Neural tube-ectoderm interactions are required for trigeminal placode formation

Abstract

Cranial sensory ganglia in vertebrates develop from the ectodermal placodes, the neural crest, or both. Although much is known about the neural crest contribution to cranial ganglia, relatively little is known about how placode cells form, invaginate and migrate to their targets. Here, we identify Pax-3 as a molecular marker for placode cells that contribute to the ophthalmic branch of the trigeminal ganglion and use it, in conjunction with DiI labeling of the surface ectoderm, to analyze some of the mechanisms underlying placode development. Pax-3 expression in the ophthalmic placode is observed as early as the 4-somite stage in a narrow band of ectoderm contiguous to the midbrain neural folds. Its expression broadens to a patch of ectoderm adjacent to the midbrain and the rostral hindbrain at the 8- to 10-somite stage. Invagination of the first Pax-3-positive cells begins at the 13-somite stage. Placodal invagination continues through the 35-somite stage, by which time condensation of the trigeminal ganglion has begun. To challenge the normal tissue interactions leading to placode formation, we ablated the cranial neural crest cells or implanted barriers between the neural tube and the ectoderm. Our results demonstrate that, although the presence of neural crest cells is not mandatory for Pax-3 expression in the forming placode, a diffusible signal from the neuroectoderm is required for induction and/or maintenance of the ophthalmic placode.

Full text

INTRODUCTION
The trigeminal ganglion, which provides sensation for much of
the face, has served as a good experimental system for inves-
tigating the development of peripheral ganglia because of its
size and accessibility (reviewed in Davies, 1988). Trigeminal
sensory neurons originate from two distinct embryonic cell
populations: the neural crest and the ectodermal placodes
(Yntema, 1942; Hamburger, 1961; Noden, 1978; Narayanan
and Narayanan, 1980; Ayer-LeLièvre and Le Douarin, 1982;
D’Amico-Martel and Noden, 1980, 1983; reviewed by Noden,
1993). The cranial neural crest population exits from the dorsal
neural tube and migrates under the head ectoderm. In addition
to contributing neurons and glia to cranial ganglia, neural crest
cells form connective tissue and bones of the face and skull
(reviewed in Le Douarin, 1982). The cranial sensory placode
population undergoes an epithelial-mesenchymal transition
from a thickened ectodermal epithelium; these cells then
invaginate, migrate, condense and differentiate into neurons,
receptors and some support cells of the peripheral nervous
system (reviewed by Webb and Noden, 1993).
Unlike most placodes, the trigeminal placode is not mor-
phologically distinct from the surrounding ectoderm. As a con-
sequence, most information about its development comes from
observations of placode cells during their migration and gan-
gliogenesis. The trigeminal ganglion comprises two lobes: the
ophthalmic lobe and the maxillomandibular lobe. Both receive
contributions from ectodermal placodes and neural crest cells.
In amphibians, the two lobes remain separate or fuse secon-
darily during development, suggesting that they are embryo-
logically and evolutionarily distinct (Hamburger, 1961;
Northcutt and Brandle, 1995). Histological analyses in the
mouse and chick indicate that placode cells leave the ectoder-
mal layer by breaking through the basal lamina as individuals
or small clusters of cells (Hamburger, 1961; D’Amico-Martel
and Noden, 1983; Nichols, 1986; reviewed by Webb and
Noden, 1993). They then migrate to the distal regions of the
trigeminal ganglion. Trigeminal placode cells become
immunoreactive for neuronal markers and exit the cell cycle
early in their development (Moody et al., 1989; D’Amico-
Martel and Noden, 1980). In contrast, the neural crest
component of the trigeminal ganglion only expresses neuronal
markers after condensation. If placode cells fail to invaginate,
they can form ectopic ganglia in the surface ectoderm
(Kuratani and Hirano, 1990), suggesting that migration and
interaction with neural crest cells are not necessary for their
neuronal differentiation.
Molecular markers of undifferentiated placodal epithelia
have not previously been described (Webb and Noden, 1993),
making it difficult to characterize the induction and cell speci-
fication of this cranial placode. In this study, we have used the
transcription factor Pax-3 and the FGF receptor FREK as
4287
Development 124, 4287-4295 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV4919
Cranial sensory ganglia in vertebrates develop from the
ectodermal placodes, the neural crest, or both. Although
much is known about the neural crest contribution to
cranial ganglia, relatively little is known about how placode
cells form, invaginate and migrate to their targets. Here, we
identify Pax-3 as a molecular marker for placode cells that
contribute to the ophthalmic branch of the trigeminal
ganglion and use it, in conjunction with DiI labeling of the
surface ectoderm, to analyze some of the mechanisms
underlying placode development. Pax-3 expression in the
ophthalmic placode is observed as early as the 4-somite
stage in a narrow band of ectoderm contiguous to the
midbrain neural folds. Its expression broadens to a patch
of ectoderm adjacent to the midbrain and the rostral
hindbrain at the 8- to 10-somite stage. Invagination of the
first Pax-3-positive cells begins at the 13-somite stage.
Placodal invagination continues through the 35-somite
stage, by which time condensation of the trigeminal
ganglion has begun. To challenge the normal tissue inter-
actions leading to placode formation, we ablated the cranial
neural crest cells or implanted barriers between the neural
tube and the ectoderm. Our results demonstrate that,
although the presence of neural crest cells is not mandatory
for Pax-3 expression in the forming placode, a diffusible
signal from the neuroectoderm is required for induction
and/or maintenance of the ophthalmic placode.
Key words: placode, cranial ganglion, neural crest, Pax-3, FREK,
chick
SUMMARY
Neural tube-ectoderm interactions are required for trigeminal placode
formation
Michael R. Stark
1,2
, John Sechrist
1
, Marianne Bronner-Fraser
1,
* and Christophe Marcelle
1
1
Division of Biology 139-74, California Institute of Technology, Pasadena, CA 91125, USA
2
Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA
*Author for correspondence

4288
molecular markers to analyze the spatiotemporal development
of the ophthalmic placode. These markers were used in com-
bination with DiI labeling and neurofilament immunocyto-
chemistry to characterize the early specification of the oph-
thalmic placode, as well as to analyze the neuronal
differentiation and subsequent gangliogenesis of placode cells
contributing to the trigeminal ganglion. We then investigated
the possible mechanisms of placode induction experimentally
and found that interactions between the neuroectoderm and
surface ectoderm, but not the neural crest, are required for
normal ophthalmic placode formation.
MATERIALS AND METHODS
Cloning of quail
Pax-3
To isolate a Pax-3 probe for use in whole-mount in situ hybridizations,
we used a fragment of the chick Pax-3 cDNA (Goulding et al., 1993)
to screen a 4-day-old quail whole embryo cDNA library (InVitrogen).
We isolated four clones, the longest of which spans 3080 bp. Com-
parison of its sequence (GenBank accession number AF000673) to
the mouse Pax-3 cDNA revealed that the quail clone spans 1442 bp
of coding sequence and around 1.6 kb of 3′ untranslated region, and
that it is missing the nucleic acid sequence corresponding to the first
5 amino acids of the Pax-3 polypeptide (Fig. 1). Comparison of the
deduced amino acid sequences of the quail Pax-3 to the mouse Pax-
3 indicates that they are 93% similar, while their nucleic acid identity
is 46% (81% in the coding region). Sequencing was performed using
an ALF automated sequencer (Pharmacia) as well as the standard
dideoxy chain termination method with radiolabeled nucleotides
(Sanger et al., 1977).
In situ hybridization
Pax-3 digoxigenin-labeled RNA probes were synthesized and used for
whole-mount in situ hybridization on fixed quail and chick embryos
as described by Wilkinson (1992) and Henrique et al. (1995).
Embryos were embedded in gelatin and prepared for cryostat sec-
tioning as described by Sechrist et al. (1995); 10-20 µm sections were
mounted on subbed slides.
DiI labeling
To label the surface ectoderm, but not the neural tube or neural crest,
whole ectoderm DiI labeling was performed following closure of the
cranial neural tube (i.e. after the 12-somite stage) as previously
described (Sechrist et al., 1995). Briefly, a small hole was made in the
vitelline membrane above the embryo, through which the DiI/sucrose
solution was applied (Cell Tracker DiI, Molecular Probes). After
allowing them to develop to the desired stage, embryos were collected,
fixed in 4% paraformaldehyde and prepared for cryostat sectioning.
Ablations and barrier placements
Fine glass needles were used to remove the dorsal third of the neural
tube as described previously (Sechrist et al., 1995) or the surface
ectoderm. For placement of barriers, glass needles were used to cut a
slit between the ectoderm and the neural folds and the barriers were
inserted with fine forceps. 7.5 µm thick tantalum foil (Goodfellow
#TA000280) was cut into pieces of approximately 250×350 µm and
shaped with fine forceps. Polycarbonate membranes (Osmotics) with
pore sizes of 0.1 µm or 0.8 µm were cut into similar sized pieces. One
day after barrier insertion, embryos were collected for in situ hybrid-
ization analysis.
Neurofilament immunoreactivity
Sections of DiI-labeled embryos were stained with a neurofilament
antibody (kindly provided by Dr Virginia Lee) as described previously
(Sechrist et al., 1993).
RESULTS
Analysis of trigeminal placode development using
ectodermal DiI-labeling
To examine the contribution of neural crest and placode cells
to the trigeminal ganglion, we selectively labeled the surface
ectoderm with DiI. Surface labeling was performed after neural
tube closure to avoid labeling the neural tube/neural crest.
Because placode cells are the only surface ectoderm cells to
invaginate, any DiI-labeled cells within the mesenchyme derive
from the ectodermal placodes. When embryos were labeled at
the 15 somite stage and collected at the 19-somite stage, we
observed several individual, DiI-positive cells within the mes-
enchyme adjacent to the midbrain and rostral hindbrain (Fig.
2A). In slightly older embryos, additional DiI-positive placode
cells were visible in this region (Fig. 2B); placode-derived cells
contributing to the ophthalmic branch ultimately aligned in a
long array extending from the rostral hindbrain up to the caudal
portion of the developing eye (Fig. 2C). As the ganglion
became morphologically distinct from the surrounding mes-
enchyme (30- to 35-somite stage), placode cells could be
observed in the distal regions of each lobe of the trigeminal
ganglion. In contrast, unlabeled neural crest cells resided in the
more proximal regions of the ganglion (Fig. 2D). These results
are in good agreement with previous studies using alternative
labeling techniques (D’Amico-Martel and Noden, 1983).
Pax-3
and FREK are molecular markers of the
ophthalmic trigeminal placode and ganglion
Although whole ectoderm DiI labeling provides an efficient
means to analyze placode cell invagination and gangliogen-
esis, this technique is not suitable to study placode develop-
ment prior to cell invagination and does not provide a means
by which to investigate placode induction and specification.
Moreover, using this technique, we cannot examine placode
cells that may invaginate prior to neural tube closure. We
therefore sought to identify molecular markers of the differen-
tiating trigeminal placode.
We found that the transcription factor Pax-3 (Goulding et al.,
1991) and the FGF receptor FREK (Marcelle et al., 1994) are
early markers of the avian ophthalmic placode and used these
to analyze the early specification of placodal cells. The earliest
indication of ectodermal Pax-3 expression in the head occurs
M. R. Stark and others
Fig. 1. Quail Pax-3 gene. Comparison of the mouse (Goulding et al.,
1991) and quail Pax-3 genes showing regions of the paired domain
(p), the octapeptide (o) sequence and the homeodomain (h). The
quail gene was isolated from a 4-day-old quail library using a chick
probe (a) (Chalepakis et al., 1993). In situ hybridizations perfomed in
the current study used probes (b and c) synthesized from two
deletion fragments of the quail Pax-3 gene. GenBank accession
number, AF000673.

4289Tissue interactions in placode formation
at the 4-somite stage, at which time Pax-3-positive ectodermal
cells are contiguous to Pax-3-positive cells in the dorsal neural
tube and neural folds (Fig. 3A,B). Ectodermal Pax-3
expression expands laterally and increases in intensity as
development proceeds. By the 6-somite stage (Fig. 3C,D), Pax-
3 is clearly visible in the dorsolateral ectoderm overlying the
head mesenchyme at the level of the midbrain and rostral
hindbrain. As neural crest cells initiate migration at the 7- to
8-somite stage, Pax-3 expression is detected in a broadening
area of surface ectoderm adjacent to the presumptive midbrain
and rostral hindbrain (Fig. 3E,F). In 12- to 16-somite-stage
embryos, the ectodermal Pax-3 expression domain becomes
restricted to a band of intensely labeled cells extending from
the rostral hindbrain towards the dorsal eye region (Fig. 3G,H).
Pax-3-positive placode cells begin to enter the mesenchyme
as early as the 13-somite stage (Fig. 4A), with the majority of
cells invaginating and becoming migratory in 18- to 26-somite-
stage embryos. As previously described (reviewed by Noden,
1993), placode cells appear to exit the ectoderm individually
or as small clusters (Fig. 3H, arrowhead; Fig. 4A,B), subse-
quently moving towards the future ophthalmic lobe of the
trigeminal ganglion. The ectoderm becomes devoid of Pax-3
expression at about the 35-somite stage as trigeminal ganglion
cells condense and differentiate. The level of Pax-3 expression
in the placodal component of the ophthalmic branch remains
significantly higher than in the neural crest component
throughout late stages of ganglion formation, allowing easy
discrimination between the placode-derived and the neural
crest-derived component of the ophthalmic lobe in the con-
densing ganglion (Fig. 4C). To determine unequivocally
whether Pax-3-positive cells present in the head mesenchyme
were derived from the placode, we combined DiI labeling and
Fig. 2. DiI labeling of the surface ectoderm reveals placode cell
migration and subsequent ganglion contributions. Sections through
the trigeminal region of embryos labeled with DiI: (A) at the 15-
somite stage and collected at 19-somite stage; (B) at 12-somite stage
and collected at 21-somite stage; (C) at 15-somite stage and collected
at 29-somite stage; (D) at 19-somite stage and collected at 34-somite
stage. Since only the surface ectoderm was labeled, all internal DiI-
positive cells are placode derived. Arrowheads indicate placode-
derived cells (A-D). The neural crest component of the trigeminal
ganglion remains unlabeled (D, arrow).
Fig. 3. Expression pattern of Pax-3 in the developing chick
ophthalmic lobe placode as revealed by in situ hybridization. (A) A
whole-mount view of a 4-somite-stage chick embryo with Pax-3
expression in the dorsal neural folds. (B) A transverse section at the
level of the yellow dotted line in A illustrates that Pax-3 expression is
confined to the dorsal neural folds and immediately adjacent
ectoderm (red arrowhead). (C) A whole-mount view of a 6-somite-
stage embryo with Pax-3 expression in the newly closed dorsal
neural tube and laterally extending ectoderm. (D) A transverse
section at the level of the yellow dotted line in C illustrates Pax-3
expression in the dorsal neural tube, prominent in premigratory
neural crest cells (arrow) and extending laterally into the adjacent
ectoderm (red arrowhead). (E) A whole-mount view of an 8-somite-
stage chick embryo. (F) A transverse section at the level of the
yellow dotted line in E illustrates that Pax-3 expression is largely
down-regulated in the neural tube; it is expressed in early migrating
neural crest cells (arrow) and prominently in the dorsolateral surface
ectoderm (red arrowhead). (G) A whole-mount view of a 16-somite-
stage chick embryo with Pax-3 expression in opthlamic lobe placode
cells. Pax-3 expression is also evident in the neural tube and in
streams of migratory neural crest cells rostral and caudal to the otic
vesicle (black arrows) (H) A transverse section at the level of the
yellow dotted line in G shows Pax-3 expression in the lateral
ectoderm and in invaginating placode cells (red arrowhead). Note
that at this caudal midbrain level, there is no obvious Pax-3
expression in the dorsal neural tube and neural crest.

4290
in situ hybridization for Pax-3. We noted numerous DiI-labeled
placode cells that were also Pax-3-positive (Fig. 5A-D), con-
firming that Pax-3-positive cells present in the mesenchyme
emanated from the placode.
Conflicting observations have been reported regarding
whether or not neural crest cells express Pax-3 (Goulding et
al., 1991; Buxton et al., 1997). To reconcile these differences,
we carefully examined its pattern of expression during cephalic
and trunk crest migration along the body axis; we observed that
Pax-3 is only transiently expressed in neural crest cells. While
neural folds express Pax-3, its expression appears to be down-
regulated to almost undetectable transcription levels soon after
neural crest cells emigrate from the neural folds and neural
tube (Fig. 3F,H). A notable exception is observed at the levels
of rhombomeres 4 and 6, where migrating neural crest cells
maintain detectable Pax-3 expression en route to the branchial
arches (Fig. 3G). Pax-3 is re-expressed later in embryonic
development in the neural crest cells as they condense in dorsal
root ganglia (Goulding, 1991; Marcelle et al., 1995) and in
some cranial ganglia, including the proximal portion of the
ophthalmic lobe as well as the maxillomandibular lobe of the
trigeminal ganglion (Fig. 4C). Pax-3 expression in cells con-
tributing to the ophthalmic lobe of the trigeminal ganglion was
recently reported (Buxton et al., 1997), although the placodal
origin of these cells was not demonstrated. In addition, we
noted Pax-3 expression in the neural folds at earlier stages than
did Buxton et al. (1997); the differences between our studies
are likely due to the different sensitivity of the probes used.
It was previously reported that FREK is expressed in the
trigeminal placode (Marcelle et al., 1995). Here, we analyzed
the temporal pattern of placodal FREK expression during
embryonic development. We found that FREK is expressed in
the ophthalmic branch of the trigeminal placode later than Pax-
3, with the first FREK-positive cells detectable at the 10-somite
stage. Robust FREK expression was observed between the 15-
to 30-somite stage (Fig. 6A,B) at which time its expression was
restricted to ophthalmic lobe placode cells within the ectoderm
and to the underlying mesenchyme, similar to the pattern of
Pax-3 expression. Unlike Pax-3, FREK expression was not
maintained after gangliogenesis.
Pax-3
expression during mouse trigeminal ganglion
formation
It was previously observed that Splotch mice, which carry a
non-functional Pax-3 gene, display a reduced ophthalmic lobe
of the trigeminal ganglion (Tremblay et al., 1995), suggesting
a failure in normal ganglion formation. Pax-3 expression in the
mouse trigeminal placode had not been previously reported and
this developmental defect was attributed to the neural crest
component of the trigeminal ganglion. However, it was
recently shown that cephalic neural crest migration and con-
tribution to the trigeminal ganglion appears normal in Splotch
mutant mice (Serbedzija and McMahon, 1997). To test whether
Pax-3 is expressed in the mouse trigeminal placode, we
M. R. Stark and others
Fig. 4. Trigeminal placode cells in
chick as revealed by Pax-3 in situ
hybridization. (A) A transverse
section through a 13 somite-stage
embryo showing invagination of
placodal epithelia (red arrowhead).
(B) A transverse section through a
20-somite-stage embryo showing a
cluster of Pax-3-expressing cells
(red arrowhead) which have
invaginated into the mesenchyme.
(C) A whole-mount view of a stage
17 chick embryo shows Pax-3
expression in the condensing
trigeminal ganglion. Pax-3 is highly
expressed in the ophthalmic
placode-derived portion of the
ganglion (red arrowhead), while the
neural crest-derived and
maxillomandibular placode-derived
portions of the ganglion expresses
Pax-3 at much lower levels.
Fig. 5. DiI-labeling of the surface ectoderm confirms that Pax-3 is a
molecular marker for invaginating trigeminal placode cells.
(A-D) Coronal sections through the trigeminal placode region of an
embryo in which the entire surface ectoderm was labeled with DiI at
12-somite stage and allowed to develop to 28-somite stage. Bright
field (A,C) shows Pax-3 expression in the ectoderm and in
invaginating placode cells. Epifluorescence view (B,D) of the same
sections reveals that the invaginated Pax-3-positive cells are also
DiI-labeled, confirming their ectodermal origin. Arrowheads (A,B)
indicate ophthalmic placode-derived cells.

4291Tissue interactions in placode formation
performed whole-mount in situ hybridization on 7- to 10-day-
old mouse embryos using a mouse Pax-3 probe. Sections
through the head show a strong Pax-3 expression domain in
the ectoderm adjacent to the midbrain neural folds (Fig. 7A,B)
in a region reminiscent of the chick trigeminal placode. These
results suggest that, as in chick, the mouse trigeminal placode
is expressing Pax-3 and that its expression might be important
for placode differentiation.
Neural fold ablation does not alter the placodal
expression of
Pax-3
The proximity of neural crest cells to the trigeminal placode
raised the possibility that the neural crest may play a role in
the induction, migration and/or differentiation of the trigemi-
nal placode. Previous experiments have demonstrated that
placode-derived ganglia form when the neural crest component
is missing or reduced (Hamburger, 1961); however, these
ablations were performed at stage 11 or later, which is after
placodal induction and the initiation of neural crest migration.
To test whether the presence of the neural crest is required for
ophthalmic placode induction, we ablated the dorsal neural
folds in the midbrain/hindbrain region either unilaterally or
bilaterally in 4- to 8-somite-stage quail and chick embryos.
This manipulation effectively removes all neural crest precur-
sors present in the neuroectoderm. The majority of ablations
were performed in 6- to 8-somite-stage embryos and collected
6 to 8 hours postsurgery, a time at which there is little or no
regeneration of the neural crest (Sechrist et al., 1995); by this
stage, Pax-3 expression had presumably expanded beyond the
lateral margins of the neural folds (Fig. 3C,D), in which case
we also removed any putative Pax-3-expressing surface
ectoderm. Embryos were collected from 1 to 48 hours after
ablation and evaluated for placode induction and ganglion
formation by in situ hybridization with the Pax-3 probe.
From 0 to 3 hours postablation (n=4), Pax-3 was absent in
the ectoderm adjacent to the neural tube shortly after ablation
(Fig. 8A), indicating effective removal of both the neural crest
and placodal precursors. However, in all embryos fixed
between 4 and 9 hours postablation (n=16), some ectodermal
Pax-3 expression was observed, with the level of expression
dependent upon the degree of healing between the neuroecto-
derm and adjacent surface ectoderm (Fig. 8B). By 11 or more
hours postsurgery (n=8), Pax-3-positive ectodermal cells could
be detected immediately adjacent to the neuroepithelium (Fig.
Fig. 6. FREK expression in the trigeminal placode as revealed by in
situ hybridization. (A) Whole-mount view of a 16-somite-stage chick
embryo with FREK expression in the opthlamic lobe of the
trigeminal placode. (B) A transverse section at the level of the yellow
dotted line in A shows FREK expression in the lateral ectoderm and
in invaginating placode cells (red arrowhead).
Fig. 7. Pax-3 surface ectoderm expression in the mouse. (A) Whole-
mount view of a 10-somite mouse embryo showing Pax-3 expression
in the dorsal neural tube and the adjacent ectoderm in the head.
(B) A transverse section at the level of the yellow dotted line in A
shows Pax-3 expression in presumptive placodal ectoderm.
Fig. 8. Effects of unilateral or bilateral neural fold ablation on
ectodermal expression of Pax-3. (A) A transverse section through a
quail embryo 3 hours after unilateral ablation of the right neural fold
at the 4-somite stage. No obvious Pax-3 expression was observed on
the operated side. (B) A transverse section through an embryo 4
hours after bilateral neural fold ablation at the 5-somite stage. Pax-3
is expressed in the ectoderm immediately adjacent to the dorsal
neural tube. (C) A transverse section through an embryo that
underwent bilateral neural fold ablation at the 3-somite stage and was
allowed to develop to the 12- to 13-somite stage. Pax-3 is highly
expressed in the dorsal ectoderm. (D) A transverse section through
an embryo after right unilateral neural fold ablation performed at 5-
somite stage and fixed at the 19-somite stage; placodal Pax-3
expression was somewhat reduced on the ablated side with fewer
cells entering the mesenchyme. However, the overall morphology
was similar to that in unoperated embryos. (E) Transverse section
through the caudal midbrain of an 11-somite embryo that underwent
bilateral neural fold ablation at the 6-somite stage and was allowed to
develop 9 hours to the 11-somite stage. Although neural tube closure
has not yet occurred, Pax-3 is expressed in the surface ectoderm.
(F) Fluorescent image of the same section after staining with the
neural crest antibody HNK-1. Neural crest cells were absent at this
axial level. (G) Transverse section illustrating the presence of
numerous HNK-1 immunoreactive neural crest cells at a more rostral
region (adjacent to the caudal portion of the optic cup) of the same
embryo shown in E and F.

4292
8C). After 16 to 24 hours, all embryos showed Pax-3
expression in both the ectoderm and invaginating placode cells
(Fig. 8D). The number of Pax-3-expressing placode cells and
their intensity of staining varied depending on the depth of
ablation and degree of healing. However, the complement of
cells was generally normal or only slightly reduced. Pax-3
expression in the dorsal neural tube also resumed in these
embryos by this stage. Embryos collected after gangliogenesis
often had trigeminal ganglia that appeared misshapen and
improperly located. Staining with the HNK-1 antibody, which
recognizes migrating neural crest cells, confirmed the absence
of neural crest cells in the region of the ablation in embryos
collected 6 to 8 hours postsurgery (Fig. 8E-G). In several
embryos, placodal Pax-3 expression was observed in the
absence of midline closure, which has recently been suggested
to be necessary for Slug expression and neural crest formation
(Buxton et al., 1997). The observation that Pax-3 is expressed
in the placodes soon after neural fold ablation and prior to the
appearance of neural crest cells suggests that induction and/or
maintenance of the ophthalmic lobe placode is independent of
the neural folds/neural crest.
The experiments described above were repeated using DiI-
labeling to mark the surface ectoderm after neural crest or
surface ectoderm ablation. Following neural crest ablation at
the 4- to 7-somite stage, chick embryos were allowed to heal
and develop to the 15- to 20-somite stage, at which time DiI
was applied. Embryos were allowed to develop for an addi-
tional 48 hours. In all cases, placode-derived ganglia developed
in the region of the trigeminal system, although most were
displaced and misshapened without an apparent neural crest
component (Fig. 9A). This result confirms that obtained with
Pax-3 and indicates that placodal cells can invaginate in the
absence of neural crest cells. To remove the trigeminal placode,
embryos underwent ectoderm ablation larger than the pre-
sumptive trigeminal placode at the 12-somite stage and were
allowed to heal and develop to the 18- to 20-somite stage prior
to DiI surface ectoderm labeling. 1 day later, the placode-
derived component of the ganglion was reduced but never
absent (Fig. 9B). Our inability to completely remove the
placode by ablation is likely to be due to the ability of the
surface ectoderm to efficiently regenerate placodal epithelium,
as suggested by Hamburger (1961).
A diffusible signal from the neural tube is required
for normal placode formation
To test whether neuroectoderm-ectoderm interactions are
necessary for proper trigeminal placode formation, we surgi-
cally separated the ectoderm from the neural folds in vivo in
2- to 9-somite-stage embryos and prevented subsequent
reclosure and contact by placing an impermeable foil barrier
between the two tissues (Fig. 10A,B). In most experiments
(n=20), a 7.5 µm thick tantalum foil barrier was used, although
we found that gold (n=4) or aluminium (n=6) foil barriers were
equally effective in preventing neuroectoderm-ectoderm
contact with no apparent toxic or teratogenic effects.
Blocking ectoderm-neural tube interactions led to a
reduction or complete loss of Pax-3 expression in the surface
ectoderm (Fig. 10A,B). The extent of placode loss varied with
the length of the barrier, its rostrocaudal location and the stage
of barrier implantation. For example, barriers placed between
the neural tube and the entire length of the presumptive oph-
thalmic placode reduced the levels of Pax-3 expression by 70-
100% (n=19). Barriers that were smaller or implanted more
caudally or rostrally resulted in a 30-70% reduction in Pax-3
expression (n=4). In several cases (n=9), the neural folds were
ablated unilaterally or bilaterally prior to barrier implantation;
no obvious differences in the amount of Pax-3 loss lateral to
the barrier were observed in the presence or absence of the
neural folds. Prior to and shortly after invaginating from the
ectoderm, some placodal cells begin to express neuronal differ-
entiation markers such as β-tubulin (Moody et al., 1989) and
become neurofilament immunoreactive (unpublished observa-
tion). Implantation of impermeable barriers (n=8) resulted in a
loss or profound reduction in neurofilament immunoreactivity
on the operated side of the embryo (Fig. 10C), confirming that
the loss of Pax-3 expression correlated with the loss of normal
differentiation of ophthalmic placode cells. As a control (n=4),
barriers were removed shortly after insertion; in these embryos,
placodal Pax-3 (n=3) or neurofilament (n=1) expression
appeared normal. These results indicate that a neuroectoderm-
ectoderm interaction is required for normal placode induction
and/or maintenance.
The ectoderm could receive signals from the neural tube
either by means of cell contact or diffusible molecules. As a
first step in determining the molecular nature of underlying
trigeminal placode induction and/or maintenance), we placed
polycarbonate barriers (Schramm et al., 1994) with pore sizes
of 0.1 µm (allowing passage of diffusible molecules; n=12) or
0.8 µm (allowing passage of both cell processes and diffusible
molecules; n=11) between the ectoderm and neural tube of 4-
to 9-somite-stage chick embryos. Pax-3 expression was
observed in the ectoderm overlying either large pore size
M. R. Stark and others
Fig. 9. Effects of neural crest or placode ablation on ganglion
formation as revealed by surface ectoderm DiI labeling. (A) Section
through a 33-somite-stage embryo which underwent neural fold
ablation at the 5-somite stage and was labeled with DiI at the 18-
somite stage. The neural crest component of the trigeminal ganglion
is largely absent while the DiI-labeled placode component comprises
the remaining ganglion (arrowhead). (B) Section through a 35-
somite-stage embryo which underwent ablation of the presumptive
placodal ectoderm at the 12-somite stage and was labeled following
healing at the 19-somite stage. The neural crest component of the
ganglion appears intact (arrow) while the DiI-labeled placode cells
are reduced in number with the majority of cells having condensed
external to the ganglion proper (arrowhead). Compare with section
through an unablated embryo shown in Fig. 2D.

4293Tissue interactions in placode formation
barriers (Fig. 11A,B) or small pore size barriers (Fig. 11C,D),
indicating that a diffusible signal from the neural tube is suffi-
cient to induce and/or maintain expression of Pax-3 in the
trigeminal placode.
DISCUSSION
Due to the lack of appropriate molecular markers, little was
known about the early inductive events leading to placode
formation and differentiation. We report that Pax-3 and FREK
are expressed in placode cells contributing to the ophthalmic
lobe of the trigeminal ganglion, from the time of early speci-
fication (Pax-3 and FREK) through ganglion formation (Pax-3
only). Analysis of Pax-3 expression indicates that placode cells
are specified early in development. We observed Pax-3
expression as early as the 4-somite stage, 10 to 15 hours before
placode cells initiate migration towards the future ganglion.
Chick/quail chimeric studies have suggested that the trigemi-
nal placodes originate in the neural folds and subsequently
translocate ventrolaterally through the ectodermal layer
(Noden, 1983; D’Amico-Martel and Noden, 1983; Couly and
Le Douarin, 1990). However, we find that the presence of a
permeable, physical barrier between the neuroectoderm and the
surface ectoderm does not prevent Pax-3 expression and sub-
sequent ganglion formation, despite the fact that it would be
expected to block lateral migration; rather than arising by
migration from the neural folds, our data suggest that the oph-
thalmic placode is induced within the surface ectoderm by a
diffusible signal from the neuroectoderm.
Although our experiments utilize Pax-3 solely as a
molecular marker for trigeminal placode cells, Pax-3 is also
likely to play a functional role in ganglion formation. The con-
servation of the distribution patterns of Pax-3 between the
chick and mouse trigeminal placode is consistent with the pos-
sibility that Pax-3 is necessary for proper trigeminal ganglion
formation. Furthermore, Splotch mutants that are deficient in
Pax-3 have significant reductions in the ophthalmic lobe of the
trigeminal ganglion (Tremblay et al., 1995). In addition, these
mice display major defects in skeletal muscle progenitor
migration towards the limb mesenchyme while muscle differ-
entiation is unimpaired (Datson et al., 1996). Like muscle pro-
genitors of the lateral somite, placode cells express high levels
of Pax-3 prior to undergoing an epithelial-mesenchymal tran-
sition and initiating migration. One intriguing possibility is that
Pax-3 may be important for placode cell migration toward the
condensing ganglion within the head mesenchyme. Another
interesting parallel between skeletal muscle precursors and
trigeminal placode cells is that they both express the fibroblast
growth factor receptor FREK following expression of Pax-3
(Marcelle et al., 1995). This raises the possibility that this
receptor may be a target of the transcription factor.
Interestingly, Pax-3 and FREK are markers of the oph-
thalmic lobe placode, while the maxillomandibular lobe
placode is devoid of their expression. Morphological studies
performed in amphibians have suggested that the ophthalmic
and the mandibular lobes of the trigeminal ganglion are embry-
ologically and evolutionarily distinct (reviewed by Hamburger,
1961; Northcutt and Brandle, 1995). Our observation that Pax-
3 and FREK are initially expressed in the ophthalmic and not
in the mandibular branch of the ganglion is consistent with this
hypothesis, since these two branches of the ganglion are mol-
ecularly distinct from one other.
The cranial ganglia have a dual origin from neural crest cells
and sensory ectodermal placodes (reviewed in Le Douarin et al.,
1986; Webb and Noden, 1993). Ectodermal placode cells share
Fig. 10. Pax-3 expression (A,B) or
neurofillament immunoreactivity
(C) in embryos after implantation of
impermeable barriers (represented
by dotted lines in B,C). (A,B) An
embryo into which a tantalum foil
barrier was placed between the
neural tube and ectoderm at the 6-
somite stage and subsequently
allowed to develop for 24 hours.
Placodal Pax-3 expression was
eliminated on the operated side, while expression on the control side was unaffected. (C) Section through an embryo into which a tantalum foil
barrier was placed between the neural tube and ectoderm at the 6-somite stage and subsequently allowed to develop for 24 hours.
Neurofilament-positive cells were absent from the operated side, whereas the control side had a normal complement of neurofilament-positive
placode cells (arrowheads).
Fig. 11. Pax-3 expression in embryos after implantation of semi-
permeable barriers. Placodal Pax-3 expression after implantation of a
0.8 (A,B) or 0.1 (C,D) µm polycarbonate barriers between the neural
tube and ectoderm. Surgeries were performed at 6- to 7-somite stage
and the embryo was allowed to develop for an additional 24 hours.
Pax-3 is expressed in the ectoderm in the presence of either pore size
barrier.

4294
many properties with neural crest cells including the ability to
undergo an epithelial-to-mesenchymal transition, migrate and
contribute to neuronal components of sensory ganglia. The
proximity of neural crest cells to the trigeminal placode raised
the prospect that the neural crest could play a role in the
induction, migration and/or differentiation of the trigeminal
placode. Our results, however, rule out this possibility since
Pax-3 expression and ophthalmic lobe formation can occur in
the absence of neural crest cells. The placode-derived trigemi-
nal ganglion of neural-fold-ablated embryos appeared
misshapen and improperly located, however, suggesting that the
normal complement of neural crest cells may at least play a role
in the organization and position of the ganglion. Hamburger
(1961) previously concluded that placode cell differentiation
was independent of neural crest cells; these experiments,
however, were performed in 12- to 20-somite-stage embryos,
after initiation of neural crest migration and placodal specifica-
tion, as established by the present results.
Previous work from our laboratory has shown that neural
crest cells regenerate after ablation of the dorsal neural folds
(Scherson et al., 1993; Sechrist et al., 1995). However, optimal
regeneration occurs when neural folds are ablated at or prior
to the 4-somite stage whereas, in the present study, the majority
of ablations were performed after the time of optimal regener-
ation. Furthermore, production of neural crest cells after
ablation is delayed compared with initial generation of neural
crest cells, with the first regenerated HNK-1-positive cells
observed at ~13-somite stage (Sechrist et al., 1995). In the
present study, we observe placodal Pax-3 expression as early
as 4 hours postablation, prior to any detectable neural crest
regeneration. Furthermore, Buxton et al. (1997) have suggested
that midline closure is necessary for re-expression of the neural
crest marker Slug after neural fold ablation. Because we
observe placodal Pax-3 expression after ablation but prior to
dorsal midline closure, our results demonstrate that neural crest
cells are not required for induction or maintenance of Pax-3 in
the placodal ectoderm.
Previous studies have shown that neural crest cells can form
via an inductive interaction between neural tissue and non-
neuronal ectoderm (Moury and Jacobson, 1989; Selleck and
Bronner-Fraser, 1995; Dickinson et al., 1995). Dorsalin-1,
BMP-4 and BMP-7 have been shown to be sufficient to sub-
stitute for the non-neural ectoderm in inducing neural crest
cells (Basler et al., 1993; Liem et al., 1995). In contrast, the
inductive interactions necessary for placode formation have
been elusive. There is some evidence that the otic placode may
be induced by the adjacent hindbrain (Waddington, 1937;
Sechrist et al., 1994) although putative inducers have not been
identified (McKay et al., 1996). In this study, we show that,
similar to the neural crest, the ophthalmic lobe placode may
arise from a neuroectoderm-ectoderm interaction. A signal
emanating from the neural tube is required for placode
formation and neuronal differentiation, as assayed by Pax-3
expression in the placode and by neurofilament expression.
Members of the TGFβ and Wnt families are expressed in the
head neuroectoderm at developmental stages compatible with
a putative role in trigeminal placode induction. Future experi-
ments will test the role of various candidate inducers in the
formation of the trigeminal placode and examine whether
similar interactions are responsible for the formation of other
ectodermal placodes.
We thank Angela Nieto for early contributions to this work and
Martin Goulding for the chick and mouse Pax-3 probe. We are
indebted to Brian Rowe, Roham Zamanian, Johnny Choi, and Parisa
Zarbafian for technical assistance and Drs Clare Baker, Andrew
Groves, Catherine Krull and Ben Murray for critical reading of the
manuscript. This work was supported by NS34671 to M. B. F. and a
grant from the Muscular Dystrophy Association to C. M.
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(Accepted 11 August 1997)