vel
vsr
ws
II
V
V1
V2,3
VII
IX
X
x1–x6
XII
posterior commissure
pharynx
posterior lateral line ganglion
posterior lateral line nerve
spinal nerve plexus
pronephric duct
pre-trematic ramus of X1
fourth pharyngeal slit
buccal ramus of the facial nerve
buccinator ramus of the facial nerve
dorsal ramus of the glossopharyngeal nerve
communicating branches of vagus nerves
maxillary ramus of the maxillomandibular nerve
posterior muscular rami of the pharyngeal ramus
pharyngeal ramus of facial nerve
recurrent nerve
thyroid ramus of the facial nerve
visceral rami of vagus nerves
velar ramus of the maxillomandibular nerve
sclerotome
supraoptic nerve
somites
taste bud
upper lip
velum
ventral spinal nerves
ciliary string of the branchial arch
optic nerve
trigeminal nerve anlage in gnathostome embryos
ophthalmicus profundus nerve
maxillomandibular nerve
facial nerve
glossopharyngeal nerve
vagus nerve
first to sixth vagal branchial branches
hypoglossal nerve
CRANIAL NERVE DEVELOPMENT IN THE LAMPREY
485
Fig. 1. Microphotographs of the whole-mount stage 25 embryos
stained with anti-acetylated tubulin antibody. A: All the branchiomeric nerve roots have developed showing metamerical organization.
Note the presence of spinal motor nerves (arrowheads). Pronephric
ducts (pnd) are also stained with the antibody. B: Enlargement of a
head portion of A. Position of the otocyst is marked by a circle. Each
branchiomeric nerve trunk is oriented rostrally toward the dorsal tip
of the pharyngeal pouches (p1–4). Proximally, the nerves are arising
from the dorsolateral fasciculus (dlt) of the hindbrain. Arrowheads
indicate the independent development of ophthalmic profundus nerve
root. The vagus nerve (X) passes between myotomes 2 (m2) and 3 (m3).
The arrow points to the communication between the facial and
maxillomandibular nerves. Scale bars 5 100 µm.
for 1 day, embryos were washed in 0.9% NaCl/distilled
water, dehydrated in a graded methanol series (50%, 80%,
100%), and stored at 220°C.
The samples to be stained were placed on ice in 2 ml
dimethyl sulfoxide (DMSO) methanol (1:1) until they sank.
Five-tenths milliliter of 10% Triton X-100/distilled water
was added, and the embryos were further incubated for 30
minutes at room temperature. After washing in TST (TST
is Tris-HCl-buffered saline: 20 mM Tris-HCl, pH 8.0, 150
mM NaCl, 0.1% Triton X-100), the samples were sequen-
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S. KURATANI ET AL.
Fig. 2. Morphological pattern of myotomes and cranial nerves. Myotomes (numbered) are superimposed over a stage 25 embryo. The glossopharyngeal nerve root develops medial to myotome 1. The vagus
nerve (X) passes between the second and third myotomes. Scale bar 5 100 µm.
Fig. 3. Graphical illustration of the neurite development at stage
26 of L. japonica based on a whole-mount embryo. Ventral (vsr) and
dorsal spinal nerve (dsr) anlagen are developing in an alternating
segmental pattern. The distal portion of the maxillomandibular nerve
(V2,3) has been bifurcated into two portions, one innervating the
upper lip (ulp) and the other (rvel) the velum. The main branch of the
facial nerve (VII), the hyomandibular nerve (hm), innervates the lower
lip (llp), showing the latter structure is a derivative of the hyoid arch.
The vagus nerve (X) has branched to the pre-trematic ramus (prtx1) in
the first branchial arch and a post-trematic nerve (x1) in the second
branchial arch. Caudally, the vagus nerve develops a bundle of nerves,
the epibranchial trunk (ebt). Dorsally, the posterior lateral line nerve
(plln) is independent of the vagus nerve anlage. Note the development
of independent bundles of fibers (asterisks) found close to the surface
ectoderm. One of these is the earliest anlage of the dorsal ramus (rdix)
of the glossopharyngeal nerve. Scale bar 5 100 µm.
tially blocked with aqueous 1% periodic acid and with 5%
nonfat dry milk in TST (TSTM).
For whole-mount immunostaining of the cranial nerves,
a monoclonal antibody raised against acetylated tubulin
(monoclonal anti-acetylated tubulin, No. T-6793, Sigma
Chemical Company, St. Louis, MO) was found to be most
suitable. Embryos were incubated in the primary antibody (diluted 1/1,000 in spin-clarified TSTM containing
0.1% sodium azide) for 2 to 4 days at room temperature while being gently agitated on a shaking platform. The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZYMED Lab.
Inc., San Francisco, CA) diluted 1/200 in TSTM. After a
final wash in TST, the embryos were preincubated with
peroxidase substrates 3,38-diaminobenzidine (DAB, 100
mg/ml) in TS (20 mM Tris-HCl, pH 8.0, 150 mM NaCl) for 1
hour and allowed to react in TS with the same concentration of DAB with 0.01% hydrogen peroxide for 20 to
40 minutes at 0°C. The reaction was stopped and simultaneously embryos were clarified with 30% glycerol in
0.5% KOH. The stained embryos were transferred to a
60% glycerol/water mixture and mounted on glass depression slides for observation. CH-1 (purchased from the
Developmental Studies Hybridoma Bank, Iowa City,
IA), which recognizes tropomyosin, was used to detect
the development of myotomes. Hardisty and Rovainen
(1971) was referred to for the anatomical identification of
muscles.
Fig. 4. Photomicrographs of whole-mount embryos of L. japonica
at stage 26. A,C: Lateral aspects of the embryos. The embryo in C is
slightly older than those in A. B,D: Enlargement of the boxes in A and
C, respectively. In B and C, the myotomes are illuminated by polarized
light. Arrowheads in B indicate the rostral and caudal surface of
myotome 2. The vagus nerve (X) develops an arch-shaped nerve trunk
(B,C). In slightly older specimens (D), late-forming vagal nerve fibers
can develop along the boundaries of myotomes (arrows), indicating
that the myotomes inhibit the neurite growth. Scale bars 5 100 µm.
Fig. 5. Sectioned specimens of a stage 26 embryo stained with
HNK-1 (an antibody that stains the nervous system). A: At the level of
the first arch. The maxillomandibular ganglion (glv2,3) is situated
beneath the surface ectoderm. B: At the level of the otocyst. Myotomes
(m) cover the otocyst (ot) and the petrosal ganglion (gldix). C:
Sclerotomes (sc) and myotomes (m) are being separated at this level.
The root and the epibranchial ganglion of the vagus nerve (X) are
located medial to the myotome. D: Trunk level. The myotome is not
separated from the sclerotome at this level. The vagal epibranchial
ganglion (gldix) lies ventral to the myotome. The posterior lateral line
nerve (plln) is located medial to the dorsomedial edge of the myotome
(arrow). Scale bar 5 100 µm.
CRANIAL NERVE DEVELOPMENT IN THE LAMPREY
489
Fig. 6. Schematic diagram to show the topographical relationships
between the nervous system and mesodermal components at stage 26.
Oral cavity and pharyngeal pouches are indicated by dashed lines.
Owing to the growth of the myotomes, the postotic nerve trunks and
ganglia are covered by myotomes. Based on the same embryo shown in
Figure 5 (A–D indicate the planes in which the sections were cut).
Scale bar 5 100 µm.
Immunostaining on sectioned specimens
RESULTS
Specimens fixed in Bouin’s were embedded in paraffin
and sectioned at 5 µm. They were deparaffinized and
treated with 1% periodic acid for 5 minutes at room
temperature, followed by washing in TST. Monoclonal
antibodies T-6793, as well as HNK-1 (Leu-7, Becton Dickinson, San Jose, CA), were used to label the developing
nervous system on the paraffin-sectioned specimens. Primary antibodies were diluted in TSTM and applied to the
sections for 1.5 hours at room temperature. After washing
with TST, secondary antibodies, HRP–anti-mouse IgG (for
those labeled with T-6793) and IgM (for those labeled with
HNK-1) (ZYMED Lab. Inc.), were diluted 1/200 in TSTM
and applied to the specimens for 40 minutes. The sections
were counterstained either with cresyl violet or hematoxylin after the peroxidase reaction.
The following description of our results deals with the
peripheral nervous system (PNS) morphology after
stage 25. The earlier development of the nerves will be
described elsewhere (Kuratani et al., unpublished observation). Briefly, the facial nerve root anlage first develops at
early stage 23 (stage 23-), followed by the maxillomandibular nerve root that appears at stage 23. The ophthalmicus
profundus nerve root is first recognized late in stage 23
(stage 231) to stage 24.
Morphology of branchiomeric nerves
Stage 25 (Figs. 1, 2). By stage 25, the four anteriormost pharyngeal pouches (pharyngeal pouches 1 through
4) are visible, and the otic vesicle has formed. The ophthalmicus profundus and maxillomandibular nerves arise
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S. KURATANI ET AL.
Fig. 7. Developing peripheral nerves in stage 27 embryos. A:
Graphical illustration of the neurite development based on a wholemount embryo. All the major branches have appeared by this stage.
The dorsal ramus of IX (rdix) is connected with the main trunk of the
glossopharyngeal nerve (IX). The thyroid ramus (rthyr) that passes
caudally in the pharyngeal floor has branched from the facial nerve.
The overall configuration of the peripheral nervous system shows
separation between the branchiomeric and somitomeric series of
nerves. Note that the main trunk of the vagus (X) and hypoglossal
nerves (XII) passes along the interface between the somitic and
branchiomeric regions, the circumbranchial region. B: Photomicrograph of a different embryo at the same stage. Scale bars 5 100 µm.
from the hindbrain with separate roots from the area
corresponding to rhombomere 2 at the preceding stage
(Fig. 1). The maxillomandibular nerve extends several
branches in front of the first pharyngeal pouch or within
the mandibular arch. The facial nerve arises from the
hindbrain, medial to the otocyst and is directed rostrally
toward the dorsal end of the first pharyngeal pouch. Dorsal
to the pouch, the facial nerve makes a connection with the
maxillomandibular nerve. Caudal to the otocyst, the glossopharyngeal nerve arises from the hindbrain at the level
of the third pharyngeal pouch (Fig. 1). Curiously, this
nerve lies medial to myotome 1 (Fig. 2). More caudally, the
vagus nerve trunk has developed and passes between
myotomes 2 and 3 (Figs. 1B, 2). The posterior lateral line
nerve primordium is present, lateral to the dorsolateral
fasciculus of the hindbrain and caudal to the otocyst, with
its rostral end close to the vagus nerve root. In the trunk
region, this nerve is found on the dorsomedial edge of
myotomes (see Fig. 4D).
Stage 26 (Figs. 3–6). Pharyngeal pouch 8 appears in
some embryos by stage 26. The ophthalmicus profundus
nerve extends its processes along the lateral aspect of the
forebrain. The maxillomandibular nerve possesses two
obvious branches by this stage, one innervating the upper
lip (maxillary ramus) and the other the velum (velar
ramus). Lateral to the facial nerve, there develops a fine
bundle of fibers, the buccal nerve, that belongs to the
anterior lateral line nerve. Rostrally, the buccal nerve
extends lateral to the maxillomandibular nerve and ventral to the ophthalmicus profundus nerve. The main
branch of the facial nerve has formed the ramus hyoideus
within the second pharyngeal arch.
The main branch of the glossopharyngeal nerve, the
post-trematicus nerve, has grown into the first branchial
CRANIAL NERVE DEVELOPMENT IN THE LAMPREY
491
Fig. 8. Semi-diagrammatic illustration of a stage 28 embryo. A:
The whole head of the embryo. Terminal branches of facial (me),
glossopharyngeal, and vagus (x1–x6) nerves are innervating lateral
line organs which are shown by circles. Note the extensive plexus
formed of rostral spinal nerve roots (plsp). From the plexus two
branches (supraoptic nerve 5 so and infraoptic nerve 5 io) arise and
pass rostrally to innervate myotomes 1 through 3 (see Fig. 10). B:
Enlargement of the branchiomeric nerve roots, showing the passage of
putative motor fibers within the ganglion. Note the development of the
recurrent nerve (rrec) connecting the facial (glf) and posterior lateral
line (pllg) ganglia. Arrow indicates the communication with the spinal
nerve plexus (plsp in A) which is cut. Scale bars 5 100 µm.
arch by stage 26. A lateral line nerve is often found close to
the glossopharyngeal nerve, provisionally called the dorsal
ramus (Fig. 3). The epibranchial ganglion of the glossopharyngeus lies medial to the myotome (Figs. 5, 6).
The vagus nerve is an arch-shaped bundle of fibers,
consisting of a dorsal part representing the proximal root
and a ventral part representing the epibranchial tract
(Fig. 3). As a whole, this nerve forms an arch that
overhangs slightly rostral to the myotome 2⁄3 border (Fig.
4). Late-forming nerve fibers can develop along this border
(Fig. 4D). No nerve branches are found on the surface of
the myotome. The proximal root passes through the posterior lateral line ganglion medial to the dorsolaterally
expanding myotomes (Fig. 6). Along the epibranchial tract
of the vagus, sensory ganglion primordia are found between each pharyngeal pouch, ventrolateral to the myo-
tome (Figs. 5, 6). Ventrally the vagus nerve possesses the
post-trematic ramus of x1 within the second branchial
arch and sometimes grows the post-trematic ramus of x2
and other posterior branches in slightly older embryos
(Fig. 3). A pre-trematic ramus can accompany the nerve x1
(Fig. 3).
Stage 27 (Fig. 7). The ophthalmicus profundus nerve
passes dorsal to the eye by stage 27, and the ophthalmic
ganglion is separated from the maxillomandibular ganglion as is the root. The maxillary ramus of the maxillomandibular nerve innervates the anterior buccal muscle in the
upper lip (not shown).
Associated with the facial nerve, a sensory ganglion
primordium is visible in the whole-mount (Fig. 7). In its
relation to the buccal nerve, this seems to represent the
anlage of the anterior lateral line ganglion in the adult
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S. KURATANI ET AL.
Fig. 9. Photomicrographs of the same stage 28 embryo as in Figure
8. A: Trigeminal innervation area. Ganglia are marked by asterisks.
The ophthalmic profundus (V1) and maxillomandibular (V2,3) nerves
still possess two separate nerve roots on the hindbrain. Note that in
the maxillomandibular nerve the neurite bundle bifurcates within the
maxillomandibular ganglion (arrows) and passes into the maxillary
(rm) and velar (rvel) branches. B: Nerve plexus formed of several
rostralmost ventral spinal nerves (arrowheads). C: The intestinal
ramus (int) of the vagus nerve. The intestinal tract is indicated by
broken lines. Note the dorsal position of the ramus in relation to the
esophagus (eso) and foregut (fg). Scale bar 5 100 µm.
(Ronan and Northcutt, 1987; Koyama et al., 1990). The
intracapsular or otic ganglion was not clearly identified in
the present study; the ganglion associated with the facial
nerve is, therefore, simply referred to as the facial ganglion in the discussion below. The ventral part of the
hyomandibular ramus has extended several smaller
branches into the lower lip. They correspond to the external mandibular ramus anlage that primarily belongs to
the lateral line nerve. The distal portion of the hyomandibular nerve, or the thyroid ramus, grows caudally traversing
the ventral aspect of the pharynx.
As the rostral vagus nerve root develops, it becomes
continuous rostrally with the glossopharyngeal nerve root.
The posterior part of the root remains separated from the
glossopharyngeo-vagal root. In many embryos the vagus
nerve possesses six post-trematic branches (x1–x6). The
pre-trematic ramus of x1 has disappeared by this stage.
Stage 28 (Figs. 8–12). Because the monoclonal antibody T-6793 does not stain the perikarya at stage 28, it is
often possible in the whole-mount embryo to identify
rami-containing motor fibers as a non-bifurcating bundle
penetrating the ganglion (Figs. 8B, 9A). The ophthalmicus
profundus nerve appears to be composed of sensory fibers.
In the maxillomandibular nerve, the maxillary nerve has
grown a medial branch, the buccinator ramus (Fig. 8A),
that passes caudally into the buccal floor. The buccal
ramus of the facial nerve appears not to contain any motor
fibers, consistent with the adult anatomy (Koyama et al.,
1990).
Our observations suggest that the glossopharyngeal
sensory fibers participate in this ramus (Fig. 8B). Most of
the motor fibers passing through the facial ganglion originate from a more ventral level of the hindbrain than that
of the sensory root (Fig. 8B), and they pass mainly into the
thyroid branch, more or less separated from the external
mandibular ramus (Fig. 8B). The latter ramus arises
directly from the facial ganglion and would also be sensory; the nerve bifurcates distally and some of the nerve
bundles terminate on the lateral line organs (Fig. 8A).
From the more proximal portion of the hyomandibular
CRANIAL NERVE DEVELOPMENT IN THE LAMPREY
493
Fig. 10. Morphology of the myotomes at stage 28. Myotomes (numbered) are superimposed over a
stage 28 embryo. Note that rostral myotomes (1 to 3) which developed postotically have grown rostrally
into the preotic region. The hypobranchial muscle (hbm) is developing along the course of the hypoglossal
nerve. Scale bar 5 100 µm.
ramus, a posteriorly oriented ramus, provisionally called
the posterior pharyngeal ramus, arises and grows along
the dorsal aspect of the pharynx (Fig. 8B).
The recurrent nerve is constantly present; the nerve
communicates between the facial and the posterior lateral
line ganglion, and also communicates with the dorsal
ramus of the glossopharyngeal nerve lateral to the otocyst
(Fig. 8A,B). Within the posterior lateral line ganglion, the
vagus nerve rootlets pass caudal to the glossopharyngeal
nerve root. The latter is also located within the ganglion
(Fig. 8B). The intestinal ramus arises from the caudal
portion of the epibranchial tract of the vagus and passes
caudally along the dorsal aspect of the esophagus and
foregut (Fig. 9C). The epibranchial tract of the vagus has
established an extensive communication with the hypoglossal nerve by this stage. The latter nerve has invaded the
ventrolateral aspect of the pharyngeal wall. Simultaneously, the hypobranchial muscle anlage appears in the
ventrolateral aspect of the pharyngeal wall which later
divides into repeating segments (Fig. 10).
Myotomes 1 and 2 grow rostrally, covering laterally all of
the cranial nerves in the preotic region (Figs. 10, 11). The
rostral group of ventral spinal nerves form an extensive
nerve plexus (Fig. 9B) and send out two nerve branches to
innervate myotomes 1 through 3, one dorsal to the eye (the
supraoptic nerve), and the other ventral to the eye (the
infraoptic nerve) (Figs. 8A, 10). Due to the position of the
muscles they innervate, these spinal nerve branches are
located lateral to the dorsal ramus of IX (Fig. 8A).
The distribution pattern of melanocytes, probably of
neural crest origin, first becomes apparent at this stage
and largely overlaps with the PNS elements (Fig. 12). The
melanocytes generally fall into two categories: those surrounding the central nervous system (CNS), and more
compact cell populations in the pharyngeal area. The
latter is further divided into three major cell populations,
i.e., the anterior population within the upper lip and in the
prechordal region, the second within the hyoid arch, and
the posterior in the postotic branchial arch region (Fig. 12).
The posterior cell population is stacked between epibranchial myotomes and the pharyngeal arches forming a
compact longitudinal cell strand corresponding to the
epibranchial tract of the vagus nerve. At the level of
epibranchial ganglia, this cell strand shows local swellings
(Fig. 12B). Slightly later, streams of melanocytes from this
cell strand extend ventrally to fill each branchial arch (Fig.
12B). It could not be determined, however, whether this
was due to the migration of the cells or delayed differentiation of the ectomesenchyme that already populated the
arches.
Morphology of branchial branches (Fig. 13)
Caudal to the glossopharyngeal nerve, the branchiomeric nerve branches of L. japonica have similar morphology in each branchial arch. Morphology of the branches is
closely associated with the branchial arch. In histological
sections of a stage 26 embryo, a typical branchial arch of L.
japonica is first seen as a simple, dorsoventral plate of the
pharyngeal wall, successively delineated by endodermal
indentations which are the pharyngeal pouches (Fig. 13A).
Each arch contains a muscle plate that occupies the caudal
aspect and a cartilage bar in the rostralateral portion.
Each post-trematic nerve passes between the muscle plate
and the cartilage (Fig. 13A).
The branchial arch grows in the rostromedial direction
at stage 27 and the muscle plate also grows in the same
direction (Fig. 13B, C). All of the arches now contain the
post-trematic nerve, which extends small branches posteriorly and also produces ventral communicating branches
(Fig. 7). The posterior ramus innervates the constrictor
muscle, the derivative of the posterior part of the muscle
plate (Fig. 13D). There is also a rostrally oriented visceral
ramus issuing from the post-trematic nerve (Figs. 8A,
13D).
494
S. KURATANI ET AL.
Fig. 11. Development of the myotome in late embryos. A: Photomicrograph of a stage 28 embryo. A whole-mount embryo stained with
anti-acetylated tubulin antibody. Myotomes are simultaneously illuminated with polarized light. Note the rostral growth of rostral myotomes (m2 and m3) that have divided into ventral and dorsal halves on
dorsal and ventral sides of the eye. The epibranchial tract (ebt) with
epibranchial ganglia develops between the myotomes and pharyngeal
arches, and the posterior lateral line nerve (plln) along the dorsal edge
of the myotomes. B: Stage 30 embryo stained with CH-1 (tropomyosin)
antibody. The hypobranchial muscle (hbm) has been divided longitudinally into pseudometamerical segments, that are associated neither
with true branchiomerism nor with epibranchial myotomes (ebm).
Note the muscles that are present in the upper lip (buccal muscle,
mbc) and in the lower lip (mvl). Scale bars 5 500 µm.
Perforation of the pharyngeal slits is completed by stage
29 (Fig. 13E). The visceral ramus innervates the rostromedial subdivision of the branchial arch, the ciliary string
(Fig. 13E), where the nerve fibers are associated with taste
buds (Fig. 13E). The anterior subdivision of the muscle
plate, the adductor muscle, is also associated with the
visceral ramus (Fig. 13D). By stage 29 the post-trematic
trunk has shifted to the lateral aspect of the branchial arch
cartilage. The main trunk of the post-trematic nerve grows
farther ventrally and bifurcates into several small branches
which are mostly innervating lateral line organs in the
ventral aspect of the pharynx (Fig. 8A).
DISCUSSION
Branchiomeric nerve morphology
Through the developmental stages examined, the ophthalmicus profundus nerve of L. japonica develops independently from the maxillomandibular nerve (Figs. 1, 3, 7, 8).
In the adult L. japonica, however, both of the roots are
fused to form a single root as seen in many gnathostomes
(Koyama et al., 1987). This indicates that the separate
profundus nerve configuration is an embryonic feature,
which has also been regarded as a plesiomorphic character
(reviewed by Goodrich, 1930).
CRANIAL NERVE DEVELOPMENT IN THE LAMPREY
495
Fig. 12. Distribution pattern of melanocytes in L. japonica. A: The
lateral view of a stage 28 embryo. Anterior is to the left. Melanocytes
form three separate populations, the rostral (arrowheads), the hyoid
arch (hy), and the glossopharyngeo-vagal or caudal cell populations. A
small number of cells surround the neural tube. The caudal cell
population is accumulated along the interface between myotomes
(illuminated with polarized light) and pharyngeal arches. B: Stage 29.
Anterior is to the right. From the accumulation of melanocytes along
the epibranchial tract, some subpopulations migrated into each pha-
ryngeal arch (a3–a6). Arrows indicate the position of vagal epibranchial ganglia surrounded by melanocytes. The arrowhead indicates
the accumulation of melanocytes between the maxillomandibular
ganglion and facial ganglion. C: HNK-1-stained section of an embryo
at stage 29 at the level of the second pharyngeal arch. Dark cells along
the arch are melanocytes (arrows). Note that the cells surround the
pharynx (ph) and the hindbrain (hb) as well as the notochord (n). Scale
bar 5 200 µm for A, B, 100 µm for C.
As found in other aquatic forms (reviewed by Haller v.
Hallerstein, 1934), branches of glossopharyngeal and vagus nerves of the lamprey appear to be in a low state of
specialization, each being similar to the others (Fig. 8A).
The main ramus of a typical branchiomeric nerve is the
post-trematic nerve. Unlike some aquatic gnathostomes
(Tanaka, 1976, 1979; Tanaka and Nakao, 1979), however,
pharyngeal and pre-trematic nerves are not found in all
the stages examined, except for a temporary pre-trematic
branch associated with x1 (Fig. 3). Each branchial arch of
L. japonica is subdivided into anteromedial and posterolateral portions and so is the branching pattern of the
post-trematic nerve. The visceral ramus innervating the
anteromedial part, or the ciliary string, presumably contains gustatory as well as motor fibers. Morphologically, no
equivalent branching was found in the maxillomandibular
or facial nerves, although gustatory fibers have been
reported in this nerve of the lamprey by Fritzsch and
Northcutt (1993). The posterolateral portion of the branchial arch is innervated by the posterior ramus. A ramus of
equivalent nature would be found in the velar ramus of the
maxillomandibular and in the thyroid ramus of the facial
nerve (Fig. 8A). The post-trematic ramus bifurcates distally to terminate on lateral line organs (Fig. 8A). The
external mandibular ramus of the facial nerve, which
primarily belongs to the anterior lateral line nerve in
gnathostomes, may be of a similar nature (Fig. 8A,B).
Each epibranchial ganglion of the vagus nerve in Petromyzon planeri sends out a minor ramus dorsal to each gill
opening, innervating epibranchial sense organs (Dohrn,
1888; Alcock, 1898). In L. japonica, similar rami were
found to arise from the epibranchial tract and terminate
on the lateral line organs by stage 29 (not shown). These
rami were barely visible in younger embryos.
The posterior ramus of the maxillomandibular nerve
that innervates the velothyroid muscle in the velum has
been termed the velar ramus (Figs. 3, 7, 8). This nerve
apparently resembles the mandibular ramus of the maxillomandibular nerve of gnathostomes. However, the other
ramus of the maxillomandibular nerve of L. japonica, the
maxillary branch, also contains motor fibers and is distributed in the upper lip. Johnston (1905) thought the maxillary branch contained a part of the mandibular ramus in
other fishes; i.e., both the rami appear to correspond to
gnathostome mandibular ramus. Immunocytochemical
data are consistent with these results. Engrailed proteinlike immunoreactivity has been detected in the mandibular arch muscle of gnathostome embryos (Hatta et al.,
1990). In the lamprey, similar immunoreactivity has been
detected in the muscle anlage of the upper lip and of the
velum, both cheek process (mandibular process) derivatives (Holland et al., 1993). The cheek process which
contains muscle anlage is probably equivalent to the
mandibular process of gnathostome embryos. This further
implies that the maxillary portion (or prominence) of the
mandibular arch is an apomorphic structure of gnathostomes.
Figure 13
CRANIAL NERVE DEVELOPMENT IN THE LAMPREY
497
Branchiomeric and somitomeric nerves
Before the addition of the anterior lateral line nerves,
initial branchiomeric nerve anlagen of L. japonica clearly
develop on the segmental scheme of branchial arches (Fig.
1). At these stages, the placodal contribution was apparent
in the epibranchial ganglia of the vagus and glossopharyngeus, and also of the facial nerves (not shown). These
ganglia appear to also follow the branchiomeric developmental scheme. A more detailed study of cranial gangliogenesis in L. japonica is now in progress.
Since Balfour (1878), it has been argued that mesodermal segments exist in the vertebrate head region, similar
to somites in the trunk. The heads of amniote embryos
show incomplete mesodermal segments called somitomeres (reviewed by Jacobson, 1993; also see Freund et al.,
1996) whose metamerical and developmental correlation
with the head cavities of chondrichthys remains unknown.
This line of ideas has primarily been based on the hypothesis that there should exist the same number of mesodermal segments, each being associated with a set of single
pharyngeal arch-derivatives (van Wijhe, 1882; Goodrich,
1930; Jarvik, 1980). However, the mesodermal metamerism, the somitomerism, does not always coincide with
metamerism of gills, the branchiomerism. It has been
observed during development of several chordate species,
including the lamprey embryo, that branchial arches and
myotomes develop at different rates and intervals (Damas,
1944; reviewed by Jefferies, 1986); this was also ascertained in the present study (see below). A group of morphologists thus proposed that somitomerism and branchiomerism are two separate metamerisms, which leads to
the dual segmental theory of the vertebrate body (reviewed by Romer, 1972).
Metamerism of the vertebrate PNS is prefigured by the
distribution pattern of crest cells (Noden, 1975; Loring and
Erickson, 1987). Primarily based on observations in the
chick embryo, the migration pathways of amniote crest
cells have been classified into two major categories, the
dorsolateral and the ventral (reviewed by Le Douarin et
al., 1984). The ventral pathway is dominant in the trunk
region of gnathostome embryos and provides the basis for
somitomeric development of spinal nerve formation (Detwiler, 1934; Keynes and Stern, 1984); both the motor root
and gangliogenic crest cells are metamerically patterned
by somites. Thus, the different nature of branchiomerism
Fig. 13. Branching pattern of post-trematic nerves. A: Horizontal
section of the stage 26 pharynx stained with the anti-acetylated
tubulin antibody. The post-trematic ramus (arrows) grows between
the branchial arch cartilage (bc) and the muscle plate (mp). B,C: Stage
27 pharynx at the vagal level. Post-trematic nerves are marked by
large arrows in C. Developing rami are labeled with small arrows in C
and arrows in B. Note that two groups of rami, rostral and caudal, are
issuing from the post-trematic nerve. D: Whole-mount embryo at stage
28, showing the branching pattern of vagal postotic nerves. Muscle
fibers are illuminated with polarized light. The posterior ramus (rmp)
innervates the constrictor muscle (mc) and the visceral ramus (rv), the
adductor muscle (ma) developing within the ciliary string (ws in E).
Asterisks indicate vagal epibranchial ganglia. E: Horizontal section of
the pharynx at the first vagal segment in a stage 29 embryo.
Immunochemically stained nerve fibers are marked with arrowheads
and the post-trematic nerve of X1 by an arrow. Note the taste bud (tb)
developing at the rostral edge of the ciliary string (ws). Scale bars 5 50
µm in C for A–C and E, 50 µm in D.
Fig. 14. Comparative morphology of the cranial nerve anlage in
vertebrate embryos. Ganglion primordia of two gnathostome species
are illustrated. For comparison with lamprey embryos, see Figure 2.
Note in both that somites inhibit development of the cranial nerve
anlage. As a result, the initial anlage of the vagus nerve forms an arch
along one of the anterior somites. A few anteriormost somites (first to
fifth somites in the shark, the first somite in the chick) allow the
development of the vagus nerve lateral to themselves. Note the similar
distribution pattern of melanocytes in the lamprey (compare with Fig.
12). The circumpharyngeal crest cells (Cc) in the chick occupy the
postotic region beneath epibranchial myotomes, which corresponds to
the site of the epibranchial tract in the lamprey. The embryos are not
drawn to scale. Modified from Goodrich (1930) and Kuratani (1997).
and somitomerism is exemplified by developmental organization of the PNS. Metamerical morphology of the lamprey
spinal nerves implies somite-dependent regulation, although the developmental process would be very different
from that of the chick. Ventral spinal nerves of the lamprey
develop at mid-somitic levels and dorsal spinal nerves at
intersomitic levels (reviewed by Goodrich, 1937).
The dorsolateral pathway characterizes the cephalic
crest cell distribution patterns; the cells pass beneath the
surface ectoderm and fill the pharyngeal arch ventrally
(reviewed by Bronner-Fraser, 1995). Sensory ganglia of
branchiomeric nerves are formed along this pathway
(Noden, 1975). Description of the crest cell migration is
very limited in lampreys (e.g., Nakao and Ishizawa, 1987).
Most of the classical works on this issue are based on
graphic reconstruction of histological sections (e.g., Veit,
1939; Damas, 1944), and no lineage markers have been
utilized. Demonstrations of the detailed distribution pattern of crest cells in early embryos would necessitate vital
labeling, which belongs to a future project. At this time,
several pieces of circumstantial evidence suggest the close
relationship between the crest cells and PNS morphology.
Fig. 15. Evolution of the peripheral nervous system (PNS) developmental pattern—a hypothesis. Both in lampreys and gnathostomes,
postotic myotomes inhibit the cranial nerve development. In the
lamprey, the vagus nerve first develops between myotomes 2 and 3
(the glossopharyngeal nerve exceptionally develops medial to the first
myotome). In later development, the secondary rostral growth of
myotomes 1 through 3 and of hypobranchial muscle covers the entire
PNS laterally. This does not take place in gnathostome development.
The origin of the lamprey hypobranchial muscle does not actually
reflect the number of segments. In terms of primary arrangement of
the paraxial mesodermal component, the pattern of cranial nerve
development in gnathostomes is less deviated than in the lamprey.
CRANIAL NERVE DEVELOPMENT IN THE LAMPREY
499
The distribution pattern of the melanocytes in L. japonica implies that cephalic crest cells distribute in a
pattern similar to that in gnathostomes; the three melanocyte populations observed at stage 28 are strikingly reminiscent of the crest cell distribution pattern in early chick
embryos (Fig. 14). Interestingly, such a pattern of melanocyte distribution partly coincides with the morphology of
the cranial nerves; the anterior cell population overlaps
the ophthalmicus profundus and maxillomandibular nerve
innervating region, the middle coincides with the facial
nerve, and the caudal cell strand colocalizes with the
glossopharyngeo-vagus as well as the hypoglossal nerves.
The caudal melanocyte population is very similar to the
caudalmost cephalic crest cell population called circumpharyngeal crest cells in the chick (Kuratani and Kirby, 1991,
1992). In both animals, the postotic epibranchial ganglia
develop in this location ventral to epibranchial somites.
In the fully grown ammocoetes larvae of Petromyzon,
Johnston (1905) described a composite nerve bundle containing the epibranchial tract and spinal nerve elements.
However, the vagus and hypoglossal nerves of L. japonica
develop primarily independently from each other (Fig. 7),
and an extensive communication between the two nerves
is only secondarily established (Fig. 8). The hypoglossal
nerve of L. japonica is solely composed of spinal motor
nerves as in other vertebrates, and it innervates the
hypobranchial muscles derived from somites (Neal, 1897),
i.e., belonging to the somitomeric part of the body. Both the
hypoglossal nerve and muscle circumnavigate the pharynx
dorsocaudally, thereby making an anteriorly opened arch
(Fig. 8A) representing the head/trunk interface. This
morphology is common to all gnathostome embryos at the
pharyngular stage of development (Kuratani, 1997 and
references therein; Fig. 15).
topographical relationships with nerves, somite 2 of the
chick embryo resembles myotome 3 of L. japonica.
The similarity of the cranial nerve configuration between lampreys and gnathostomes is seen only at the early
stages of development, before the secondary modification
by myotomes. Such morphology can be characterized as
the coexistence of branchiomerism and somitomerism in
the postotic region; both make an S-shaped interface.
From an evolutionary point of view, cranial nerve and
mesodermal development in gnathostome embryos may
maintain a plesiomorphic or less-derived configuration
than the lamprey embryo (Fig. 15).
The arched shape of the vagus and hypoglossal nerve
exemplifies the head/trunk interface, or the interface
between two distinctive metamerisms, each of which has
an independent patterning mechanism. The alternating
metamerical pattern of the peripheral nerve (spinal dorsal
nerve) in amphioxus is suggestive of myotome-dependent
axonal growth (reviewed by Franz, 1927). However, they
have not attained the arched shape in any of the nerves,
i.e., the amphioxus probably lacks the distinction between
the branchiomerism and somitomerism in the nervous
system. The lamprey does not represent the intermediate
state between the acraniates and gnathostomes: Preotic
pharyngeal arches are highly modified, and dual metamerism is completely established. Possession of S-shaped
nerve morphology, therefore, serves as a synapomorphy
that establishes either Vertebrata or Craniata. It now
remains to examine whether or not myxinoids, the sister
group of vertebrates, show PNS morphogenesis reflecting
the most basic plan of the vertebrate body—the dual
metamerism.
Myotomes and cranial nerves
We are grateful to Nobutoshi Maeda and Miyuki Yamamoto in the Institute for Laboratory Animals, Niigata
University School of Medicine, for maintenance of animals. We also thank Dr. Hiromichi Koyama for critical
reading of the manuscript. Sincere gratitude is extended to
Dr. Glenn Northcutt for his valuable comments on the
manuscript. The CH-1 antibody developed by Dr. J. Lin
was obtained from the Developmental Studies Hybridoma
Bank maintained by the Department of Pharmacology and
Molecular Sciences, Johns Hopkins University School of
Medicine, Baltimore, MD, and the Department of Biological Sciences, University of Iowa, Iowa City, under contract
N01-HD-2-3144 from the NICHD. This work was supported by grants-in-aid from the Ministry of Education,
Science and Culture of Japan (Specially Promoted Research) and the Science and Technology Agency of Japan.
ACKNOWLEDGMENTS
The most intriguing difference in the peripheral morphology of cranial nerves between lampreys and gnathostomes
is the primary topographical relationship between the
nerves and myotomes (Figs. 2, 13). In gnathostome development, branchiomeric nerve anlagen are essentially located superficially. This condition lasts well beyond the
state of pharyngula (Kuratani, 1997 and references
therein). In the lamprey, in contrast, some rostralmost
myotomes grow rostrally and entirely cover the cranial
nerves laterally by stage 28 (Figs. 2, 10, 11). Obviously, the
myotomes found in the preotic region at stage 28 originate
from postotic somites that have nothing to do with the
preotic head cavities or head somites illustrated by Neal
(1918b). Earlier development of myotomes in relation to
the cranial nerves will be described elsewhere.
Dermomyotomes with the exception of several rostralmost ones, have been shown in the chick embryo to inhibit
the dorsolateral migration of crest cells, and subsequently
cranial nerve formation (Oakley et al., 1994; Kuratani and
Aizawa, 1995), suggesting that a somite-free environment
is a prerequisite for cranial nerve patterning. The vagus
nerve trunk consistently circumnavigates the rostral aspect of somite 2 (Kuratani and Kirby, 1992; Shigetani et
al., 1995). The same seems to be true for the lamprey
embryo; the early vagus nerve passes between myotomes 2
and 3, and no nerve fibers were formed lateral to myotomes at early stages (Figs. 3C, 4). In terms of the
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