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Morphological and mapping studies of the paranodal and postnodal levels of the neural plate during chick neurulation.

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THE ANATOMICAL RECORD 233:281-290 (1992)
Morphological and Mapping Studies of the Paranodal and Postnodal
Levels of the Neural Plate During Chick Neurulation
GARY C . SCHOENWOLF
Department of Anatomy, University of Utah, School of Medicine, Salt Lake City,
Utah 84132
ABSTRACT
The morphology of the paranodal and postnodal levels of the neural plate as well as the fate of its cells was examined in chick embryos at stages
3-11. The morphology of the paranodal and postnodal levels of the neural plate
closely resembles that of the prenodal neural plate. Furthermore, during shaping
and bending of the neural plate, these levels undergo changes similar to those of
the prenodal level. In short, the paranodal and postnodal levels of the neural plate
consist of a pseudostratified columnar epithelium that thickens dorsoventrally and
narrows mediolaterally and then undergoes localized furrowing and folding. Fate
mapping revealed that a t mid-neurula stages, the prospective hindbrain and spinal cord levels of the neuraxis flank the primitive streak. Hensen’s node moves
caudally with respect to these future neuraxial levels as it regresses during the
latter stages of gastrulation. Cells of the medullary cord, the rudiment of the
secondary portion of the neural tube, arise in the vicinity of the cranial portion of
the primitive streak, near the caudal end of the postnodal levels of the neural
plate. Thus, during stages of gastrulation and primary neurulation, the precursor
cells of the primary and secondary portions of the neural tube (spinal cord) lie in
close proximity to one another. This study provides new information on the morphology and extent of the paranodal and postnodal levels of the neural plate, the
changes these areas undergo during shaping and bending of the neural plate, and
the contributions of its cells to the primary and secondary levels of the neural tube,
increasing our understanding of the complex events underlying avian gastrulation
and neurulation. o 1992 Wiley-Liss, Inc.
The early neural plate of avian embryos consists of a
pseudostratified columnar epithelium, which is first
detectable near the time of primitive streak formation
(e.g., see reviews by Schoenwolf and Smith, 1990a;
Schoenwolf and Alvarez, 1992). Accordingly, neurulation begins while gastrulation is still underway, and
the subsequent events of neurulation, namely shaping
and bending of the neural plate, occur in close spatial
and temporal association with regression of Hensen’s
node-the caudally directed movement of the cranial
end of the primitive streak. Based on the proximity of
the early neural plate and Hensen’s node, three levels
of the neural plate can be distinguished: a single prenodal level spanning the midline cranial to Hensen’s
node (usually referred to as the neural plate), paired
paranodal levels lying directly lateral to Hensen’s
node, and paired postnodal levels lying caudal to Hensen’s node and lateral to the more caudal levels of the
primitive streak.
Most previous studies on the avian neural plate have
focused primarily on its prenodal level (e.g., Bancroft
and Bellairs, 1974, 1975; Nagele and Lee, 1979,
1980a,b; Schoenwolf, 1982,1983,1985). Consequently,
much has been learned about the prenodal level, but
little is known about the paranodal and postnodal levels. The contributions of these areas to the central nervous system have been largely overlooked by previous
0 1992 WILEY-LISS, INC.
studies, mainly because of the belief that most of the
epiblast flanking the streak ingresses into the interior
to form mesoderm and endoderm, rather than remaining on the surface as ectoderm. Recent studies suggest,
in contrast, that a considerable extent of the primitive
streak is flanked by neural plate (Schoenwolf et al.,
1989; Schoenwolf and Sheard, 19901, and that less ingression occurs through the cranial part of the streak
than formerly believed (Schoenwolf et al., 1992).
The purpose of this study was to characterize the
morphological features of the paranodal and postnodal
levels of the avian blastoderm throughout the period of
neurulation, as well as to determine what changes occur at these levels during shaping and bending of the
neural plate. In addition, a fate mapping technique has
been used to determine what future axial levels of the
neural plate are comprised by the paranodal and postnodal levels a t progressively more advanced stages of
neurulation. The results of this study provide important new baseline data on the changing structure and
distribution of the caudal neural plate and on the fate
of the cells flanking the cranial part of the primitive
streak during its regression, increasing our under-
Received October 7, 1991; accepted December 2, 1991
282
G.C. SCHOENWOLF
standing of the complex events underlying gastrulation and neurulation in avian embryos.
MATERIALS AND METHODS
Morphological Studies
Fertile White Leghorn chicken eggs were incubated
in forced-draft, humidified incubators a t 38°C until embryos reached stages 3-11 (Hamburger and Hamilton,
1951). Blastoderms were removed from the yolks and
vitelline membranes, washed with saline (123 mM sodium chloride), and fixed for 2 hours at room temperature with 2% glutaraldehyde, 2% paraformaldehyde
in 0.1 M sodium cacodylate buffer containing 0.05%
calcium chloride and 0.1 M sucrose at pH 7.2. After
washing with buffer, blastoderms were postfixed for 1
hour with 1%osmium tetroxide (in cacodylate buffer),
dehydrated with ascending concentrations of ethanol,
cleared with propylene oxide, and embedded in an
Epon-Araldite mixture (Kushida, 1971). The entire extent of the paranodal and postnodal levels of each blastoderm (i.e., the level immediately flanking Hensen’s
node and the level caudal to Hensen’s node, respectively) were serially sectioned transversely at 5 pm
and mounted onto glass slides as described previously
(Schoenwolf and Chandler, 1983). Unstained sections
from 39 embryos were subsequently examined with differential interference contrast (DIC) optics after extraction of the plastic (Schoenwolf and Chandler, 1983)
and mounting of coverslips. Five-micrometer sections
were used rather than thinner sections to make it practical to serially section a large number of specimens.
For morphological studies, plastic sections were used
instead of paraffin sections to avoid the distortion of
the neural plate that is inherent with the latter method
(Schoenwolf and Franks, 1984).
fate Mapping Studies
Blastoderms containing embryos at stages 6-8 were
removed from the yolks and vitelline membranes,
washed with saline, and cultured dorsal-side-up in
Spratt culture (Spratt, 1955, modified as described by
Schoenwolf, 1988). The epiblast of each embryo was
microinjected a t one of three sites (Fig. l),1 paranodal
(site 1) and 2 postnodal (sites 2 and 31, with rhodamineconjugated horseradish peroxidase (R-HRP). Methods
were similar to those described previously in which the
prenodal, paranodal, and postnodal levels of the neural
plate (and adjacent areas of the epiblast) of embryos at
stages 3-5 were microinjected (Schoenwolf and
Sheard, 1989,1990). Briefly, about 1 nl of R-HRP was
injected with the aid of a micropipette (tip 0.d. of approximately 10 Fm) mounted on a Narishige hydraulic
micromanipulator and attached to a Picospritzer I1
(General Value Corp., Fairfield, New Jersey). The injection quality was assessed immediately after the injection by viewing blastoderms with a Nikon epifluorescence microscope. Cultures were returned to the
incubator for an additional 24 hours, after which embryos were washed in saline and fixed for 2 hours a t
room temperature with 1% glutaraldehyde in 0.1 M
phosphate buffer at pH 7.2. They were then rinsed with
the same buffer, placed on 0.125% diaminobenzedine
tetrahydrochloride dihydrate (DAB) in buffer for 1/2
hour, transferred to DAB containing hydrogen peroxide (0.075%)for 1/2 hour, washed with buffer, dehy-
Fig. 1. Diagram showing the three injection sites, one paranodal (1,
lateral to the center of Hensen’s node) and two postnodal (2, 250 pm
caudal to the center of Hensen’s node; 3, 500 pm caudal to the center
of Hensen’s node), at stage 6. The relative position of the three sites
with respect to Hensen’snode was maintained at stages 7 and 8. At all
stages, each site was located 250 pm lateral to the midline on the
right side of the embryo and near the expected neural plateisurface
epithelium juncture (long horseshoe-shapedcurvature).
drated with ethanol, cleared with Histo-Clear, and embedded in Paraplast X-tra. Seven-micrometer serial
transverse sections were cut from blocks and viewed
unstained with DIC optics. Thick sections were used to
increase the likelihood that whole labeled cells would
be viewed in single sections, facilitating their detection. Serial sections from 43 microinjected embryos
were subsequently studied.
RESULTS
Morphological Characteristicsof the Paranodal and
Postnodal Levels of the Neural Plate
Examples of the morphology of the paranodal and
postnodal levels of the neural plate during their shaping and bending are shown in Figures 2-5. The neural
plate at these levels has a structure identical to that
described previously for the prenodal neural plate
(Schoenwolf, 19851, and changes similar to those that
occur in the prenodal neural plate occur in their structure during shaping and bending. The paranodal and
postnodal levels of the neural plate, like the prenodal
level, is not well delineated from the surrounding epiblast at stages 3-6 (Fig. 2). Near the level of Hensen’s
node, the prospective neural plate consists of high columnar cells arranged in a pseudostratified epithelium
(Fig. 5A) with apically positioned mitotic figures, and
the dorsoventral (apicobasal) thickness of the epithelium gradually tapers laterally where it merges without a distinct boundary with the flattened epiblast constituting the prospective surface epithelium (Fig. 2A).
At the level of Hensen’s node, the thickened zone of
epiblast extends about 600 pm bilateral from the midline and has a thickness of 30-35 pm. As sections are
PARANODAL AND POSTNODAL NEURAL PLATE
Fig. 2. Plastic transverse sections (5 pm) of a single chick embryo at
stage 6 viewed with differential interference contrast (DIC) optics.
The approximate level of Hensen’s node is shown in A; B, about 250
pm caudal to A, C, about 500 pm caudal to A, D, about 750 pm caudal
to A; E, about 2 mm caudal to A and just caudal to the primitive
streak. Each section shows the midline and one side of the epiblast,
283
with the exception of E, which is centered on the midline. e, endoderm; m, mesoderm; np, prospective neural plate; ps, primitive streak;
se, prospective surface epithelium; filled arrow, approximate location
of site 1 injections; open arrow, approximate location of site 2 injections; arrowhead, approximate locate of site 3 injections. Bar = 40
pm, and applies t o A-E.
traced caudally, the thickness of the epiblast also is between sections E and F). This distance has been reseen to taper, and just caudal to the primitive streak, duced from that of younger stages by the regression of
the thickened epiblast merges imperceptibly with the Hensen’s node and the concomitant shortening of the
most caudal epiblast constituting the prospective sur- primitive streak; hence, what was designated a t
face epithelium (Fig. 2B-E). The thickened epiblast younger stages as the paranodal neural plate and the
can be traced for a distance of about 2 mm caudal to cranial levels of the postnodal neural plate has now
Hensen’s node (i.e., it ends between sections D and El. become prenodal neural plate, and a portion of the forDuring stages 6-8, the paranodal and postnodal lev- merly postnodal neural plate has now become paraels of the neural plate undergo shaping (Figs. 3, 5B). nodal neural plate.
Bending of the paranodal and postnodal levels of the
Near the level of Hensen’s node, the prospective neural
plate narrows mediolaterally (transversely) and thick- neural plate is underway as early as stage 9 and conens dorsoventrally, owing to an increase in the height tinues until the posterior neuropore closes during
of its cells (cf. Figs. 2A and 3A; Fig. 5A and B). The stages 10 and 11 (Schoenwolf, 1979). Bending at these
condensed neural plate extends about 225 pm bilateral levels involves the elevation of the neural folds toward
from the midline and has a thickness of approximately the dorsal midline, with the axis of rotation of each
40 km. At more caudal levels, the thickness of the pro- lateral half of the neural plate being centered a t the
spective neural plate gradually tapers both laterally groove overlying the cranial part of the primitive
and caudally as it merges with the flattened cells con- streak (Fig. 4A). Thus, elevation of the neural folds in
stituting the prospective surface epithelium (Fig. 3B- this area begins prior to the formation of the correF). The thickened epiblast can be traced for a distance sponding level of the notochord. The condensed neural
of about 1.5 mm caudal to Hensen’s node (i.e., it ends plate extends about 125 pm bilateral from the midline
284
G.C. SCHOENWOLF
Fig. 3. Plastic transverse sections (5 pm) of a single chick embryo a t
stage 8 viewed with DIC optics. The approximate level of Hensen’s
node is shown in A, B, about 250 pm caudal to A; C. about 500 pm
caudal to A; D, about 750 pm to A; E, about 1 mm caudal to A; F,
about 1.5 mm caudal to A and just caudal to the primitive streak. np,
prospective neural plate; ps, primitive streak, se, prospective surface
epithelium; filled arrow, approximate location of site 1 injections;
open arrow, approximate location of site 2 injections (the location of
site 3 injections is out of the view shown in C). C-F are centered on the
midline. Bar = 40 pm, and applies to A-F.
and has a thickness of 50-55 km; well-defined neural
folds have formed (Fig. 4A).
As shown in previous studies, the neural plate
within the sinus rhomboidalis region (i.e., the dilated
area of neural groove just cranial to Hensen’s node, and
flanking Hensen’s node, at stages 8-10) furrows at
three characteristic sites: the midline overlying the notochord, and paired dorsolateral regions associated
with the neural folds (Schoenwolf and Franks, 1984).
These three furrows subsequently extend caudally into
the paranodal and postnodal levels as the posterior
neuropore closes (see Figs. 19,20 of Schoenwolf, 1982).
Additional shaping of caudal levels of the neural
plate occurs during their bending. Thus, these levels
continue to thicken dorsoventrally and narrow mediolaterally (cf. Figs. 3A and 4A; Fig. 5B and C). At more
caudal levels, the thickness of the prospective neural
plate tapers both laterally and caudally as it merges
with the flattened cells constituting the prospective
surface epithelium (Figs. 4B-F). The thickened epiblast can be traced caudally for a distance of about 1
mm caudal to Hensen’s node. This distance has been
reduced from that of younger stages by the continued
regression of Hensen’s node and the shortening of the
primitive streak.
Neuraxial Levels Formed From the Paranodal and
Posfnodal Levels of the Neural Plate
Paranodal injections (site 1)
Examples of injections a t the paranodal level (site 1;
Fig. 1)are shown in Figs. 6-8. At stages 6 and 7, these
injections resulted in labeled cells in the auditory placode (Fig. 6A), neural crest a t the level of the hindbrain
(Figs. 6B, 7) (with labeled cells sometimes distributed
bilaterally), surface epithelium overlying and immediately lateral to the hindbrain (Fig. 6C), dorsal portion
of the hindbrain (Fig. 71, and cranial levels of the spinal cord (the spinal cord is believed to begin at the level
of the 5th somite in the chick). At stage 8, such injections resulted in labeled cells in cranial levels of the
spinal cord (not shown) and in the surface epithelium
adjacent to the cranial levels of the spinal cord (Fig. 8).
In contrast to data obtained from fate-mapping studies
of younger stages (i.e., stages 3-51 in which labeled
cells extended throughout much of the craniocaudal
extent of the embryo during the subsequent 24 hours of
PARANODAL AND POSTNODAL NEURAL PLATE
Fig. 4. Plastic transverse sections (5 pm) of a single chick embryo at
stage 10 viewed with DIC optics. The approximate level of Hensen’s
node is shown in A; B, about 250 pm caudal to A; C, about 500 p,m
caudal to A D, about 750 pm caudal to A; E, about 1 mm caudal to A;
F, about 1.25 mm caudal to A and just caudal to the primitive streak.
np, prospective neural plate; ps, primitive streak, se, prospective sur-
285
face epithelium; filled arrow, approximate location of site 1; open
arrow, approximate location of site 2 (the location of site 3 is out of the
view shown in C; injections were not done at this stage owing to the
closeness of the condensed epiblast to the primitive streak). C-F are
centered on the midline. Bar = 40 pm, and applies to A-F.
incubation (Schoenwolf and Sheard, 1990), labeled cord formed from the tail bud during secondary neurucells from embryos injected a t stages 6-8 occupied rel- lation; e.g., see Schoenwolf and DeLongo, 1980), and
atively short craniocaudal extents 24 hours later. For cells in the adjacent surface epithelium (Fig. 10A).
example, labeled cells contributing to the neural tube Neural crest cells had not yet emigrated a t this level;
were confined to the hindbrain, to the cranial levels of presumably, some of the labeled cells in the roof of the
the spinal cord, or to an expanse that bridged the hind- spinal cord were prospective neural crest. As for site 1
brain-spinal cord interface.
injections, labeled cells from embryos injected at site 2
A total of thirteen embryos was sectioned after in- a t stages 6-8 occupied relatively short craniocaudal
jection a t site 1. Of these, 12 had labeled cells in the extents 24 hours later. For example, labeled cells conneural tube, 8 had labeled cells in the surface epithe- tributing to the neural tube were confined to the midlium, 2 had labeled cells in the auditory placodes, and and caudal levels of the spinal cord and to the medul7 had labeled cells in the neural crest. In 1 embryo, no lary cord.
labeled cells were detected.
A total of thirteen embryos was sectioned after injection a t site 2. Of these, 10 had labeled cells in the
Postnodal injections (sites 2 and 3)
neural tube and 7 had labeled cells in the surface
Examples of injections at the postnodal levels (sites 2 epithelium. In 3 embryos, no labeled cells were deand 3; Fig. 1) are shown in Figs. 9-11. These injections tected.
Injections at site 3 a t stages 6-8 labeled cells in the
labeled ectodermal structures at levels caudal to those
labeled by injections at paranodal areas. Injections at surface epithelium associated with caudal levels of the
site 2 a t stages 6-8 labeled cells in the mid- to caudal spinal cord (Fig. l l A ) , as well as cells in caudal levels
levels of the spinal cord (Figs. 9, lOB),including the of the spinal cord and the medullary cord at their level
medullary cord (Fig. 1OC) (i.e., the portion of the spinal of overlap (Fig. 11B). As for site 1 and 2 injections,
286
G.C. SCHOENWOLF
DISCUSSION
Morphological Studies
Fig. 5. Enlargements of the prospective neural plate from levels
shown in Figures 2A, 3A, and 4A (i.e., at stages 6,8, and 10, respectively). m, mesoderm subjacent to the prospective neural plate; asterisks, mitotic figures. Bar = 10 hm, and applies to A-C.
labeled cells from embryos injected at site 3 at stages
6-8 occupied relatively short craniocaudal extents 24
hours later. For example, labeled cells contributing to
the neural tube were confined to caudal levels of the
spinal cord, to the medullary cord, or to caudal levels of
the spinal cord and the medullary cord.
A total of 17 embryos was sectioned after injection at
site 3. Of these, 9 had labeled cells in the neural tube,
6 had labeled cells in the surface epithelium, and 3 had
labeled cells in the caudal endoderm and mesoderm. In
4 embryos, no labeled cells were detected.
The morphological studies provide new information
on the structure of the paranodal and postnodal levels
of the neural plate and reveal that coordinated changes
occur in the width and height of the neural plate at
these levels during shaping. The structure of the caudal levels of the neural plate and the changes they
undergo during shaping and bending closely mimic
those of the prenodal neural plate. The prenodal neural
plate consists of a pseudostratified columnar epithelium, whose cells exhibit interkinetic nuclear migration (reviewed by Watterson, 1965).During its shaping
and bending several events occur (Schoenwolf, 1982,
1983, 1985; Schoenwolf and Franks, 1984; Schoenwolf
and Smith, 1990b). During shaping, the prenodal neural plate (on the average) thickens dorsoventrally, narrows mediolaterally, and lengthens craniocadually.
Bending of the prenodal neural plate also involves multiple events. As shaping is underway, areas called
hinge points first appear. Each hinge point consists of
a circumscribed area of neurepithelium attached to underlying tissues. Three hinge points form: a median
hinge point (MHP) and paired dorsolateral hinge
points (DLHPs). The neurepithelial cells within each
hinge point change their shape from spindle-like to
wedge-like, and the neurepithelium concomitantly furrows, forming a shallow longitudinal groove. Folding of
the neural plate involves its rotation around the furrows. The neural folds elevate dorsally during folding,
rotating around the MHP, and then turn inwardly (medially), with each rotating around its corresponding
DLHP. The DLHPs do not form at all levels of the
neuraxis: most of the extent of the future spinal cord
(with the exception of the sinus rhomboidalis region)
lacks DLHPs. Consequently, elevation of the neural
folds to the dorsal midline at the future spinal cord
level results in the midline apposition of the neural
folds and of the apical sides of the two lateral walls of
the incipient neural tube, thereby transiently obliterating the neurocele at this level (i.e., the neurocele
becomes occluded; Schoenwolf and Desmond, 1984).
The results of the present study demonstrate that
each half of the neural plate at paranodal and postnodal levels has a structure identical to that of the
prenodal level, and that similar changes occur during
shaping and bending. The paranodal and postnodal levels of the neural plate consist of thickened epiblast constituting a pseudostratified epithelium. The presence
of apical mitotic figures suggests that interkinetic nuclear migration occurs at these levels as it does at the
prenodal level. Furthermore, the present study shows
that the paranodal and postnodal levels of the neural
plate undergo shaping and, particularly, dorsoventral
thickening (owing to increase in cell height) and concomitant mediolateral narrowing. Finally, the paranodal and postnodal levels of the neural plate exhibit
both furrowing and folding during bending. Furrowing
occurs in the midline, overlying the primitive streak,
and dorsolaterally, as the DLHPs extend caudally into
the area of the closing posterior neuropore. Folding occurs in relation to the furrows, such that the neural
plate rotates around each of these areas. Collectively,
these morphological similarities suggest that the pre-
PARANODAL AND POSTNODAL NEURAL PLATE
Fig. 6 . Paraffin transverse sections (7 pm) of a single chick embryo
whose epiblast was microinjected with rhodamine-conjugated horseradish peroxidase (R-HRP) at site 1 (i.e., paranodally), 24 hours earlier when the embryo was at stage 6. Labeled cells occupy the auditory
placode (ap), neural crest (nc), surface epithelium be), and dorsal
neural tube (labeled cells not shown) at the hindbrain (hb) level. Sections are lettered in cranial to caudal sequence. Bar = 20 km, and
applies to A-C.
287
hours earlier when the embryo was at stage 6. Labeled cells occupy
the dorsal hindbrain (hb) and associated neural crest (nc).Bar = 20
pm.
Fig. 8. Paraffin transverse section (7 pm) of a chick embryo whose
epiblast was microinjected with R-HRP at site 1 (i.e., paranodally),
24 hours earlier when the embryo was at stage 8. Labeled cells occupy the surface epithelium (se) at the spinal cord (sc) level. Bar =
20 pm.
Fig. 7. Paraffin transverse section (7 pm) of a chick embryo whose
epiblast was microinjected with R-HRP at site 1 (i.e., paranodally), 24
nodal, paranodal, and postnodal levels of the neural
plate merely represent different regions of a structure
with uniform characteristics through its entire extent.
Thus, all craniocaudal levels of the neural plate seem
to be structurally equivalent.
Aside from providing baseline data important for understanding gastrulation and neurulation, these stud-
ies on the structure of the caudal levels of the neural
plate provide a necessary morphological context in
which to interpret fate mapping
_ _
- studies.
Fate Mapping Studies
The fate mapping studies provide new information
on the fate of cells in the epiblast, and they reveal that
288
G.C. SCHOENWOLF
Fig. 9. Paraffin transverse section (7 pm) of a chick embryo whose
epiblast was microinjected with R-HRP at site 2 (i.e., postnodally), 24
hours earlier when the embryo was at stage 7. Labeled cells occupy
the dorsal spinal cord (sc). Bar = 20 pm.
neurulation overlap zone. At the level of overlap between the caudal
spinal cord and medullary cord, labeled cells contain small packets of
R-HRP (arrows),rather than being fully filled. Sections are lettered in
cranial to caudal sequence. Bar = 20 pm, and applies to A-C.
Fig, 10.Paraffin transverse sections (7 pm) of a single chick embryo
whose epiblast was microinjected with R-HRP at site 2 (i.e., postnodally), 24 hours earlier when the embryo was at stage 8. Labeled cells
occupy the caudal surface epithelium be), spinal cord (sc), and dorsal
part of the medullary cord (mc; the rudiment of the secondary neural
tube, the level of the neural tube formed from the tail bud) in the
Fig. 11. Paraffin transverse sections (7 pm) of a single chick embryo
whose epiblast was microinjected with R-HRP a t site 3 (i.e., postnodally), 24 hours earlier when the embryo was at stage 6. Labeled cells
occupy the caudal surface epithelium (se) and spinal cord (sc). mc,
medullary cord; arrows, packets of R-HRP. Sections are lettered in
cranial to caudal sequence. Bar = 20 pm, and applies to A and B.
changes occur over time in the disposition of groups of
epiblast cells in relation to Hensen’s node. The extent
of the prospective neural plate in the avian epiblast has
been a matter of controversy for over 50 years (see
Discussions in Spratt, 1955; Schoenwolf et al., 1989;
Schoenwolf and Sheard, 1990). The present results
show that at stages 6-8, the caudal end of the neural
plate lies at about 500 pm caudal to the center of Hensen’s node (that is, at all three stages, site 3 injections
typically labeled cells contributing to the most caudal
levels of the neural tube). Moreover, these results reveal that future hindbrain levels of the neuraxis (and
PARANODAL AND POSTNODAL NEURAL PLATE
sometimes cranial levels of the spinal cord as well) lie
directly lateral to Hensen’s node (i.e., at site 1)at stage
6, and that, by stage 8, the node is flanked by prospective spinal cord. Little cell displacement occurs down
the length of the neuraxis during the subsequent 24
hours of development after labeling at stages 6-8, suggesting that cells are already allocated to specific craniocaudal subdivisions of the neuraxis by mid-neurula
stages. Thus, Hensen’s node moves caudally relative to
these structures as it undergoes its regression. Additionally, the fate mapping studies reveal that the secondary neural tube, the portion of the spinal cord derived from the medullary cord (a tail bud derivative) by
cavitation, arises about 250-500 pm caudal to the center of Hensen’s node and near the caudal end of the
postnodal levels of the neural plate (i.e., a t sites 2 and
3). Labeled cells in the medullary cord were marked
with isolated packets of HRP reaction product, rather
than being fully filled. A similar pattern of marking
was observed in a previous study of stage 3-5 blastoderms injected 750-1,000 pm caudal to the center of
Hensen’s node (Schoenwolfand Sheard, 1990). The reason for this pattern is unclear, but it may be related to
a faster cell division rate for caudal epiblast cells,
which might dilute and fragment the cytoplasmic HRP.
The morphology of the cells of the epiblast does not
conform directly a t stages of neural plate shaping and
bending with the ultimate fate of the corresponding
cells. For example, in contrast to one’s expectations,
relatively tall cells within the epiblast can eventually
contribute to surface epithelium, and relatively short
cells can contribute to the neural plate. This is evident
when the widths and lengths of the thickened epiblast
(see Figs. 2A, B, C; 3A, B) are compared with the fates
of cells at the three injection sites. Such comparison
reveals that the width and length of the thickened area
exceed those giving rise to neural plate (i.e., width:
thickened epiblast at site 1a t stages 6 and 7 [see filled
arrow: Fig. 2Al forms both neural plate and surface
epithelium, and thin epiblast a t site 1 at stage 8 [see
filled arrow: Fig. 3A] forms both neural plate and surface epithelium; length: the thickened epiblast at
stages 6 and 8 extends approximately 2 and 1.5 mm,
respectively, caudal to the center of Hensen’s node, and
injections placed at site 3 [500 pm caudal to the center
of Hensen’s node] label the caudal end of the neural
tube near the overlap zone). Thus, it is impossible to
draw a boundary to delimit the absolute transverse and
longitudinal extents of the neural plate at these stages;
much intermingling must occur along the mediolateral
and craniocaudal junctures. Nevertheless, it is clear
that the area lying caudal to the prenodal neural plate
and extending at least 250 km bilateral from the midline and 500 pm in length contributes cells to either
the primary or secondary level of the neural tube. This
fact establishes the important contributions of the
paranodal and postnodal levels of the epiblast to the
development of the central nervous system, and emphasizes the need to understand the changes they undergo during neurulation.
CONCLUSIONS
In conclusion, the results of this study reveal that the
paranodal and postnodal levels of the neural plate have
structures identical to that of the prenodal level of the
289
neural plate, and that caudal levels of the plate undergo shaping and bending in a manner closely resembling that of the prenodal level of the neural plate.
Moreover, by stage 6, ectodermal cells have become
restricted in their previous ability to rearrange extensively and to move down the length of the epithelial
sheet. Thus, the positions of the future hindbrain and
cranial, mid-, and caudal levels of the spinal cord in
relationship to Hensen’s node are spatially established
by mid-neurulation stages. Finally, the labeling of
medullary cord cells after injection a t sites 2 and 3,
suggests that the secondary portion of the neural tube
originates in close proximity to the prospective neurepithelial cells of the neural plate (i.e., the precursor of
the primary neural tube). Collectively, these results
provide a framework for interpreting future experimental studies on the paranodal and postnodal levels of
the neural plate.
ACKNOWLEDGMENTS
I acknowledge the superb technical assistance of
Nancy B. Chandler and Jodi L. Smith and the secretarial assistance of Jennifer Parsons. The research was
supported by grant NS 18112 from the National Institutes of Health.
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