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Developmental origin and kit-dependent development of the interstitial cells of cajal in the mammalian small intestine

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Rostral Truncation of a Cyclostome, Lampetra japonica,
Induced by All-Trans Retinoic Acid Defines the Head/Trunk
Interface of the Vertebrate Body
of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine,
Kumamoto, Japan
2Department of Anatomy, Niigata University School of Medicine, Niigata, Japan
The effect of all-trans retinoic
acid on embryogenesis was studied in a cyclostome, Lampetra japonica. Treatment with 0.05?
0.5 mM retinoic acid on early gastrula and early
neurula resulted in loss of the pharynx and in the
rostral truncation of the neural tube. The mouth,
pharynx, esophagus, heart, endostyle, and rostral
brain were missing with graded severity. In the
severest case, the embryo consisted only of trunk
segments, especially myotomes that extended to
the rostral end of the axis. The effect appeared to
be dose- and stage-dependent: Rostral pharyngeal arches were more vulnerable to a lower
amount of retinoic acid, and earlier treatment
resulted in severer defects. The initial protrusion
of the anterior axis started equally in control and
retinoic acid-treated embryos, implying that the
head morphogenesis is omitted in treated embryos. By identifying the number of myotomes
based on the differentiation of hypobranchial
muscles, there seemed to be no myotomes lost by
retinoic acid-induced truncation. The rostral
truncation, therefore, was not simply a limitation
of the anterior axis but was restricted to the
ventral portion; only the branchial arches disappeared with normally developing myotomes dorsally. The absent region can be defined as the
vertebrate head in a morphological sense, including the branchiomeric and preotic paraxial regions as well as the heart. The results suggest the
presence of distinct programs between somitomeric and branchiomeric portions of the body,
providing a developmental basis for the dualmetamerical body plan of vertebrates. Dev. Dyn.
1998;211:35-51. r 1998 Wiley-Liss, Inc.
Key words: branchial region; metamerism; cranial nerves; neural crest; all-trans
retinoic acid; lampreys
comprise the most basic body plan of the common
ancestor of cephalochordates and craniates. Among the
classes of vertebrates, lampreys belong to a sister group
of gnathostomes. Fossil records suggest that the dichotomy of these two major lines may have taken place
quite early in the vertebrate evolution (for review, see
Janvier, 1993). Regardless of the specific characteristics evolved during their own history, lampreys share
common morphological features with gnathostomes,
i.e., anteroposterior as well as dorsoventral dissociation
of branchiomerism and somitomerism and transient
neuromeres in the rostral neural tube (Kuratani, 1997;
Kuratani et al., 1997a). These synapomorphies are
apparently missing in amphioxus, in which the two
metamerisms are separated only dorsoventrally.
The metamerical body plan becomes distinct through
the organized and hierarchical processes of cell growth
and differentiation, for which spatiotemporally regulated gene expression is a prerequisite. Alternatively,
this body plan is profoundly related to such gene
expression patterns. All-trans retinoic acid (RA) has
been shown to affect diverse aspects of cellular development (for review, see Lotan, 1980) as well as the
patterning processes of vertebrates (for review, see
Hofmann and Eichele, 1994): In several different species, administration of RA results in the truncation of
the rostral brain and posteriorization of structures
caudal to a certain level of the hindbrain (Holder and
Hill, 1991; Morriss-Kay et al., 1991; Papalopulu et al.,
1991; for reviews, see Morriss-Kay, 1993; OsumiYamashita, 1996). Advances in molecular genetics have
shed light on regulatory gene functions in embryonic
patterning. In this respect, the effect of RA on axial
development has drawn the attention of molecular
embryologists, because this molecule appears to alter
the axial value of the body by shifting the genetic code,
as exemplified by Hox gene expression pattern: Morphological changes depending on RA dosage roughly coin-
The vertebrate body is characterized by possession of
segmentally arranged units along the anteroposterior
axis: myotomes, pharyngeal arches and peripheral
nerves. All of these characters are also present in
amphioxus, a close relative of vertebrates, and probably
Grant sponsor: Ministry of Education, Science, and Culture of Japan
(Specially Promoted Research); Grant number: 08102009.
*Correspondence to: Shigeru Kuratani, Department of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto
University School of Medicine, 4-24-1 Kuhonji, Kumamoto 862, Japan.
Received 14 August; Accepted 25 September 1997
Fig. 1. External appearance of retinoic acid (RA)-treated embryos in
experiment 1 at 2 days (A?C), 3 days (D?F), and 4 days (G?I) of RA
treatment. A: Control embryo has grown to stage 21 with the protrusion of
the head (arrowhead). B: The 0.01 然 RA-treated embryos. The morphology of the embryos is similar to that of the control. The arrowhead
indicates the head protrusion. C: The anterior axis begins to protrude in
0.1 然 RA-treated embryos (arrows). D,E: The control and 0.01 然
RA-treated embryos are at stage 23, showing the rolling of the head
(arrowhead). F: The difference is clear in the thickness of the rostral
protrusion (arrows) of 0.1 然 RA-treated embryos compared with the
embryos shown in D and E. G, H: On day 4 of RA treatment, control and
0.01 然 RA-treated embryos have extended their head and neck in a
straight line. I: Due to the lack of the pharynx, the rostral portion of the 0.1
然 RA-treated embryos rolls downward and is much thinner than in the
other two. Scale bar 5 1 mm.
cide with the shift of the Hox code as well as the
expression domains of other regulatory genes (Kessel,
1992; Lo?pez et al., 1995; Marshall et al., 1992; MorrissKay et al., 1991; for review, see Hofmann and Eichele,
To investigate the evolutionary process of the vertebrate body plan, comparative analyses are essential not
only by looking at the normal developmental processes
but also by examining the effects of shifts in patterning
programs. The effects of RA have been examined in a
variety of gnathostomes and amphioxus (Holland and
Holland, 1996), but nothing has been reported on
agnathan animals. It is intended here to evaluate the
morphological change in lamprey embryos after the
administration of RA, thereby comparing the developmental plan of this animal with that of gnathostomes
and cephalochordates. By controlled treatment with
various concentrations of RA, we successfully obtained
embryos with altered patterns. Here, we demonstrate
that RA affects the lamprey development in a similar
but distinct way to that shown by the experiments in
gnathostomes. Long-term viability after RA treatment
and the many branchial arches possessed by this
animal helped to elucidate some previously unrecognized aspects of vertebrate body architecture.
Experiment 1
External features. Development of the embryos
treated with 1 然 and 5 然 RA at early gastrula
stopped within the first 2 hours. Other embryos treated
with lower concentrations of RA survived. No substantial difference was detected morphologically between
the control embryos and those treated with 0.01 然 RA.
On the second day of treatment, the anterior protrusion
began to appear in both control and RA-treated embryos (Fig. 1A?C). The protrusions of 0.1 然 RAtreated embryos were more pointed at the tip than the
control embryos, and, on day 3, the head portion was
more slender (Fig. 1D?F). There were no apparent
differences between the control and 0.01 然 RAtreated embryos (Fig. 1D,E). By 4 days of treatment
(around stage 25), control and 0.01 然 RA-treated
embryos had developed normally, whereas 0.1 然
RA-treated embryos showed a truncated head and
strong ventral bending of the body axis, which lacked
the pharynx (Fig. 1G?I).
At 5 days, roughly corresponding to stage 26, melanocytes were first seen in the embryos. Normally, they
appeared around the otocyst, forming a couple of cell
streams both rostral and caudal to the otocyst (Fig. 2)
that corresponded to rhombomere 4 (r4) on the neuraxis
(Kuratani et al., 1997b). In the 0.1 然 RA-treated
embryos, the melanocyte also appeared and was distributed in the rostralmost portion of the body (Fig. 2C). In
5 of 16 embryos of this group at all stages, an epithelial
cyst that was reminiscent of an otocyst was associated
with the melanocytes, showing that the neuraxis of
these embryos was truncated at the level of the anterior
Anatomical features. At day 3 of RA treatment,
when the control embryo had grown to stage 21 (Fig.
3B), nervous tissue was detected by using the monoclonal antibody (MAb), T-6793, which recognizes the acetylated tubulin. In the neural tube of control and 0.01 然
RA-treated embryos, Rohon-Beard cells formed the
dorsolateral fasciculus (DLF), which had a rostral tip
that reached the caudal hindbrain, and commissure
neurons grew axons ventrally to form the ventrolateral
fasciculus (VLF); in the middiencephalic region, the
interstitial nucleus, the anlage of the medial longitudinal fasciculus, was observed (Fig. 3B; Kuratani et al.,
1997b). The otocyst was already present, and the DLF
reached a level caudal to the otocyst (Fig. 3B).
In all of the 0.1 然 RA-treated embryos (3 of 3), the
DLF and VLF were found up to the anterior end of the
neural tube (Fig. 3A,C). An otocyst-like cyst was often
observed nearby (see below). The interstitial nucleus,
on the other hand, could not be identified.
Consistent with the RA-induced changes at stage 21,
older embryos also lacked a number of head-specific
structures in the nervous system. At day 3.5 of RA
treatment, when control embryos grew to stage 24, the
posterior commissure and some neuromeres were recognized in the neural tube in control and 0.01 然
RA-treated embryos. The eye primordium was visible,
and the maxillomandibular and facial nerve roots were
present. Around the eye stalk, supraoptic and postoptic
tracts were developing. In the anterior head ectoderm,
olfactory epithelium had appeared as a compact sheet
of cells showing strong acetylated tubulin (AT) immunoreactivity (not shown). In the 0.1 然 RA-treated embryos, on the other hand, no neuronal structures rostral
to the otocyst could be identified, including the eye, the
cranial nerve roots, the nerve tracts, or the olfactory
epithelium (2 of 2 embryos).
Fig. 2. Initial distribution patterns of melanocytes (experiment 1).
Wholemount fixed embryos on day 5 of RA treatment. Myotubules are
illuminated by the polarized light. A: Control embryo. There are two
melanocyte populations (arrows) rostral and caudal to the otocyst (ot). B:
An embryo treated with 0.01 然 RA. The distribution pattern of melanocytes (arrows) is similar to that in the control embryo. C: An embryo
treated with 0.1 然 RA. The melanocyte population is accumulated in the
rostralmost part of the axis (small arrows). A large arrow indicates the
position of an epithelial cyst. Scale bar 5 200 痠.
Defects of the peripheral nervous system (PNS) in 0.1
然 RA-treated embryos were correlated with the truncation of the central nervous system (CNS) as well as
with the absence of the pharynx. By day 9 of RA
administration, the control and 0.01 然 RA-treated
embryos had grown to stages 27?28, and all of the
branchiomeric nerves were present (Fig. 4); in contrast,
the 0.1 然 RA-treated embryos apparently lacked the
branchiomeric nerves (Fig. 5B), i.e., all of the peripheral nerves were developing either between or within
myotomes, a feature of the spinal nerves of this animal
Fig. 3. Comparison of the nervous system (experiment 1). A: An
embryo treated with 0.1 然 RA at 3 days of RA treatment wholemounted
and stained with the antiacetylated tubulin antibody. Note the rostral extension
of the dorsolateral fasciculus (DLF) and the presence of Rohon-Beard
cell-like immunostaining (arrowheads). Pronephric ducts (prd) are stained
with the antibody. B,C: Illustrations based on similarly stained wholemount embryos. An embryo treated with 0.01 然 RA (B) shows the normal
development of the nervous system. The DLF and ventrolateral fasciculus
(VLF) are present caudal to the posterior hindbrain level. Note the
development of the otocyst (ot) as well as the interstitial neurons (isn). An
embryo treated with 0.1 然 RA is shown in C. Otocyst was not found in
this specimen. The VLF and DLF extend almost the entire length of the
neural tube. In B and C, compare the levels at which the rostral end of
each tracts (arrows and arrowheads in B and C). Some axonal bundles
are sprouting from the ventral aspect of the neural tube, reminiscent of the
ventral spinal nerves (asterisks). Note that, in B and C, that the anterior
protrusions are of similar length. Scale bars 5 100 痠.
(Kuratani et al., 1997a). Of those, three anteriormost
nerves passed the intermyotomic space and fasciculated distally to supply the gut and the putative pharyngeal region (Fig. 5B).
Concomitant with the loss of the head, the rostral end
of the notochord bent ventrally and reached the rostral
end of the body (Figs. 5B, 6). The pharyngeal pouches
were never visible, but endodermal tissue extended
rostrally to the rostral tip of the notochord, where two
tissues were mixed (Figs. 5B, 7A). No mouth opening or
stomodaeum was apparent. Myotomes in the lamprey
developed caudal to the otocyst and grew secondarily in
a rostral direction (Fig. 4), whereas, in the 0.1 然
RA-treated embryo, the rostralmost one developed
slightly caudal to the rostral end of the body (Figs. 6, 7).
The space between the otocyst and the first myotome
was filled with the rostral endodermal tissue, which, by
this stage, had become greenish, similar to the tint of
the liver primordium in the normal embryo of this stage
(not shown). In the histological sections, the heart was
lacking in 0.1 然 RA-treated embryos, although pericardium as well as pronephric ducts were present (Fig.
7A,B; see also Fig. 3A). The number of the ducts varied
in each RA-treated embryo. In the total of 18 embryos
treated with 0.1 然 RA, 15 embryos lacked the entire
pharynx as well as the esophagus, three embryos
possessed only the esophagus and the posterior portion
of the pharynx, and one embryo developed a normal
endodermal configuration (Table 1).
For the analyses of muscle morphology, embryos were
stained with CH-1 MAb, which recognizes tropomyosin.
In normal development, the first postotic myotome
becomes the infraoptic myotome, and the second becomes the supraoptic myotome (Fig. 4). Caudally, a
certain number of occipital level myotomes were involved in the formation of the hypobranchial muscle,
which is homologous with gnathostome tongue muscles
(Fig. 4; Kuratani et al., 1997a). The myotomic origin of
the hypobranchial muscle could not always be determined in the normal embryos due to the dorsocaudal
expansion of the pharynx. In the RA-treated embryos,
the first myotome was found slightly caudal to the
rostral tip of the notochord (3 of 3), and the ventral
portions of myotomes 7?12 were detached and had
migrated ventrally, apparently forming the hypobranchial and hypaxial muscles (Fig. 6A).
Experiment 2
In the second series of experiments, RA treatment
was performed at the early-neurula stage (Tahara?s
stage 18). The morphology of the treated embryos
showed a graded series of severity, i.e., more extensive
loss of the head structures with a higher dose of RA.
The nervous system was the main focus of the analyses.
The development of the nervous system at stages 26
and 27 was consistent with regression of the pharynx
(Figs. 8, 9). In the control embryos at stage 26, pharyngeal pouches 2?7 were present, and the first pouch was
made vestigial by the modification of the second pharyn-
Fig. 4. Normal development of the lamprey embryo at stage 28. A:
Microphotograph of a wholemounted, stained normal embryo with the
antiacetylated tubulin antibody. Note in A that the pronephric duct (prd)
stains with the antibody. B: Illustration of the nervous system and
myotomes of a similar embryo. Myotomes are numbered. Rostral myotomes (1?3) have grown rostrally over cranial nerves. On the ventrolateral
aspect of the pharynx, the hypobranchial muscle (HBM) is forming. Due to
the normal development of the pharynx, the origin of the myotubules of
the HBM cannot be determined precisely. ds, spinal dorsal nerves; e, eye;
lat, posterior lateral line nerve; llp, lower lip; oph, ophthalmic nerve; ot, otic
vesicle; p1?p8, pharyngeal pouches; ulp, upper lip; vs, spinal ventral
nerves; IX, glossopharyngeal nerve; V2,3, maxillomandibular nerve; VII,
facial nerve; X, vagus nerve; x1?x6, vagal nerve branches innervating
each branchial arch; XII, hypoglossal nerve. Scale bar 5 100 痠.
geal arch (Fig. 8A). The notochord rostrally ended at
the level of diencephalon. In the PNS, each pharyngeal
arch was innervated by a branchiomeric nerve branch.
When it was stained with T-6793 antibody, the olfactory
epithelium was seen at the tip of the head, and the
endostyle was darkly stained at the floor of the pharynx
at stage 27 (Figs. 8A).
In some of the 0.05 然 RA-treated embryos (Figs. 8B,
9C,D), three rostral nerves, the ophthalmic, maxillomandibular, and facial nerves, were developmentally
altered, but others showed normal PNS morphology.
The ophthalmic nerve was apparently lost or fused with
the trigeminal nerve, and the development of the facial
nerve was arrested. The endostyle was always present
(4 of 4 embryos; Fig. 9C). In 2 of 4 embryos, the lower lip
failed to differentiate (Fig. 9D), whereas, in the other
two embryos, the endodermal portion corresponding to
the first pharyngeal pouch was missing (not shown). At
stage 26, the vagal nerve root grew more caudally on
the hindbrain than in the control (Figs. 8B). The eye
was lacking in 1 of 4 embryos. Olfactory epithelium was
always present.
The pharyngeal defects of embryos treated with 0.1
然 RA extended caudally to the third pharyngeal arch
(1 of 3 embryos; Figs. 6B, 8E,F). The second pouch was
usually absent (3 of 4). Concomitantly, the facial and
glossopharyngeal nerves were fused distally. The second pharyngeal arch usually failed to develop, and
several irregular connections were seen between the
glossopharyngeal nerve and the vagus nerve. The olfactory epithelium, heart, and otocyst were always present
(3 of 3), but the endostyle was missing (Fig. 8E).
Fig. 5. Development of the nervous system and myotomes in an
RA-treated embryo (experiment 1). A: Microphotograph of a wholemounted, stained 0.1 然 RA-treated embryo with the antiacetylated
tubulin antibody on the 9th day of RA treatment, when a control embryo
has grown to stage 27/28. B: Illustration of the nervous system of the
same embryo shown in A. The nerve roots are drawn with heavy lines.
Note that typical ventral spinal nerves that pass the middle part of
myotomes are seen caudal to the third myotome, whereas the first three
roots associated with the rostral two myotomes are passing along the
intermyotomic pathway, reminiscent of the vagus nerve in the normal
embryo (Kuratani et al., 1997a). Consistently, they are fasciculated
distally to innervate the gut. Endodermal portions are shaded by dots.
Note that the rostral end of the notochord is closely associated with the
endoderm. Scale bar 5 100 痠.
In the 0.5 然 RA-treated embryos, most of the
pharynx as well as the endostyle were missing (3 of 4),
except for the presence of a few pharyngeal arches
belonging to the vagal segment in one embryo (Figs. 8D,
9G). The esophagus could be seen in 4 of 5 embryos. Due
to the similar appearances of the postotic arches, the
numbering of these pouches was not always possible
(see Table 1). Nevertheless, the nerve innervating these
pouches resembled the vagus of the control embryo, in
that the nerve root formed an arch rostral to myotomes
after issuing from the hind brain (Fig. 8D). The vagal
branch was distributed on the dorsal aspect of the gut,
like the intestinal branch of the vagus nerve (Kuratani
et al., 1997a). The rostral end of the notochord was
Fig. 6. Changes in myotomes (experiment 1). A: Illustration of a 0.1
然 RA-treated embryo 9 days after the treatment. The rostral head, as
well as the entire pharynx are missing. Note the positions of the rostral tip
of the notochord (double arrows) and the otocyst (ot) with those structures. Myotomes are numbered from rostral to caudal direction. Seventh
through twelfth myotomes are releasing myotubules ventrally (small
arrows; compare with Fig. 4B). They appear to represent the autonomous
development of the HBM in the absence of the pharynx. The line indicates
the plane of section shown in Figure 7A. B: Microphotograph of the same
embryo as A. Wholemounted and stained with the antibody, CH-1.
Hypobranchial muscle-like myotubules are indicated by arrowheads.
Scale bar 5 100 痠.
found almost at the rostral tip of the body, and there
was no olfactory epithelium except in 1 of 4 embryos.
The otocyst was missing in some embryos. The pericardium and pronephros were present in all of the embryos, but the heart was missing in 1 of 3 examined.
1992; Marshall et al., 1992; for review, see Marshall et
al., 1996). Ectopic expression of a Hox gene in a more
rostral region can actually mimic the RA effect in the
head of zebrafish (Alexander et al., 1996). Furthermore,
several models of Hox gene regulation mediated by
endogenous RA have been proposed (Dekker et al.,
1993; Hogan et al., 1992; Simeone et al., 1990, 1991; for
reviews, see Hofmann and Eichele, 1994; Langston and
Gudas, 1994). It must be noted, however, that the
above-proposed scheme inherently tends to deal with
the vertebrate body too simply, as though it had only a
single metamerical body axis. To circumvent the idiosyncrasy obtained from a rather compact group of animals
(gnathostomes), it is necessary to examine a sister
group of gnathostomes, the lamprey.
Many studies have described the developmental
changes caused by exogenous all-trans RA in various
embryos. The consensus on how RA affects embryogenesis can be summarized as rostral truncation of the axis
and concomitant posteriorization of segmental values.
Typically, this phenomenon is realized by homeotic
transformation of segments (e.g., vertebrae), which
accompanies the anteriorly shifted Hox code (Kessel,
Fig. 7. Histology of RA-treated embryos (experiment 1). A: Semihorizontal section of the same experimental embryo shown in Figure 6 (for the
plane of sectioning, see Fig. 6B). The neural tube ends slightly rostral to
the otocyst (ot). A crest cell population is accumulated on the dorsal
aspect of the neural tube (arrows). The rostral end of the notochord is
sectioned. Note the close association of endodermal cells (containing yolk
granules; arrowheads) and notochordal cells (n; vacuolated) in this
region. my, myotome. B: Transverse section of the control embryo at the
corresponding stage cut at the level of the pericardial cavity (pc). The
pronephric duct (prd); the heart, consisting of myocardium (mc) and
endocardium (ec); the dorsal aorta (da); and the esophagus (eso) are
recognized. C: The same 0.1 然 RA-treated embryo shown in A
sectioned at the corresponding level to B. Note the absence of the heart
and esophagus and expansion of the dorsal aorta. Scale bars 5 100 痠.
RA Eliminates the Rostral Identity
With a high dose of RA at the time of gastrulation, the
rostral neural tube of the lamprey embryo was lost. The
apparent rostral extension of the notochord in these
embryos is consistent with the rostral truncation. Based
on the frequent development of the otocyst and melanocyte distribution pattern, the caudal limit of the truncation seemed to fall close to r4.
TABLE 1. Results Obtained in Experiments 1 and 2a
Olfactory placode
Pronephric duct
Pouch 1
Pouch 2
Pouch 3
Pouch 4
Pouch 51
Experiment 1:
Tahara?s stage 12 (early gastrula)
0.01 然
0.1 然
Experiment 2:
Tahara?s stage 18 (early neurula)
Control 0.05 然 0.1 然 0.5 然
aThe table shows the ratio of embryos among all specimens in which the presence or absence of each
structure was able to be determined. Not all structures were able to be observed in each embryo.
RA, retinoic acid.
bPresence of pouches 5 and 6 was assumed in one embryo in which two posterior pouches were
innervated by the vagus nerve. No pharyngeal pouch was found in the other embryos at this dose.
The rostral truncation of the CNS is apparently
analogous with the results in gnathostome species. The
severest phenotype obtained in the lamprey resembles
the zebrafish embryo treated with 9-cis RA (Zhang et
al., 1996); only a mild truncation can be obtained in the
zebrafish by all-trans RA (Hill et al., 1995; Holder and
Hill, 1991). In mouse and Xenopus, similar truncation
has been obtained with RA (Morriss-Kay et al., 1991;
Papalopulu et al., 1991) in which the brain rostral to
the anterior hindbrain is truncated.
RA has been shown to down-regulate some rostrally
expressed genes, resulting in the loss of anterior brain
identities (Bally-Cuif et al., 1995; Simeone et al., 1995;
Taira et al., 1994). Being expressed in the chordal- and
mesodermal substrate, Otx2 appears to be one such
gene responsible for anterior specification of the neurectoderm (Ang et al., 1994). Actually, homozygous disruption of the Otx2 gene results in the rostral truncation of
the CNS at a similar neuraxial level (Acampora et al.,
1995; Ang et al., 1996; Matsuo et al., 1995). In the
disruption of Lim1 as well, a similar level of the rostral
head is lost (Shawlot and Behringer, 1995). Obviously,
the next step is to examine the normal and shifted
expression patterns of these anteriorly expressed regulatory genes.
The heart of a lamprey embryo was often lost by a
rather high concentration of RA (Table 1). In gnathostomes as well, the heart is included in the target of
teratogenic effects of retinoids or vitamin A deficiency
(Wilson and Warkany, 1949), involving the malformation of mesoderm-derived myocardium and aorticopulmonary septation to which the neural crest contributes
(Kirby and Waldo, 1990; Kirby et al., 1983). The heart is
known to be affected by exogenous RA in the chick and
zebrafish (Osmond et al., 1991; Stainier and Fishman,
1992). In the mouse, retinoid receptor-mediated path-
ways have been shown to be prerequisite for normal
cardiogenesis (Sucov et al., 1994; for review, see OsumiYamashita, 1996). The intimate relationships between
the heart and the pharynx during embryogenesis (for
review, see Kirby and Waldo, 1990) might explain the
codistribution of defects in the two structures in the
lamprey embryos. Also, the pharyngeal endoderm, which
is sensitive to RA in the lamprey (see below), has been
suggested to be responsible for myocardial differentiation (Sugi and Lough, 1994).
RA Induces the Loss of Pharyngeal Arches
Most intriguing was the graded loss of the pharynx;
the more rostral part tends to disappear with lower
concentration of RA (Figs. 8, 9), indicating that more
anterior pharyngeal pouches are more sensitive to RA.
Pharyngeal pouches in the lampreys develop from
anterior to posterior, as in gnathostomes (Damas, 1944;
Kuratani et al., 1997b; Veit, 1939). The treatment at
early stages seemed to have more impact than a higher
concentration given at later stages; the former phenotype was more severe than the latter (Figs. 5, 6, 8, 9). In
particular, the heart was usually present in 0.5 然
RA-treated embryos in the second series, whereas it
was totally absent from the 0.1 然 RA-treated embryos
in the first series (Figs. 5, 7). Therefore, the sensitivity
of the pharyngeal arches and also of the heart to RA is
stage-dependent as well as dose-dependent (Fig. 10).
The possible homeotic transformation of pharyngeal
arches has been observed in gnathostomes (Lee et al.,
1995; Marshall et al., 1992). Unlike vertebrates, branchial skeletons are hard to transform homeotically by
RA; they are more likely to disappear (see, e.g., Mallo,
1997). In the lamprey, a similar change would only be
found, if present, in the second arch; it failed to expand
as the lower lip in a few specimens with low-dose
Fig. 8.
treatment at the late stage (Fig. 9D). Even if it was the
case, the developmental window and the RA concentration that allow the transformation would be very narrow.
It has not yet been demonstrated in any experiment
using gnathostomes that pharyngeal arches disappear
as dramatically as they do in the lamprey. This may be
partly because gnathostome embryos cannot survive
long enough to develop the pharyngeal arch. Lee et al.
(1995) found that RA treatment at the neural plate
stage of the rat results in the fusion of the first and
second arches. The developmental stage at which they
administered RA was similar to experiment 2 of the
present study. The two results, however, are probably of
a different nature: The fused branchial arch of the rat
still contains a pharyngeal pouch. The loss of endodermal pharyngeal pouches has never been reported in
gnathostomes, although it has been reported in the
amphioxus (see below).
The branchiomeric nerve anomalies (Figs. 8, 9) might
be secondary to those of the pharynx. Among gnathostomes, the RA treatment at midgastrula results in the
partial posteriorization of the branchiomotor neurons
in the mouse, with no substantial changes in the
peripheral morphology (Kessel, 1993; Marshall et al.,
1992). In Xenopus (Papalopulu et al., 1991) and zebrafish (Holder and Hill, 1991), however, a disturbed
pattern of the PNS has been illustrated. Especially in
Xenopus, cranial nerves tended to fuse with each one
another, as in the lamprey, although the relationship
between the nerve and the pharyngeal arches has not
been reported (Papalopulu et al., 1991).
The morphology of the PNS depends largely on the
distribution pattern of neural crest cells in amniotes
(Kuratani and Eichele, 1993; Loring and Erickson,
1987; Noden, 1975) and also in the lamprey (Kuratani
Fig. 8. Development of the cranial nerves (experiment 2). Wholemount embryos stained with the antiacetylated tubulin antibody to show
the nervous system. The developmental stage of these embryos corresponds to stage 26 of normal development. Anterior is to the right except
in D. Semidiagrammatic drawings of the same embryos are shown at right
(that in D is reversed horizontally for comparison). A: In a control embryo,
all of the branchiomeric nerves have developed, growing axons to each
pharyngeal arch. Pharyngeal arches are divided by pharyngeal pouches
(p2?p7). The mouth (arrow) is formed between the upper (ulp) and lower
(llp) lips. The vagus nerve (x) is distally innervating the posterior
pharyngeal arches caudal to pouch 3, and, dorsally, it is associated with
the posterior lateral line nerve (lat) at the exit point from the hindbrain
(arrowheads). Olfactory epithelium (ol) stains dark. B: In a 0.05 然
RA-treated embryo, the first and second pharyngeal arches are deformed. Note the shift of the mouth opening (large arrow) and the change
of the upper lip (ulp). The trigeminal nerve (v) is partly fused with the facial
nerve (vii). In the vagus nerve, an additional nerve root is visible (small
arrows). Note the caudal elongation of the vagus nerve root on the
hindbrain (arrowheads). C: In a 0.1 然 RA-treated embryo, the fusion and
disturbance of the cranial nerves extend to the glossopharyngeal nerve
(ix). Identification of the cranial nerves was based on the position of the
otocyst (ot). The second pharyngeal pouch has been diminished, and the
preliminary pouches are numbered by asterisks (p3*?p6*). The mouth
opening is absent. The proximal root of the vagus nerve (x) has been
extensively disturbed, and its nerve root extended farther posteriorly
along the hindbrain (arrowheads). Arrows indicate vagus nerve trunks
developing between myotomes. D: In a 0.5 然 RA-treated embryo, the
only cranial nerves left are the vagus nerve (x) and vestigial nerve fibers,
possibly representing the glossopharyngeal nerve (ix). The vagus nerve is
associated with only two pharyngeal arches. In the rostralmost portion of
the head, there is an unorganized reticulum of neurites, in which
segmental identity is no longer distinguishable. The vagus nerve root has
extended more caudally than other embryos (arrowheads). Arrows indicate vagal nerve trunks developing between two successive myotomes.
Scale bar 5 100 痠.
et al., 1997a,b). In the pharynx and hindbrain region,
cephalic crest cells in the chick embryo adhere to
even-numbered rhombomeres, providing the bases for
the nerve root metamerism (Kuratani and Eichele,
1993). In the lamprey, a similar, shared pattern seems
to be present; the rhombomere develops only transiently around stage 23, and nerve roots arise on
even-numbered rhombomeres (Kuratani et al., 1997b).
RA treatment at stage 10 chick embryo results in the
disturbed migration of both crest cells and cranial
nerve morphology (Gale et al., 1996). A similar alteration may be involved in the RA-treated lamprey
embryos, especially in those cranial nerves that were
associated with the lost pharyngeal arches (Fig. 8).
Myotomes Do Not Disappear but Shift Rostrally
The initial rostral protrusion took place similarly in
control and RA-treated embryos (Fig. 1), but the protrusion contained neural elements of quite different axial
natures (Fig. 3). It might be because of the so-called
posteriorization of embryonic materials that RA treatment leads to the elimination of anterior identities, in
the sense that trunk-type specification is executed by
skipping the accomplishment of the head.
In the preotic region of cyclostomes as well, there is
the cephalic mesoderm, which is not segmented and
does not develop into myotomes (Damas, 1944; Veit,
1939; our unpublished data on the lamprey; see Fig.
11A). This mesoderm, as a result of a posteriorizing
effect, appears specifically to be truncated by a high
concentration of RA (Figs. 5B, 6A). On the other hand,
as indicated by the hypobranchial muscle formation,
the postotic elements are likely to be simply posteriorized by RA treatment.
The first myotome, as the result of rostral truncation
of the lamprey, was found near the rostral end of the
head, as has been observed in RA-treated gnathostome
embryos (Morriss-Kay et al., 1991; Sundin and Eichele,
1992; Sundin et al., 1993). This raises the question of
what numbers of segments were actually shifted rostrally. In the trunk of the lamprey, myotomes go through
developmental modifications in a segmental, levelspecific manner; myotomes 1 (m1)?m3 grown secondarily into the preotic region, and some occipital level
myotomes differentiate into hypobranchial muscles (Fig.
The numbers and levels of myotomes involved in the
hypobranchial muscle are not clear in the control
embryos due to the expansion of the pharynx, and
observation of slightly younger normal embryos did not
allow us to see clearly the developing hypobranchial
muscles. In light of the number of spinal motor nerves
forming the hypoglossal nerve, myotomes involved in
the hypobranchial muscle formation probably range
from m5 through m10 in normal development (Kuratani et al., 1997a,b). Importantly, the segmental levels
of myotomes involved in the putative hypobranchial
muscle in RA-treated embryos are very close to those of
the control embryo (Fig. 11). Therefore, the rostral
Fig. 9. Development of the cranial nerves (experiment 2). Photomicrographs of the control (A,B), 0.05 然 RA-treated (C,D), 0.1 然 RA-treated
(E,F), and 0.5 然 RA-treated (G,H) embryos correspond to stage 27 of
normal development. Myotomes are illuminated by the polarized lighting
in B, F, and H. A,B: The first myotome (arrow in B) develops ventral to the
otocyst (asterisk in A). Note the development of the olfactory epithelium
(ol) and the endostyle (es). The mouth opens between the upper (ulp) and
the lower (llp) lips (arrow in A). C,D: The lower lip is often obliterated (D),
and the mouth (arrow in D) opens widely caudal to the upper lip (ulp).
E?H: The morphology of 0.1 然 and 0.5 然 RA-treated embryos can be
very similar. In both groups, only the vagus nerve is seen in the head
(arrows in E and G). In G, the vagus nerve roots are arranged metamerically with the rostral myotomes (arrows; compare with F). Only two
pharyngeal pouches (p) are present in E, whereas none are seen in G.
The distal portion of the vagus nerve in G innervates the putative
pharyngeal region. In both embryos, the anteriormost myotome develops
close to the rostral end (single arrow in F,H). The rostral tip of the
notochord is indicated by double arrows (F,H). Note the absence of
endostyle and otocyst in these embryos. Scale bar 5 100 痠.
Fig. 10. Graded sensitivity of pharyngeal pouches to exogenous RA.
Three-dimensional graph shows the ratio by number of embryos that
lacked each pharyngeal pouch at each RA concentration in experiment 2.
Note that the more anterior pouches are apt to disappear more frequently,
and the absence of the pouches becomes more frequent as the concentration of RA rises.
myotomes seem to develop normally in RA-treated
embryos, whereas the branchial arches disappear ventrally at the same axial levels.
In summary, RA induces what is known as rostral
truncation in the lamprey, but it is not a simple
posteriorization of segmental values. The RA-induced
anomalies are not merely axis-specific but, rather, are
body part-specific, i.e., even when all of the pharyngeal
pouches disappeared, all myotomes were present, and
there appeared to be none clearly lost by the RA
treatment. The anomalies in the pharyngeal structures, on the other hand, are more likely to be the
simple disappearance of endodermal pharyngeal
pouches. Thus, the developmental disturbance of segmental units is not equally coextensive for the paraxial
and pharyngeal structures, but RA appears to affect
different axial levels in each system. It seems more
appropriate to postulate that independent developmental programs may exist for paraxial and endodermal
systems that are isolated from one another.
RA and the Vertebrate Body Plan
Some comparative morphologists and embryologists
have assumed a complete set of head segments, each
possessing one branchiomere and one somitic metamere (Balfour, 1878; Bjerring, 1977; Goodrich, 1930;
Jarvik, 1980; van Wijhe, 1882). Others have realized,
however, that the branchiomerism and somitomerism
do not develop in a coordinated manner but are shifted
away from each other in actual developmental processes (Hatschek, 1892; Damas, 1944; de Beer, 1922).
The distribution pattern of crest cells as well as the
actual morphology of paraxial mesoderm are consistent
with the latter hypothesis, the dual metamerism put
forth by Romer (1972; see also Jefferies, 1986; Kuratani, 1997; Starck, 1975).
The dual-metamerical model is well reflected in the
developmental pattern of the PNS in vertebrate embryos as well, i.e., the branchiomeric nerves are associated segmentally with rhombomeres and pharyngeal
arches, whereas each spinal nerve is associated with
somite derivatives. Different embryonic environments
are shown experimentally to function as distinct constraints in patterning of each group of nerves (Detwiler,
1934; Keynes and Stern, 1984; Keynes et al., 1991;
Kuratani and Eichele, 1993; Rickmann and Fawcett,
1985; Teillet et al., 1987). The early developmental
pattern of the lamprey PNS has shown that the dualmetamerical scheme is also applicable in this animal
(Kuratani et al., 1997a). Interestingly, in this dualmetameric model, it is the head of the lamprey that was
almost selectively lost by RA treatment (Fig. 11A,B).
The interface between the affected and nonaffected
regions corresponds roughly, if not exactly, to the head/
trunk interface based on the distribution pattern of
cephalic crest cells (Fig. 11C; Kuratani, 1997).
The evolutionary origin of the dual-metamerical developmental plan is an intriguing subject for speculation. In this context, tunicate embryos have been
treated with RA, and rostral truncation has been
observed in the tadpole larvae (Katsuyama et al., 1995).
Because the possible somitomeric and branchiomeric
segmentations are found in this organism (Wada et al.,
1996; for review, see Gee, 1996), it would be interesting
to know whether there would be a condition in which
only the endodermal derivatives were affected.
The configuration of the notochord in RA-treated
embryos (Figs. 4, 5, 8E?H) as well as the apparent
absence of nerves with typical branchiomeric features
are reminiscent of the anatomy of amphioxus (Fig. 6;
Franz, 1927; Hatschek, 1892). The development of the
amphioxus PNS is not well known, although the adult
anatomy has shown the absence of distinction between
the branchiomeric and spinal nerves, and all of the
segmental dorsal nerves pass along the intermyotomic
pathway (for reviews, see Franz, 1927; Fritzsch and
Northcutt, 1993; Jefferies, 1986). Moreover, the brain of
this animal is reported to have some features common
to the vertebrate rostral brain (Lacalli et al., 1994) that
are clearly lost by RA treatment (Fig. 11A,B). Therefore, the resemblance of the RA-treated lamprey embryo to the amphioxus larva may be no more than
superficial and may be caused merely by truncation of
the unsegmented head mesoderm.
The idea of selective loss of the morphological head by
RA is strengthened by a similar experiment made on
the amphioxus. Holland and Holland (1996) have shown
that, in this animal, RA induces the absence of anterior
endodermal structures, the mouth and pharyngeal
slits, a result that is reminiscent of the present study.
Although the pharyngeal and mouth formation processes differ greatly between the two animals (Willey,
Fig. 11. Diagram summarizing the effects of RA on lamprey development. A: Pharyngula stage of normal embryo. On the neural tube, the
posterior commissure (pc) divides the forebrain and midbrain, and the
midhind brain border (prm) is located between midbrain (m) and hindbrain. The notochord extends rostrally to the level of diencephalon. The
dorsolateral fasciculus (DLF) terminates rostrally at the level of the rostral
hindbrain. The rostral end of the forebrain is associated with the olfactory
epithelium (olep). Paraxial mesoderm is segmented as myotomes caudal
to the otocyst but is not clearly segmented preotically (phm). Myotomes
caudal to m7 are likely to differentiate into hypobranchial musculature
(HBM). Endodermal pharyngeal pouches (solid circles) penetrate the
pharyngeal wall, resulting in nine pharyngeal arches. The rostralmost
pouch is vestigial. The endostyle (es) develops on the floor of the pharynx.
The heavy dashed line indicates the border between the truncated and
unaffected regions after RA treatment (myotomes 1?3 and HBM second-
arily grow rostrally beyond the border). ep, epiphysis; oc, oral cavity; ot,
otocyst; phm, preotic head mesoderm. B: RA-treated embryo, representing a generalized severest case. The dorsolateral fasciculus is associated
with almost the entire length of the neural tube. The otocyst can be
present at the rostral tip of the head, close to the rostral tip of the
notochord. The notochord is associated rostrally with the detached endoderm
(arrow). The pharynx and esophagus are missing. The paraxial space of
this embryo is occupied by myotomes, the seventh and caudal of which
differentiate into presumptive HBM. The pericardium develops but does
not contain the heart. C: A pharyngula embryo of a shark (modified from
Goodrich, 1930). There appears to be an S-shaped ??head/trunk interfaces?? (dashed line) along the caudal limit of the cephalic crest cell distribution
in this animal as well as in other gnathostome embryos (Kuratani, 1997).
Note that the dashed line resembles the border shown in A.
1891), both depend on normal pharyngeal arch development.
Due to the small amount of yolk, RA-treated, mouthless amphioxus larvae cannot live longer than 4 days,
and there is no knowing how normally their somitomeric part can develop or whether there is dosedependent sensitivity at each pharyngeal level. Nevertheless, the apparent specific loss of pharyngeal
structures commonly observed in the lamprey and
amphioxus might represent shared mechanisms of
dual-metamerical patterning, like the synplesiomorphies that may be obscured in many gnathostomes.
Adult male and female lampreys (Lampetra japonica) were collected in Miomote River (Niigata, Japan) during the breeding season (late May through
June) in 1995. They were brought into the laboratory,
where eggs were artificially fertilized and kept in fresh
water at 18蚓. Embryos were fixed either with 4%
paraformaldehyde in 0.1 M phosphate-buffered saline
(PFA/PBS) for immunohistochemistry or with Bouin?s
fixative for histological preparations. For the staging of
embryos, developmental sequence of a brook species, L.
reissneri (Tahara, 1988), was applied.
RA Administration
A stock solution of the all-trans RA (R-2625, Sigma,
St. Louis, MO) was made at a concentration of 0.01 M in
dimethylsulfoxide (DMSO) and was stored at 220蚓 in
the dark. The solution was diluted with DMSO, and the
same amount of RA/DMSO solution was added to 50 ml
of Steinberg?s solution (Steinberg, 1957) diluted 1/10,
into which the lamprey embryos were transferred. For
the control treatment, the same amount of DMSO alone
was added. Embryos were left in the solution for 1 hour
at 23蚓 in the dark. After extensive washing with 1/10
Steinberg?s solution, they were allowed to grow at room
temperature in the same buffer. Two series of experiments were made. In the first, embryos at Tahara?s
stage 12 (early gastrula) were treated with different
concentrations of RA. Concentrations were 0.01 然, 0.1
然, 1 然, and 5 然. The second experiment was made
at stage 18 (early neurula) with concentrations of 0.05
然, 0.1 然, and 0.5 然.
Wholemount Immunostaining
Wholemount embryos were prepared as described by
Kuratani et al. (1997a) with minor modifications. After
fixation with PFA/PBS at 4蚓 for 1 day, embryos were
washed in 0.9% NaCl/distilled water, dehydrated in a
graded series of methanol (50%, 80%, 100%), and stored
at 220蚓. The samples to be stained were placed on ice
in 2 ml DMSO/methanol until they sank. Five tenths of
a milliliter of 10% Triton X-100/distilled water was
added, and the embryos were incubated for another 30
minutes at room temperature. After washing in TrisHCl-buffered saline (20 然 Tris-HCl, pH 8.0; 150 然
NaCl; and 0.1% Triton X-100; TST), the samples were
sequentially blocked with aqueous 1% periodic acid and
with 5% dry nonfat milk in TST (TSTM).
For wholemount immunostaining of the nervous system, a MAb raised against acetylated tubulin (monoclonal antiacetylated tubulin; no. T-6793; Sigma) was
used. Embryos were incubated in primary antibody
(diluted 1/1,000 in spin-clarified TSTM containing 0.1%
sodium azide and 5% DMSO) for 2?3 days at room
temperature while being gently agitated on a shaking
platform. The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat antimouse IgG
(Zymed Laboratories, San Francisco, CA) diluted 1/200
in TSTM. After washing in TST, the embryos were
preincubated with peroxidase substrates 3,38-diaminobenzidine (DAB; 100 mg/ml) in Tris-HCl-buffered saline
(TS) for 1 hour and allowed to react in TS with the same
concentration of DAB and hydrogen peroxide dissolved
at 0.01% for 20?40 minutes. After stopping the reaction
with TS, some embryos were clarified in 30% glycerol
containing 0.5% potassium hydroxide (KOH). These
embryos were stored in 60% glycerol/water. Others
were dehydrated in a graded series of methanol and
cleared in benzyl alcohol/benzyl benzoate mixture (1:2).
Embryos of both groups were mounted on depression
slide glass for observation under the light microscope
with Nomarski optics. Another MAb, CH-1 (purchased
from Developmental Studies Hybridoma Bank, Iowa
City, IA) raised against tropomyosin, was used to stain
myotomes in wholemount embryos.
Embryos fixed with Bouin?s fixative were dehydrated
and embedded in paraffin. Sections were cut at 5 痠
and stained with Gill?s hematoxylin and eosin.
We are grateful to Nobutoshi Maeda and Miyuki
Yamamoto at the Institute for Laboratory Animals,
Niigata University School of Medicine for maintenance
of animals and to Atsuo Nishino and Naoto Horigome
for their technical assistance. We also thank Dr. Norimasa Miyamoto for technical advice and Drs. Noriko
Osumi and Kazuhiko Umesono for critical reading of
the paper. 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 21205, and the
Department of Biological Sciences, University of Iowa,
Iowa City, IA 52242, under contract N01-HD-2-3144
from the NICHD.
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development, mammalia, intestinal, interstitial, small, kit, dependence, origin, cajal, cells
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