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4547
Development 126, 4547-4556 (1999)
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DEV6419
Neural tube closure in Xenopus laevis involves medial migration, directed
protrusive activity, cell intercalation and convergent extension
L. A. Davidson* and R. E. Keller
Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22903, USA
*Author for correspondence (e-mail: [email protected])
Accepted 21 July; published on WWW 27 September 1999
SUMMARY
We have characterized the cell movements and prospective
cell identities as neural folds fuse during neural tube
formation in Xenopus laevis. A newly developed wholemount, two-color fluorescent RNA in situ hybridization
method, visualized with confocal microscopy, shows that
the dorsal neural tube gene xpax3 and the neural-crestspecific gene xslug are expressed far lateral to the medial
site of neural fold fusion and that expression moves
medially after fusion. To determine whether cell
movements or dynamic changes in gene expression are
responsible, we used low-light videomicroscopy followed by
fluorescent in situ and confocal microscopy. These methods
revealed that populations of prospective neural crest and
dorsal neural tube cells near the lateral margin of the
neural plate at the start of neurulation move to the dorsal
midline using distinctive forms of motility. Before fold
fusion, superficial neural cells apically contract, roll the
neural plate into a trough and appear to pull the superficial
epidermal cell sheet medially. After neural fold fusion,
lateral deep neural cells move medially by radially
intercalating between other neural cells using two types of
motility. The neural crest cells migrate as individual cells
toward the dorsal midline using medially directed
monopolar protrusions. These movements combine the two
lateral populations of neural crest into a single medial
population that form the roof of the neural tube. The
remaining cells of the dorsal neural tube extend
protrusions both medially and laterally bringing about
radial intercalation of deep and superficial cells to form a
single-cell-layered, pseudostratified neural tube. While
ours is the first description of medially directed cell
migration during neural fold fusion and re-establishment
of the neural tube, these complex cell behaviors may be
involved during cavitation of the zebrafish neural keel and
secondary neurulation in the posterior axis of chicken and
mouse.
INTRODUCTION
1990a) where these cell behaviors (apical contraction and
interkinetic nuclear migration) result in the formation of a
‘medial hinge’ overlying the notochord and a ‘dorsal lateral
hinge’ near the prospective sulcus limitans, that together bring
the neural folds into opposition. The epidermis then fuses, the
neural ectoderm fuses and the neural crest is released from
neural epithelium of the newly formed neural tube. In this
manner, it is thought that the flat neural plate rolls into a tube
with the lumenal face of the neural tube forming from the
apical face of the neural plate.
However, additional processes appear to be involved in chick
neurulation. By following the rapid events at the start of
neurulation, van Straaten and coworkers (1996) found that the
lateral face of the two neural folds ‘zip’ into apposition,
starting near the floorplate and proceeding dorsally. They
found that the lumen of the neural tube nearly disappears after
apposition and then re-opens to form the lumen after the neural
Neurulation is the process by which progenitors of the central
nervous system are shaped, separated from and brought
beneath the epidermis. The cellular basis of these movements
have been investigated in amphibians (Jacobson, 1981;
Jacobson and Gordon, 1976; Keller et al., 1992b), chicken
(Schoenwolf and Smith, 1990b; Smith and Schoenwolf, 1997)
and mice (Bush et al., 1990; Sausedo and Schoenwolf, 1994;
Smith et al., 1994) but remains poorly understood. In the chick,
which has been used extensively as a paradigm for vertebrate
neurulation, a broad neural plate folds and the margins of the
plate are raised and brought into apposition at the neural folds
as cells take on stereotypical shapes. Analysis of serial sections
and electron microscopy have identified cell movements and
shape changes accompanying neural tube formation in chick
(Schoenwolf and Alvarez, 1989; Schoenwolf and Smith,
Time-lapse sequences online:
http://www.people.virginia.edu/~lad4x/tubeclosure.html and
http://www.biologists.com/Development/movies/dev6419.html
Key words: Tyramide, Fluorescent in situ hybridization, Wholemount confocal microscopy, xk81, Epidermal cytokeratin, xslug,
Neural crest, xpax3, n-tubulin, neuron, xash3, Mediolateral
intercalation, Radial intercalation, Directed protrusive activity,
Convergent extension
4548 L. A. Davidson and R. E. Keller
folds fuse. Another group, using high-resolution electron
microscopy have identified distinctive cell morphologies at the
time of fusion of the neural folds (Lawson and England, 1998).
Thus, while it is likely that cell shape changes and neural plate
bending establishes the ventral and intermediate aspects of the
neural tube, more complex and as yet undefined events are
involved in forming the dorsal neural tube.
The revelations in chick inspired us to re-investigate the
mechanisms of neurulation in the frog. Based on sectioned
material and light and electron microscopy, Schroeder (1970,
1971) outlined four processes that he thought would ‘constitute
the ultimate mechanisms of neurulation’. (1) Superficial cells
of the neural epithelium change from cuboidal to bottle-shaped
initiating the formation of the neural groove. (2) Presomitic
mesoderm and lateral deep neural plate cells elongate to help
elevate the neural folds. (3) Both superficial and deep
epidermis ‘migrate’ medially bringing the neural folds into
apposition thus aiding closure of the neural tube. (4) Extension
of the underlying notochord prevents an anteroposterior
shortening of the neural plate that would result after apical
contraction of superficial neuroepithelial cells.
However, the closure and formation of the neural tube also
involves complex movements of radial intercalation of several
layers of deep cells with the superficial layer of the
multilayered neural anlagen of the frog (Schroeder, 1971).
These cell movements and the identities of the cells involved
are poorly understood, particularly at the lateral margin of the
neural plate, which is the source of three distinct tissues, the
epidermis, which will form the dorsal skin, the neural crest,
and the dorsal neural tube (Schroeder, 1970). The role of these
dorsolateral populations in neural tube formation are difficult
to evaluate because of the low spatial resolution of current
molecular marker methods. Correlation of cell identity with
motility is difficult because fluorescence-based imaging of cell
behaviors and tissue movements is largely incompatible with
RNA in situ hybridization techniques.
To solve this problem, we have modified the RNA in situ
hybridization protocol (Harland, 1991) using a recently
synthesized fluorescent substrate of peroxidase (Kerstens et al.,
1995) and have developed a streamlined whole-mount
preparation for confocal microscopy. Using this method allows
epidermal, neural crest and dorsal neural tube gene expression
patterns to be visualized with the same high-resolution
confocal techniques used for immunofluorescence and lineage
analysis and is generally useful to analyze gene expression
patterns in the context of cell and tissue shapes.
We show that the mediolateral organization of the early
neural plate is thrown into disorder by the rapid rolling
movements of the superficial neural ectoderm and fusion of
the neural folds over the neural groove. Immediately after
fusion, markers of the prospective dorsal neural tube cells are
still found far lateral to the medial site of fusion, whereas at
the midline, the expected site of the neural lumen is occupied
by multiple layers of deep mesenchymal cells lying above the
floorplate. The dorsal neural tube with a well-defined lumen
and roof is reformed after a complex set of cell movements:
medially directed migration and intercalation of neural crest,
and radial intercalation of deep neural cells. These
movements occur for several hours after neural fold fusion to
create a single-cell-layered neural tube characteristic of
vertebrates.
MATERIALS AND METHODS
Embryos
Embryos were obtained by standard methods (Kay and Peng, 1991)
and staged according to Nieuwkoop and Faber (1967). Albino
embryos were used for all in experiments. For fluorescence-based
imaging, it is important not to use Nile blue to add contrast for
staging. Nile blue contains the dye Neutral red, which fluoresces in
the rhodamine channel and does not wash out of embryos (in
contrast to the blue components of Nile blue) in organic solvents.
For general histology of cell and tissue shapes, all the cells in the
embryo were labeled by injection of 1.5 nL from a stock solution
(25 mg/ml of water) of rhodamine-dextran amine (RDA; anionic
lysine-fixable; Molecular Probes) at the 1-cell stage. Scattered,
labeled cell populations were made by injecting single blastomeres
at stages 6 or 7 when there are between 100 and 300 cells. Embryos
used for both histology and in situs were fixed overnight at 4°C in
MEMFA (0.1 M MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO4 and
3.7% formaldehyde) and stored at −20°C in 100% methanol. For
time-lapse recordings, embryos were mounted in custom chambers
at stage 17 in 1/3× Modified Barth’s Solution with 0.1% bovine
serum albumin. Single embryos were pierced transversely on a short
piece of a thin plastic ‘spear’ (1 mm long by 20 µm in diameter).
The ends of this plastic spear were pressed into silicone grease (Dow
Corning) by coverglass fragments producing an embryo solidly fixed
in position whose dorsal surface is centered and slightly pressed
against a coverslip. All embryos pierced in this manner develop
normally.
Videomicroscopy and morphometric analysis
Low-light, time-lapse recordings were collected using a Hamamatsu
C2400-008 SIT camera, an Olympus IX-70 inverted microscope, a
Uniblitz shutter and a Metamorph imaging system (Universal Imaging
Corp.). From these time-lapse recordings, we quantified the angular
protrusive activity of individual cells (see Elul et al., 1997). Briefly,
the outline of a single cell was traced over the course of a time-lapse
recording (from 40 to 100 frames). The pixel areas of cellular
protrusions from subsequent frames were accumulated into each of
twelve 30° sectors centered on the center of mass of the cell. The
angular protrusive activity for a cell was calculated as the percentage
of the total ‘protrusion’ area falling within each sector. The angular
protrusive activity of cells within the same domain of gene expression
was calculated as the mean of the percentages for all the cells within
that domain. This analysis was carried out using macros written by
the authors for NIH-Image (version 1.61; http://rsb.info.nih.gov/nihimage/).
Variations on the Harland protocol for RNA in situ
hybridization
We have made a number of modifications to the Harland protocol
(Harland, 1991). Antisense RNA probes were transcribed (Ambion),
labeled with either digoxigenin or fluorescein UTP (BMB) and used
without being hydrolyzed. Embryos in 100% methanol were
rehydrated through a series of 25:75, 50:50 and 75:25 PBS:methanol
washes. Proteinase K digestion was not done. Hybridization was at
60°C overnight and the post-hybridization RNase treatment was
skipped with little or no change in sensitivity. In several trials, the
potential activity of endogenous peroxidases was reduced by
incubating embryos for 1 hour with 1% H2O2 in PBS, but no change
in background was observed. Embryos were then washed, blocked and
incubated with peroxidase-coupled (POD) fab fragments (BMB)
directed against digoxigenin or fluorescein in maleic acid buffer with
2% BMB blocker and 20% heat-inactivated goat serum. With the
substitution of POD for alkaline phosphatase, this protocol was the
same as that used in a recently revised protocol (Knecht and Harland,
1997).
Neural tube closure in Xenopus 4549
Fluorescent-color POD reaction
Samples are equilibrated in POD reaction buffer (PBS, 0.1 M
Imidazole pH 7.6, and 0.001% H2O2) for an hour and reacted with
either tyramide-fluorescein or tyramide-rhodamine (TSA-Direct
GreenFISH and RedFISH, NEN Lifesciences) for 30 to 60 minutes.
Other tyramide-conjugated substrates are available commercially as
kits or may be synthesized in the laboratory (Hopman et al., 1998;
Jacobs et al., 1998). Fluorescence signal was increased with one to
three rounds of POD-mediated deposition with fresh tyramidefluorophore in buffer. After deposition of insoluble substrate, the
embryos were washed extensively for up to 2 days in PBS to remove
unreacted soluble tyramide-fluorophore revealing the expression
pattern. A second fluorophore may be used by incubating the embryos
for 45 minutes with 1% H2O2 in PBS to deactivate the first POD,
followed by repeating the above protocol with an appropriate PODconjugated fab fragment against the epitope carried by the second RNA
probe. Prior to confocal sectioning, preliminary assessments of the
outcome of the in situ reaction were made in whole embryos in PBS
using an epifluorescence equipped stereoscope (Olympus, SZH10).
‘Half’-mount preparation for optical sectioning
Embryos from fixative or 100% methanol were ‘softened’ by
incubating for 20 minutes in PBS and 0.01% Tween-20 (embryos
processed for RNA in situs do not require softening) and then bisected
transversely with a scalpel under a standard stereoscope. Tailbudstage embryos could be cut into several pieces. These pieces were then
dehydrated in 100% methanol and placed in disposable chambers
constructed by stacking 10 to 15 clear plastic paper reinforcement
rings (Avery cat. no. 05722) to form a well on a no. 1g thickness
(0.17 mm) coverslip. The embryos were then cleared by replacing the
methanol with benzyl benzoate and benzyl alcohol (BB:BA; 2:1).
Samples were oriented as they cleared with their cut side facing the
microscope objective and the chamber sealed with another coverslip.
This chamber remains intact for only a few hours as the BB:BA slowly
dissolves the adhesive. For future confocal sessions, embryos can be
retrieved immediately from these chambers, washed and stored in
100% methanol. Samples were optically sectioned immediately after
mounting using a confocal scanning laser system attached to an
inverted compound microscope (20×, 0.70 n.a. objective, Olympus or
Nikon) at the W. M. Keck Center for Cellular Imaging (Biology
Department, University of Virginia).
Probes
n-tubulin encodes a class II β-tubulin that is expressed in prospective
motor neurons and prospective dorsal sensory neurons, including
Rohon-Beard cells during midgastrulation in two to three mediolateral
stripes (Chitnis et al., 1995). Xpax3 encodes a transcription factor that
is expressed in prospective neural crest, dorsal neural tube, somitic
and lateral plate mesoderm in chick, mouse and frog (see figure 2D
in Bang et al., 1997; Goulding et al., 1991, 1993). xash3 encodes a
transcription factor that is expressed in cells marking the prospective
sulcus limitans (Ferreiro et al., 1994; Turner and Weintraub, 1994;
Zimmerman et al., 1993). xk81 encodes a cytokeratin that is expressed
in prospective epidermal cells (Fouquet et al., 1988; Jamrich et al.,
1987; Jonas et al., 1985).
RESULTS
Cell morphology and arrangement during fusion of
the neural folds over the neural groove
Shortly after the completion of gastrulation (stage 13), the
neural plate in the prospective trunk region consists of two
layers of cells (Fig. 1A). At this stage, there is no clear
morphological boundary at between the prospective neural
plate and the prospective epidermis in sectional view. At late
neural groove stages (stage 17), the neural folds rise above the
notoplate (see Jacobson and Gordon, 1976), that
part of the neural plate in contact with the
notochord, forming a groove (Fig. 1B). These folds
come into close apposition over the neural groove
(stage 18; Fig. 1C) and fuse, enclosing a very small
ventral neural lumen (stage 19; Fig. 1D). In some
cases the entire lumen disappears after fold fusion
(data not shown). Over the next several hours, the
Fig. 1. Transverse confocal sections at the level of the
trunk from late gastrula to early tailbud. Embryos labeled
at the 1-cell stage with 10 kDa lysinated rhodamine
dextran amine and fixed at various stages clearly show
tissue morphology. (A) Late gastrula (stage 13) showing
the three germ layers (en, endoderm; no, notochord; so,
prospective somites; ne, neural ectoderm). (B) Late
neural groove stage (stage 17) shows the medial groove
has formed and the neural folds are rising. (C) Neural
fold apposition (stage 18) shows the lips of the neural
fold (arrows) nearly in contact. (D) Shortly after fusion
of the neural folds, a slight groove remains in the
ectoderm (arrow). Cells above the small incipient lumen
(asterisk) are in disarray. Cells in the floorplate region
have begun to radially intercalate. (E) The lumen is reopened as radial intercalation proceeds from the ventral
floorplate into more intermediate regions of the neural
tube (stage 20/21). (F) Radial intercalation produces a
single-cell-layered neural tube by the time the dorsal fin
begins to form (stage 24/25). The deep layer of the
dorsal epidermis has been re-established over the neural
tube (arrowheads).
4550 L. A. Davidson and R. E. Keller
Fig. 2. Dorsal gene expression before and after neural fold
fusion. Transverse confocal sections in the trunk region (50150 µm posterior of the hindbrain) of fluorescent RNA in
situs showing gene expression in the forming neural tube.
(A) Epidermal marker xk81 shows the cells at the contacting
lips of the neural folds are epidermal. (B) xk81 expression
over the neural tube as the lumen reforms. (C) Neural crest
marker xslug shows the neural crest is far lateral as the
neural folds come into apposition. (D) xslug expression is
found medially in the dorsal neural tube after the tube has
formed. (E) A dorsal neural tube marker, xpax3, is also
found laterally as the folds come into apposition. (F) After
the neural tube forms xpax3 is found in the dorsal aspect of
the tube. (G) A marker for prospective neurons, n-tubulin, is
expressed in two domains, a medial one marking prospective
primary motorneurons at the borders of the floorplate and
lateral one marking the dorsal primary sensory neurons.
Again the prospective dorsal gene expression pattern is
found far lateral to the site of apposition. (H) n-tubulin is
expressed in a broad domain after the neural tube had
formed. (I) A marker for an intermediate position in the
neural tube, xash3 is found much closer to the midline.
(J) xash3 is expressed in a narrow stripe midway between the
floorplate and the roofplate in the single-cell-layered neural
tube.
the neural folds are epidermal (Fig. 2A) and that xk81
expression is limited to the outermost, single cell layer
over the open neural plate and early neural tube. The deep
layer of the epidermis is restored over the neural tube only
later (see arrowheads in Fig. 1F). There are occasional,
single-cell gaps in the expression of xk81 in the epidermis
(data not shown) that may mark prospective epidermal
ciliary cells (Chen and Grunz, 1997).
lumen is reconstructed from this rudiment, progressing from
ventral to dorsal as radial intercalation (i.e. deep cells
extending to the lumenal surface and superficial cells extending
to the basal surface of the neural tube) brings the two layers of
the early neural plate (Fig. 1E) into a single-layered neural tube
(Fig. 1F).
Cells participating in neural fold fusion over the
neural groove are prospective epidermis
Fusion of the neural folds over the neural groove creates a jumble
of cells that can now be identified. Expression of xk81, an
epidermal cytokeratin, shows that the cells at the fusing lips of
Neural fold fusion occurs while the future
dorsal cells of the neural tube are far lateral
xslug, xpax3, n-tubulin and xash3 gene expression
patterns (Fig. 2C,E,G,I) were used to identify other
dorsal cell types at neural fold fusion. Before fold
fusion, genes such as xslug and xpax3 are expressed far
lateral to the site of fusion (Fig. 2C,E) but later are
expressed in the dorsal aspect of the neural tube (Fig.
2D,F). The same is true of the dorsal limit of n-tubulin
expression (Fig. 2G,H). In contrast, a mediolateral
marker of the neural plate, xash3, is found much closer
to its eventual position in the neural tube (Fig. 2I,J).
Immediately at apposition or just after neural fold
fusion, in each of these cases, the cells over the neural
tube lumen do not express genes normally associated
with the dorsal neural tube. Later, however, after the neural
tube has formed a complete lumen, the dorsal aspect of the
neural tube expresses its definitive pattern of gene expression.
Thus, either gene expression is shifting from lateral to more
dorsal cells or the cells themselves are moving.
Medial shift in gene expression is due to medial
migration of cells
Time-lapse videorecordings of scattered, fluorescently labeled
deep cells at the margin of the neural plate in the whole embryo
revealed medially convergent movements of large groups of
individual cells (Fig. 3A) just below the epidermis. The embryo
Neural tube closure in Xenopus 4551
was fixed after the last frame of the videorecording and
medially migrating cells were visualized in transverse section
using a confocal microscope (Fig. 3B). A comparison with the
fluorescent in situ revealed that these cells express xslug (Fig.
3C). A dual image of the cells and xslug expression shows that
the leading cell in this section approached the midline by the
end of the time-lapse recording (Fig. 3D). Mapping these cells
back to their location when the neural folds fused shows that
the shift in gene expression reflects the medial movement of
prospective neural crest cells after the folds fuse.
Medial migration of neural crest cells takes place by
monopolar, medially directed protrusive activity
The thin epidermis allowed us to record the medial movements
of several cells within the xslug-expressing neural crest domain
(Fig. 4A). Cells within this domain exhibit monopolar
protrusions directed toward the midline (Fig. 4B) as they
migrate between the basal surface of the epidermis and more
ventral neural tube cells. At the completion of neural fold
fusion, these cells are isodiametric (data not shown) but soon
become mediolaterally elongated (i.e. transverse to the axis of
the embryo). Once they approach the midline, they return to a
more isodiametric shape (0:30 panel of Fig. 4A). These neural
crest cells remain quiescent in this medial location until they
start to emigrate from the tube (data not shown). Occasionally,
neural crest cells overshoot their midline target and remain on
the other side (Fig. 4C).
Medial migration and radial intercalation of dorsal
neural cells takes place by bipolar, mediolaterally
directed protrusive activity
In this period of medial migration and convergent extension,
more medial (i.e. ventral) cells of the neural tube show bipolar
protrusive activity. Prospective dorsal neurons (expressing ntubulin) immediately beneath the neural crest, are also medially
protrusive even though their lateral face is bound to the outer
surface of the neural tube (Fig. 5A). These two cells extend
protrusions in a bipolar manner directed mediolaterally (Fig.
5B). These tracked cells represent the dorsalmost population of
the neural tube, a domain of n-tubulin-expressing cells (Fig. 5C),
with their lateral end on the outer surface of the neural tube and
their medial end protruding beneath neural crest cells (Fig. 5D).
Relumenation involves simultaneous medial
migration, radial intercalation and reconstruction of
the lumen from ventral to dorsal
The medial movement of deep dorsal cells is paralleled by the
radial intercalation of more ventral cells in the neural tube.
Before neural fold fusion only the most ventral neural deep
cells have interdigitated but have not yet intercalated radially
between the more superficial cells surrounding the neural
groove (Fig. 6A). Radial intercalation begins first among cells
in the ventral tube (Fig. 6B) and progresses to more dorsal cells
after fusion (Fig. 6C). This progression of radial intercalation
coincides with the ventral-to-dorsal reconstruction of the
neural lumen. The ventral floorplate is the first stable portion
of the small round lumen immediately after the neural folds
fuse (Fig. 6D). The lumen becomes flask shaped with new deep
cells intercalating into the wall of the neural tube extending the
dorsal aspect of the lumen (Fig. 6E). Finally, the dorsalmost
surface of the lumen is completed and the single-cell-layered
pseudostratified neural tube is formed (Fig. 6F).
Medial migration results in convergent extension of
the dorsal neural tube
Time-lapse recordings of medial migration of dorsal cells of
the neural tube and transverse confocal sections over the course
of neural tube formation reveal that medial migration of dorsal
neural tube cells contribute to the convergent extension of the
dorsal neural tube. Convergent extension movements have
previously been characterized in the neural plate by an
anteroposterior dispersal of cells as local protrusive activity
brings the cells medially (Elul et al., 1997; Keller et al., 1992a).
These same features are seen in the time-lapse video(Fig. 3A)
as an initially solidly labeled group of cells break up (unlabeled
cells intercalate between labeled cells) as the group nears the
midline. The results of convergent extension are also revealed
in the decreasing number of cells in the neural tube visible in
transverse section over the course of neural fold fusion and
relumenation (compare the neural anlagen in Figs 1B,F, 6D,F).
Fig. 3. Neural crest cells migrate medially and do so
as individuals. (A) A series of frames selected at 20
minute intervals from a low-light time-lapse
videorecording begun during neural fold fusion. A
patch of rhodamine-dextran-labeled cells is seen at the
left of the frame (dark cells) against an otherwise
unlabeled embryo (light). A dotted line in the :00
frame marks the midline. The solid line in the 1:20
frame marks the confocal section shown in B-D. An
arrow in the 1:20 frame marks an individual cell that
has migrated away from its labeled neighbors. (B) A
confocal section transverse to the axis in the
rhodamine channel at the line marked in the 1:20
frame of A′. The labeled cells are shown in red and an
outline of the tissue boundaries of the early neural
tube, somites and notochord are shown in blue.
(C) xslug expression in the same confocal section is
shown in the fluorescein channel. (D) Dual image
showing xslug (green) is expressed by the most medial
group of cells whose edges were recorded in the lowlight videorecording (red). Scale bar in (A) is 50 µm.
4552 L. A. Davidson and R. E. Keller
Fig. 4. Neural crest cells migrate with medially directed monopolar
protrusive activity. (A) A series of frames selected at 10 minute
intervals from a low-light time-lapse videorecording begun shortly
after neural fold fusion. A single rhodamine dextran-labeled cell
(dark) is visible against an unlabeled background as it migrates to the
right toward the midline. (A′) Drawings of the panels in A showing
with the migrating cells (outlined in grey) and the midline (dashed
line). Fine structure details of protrusive activity such as filopodia
and lamellae are not resolvable when cells are recorded through the
epithelium. (B) A plot averaging the directional protrusive activity of
nine migratory cells tracked in the xslug-expressing domain.
(C) Transverse confocal section at the trunk region of an embryo
labeled in a single blastomere at the 2-cell stage and fixed shortly
after fold fusion. Such labeled embryos are labeled in the left or right
halves. Labeled cells have crossed over the midline to the opposite
unlabeled half (arrows). The juncture between labeled and unlabeled
epidermis marks the site of neural fold fusion (arrowhead). Scale bar
in (A) is 50 µm.
DISCUSSION
Analysis of neural tube closure, or its failure (Copp, 1994;
Copp et al., 1990), has been hindered by poor definition of the
cellular motility involved in this process. Here we use confocal
microscopy, a newly developed whole-mount fluorescent in
situ RNA hybridization method and correlated time-lapse
recording of cell behaviors to characterize cell motility
involved in neural tube closure in Xenopus laevis.
Formation of the dorsal neural tube involves
previously unexpected types of cell motility: medial
migration, polarized, directed protrusive activity, cell
intercalation and convergent extension
Whole-mount RNA in situs showed prospective neural crest
lies far lateral to the point of fusion of the neural folds and
time-lapse recordings revealed the behaviors used by these
cells to move to their definitive positions in the neural tube
(Fig. 7). Immediately after fusion of the neural folds, the dorsal
neural tube consists of mesenchymal cells derived largely from
the deep cell layer of the neural plate. Then, the neural crest,
arising from the lateral deep cell layer of the two-layered neural
plate, begins migrating medially, using monopolar, medially
Fig. 5. Other deep dorsal cells extend protrusions medially but are
not monopolar. (A) A series of frames selected at 10 minute intervals
from a low-light time-lapse recording begun shortly after neural fold
fusion. Two rhodamine dextran-labeled cells in the domain of dorsal
sensory neurons extend protrusions medially. (A′) Drawing of the
panels in A outlining the prospective neurons and indicating the
direction of the midline (arrow). (B) A plot averaging the protrusive
activity of these two cells. (C) Transverse confocal section showing
n-tubulin expression and the dorsalmost limit (arrow) of expression.
Scale bar in (A) is 50 µm.
directed protrusive activity. As the crest and other lateral cells
migrate medially they intercalate between more medial cells,
predominately along the anteroposterior axis, and thus elongate
and narrow the future dorsal aspect of the neural tube. This
pattern is reminiscent of the mediolateral cell intercalation seen
earlier in the neural plate (Fig. 7A; Elul et al., 1997; T. M. Elul
and R. E. K., unpublished data) and in the mesoderm (Keller
and Winklbauer, 1992), which in each case produces
convergent extension. At the outset of medial migration, the
entire population of cells involved consist of a multilayered
mesenchyme without a lumen. Through the process of
relumenation, involving medial migration and radial
intercalation (Fig. 7C; see below for discussion) the dorsal
neural tube forms a single-cell-layered, pseudostratified
epithelium surrounding a lumen (Fig. 7D).
Monopolar medially directed protrusive activity of
neural crest cells drives convergent extension of the
dorsal neural tube and formation of the roof plate
Without medial migration and narrowing in the mediolateral
direction, the very wide anlagen at the time of neural fold
fusion could not form a properly shaped neural tube. The
monopolar, medially directed protrusive activity seen in the
neural crest cells is consistent with the directed movement of
these cells toward the dorsal midline and, indeed, such activity
is always correlated with convergence movements. Thus, this
Neural tube closure in Xenopus 4553
protrusive activity in the dorsal neural tube
is directed toward the future roofplate.
However, even that notion of directionality
is not entirely accurate as we occasionally
observe neural crest cells overshooting and
crossing the midline. Such behavior is not
consistent with a dorsal midline cue that
attracts or directs protrusive activity
medially. However, the few cells seen
crossing the midline might represent the
earliest stages of neural crest cell emigration
from the neural tube, which is known to
involve crossing the midline (Krotoski et al.,
1988).
The potential cues for the monopolar,
directed migration could come from the
overlying epidermis, from the cells initially
at the dorsal midline of the neural anlagen,
or from cells still lateral to the neural tube.
In the first case, one would envision the cue
being a directional one on the undersurface
of the single-cell-layered epidermis in this
region. In the second case, one could
envision a signal emanating, probably
diffusing, from cells initially at the midline,
or perhaps even from the neural cells last in
contact with the epidermis. In the last case,
one could envision an inhibitory signal,
rather than a protrusion-stimulating signal,
from tissues lying off the lateral margin of
the neural plate, including the deep layer of
the epidermis, or the lateral plate mesoderm,
or at later stages, the somitic mesoderm.
Relumenation involves medial
migration, and progressive radial
Fig. 6. Neural tube lumen reconstruction and radial intercalation progress from ventral to
interdigitation and radial
dorsal after neural fold fusion. (A) Several transverse confocal sections in the trunk region
intercalation
showing scattered labeled cells as the neural folds converge over the groove. Ventral cells
Surprisingly, immediately after the
are interdigitated (arrow and v) while more lateral, prospective dorsal, cells are not (arrow
epidermis of the neural folds meet and fuse,
and d). (B) After fold fusion, ventral cells complete radial intercalation (arrow v) while
the bulk of the neural anlagen consists of a
intermediate cells begin radial intercalation and dorsal cells interdigitate (arrow i and
arrow d, respectively). (C) As the lumen reaches its full ventral-to-dorsal extent, radial
large population of deep mesenchymal cells
intercalation is complete as all cells span the wall of the neural tube. (D-F) Transverse
that do not immediately form a lumen. It is
confocal sections of embryos labeled with RDA as the neural tube lumen is reconstructed.
only after neural fold fusion that a lumen
(D) The ventral lumen is the first part of the neural tube lumen to fully reopen after fold
reforms. Relumenation involves two general
fusion. (E) The lumen extends dorsally as intermediate cells intercalate, restoring the
forms of motility. First, the medially
lumenal surface. (F) The roofplate forms as the lumen reaches its full extent.
directed monopolar motility described
above reforms the roofplate. Second, radial
specific type of directed motility is likely responsible for
interdigitation and subsequent radial intercalation of the two
shaping the dorsal neural tube.
cell layers of the neural anlagen occur during relumenation of
A medially directed, monopolar protrusive activity strongly
the ventral aspect of the neural tube and result in formation of
resembling that seen here, is expressed earlier by deep cells in
a single-layered tube.
the open neural plate and is thought to underlie the
Interestingly, this process of radial intercalation, as well as
mediolateral intercalation movements of cells that produce
the radial interdigitation that precedes it, begins ventrally at the
convergent extension (T. M. Elul and R. E. K., unpublished
midline, and progresses dorsally. Whether it is coincidence that
data). Whether the activity seen in the dorsal neural tube is a
radial intercalation begins ventrally where the lumen of the
continuation of this earlier activity or a new and independently
neural anlagen persists or whether the lumen cues the process
regulated behavior is not known. Although similar in
of radial intercalation is not known. It appears that radial
appearance, the two behaviors may have different mechanisms
intercalation occurs at the same time as relumenation. How and
of directionality; the earlier activity is directed toward the
why these processes are linked is the subject of further
midline of the open neural plate, i.e. the notoplate, while the
investigation.
4554 L. A. Davidson and R. E. Keller
behavior is not used in this region is not known. Bipolar
protrusive activity is expressed by deep neural cells as they
mediolaterally intercalate during their convergent extension
under conditions of planar induction (Elul et al., 1997);
however, the bipolar form of intercalation may not be used
during convergent extension under both planar and vertical
inducing signals (T. M. Elul and R. E. K., unpublished data).
Fig. 7. Neurulation in Xenopus laevis. (A) Medial migration of deep
cells shape the early neural plate. Cells exhibit different patterns of
motility in the notoplate and lateral neural plate. (B) As neurulation
progresses, cells in the superficial neurectoderm apically contract
shaping the neural groove. Continued cell shape change in the neural
plate, notochord and prospective somites cause the neural folds to
rise and come into apposition. Cells in the deep cell layer of the
prospective floorplate and adjacent regions interdigitate with cells in
the superficial layer. (C) After closure, medial migration and radial
intercalation remodel the dorsal tube and establish the lumen. Deep
cells in the ventral neural tube are the first to extend completely
across the neural tube. Radial intercalation then proceeds dorsally.
(D) Neural crest cells take up residence in the dorsal neural tube
above the roof plate as medial migration and radial intercalation
establish the ‘definitive’ neural tube.
The progression of radial intercalation behavior from the
ventral midline towards the dorsal was somewhat of a surprise
and has implications for patterning. The directed cell protrusive
activity driving convergent extension of the neural plate begins
anteriorly and laterally near the lateral border of the hindbrain,
and proceeds medially (future ventral) and posteriorly (Elul
and R. E. K., unpublished data). This is, of course, the reverse
of the progression seen in radial intercalation. If the
progression of these behaviors reflects when the participating
cells received the signals to execute the movements, then the
earlier mediolateral cell intercalation behaviors are organized
by signals emanating from lateral and anterior and progressing
medially (ventrally with regard to the future tube) and
posteriorly. In contrast, it could be that the radial intercalation
described here is organized by a signal emanating from the
ventral midline. Alternatively, it could simply be dependent on
conditions that mature progressively in the neural anlagen from
ventral to dorsal. Previous work by Hartenstein (1989)
described a progression of both cell division and radial
intercalation from dorsal (lateral) to ventral (medial).
Bipolar protrusive activity may be involved in radial
intercalation of the thick neural anlagen
Time-lapse recordings of cell behavior in the thick dorsal
region below the neural crest in a region undergoing radial
intercalation shows bipolar protrusive activity directed both
medially and laterally. Such behavior could be the principle
form of motility underlying the radial intercalation of multiple
layers of deep cells found in this region. Why the monopolar
Relumenation likely involves re-epithelialization
The reformation of the lumen of the neural tube appears to
involve a transition from a deep mesenchymal cell to an
epitheloid cell type. The dorsal neural anlagen is a jumble of
interlaced deep cells after neural fold fusion but as
relumenation occurs, the cells establish what appear to be
stable surfaces at their lumenal and basal faces, forming a
single-layered array of cells. The behavioral and
morphological evidence presented above suggests that a
mesenchymal-to-epithelial transition is a major process in
relumenation and thus closure of the Xenopus neural tube. We
are examining this hypothesis with an ultrastructural and
immunocytochemical analysis of junctional complexes during
neural tube formation.
What is the signal for radial intercalation and
relumenation?
We believe that radial intercalation and correlated
relumenation depends on neural fold fusion. In previous work,
‘giant explants’ of the neural anlagen converged, extended and
formed neural folds and grooves under both planar and vertical
neural-inducing signals through the late midgastrula stage, but
the neural folds produced by these explants would not fuse and
radial intercalation did not occur (Poznanski et al., 1997). Here
we show that radial intercalation does not occur until after
neural fold fusion. In another study A. Edlund and R. E. K.
(unpublished data) show that neural plates held open beyond
the time of normal neural fold fusion likewise do not show
radial intercalation of deep and superficial cells. These facts
argue that there is some signal or condition associated with
neural fold fusion that cues or enables radial intercalation. The
fact that radial intercalation fails when folds are mechanically
held open argues that radial intercalation depends directly on
fold fusion and eliminates the possibility that both processes
depend on yet a third signal.
Medial migration, radial intercalation and
relumenation appear to underlie the diverse
mechanisms driving neurulation among other
vertebrates
Chordates neurulate using a variety of mechanisms. In the
amphioxus, Branchiostoma floridae, the epidermal cells and
neural ectoderm completely separate well before the neural
tube forms and epidermal cells begin to crawl over the surface
of the neural ectoderm (Holland et al., 1996). As the
epidermis moves over the neural ectoderm, the neural plate
itself begins to fold. Once the epidermis has fused, the neural
plate bends at the midline and brings the edges of the plate
into contact and fusion. In this case, the processes of fusion
of the epidermis and fusion of the neural ectoderm have been
uncoupled from each other. It is unclear whether cells in the
neural plate begin directed medial migration of the type seen
in Xenopus, or whether a mass tissue movement such as
Neural tube closure in Xenopus 4555
convergent extension or folding takes place under the already
fused epidermis.
In zebrafish, neurulation involves transformation of the
neural plate into the neural keel (Papan and Campos-Ortega,
1994). Keel formation appears to proceed in a ‘cell-by-cell’
manner as cells on opposite sides of the neural plate move
medially, come into apposition and then move ventrally
together, side-by-side to form the neural keel. Two apposing
rows of cells lie on either side of the midline, but cells may
divide with daughter cells crossing over to the other side of the
keel (Kimmel et al., 1994). The keel appears to be a
mesenchymal mass of cells, which subsequently cavitates to
form the neural lumen. Relumenation, as in Xenopus, appears
to start ventrally at the floorplate and proceed dorsally to the
roofplate. It is unclear when the epidermis fuses over the top
of the neural keel.
Both chick and mouse form their posterior neural tube
(secondary neurulation) by cavitation (Costanzo et al., 1982;
Schoenwolf, 1984; Schoenwolf and Delongo, 1980). The
morphogenetic movements during this secondary neurulation
in mouse and chick might bear some resemblance to the sorting
out of mesenchymal cells in Xenopus. Moreover, the process
of cavitation in these embryos may share mechanisms with the
process of relumenation seen in Xenopus. Although, in the case
of secondary neurulation, the lumen is reconstructed from the
roofplate down to the floorplate.
Our work in Xenopus is the first instance in which the cell
behaviors underlying neural tube closure have been descibed
in detail. It is likely that, when broken down into their
constituent cell behaviors, these other diverse forms of
neurulation may begin to resemble neural tube closure in
Xenopus. The separation and fusion of the epidermis in
amphioxus might resemble the separation and fusion of the
neural folds over the neural groove in Xenopus. The
mesenchymal mass of neural keel cells in zebrafish may
resemble the disorganized mass of mesenchymal cells in the
dorsal aspect of the neural tube in Xenopus. The relumenation
in Xenopus from the ventral floorplate to the dorsal roofplate
might also resemble cavitation in the zebrafish neural tube as
well as cavitation in secondary neurulation in mouse and chick.
Just as the various forms of neurulation are driven by similar
cell behaviors, the cell behaviors themselves may be driven by
similar molecular pathways. The identification of these
molecular components and their relationship among the
vertebrates is only just beginning.
Fluorescent detection of gene expression and
improvements in protein localization
Further elaboration of these molecular components and their
roles in driving neurulation require both sophisticated
histology and fluorescence-based imaging techniques that
allow characterization of cell behaviors, tissue movements and
protein localization. Our fluorescent in situ RNA hybridization
methods have allowed us to visualize epidermal, neural crest
and dorsal neural tube gene expression patterns set in the
context of cell and tissue shapes. However, in situs merely
reflect gene expression and say little about the localization and
function of their encoded proteins. The next steps that we take
will involve expressing constructs encoding fluorescent
chimeras of candidate molecules to dynamically image these
molecules and explore their role in neurulation.
We would like to thank Anna Edlund, Tamira Elul, Max Ezin, Paul
Skoglund, and Dave Shook for critical discussions and assistance. We
would also like to thank C. Kintner for the pax3 and n-tubulin probes,
D. Anderson for the xash3 probe, R. Grainger for the xslug probe and
T. Sargent for the xk81 probe. This work was supported by NIH grant
HD25595 to Ray Keller and an American Cancer Society Postdoctoral
Fellowship to Lance Davidson.
REFERENCES
Bang, A. G., Papalopulu, N., Kintner, C. and Goulding, M. D. (1997).
Expression of Pax-3 is initiated in the early neural plate by posteriorizing
signals produced by the organizer and by posterior non-axial mesoderm.
Development 124, 2075-2085.
Bush, K. T., Lynch, F. J., DeNittis, A. S., Steinberg, A. B., Lee, H. Y. and
Nagele, R. G. (1990). Neural tube formation in the mouse: a morphometric
and computerized three-dimensional reconstruction study of the relationship
between apical constriction of neuroepithelial cells and the shape of the
neuroepithelium. Anat. Embryol. 181, 49-58.
Chen, Y. and Grunz, H. (1997). The final determination of Xenopus ectoderm
depends on intrinsic and external positional information. Int. J. Dev. Biol.
41, 525-528.
Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. and Kintner, C.
(1995). Primary neurogenesis in Xenopus embryos regulated by a
homologue of the Drosophila neurogenic gene Delta [see comments].
Nature 375, 761-766.
Copp, A. J. (1994). Genetic models of mammalian neural tube defects. Ciba
Found. Symp. 181, 118-34; discussion 134-143.
Copp, A. J., Brook, F. A., Estibeiro, J. P., Shum, A. S. and Cockroft, D. L.
(1990). The embryonic development of mammalian neural tube defects.
Prog. Neurobiol. 35, 363-403.
Costanzo, R., Watterson, R. L. and Schoenwolf, G. C. (1982). Evidence that
secondary neurulation occurs autonomously in the chick embryo. J. Exp.
Zool. 219, 233-240.
Elul, T. M., Koehl, M. A. R. and Keller, R. E. (1997). Cellular mechanism
underlying neural convergence and extension in Xenopus laevis embryos.
Dev. Biol. 191, 243-258.
Ferreiro, B., Kintner, C., Zimmerman, K., Anderson, D. and Harris, W.
A. (1994). XASH genes promote neurogenesis in Xenopus embryos.
Development 120, 3649-3655.
Fouquet, B., Herrmann, H., Franz, J. K. and Franke, W. W. (1988).
Expression of intermediate filament proteins during development of
Xenopus laevis. III. Identification of mRNAs encoding cytokeratins typical
of complex epithelia. Development 104, 533-548.
Goulding, M. D., Chalepakis, G., Deutsch, U., Erselius, J. R. and Gruss,
P. (1991). Pax-3, a novel murine DNA binding protein expressed during
early neurogenesis. EMBO J. 10, 1135-1147.
Goulding, M. D., Lumsden, A. and Gruss, P. (1993). Signals from the
notochord and floor plate regulate the region-specific expression of two Pax
genes in the developing spinal cord. Development 117, 1001-1016.
Harland, R. M. (1991). In situ hybridization: an improved whole-mount
method for Xenopus embryos. Methods Cell Biol. 36, 685-695.
Hartenstein, V. (1989). Early neurogenesis in Xenopus: the spatio-temporal
pattern of proliferation and cell lineages in the embryonic spinal cord.
Neuron 3, 399-411.
Holland, N. D., Panganiban, G., Henyey, E. L. and Holland, L. Z. (1996).
Sequence and developmental expression of AmphiDll, an amphioxus Distalless gene transcribed in the ectoderm, epidermis and nervous system:
insights into evolution of craniate forebrain and neural crest. Development
122, 2911-2920.
Hopman, A. H. N., Ramaekers, F. C. S. and Speel, E. J. M. (1998). Rapid
synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled
tyramides and their application for in situ hybridization using CARD
amplification. J. Histochem. Cytochem. 46, 771-777.
Jacobs, W., Dhaene, K. and Van Marck, E. (1998). Tyramine-amplified
immunohistochemical testing using ‘homemade’ biotinylated tyramine is
highly sensitive and cost-effective [see comments]. Arch. Pathol. Lab. Med.
122, 642-643.
Jacobson, A. G. (1981). Morphogenesis of the neural plate and tube. In
Morphogenesis and Pattern Formation, (ed. T. G. Connelly, L. L. Brinkley
and B. M. Carlson), pp. 233-263. New York: Raven Press.
Jacobson, A. G. and Gordon, R. (1976). Changes in the shape of the
4556 L. A. Davidson and R. E. Keller
developing vertebrate nervous system analyzed experimentally,
mathematically, and by computer simulation. J. Exp. Zool. 197, 191-246.
Jamrich, M., Sargent, T. D. and Dawid, I. B. (1987). Cell-type-specific
expression of epidermal cytokeratin genes during gastrulation of Xenopus
laevis. Genes Dev. 1, 124-132.
Jonas, E., Sargent, T. D. and Dawid, I. B. (1985). Epidermal keratin gene
expressed in embryos of Xenopus laevis. Proc. Natl. Acad. Sci. USA 82,
5413-5417.
Kay, B. K. and Peng, H. B. (1991). Xenopus laevis: Practical Uses in Cell
and Molecular Biology. New York: Academic Press.
Keller, R., Shih, J. and Sater, A. (1992a). The cellular basis of the
convergence and extension of the Xenopus neural plate. Dev. Dyn. 193, 199217.
Keller, R., Shih, J. and Sater, A. (1992b). The cellular basis of the
convergence and extension of the Xenopus neural plate. Dev. Dyn. 193, 199217.
Keller, R. and Winklbauer, R. (1992). Cellular basis of amphibian
gastrulation. Current Topics In Developmental Biology, Vol. 27. Xii+397p.
(ed. R. A. Pedersen). pp. 39-89. San Diego, California, USA; London,
England: Academic Press Inc.
Kerstens, H. M., Poddighe, P. J. and Hanselaar, A. G. (1995). A novel in
situ hybridization signal amplification method based on the deposition of
biotinylated tyramine. J. Histochem. Cytochem. 43, 347-352.
Kimmel, C. B., Warga, R. M. and Kane, D. A. (1994). Cell cycles and clonal
strings during formation of the zebrafish central nervous system.
Development 120, 265-276.
Knecht, A. K. and Harland, R. M. (1997). Mechanisms of dorsal-ventral
patterning in noggin- induced neural tissue. Development 124, 2477-2488.
Krotoski, D. M., Fraser, S. E. and Bronner-Fraser, M. (1988). Mapping of
neural crest pathways in Xenopus laevis using inter- and intra-specific cell
markers. Dev. Biol. 127, 119-132.
Lawson, A. and England, M. A. (1998). Neural fold fusion in the cranial
region of the chick embryo. Dev. Dyn. 212, 473-481.
Nieuwkoop, P. D. and Faber, J. (1967). Normal Tables of Xenopus laevis
(Daudin). Amsterdam: Elsevier North-Holland Biomedical Press.
Papan, C. and Campos-Ortega, J. A. (1994). On the formation of the neural
keel and neural tube in the zebrafish Danio (Brachydanio) rerio. Roux’s
Arch. Dev. Biol. 203, 178-186.
Poznanski, A., Minsuk, S., Stathopoulos, D. and Keller, R. (1997).
Epithelial cell wedging and neural trough formation are induced planarly in
Xenopus, without persistent vertical interactions with mesoderm. Dev. Biol.
189, 256-269.
Sausedo, R. A. and Schoenwolf, G. C. (1994). Quantitative analyses of cell
behaviors underlying notochord formation and extension in mouse embryos.
Anat. Rec. 239, 103-112.
Schoenwolf, G. C. (1984). Histological and ultrastructural studies of
secondary neurulation in mouse embryos. Am. J. Anat. 169, 361-376.
Schoenwolf, G. C. and Alvarez, I. S. (1989). Roles of neuroepithelial cell
rearrangement and division in shaping of the avian neural plate.
Development 106, 427-439.
Schoenwolf, G. C. and Delongo, J. (1980). Ultrastructure of secondary
neurulation in the chick embryo. Am. J. Anat. 158, 43-63.
Schoenwolf, G. C. and Smith, J. L. (1990a). Mechanisms of neurulation:
traditional viewpoint and recent advances. Development 109, 243-270.
Schoenwolf, G. C. and Smith, J. L. (1990b). Mechanisms of neurulation:
traditional viewpoint and recent advances. Development 109, 243-270.
Schroeder, T. E. (1970). Neurulation in Xenopus laevis. An analysis and
model based upon light and electron microscopy. J. Embryol. Exp. Morphol.
23, 427-462.
Schroeder, T. E. (1971). Mechanisms of morphogenesis: the embryonic neural
tube. Int. J. Neurosci. 2, 183-197.
Smith, J. L. and Schoenwolf, G. C. (1997). Neurulation: coming to closure.
Trends Neurosci. 20, 510-517.
Smith, J. L., Schoenwolf, G. C. and Quan, J. (1994). Quantitative analyses
of neuroepithelial cell shapes during bending of the mouse neural plate. J.
Comp. Neurol. 342, 144-151.
Turner, D. L. and Weintraub, H. (1994). Expression of achaete-scute
homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate.
Genes Dev. 8, 1434-1447.
Van Straaten, H. W., Janssen, H. C., Peeters, M. C., Copp, A. J. and
Hekking, J. W. (1996). Neural tube closure in the chick embryo is
multiphasic. Dev. Dyn. 207, 309-318.
Zimmerman, K., Shih, J., Bars, J., Collazo, A. and Anderson, D. J. (1993).
XASH-3, a novel Xenopus achaete-scute homolog, provides an early marker
of planar neural induction and position along the mediolateral axis of the
neural plate. Development 119, 221-232.