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DEVELOPMENTAL DYNAMICS 207:30%318
(1996)
Neural Tube Closure in the Chick Embryo Is Multiphasic
HENNY W.M. VAN STRAATEN, HENNIE C.J.P. JANSSEN, MARIAN C.E. PEETERS, ANDREW J. COPP, AND
JOWAN W.M. HEKKING
Department AnatornylErnbryology, University of Limburg, Maastricht, Netherlands (H.W.M.V.S.,H.C.J.P.J., M.CE P.,
J.W.M.H.);Division of Cell and Molecular Biology, Institute of Child Health, University of London, London W C l N LEH,
United Kingdom (A.J.C.)
ABSTRACT
Progression of neurulation in
the chick embryo has not been well documented.
To provide a detailed description, chick embryos
were stained in ovo after the least manipulation
possible to avoid distortion of the neural plate and
folds. This allowed a morphological and morphometric description of the process of neurulation in
relatively undisturbed chick embryos. Neurulation comprises several specific phases with distinct closure patterns and closure rates. The first
closure event occurs, de novo, in the future mesencephalon at the 4-6 somite stage (sst 4-6). Soon
afterwards, at sst 8-7, de novo closure is seen at
the rhombocervical level in the form of multisite
contacts of the neural folds. These contacts occur
in register with the somites, suggesting that the
somites may play a role in forcing elevation and
apposition of the neural folds. The mesencephalic
and rhombocervical closure events define an intervening rhombencephalic neuropore, which is
present for a brief period before it closes. The remaining pear-shaped posterior neuropore (PNP)
narrows and displaces caudally, but its length remains constant in embryos with seven to ten
somites, indicating that the caudal extension of
the rhombocervical closure point and elongation
of the caudal neural plate are keeping pace with
each other. From sst 10 onward, the tapered cranial portion of the PNP closes fast in a zipper-like
manner, and, subsequently, the wide caudal portion of the PNP closes rapidly as a result of the
parallel alignment of its folds, with numerous button-like temporary contact points. A role for convergent extension in this closure event is suggested. The final remnant of the PNP closes at sst
18. Thus, as in mammals, chick neurulation involves multisite closure and probably results from
several different development mechanisms at
varying levels of the body axis.
0
1996 WtIey-Lisa, Ine.
Key words: Chick, Embryo,Neurulation, Closure
rate, Neuropore, Morphology, Morphometry
INTRODUCTION
Neurulation, the process of neural tube formation,
involves the concerted action of a variety of intrinsic
0
1996 WILEY-LISS, INC.
and extrinsic forces, resulting in the complex shaping
and folding of the neural plate and closure of the neural
tube. In view of the complexity of the process, it is not
surprising that the underlying mechanisms are only
partially understood. Work with amphibian and chick
embryos has defined the principal morphogenetic
events that comprise neurulation (for review, see Karfunkel, 1974; Schoenwolf, 1982, 1994; Gordon, 1985;
Copp et al., 1990; Schoenwolf and Smith, 1990; Jacobson, 1991). The neural plate extends longitudinally,
narrows transversely, and thickens apicobasally during the process of shaping. This elongation appears to
result mainly from cell rearrangement and directed
cell proliferation. Simultaneously, the lateral borders
of the neural plate elevate, and the arising neural folds
converge to the midline. The mechanisms of elevation
and convergence have been suggested to include apical
constriction of neuroepithelial cells, the formation of
medial and dorsolateral hinge points, and extrinsic factors, such as expansion of underlying mesoderm and
the medial extension of the surface ectoderm. Finally,
the neural folds appose, adhere, and fuse in the midline, processes that are very poorly understood in
mechanistic terms.
Relatively little attention has been paid to the sequential events of neural tube closure along the body
axis in the chick embryo. Closure has been described as
proceeding in an orderly sequence from the initial closure site in both rostra1 and caudal directions, in a
zipper-like manner (Portch and Barson, 1974; Bancroft
and Bellairs, 1975; Silver and Kerns, 1978; Schoenwolf, 1982, 1985). However, a recent detailed study
using the high-definition microscope has identified closure occurring as separate de novo events a t mesencephalic and rhombencephalic levels (Jaskoll et al.,
1991). Indeed, multisite closure was suggested to occur
in the chick in an analogous manner to that described
in mammals, in which up to five different closure sites
have been identified (for review, see Golden and Chernoff, 1993).
High-magnification observations by Jaskoll et al.
(1991) suggest that neural tube closure may be a more
subtle process than previously recognized. ARer initial
Received March 11, 1996;accepted June 7, 1996.
Address reprint requestdcorrespondence to H.W.M. Van Straaten,
Department AnatomylEmbryology,University of Lirnburg, P.O.Box
616,6200 MD Maastricht, Netherlands.
310
VAN STRAATEN ET AL.
Fig. 1. A: Visualization of a chick embryo with seven somites after
staining with toluidine blue via the vitelline membrane. 6: The shape and
size of the neuropores of the embryo in dorsal view following careful
removal of the vitelline membrane. Structures that were in contact with
the vitelline membrane (e.g., the neural folds) are stained most intensely,
whereas deeper structures like floor plate area (f), the primitive streak
(ps), and the contacts between the neural folds are not stained. Somites
1 and 7 are indicated by arrows. Bulgings of the neural folds or fold
contacts are seen in register with the somite pairs. A rhombencephalic
neuropore (RNP) is present; its neural folds and the adjacent ectodem
are stained only lightly due to their deep location. Caudally, a pearshaped posterior neuropore (PNP) is present. Note that few morphological differences exist between the embryo before (A) and after (6) removal of the vitelline membrane. The length is reduced by 4%, and the
RNP is slightly widened. Thus, the preparation method used is relatively
free from attifacts. ANP: anterior neuropore; H: Hensen’s node. Scale bar
= 250 pm.
using scanning electron microscopy. Using the highdefinition microscope we recently found (Van Straaten
et al., 1993b) that, during PNP closure, numerous button-like contacts arise, not only during fusion, but even
as early as stages of apposition and adhesion of the
neural folds. This finding suggests that a zipper-like
model of neural tube closure may be an oversimplification. In order to shed more light on this morphogenetic process, the present study was undertaken to document the sequence of de novo closure points, the
changes in shape, size, and position of the neuropores,
and the presence and extension of the various types of
neural fold contact. Care was taken to avoid manipulating the embryo during preparation because our previous study had shown that artefacts of PNP length
and width and reopening of de novo adhesion sites can
easily be introduced. The detailed description reveals
that closure in the chick embryo is multiphasic and
based on distinct closure patterns at different levels of
the body axis.
Fig. 1 (legend in facing column).
contact between the mesencephalic folds, apposition occurred as an imperfect “zipping up,” in which several
nonfused sites were interspersed with fused sites (Jaskoll et al., 1991).Bancroft and Bellairs (1975) were the
first to mention such focal fusion sites, which they observed during closure of the posterior neuropore (PNP)
RESULTS
A total of 117 embryos between somite stages 4 and
18 (sst 4-18) were used to gather data on several morphological and morphometric parameters of neurulation in the chick. The embryos were studied in the dorsal view with the least possible manipulation (Fig. 1)in
order to preserve the natural morphology and sequence
of neurulation events. A representative series of embryos is shown in Figure 2, and the morphometric data
on neuraxis elongation, neural fold contacts, and neuropore size are presented in Figures 3-6. The morphometric data, combined with the drawings of the changing shape of neuropores and neural folds, are
summarized in a general schematic representation of
neural tube closure (Fig. 7). This drawing reveals a
multiphasic pattern of neurulation in the chick embryo.
Mesencephalic Closure
At the earliest stage studied (sst 4), the mesencephalic neural folds are in contact over a short distance.
311
MULTIPHASIC NEURAL TUBE CLOSURE
length (pm)
7000
0
6500
0
6000
5500
5000
4500
4000
3500
0
0
3000
0
2500
2000
2
4
6
8
10
12
development (somites)
14
16
18
20
Fig. 3 Elongation of the neuraxis during neurulation in the chick embryo. Neuraxial length was measured between the anterior extremity of
the embryo and the caudal extremity oi the PNP, as defined in Figure 8.
The neuraxis elongates progressively, but its data are described better by
a polynomial distribution than by a linear regression. The polynomial
indicates a relatively enhanced rate of elongation between sornite stages
(sst) 7 and 11 and delay between sst 11 and 14.
Fig. 2 Changes in the shape of the neuropores during neurulation.
Chick embryos were stained with toluidine blue via the vitelline membrane, which was removed subsequently for clarity of observation in this
figure. The numbers of somites are indicated. Arrowheads indicate the
caudal extremity of the neuraxis, as defined in Figure 8. A,B: Mesencephalic contact (rn) progresses, and the PNP is narrowed at the rhombocervical level (r). C,D The PNP is pear-shaped with tapered (t) and
wide (w) portions. The PNP narrows progressively but retains its length.
E: The tapered portion of the PNP is almost closed. F: The wide portion
of the PNP is closing, and its neural folds are oriented in parallel. Scale
bar = 400 pm.
Anterior to this closure site is the anterior neuropore
(ANP). Caudally, a large posterior neuropore (PW) is
present, which is narrowed a t the level of the somites
(Fig. 2A). The mesencephalic contact extends in both
rostra1 and caudal directions and, a t this stage, comprises apposition and adhesion but not fusion of the
neural folds (for definitions of these terms, see Experimental Procedures). Fusion apparently lags behind,
because it could be detected for the first time only in sst
6 embryos (Fig. 4). The length of the ANP gradually
decreases to zero as the prosencephalic neural tube is
formed (Table 1, Fig. 5). The closure point of the PNP
progresses in a caudal direction (Fig. 7) at a rate (relative to somite 1) of about 200 pdsomite stage between sst 4 and 6.
Rhombocervical Closure
Multisite de novo closures at the rhombocervical
level occur a t sst 6-7. Preceding closure, the neural
folds show local bulges, which are in register with the
somites (Fig. 2B)and which result in a number of separate contact sites that are first visible at the level of
somites 3-4(‘%utton-like”closure; Fig. 1B). In between,
312
VAN STRAATEN ET AL.
length (pm)
7000
length (pm)
3500
'
I
0
r
6000
3000
I
5000
2500
-
.~ .
.
oPNP
I
- PNP, wide portion 1
+RNP
oANP
~
0
4000
0
2000
0
0
3000
1500
2000
0
1000
O
\
1000
+
0
0
0
0
a
a
\&a
500
0
0
2000
2
4
6
8
10
12
14
16
18
20
development (somites)
1000
0
2
4
6
8
10
12
14
16
18
20
development (somites)
Fig. 4 Length of neural fold contacts during neurulation in the chick
embryo. Data from apposition and adhesion were taken together, because distinction between them was not always clear. When a specific
contact was scattered throughout an embryo, the values were added.
Regression lines are drawn for clarity only. The average length of apposition and adhesion shows a roughly biphasic pattern, with relatively high
values at sst 7-8 and 13-14; at sst 9-10, both high and low values are
seen. Fusion is seen at sst 6 for the first time, and the length of fused
neural tube increases from that stage onward, although it lags behind that
of apposition and adhesion by about three somite stages. A marked
threefold increase in fusion occurs between sst 8 and 11. The rate of
increase slows up to sst 14 but increases afterwards. At sst 18, the neural
folds are in contact over their full length, but not all contacts are transformed into fusion yet. Each data point represents a single embryo, with
the exception of the solid circles, which represent data combined from 7
embryos (at sst 4), from 11 embryos (at sst 5),and from 13 embryos (at
sst 6).
Fig. 5 Reduction in length of neuropores during neurulation in the
chick embryo. Data for the three major neuropores, ANP, RNP, and PNP,
are shown. The ANP and PNP are present from sst 4 onward. The ANP
exhibits a gradual reduction of its length and no longer seen after sst 9.
The RNP is present briefly between sst 6 and 8. The PNP is the most
pronounced and long-lived neuropore. The length of the PNP during sst
6-7 also includes the lengths of the several small intermediate neuropores. The length of the wide portion of the PNP is plotted separately.
Two major length reductions occur during development, at sst 6-7 and at
sst 10-14, whereas, between sst 7 and 10, no reduction in PNP length is
seen. Variation between embryos at a single somite stage is limited. This
is especially evident from the close correlation between PNP length and
sst during the rhombocervicalclosure and the closure between sst 10 and
14. Each data point represents a single embryo and is included in the
regression lines (with the exception of the solid circles).
has a rhomboid shape, and a pear-shaped PNP (Fig.
lB), which has distinguishable tapered and wide portions (see Fig. 8). The RNP is transiently present for
1.5 somite stages on average, predominantly at sst 6-7
(Table 1, Fig. 5). The separation of the original PNP
explains its dramatic length reduction from 2,500 to
1,500 pm between sst 6 and 7 (Fig.5).
small neuropores arise temporarily. The rhombocervi- Narrowing and Caudal Shifting of the PNP
During sst 7-10, the width of the PNP (measured at
cal closure occurs fast: caudal progression of the PNP
closure point now occurs a t a rate of 1,200 pdsomite its wide portion) shrinks steadily (Fig. 61,but its length
stage between sst 6 and 7. This abrupt increase in con- remains constant, at about 800 pm for the wide portion
tact is predominantly based on apposition and adhesion and about 700 pm for the tapered portion (Figs. 2C,D,
(Fig. 4),whereas fusion again lags behind, because a 5). This means that the elongation rate of the neuraxis
marked increase in fusion length (from 1,000 to 3,000 and the rate of caudal progression of the PNP closure
pm) does not occur until sat 8-11 (Fig. 4). The rhomb- point both must be equal at about 300 pm/somite stage.
ocervical closure results in separation of the original Because the rate of somite gain is only 140 pdsomite
PNP into a rhombencephalic neuropore (RNP), which stage, the PNP as a whole is seen to be displaced in a
MULTIPHASIC NEURAL TUBE CLOSURE
width (pm)
700
600
1
I
!
ca
0
0
0
$
0
0
I
100
0
temporary button-like closure sites evident, mostly adhesion points. A small aperture remains.
The caudal progression of the PNP closure point is
rapid between sst 11 and 13, with a maximum rate of
480 pdsomite stage. This coincides with an increase
in apposition and adhesion (between sst 10 and 141,
whereas an increase of neural fold fusion occurs later,
between sst 14 and 16 (Fig. 4).
The rapid reduction in PNP length from sst 10 onward is shown dramatically in Figure 5, but the increase of closure rate (Fig. 7) during this period appears to be less dramatic: from 300 to 480 pdsec. This
is due to a simultaneous decrease in the rate of neuraxial elongation (Fig. 3).
I
4
500
200
313
-I
b
1
1
O
0
&
0
O
Q
)
@
o
0
Closure of the PNP Remnant
From sst 14 onward, the small remnant of the PNP
further narrows and shortens, concluding in its closure.
This opening appears to be localized mostly a t the caudal extremity of the PNP, because, from this aperture
onward, reopening was successful in the cranial direction but failed in the caudal direction. Apposition and
adhesion decrease, indicating that the apposed walls of
the previously tapered and wide portions of the PNP
are becoming fused. After sst 18, the PNP is invariably
closed.
I
2
4
6
8
10
12
14
16
18
20
development (somites)
Fig. 6 Reduction in width of the PNP during neurulation in the chick
embryo. The width was measured at the wide portion of the PNP. Note
that reduction of width of the PNP, as measured in the dorsal view, results
both from narrowing due to elevation and convergence and from reduction in width of the neural plate. A relatively uniform rate of width reduction
is seen up to sst 12 followed by a slow rate of reduction until the PNP
finally closes.
DISCUSSION
Possible Distortion of the Neural Plate During
Embryo Preparation
Descriptions of neurulation in the chick embryo have
been based mostly on studies in which the embryo was
manipulated to a greater or lesser extent, which could
caudal direction as development progresses. The most have resulted in distortion of the neural plate (Van
recently formed somite is passed by the PNP closure Straaten et al., 1993b). In the present study, we took
point at sst 8 (Fig. 7). The reduced rate of progression great care to avoid manipulating the embryos in order
of the PNP closure point (300 vs. 1,200 pdsomite stage to prevent artifactual distortion. It is possible that
at sst 7-10 vs. sst 6-7) is reflected in a reduction of opening the egg and immersion of embryos in saline
apposition and adhesion lengths until sst 11 (Fig. 4). can introduce artefacts, but our experience has shown
Button-like adhesion and fusion points, as observed a t that even pushing on the vitelline membrane does not
the rhombocervical closure, are present in register deform the embryo; therefore, we assume that distorwith the somites during caudal progression of the PNP tion is unlikely a t this stage of preparation. The viclosure point.
telline membrane probably acts as a protective cover
for the embryo against mechanical forces. On several
Closure of the PNP
occasions, removal of the vitelline membrane did cause
A second major phase of reduction in PNP length distortion of the embryo, but these artefacts could be
(from 1,500to 165 pm) occurs between sst 10 and 14 recognized and the embryos omitted from the analysis.
(Fig. 5). While the PNP narrows, the neural folds of the We assume that the description of the chick embryo
more rostra1 tapered portion appose (Fig. 2D) and sub- given in this paper reflects naturally occurring events
sequently close over their full length between sst 10 of neural plate morphogenesis during neurulation.
and 12 (Fig. 2E). This causes the length of the PNP to
reduce from 1,500 to 800 p m (Fig. 5 ) ; relatively few Multiphasic Neural Tube Closure
We found a multiphasic pattern of closure of the PNP
button-like contact points are seen during this closure
event. The wide portion of the PNP does not change in with varying morphology of closure and varying rates
length until sst 12 (Fig. 5 ) , although its folds continue of neural tube closure progression a t specific locations
to approach the midline and become oriented almost along the body axis. This nonuniform pattern of neuparallel t o each other. Between sst 12 and 14, the folds rulation contrasts with the traditional view of a smooth
in this region close rapidly (Fig. 2F) with numerous progression of closure from the mesencephalic region
314
VAN STRAATEN ET AL.
0
1
neural tube:
....... open
c3closed
-
--
apposition
adhesion
fusion
somites
18
Fig. 7 Schematic representation of the entire neurulation process in
the chick embryo. The drawing is based on morphometrical data on
neuraxis elongation, neural fold contacts, and neuropore sizes and also
incorporates morphological data with respect to the shape of neuropores
and neural folds. Neuropore outlines are indicated by the thick lines.
Apposition, adhesion, and fusion of the neural folds are based on an
average impression of their location. Progression of the ANP and PNP
closure points are indicated by the curved border line between heavy and
light shading. This drawing reveals a multiphasic pattern of neural tube
closure with distinct phases, as seen at the mesencephalic level at sst 4,
at the rhornbocervical level (sst 6-7), and for the PNP in two steps (sst
10-14) and for its remnant (sst 14-18).
onward (Portch and Barson, 1974; Bancroft and Bellairs, 1975; Schoenwolf, 1979, 1982). Even the detailed
longitudinal study of Schoenwolf (1985) did not report
the enhanced rate of final PNP closure, although Figure 9 of that study does indicate a marked rate of PNP
closure between Hamburger and Hamilton (HH) stages
10 and 11(Hamburger and Hamilton, 1951). Jaskoll et
al. (1991) described independent closure events at the
mesencephalic and rhombocervical levels that appeared to result from different morphogenetic mechanisms. These findings have been confirmed and extended in the present study. In the following sections,
we discuss the mechanisms that may be involved pre-
dominantly in each phase of the neurulation process in
the chick embryo.
Possible Role for the Somites in
Rhombocervical Closure
The pattern of the rhombocervical closure suggests
that the somites may be involved in closure of the neural folds. Progression of neurulation appears enhanced
at the future rhombocervical level, as indicated by the
local narrowing of the PNP, and subsequent buttonlike contact points between the neural folds are in register with the somites. Both observations suggest that
somite expansion could be aiding in the dorsomedial
315
MULTIPHASIC NEURAL TUBE CLOSURE
TABLE 1. Presence of the Anterior Neuropore (ANP) and the Rhombencephalic
Neuropore (RNP) With Increasing Somite Numbers During Development
Presence of ANPI
no. of embryos
Percentage
4
616
100
5
6
10110
11112
7
516
119
100
92
83
Somites
8
9
016
10
016
closure
point
11
0
0
-
t
n
z
n
neural
fold
TI
f!
P
1
t
notochord
n
z
n
aa,
Hensen's
node
3
caudal
neuraxis
I
.
Prlm"'''n
.I,
streirn
1LIV-z
I
Bff
u
u
Fig. 8 Drawing of the PNP illustrating the definition of its length from
the cbsure point to the caudal extremity of the neuraxis and the arrangement of its wide and tapered portions.
movement of the neural walls and folds. Indeed,
Schroeder (1970) proposed that expansion of paraxial
mesoderm could play a role in neural plate morphogenesis in amphibian embryos. Several observations support this idea for rhombocervical closure in the chick.
First, the enhanced rhombocervical narrowing of the
PNP and subsequent closure initiates at the level of
somites 3-4, that is, midway along the row of 6-7
somites. This may indicate that the combined action of
several pairs of somites is necessary to force the neural
walls to elevate sufficiently for contact to be initiated.
Second, there is considerable supportive evidence to
validate the existence of the button-like, segmental closure pattern. This has been described in detail by Jas-
Presence of RNP/
no. of embryos
016
017
4115
618
1/14
017
016
Percentage
0
0
27
75
7
0
0
koll et al. (1991) and mentioned by other authors
(Gouda, 1974; Bancroft and Bellairs, 1975; Nagele and
Lee, 1987; Nagele et al., 1989). Furthermore, at later
stages of development, the neural tube exhibits evidence of morphological segments. These periodic undulations do not match a specific spatiotemporal pattern
of neuroepithelial proliferation and differentiation and
are regarded as being the result of mechanical moulding of the neuroepithelium by the somites (Lim et al.,
1991). Corresponding cell lineage restrictions within
the neural tube have been suggested to be imposed
secondarily by the somites (Stern et al., 1991). The
present study indicates that the periodic bulging of the
neural tube originates before neural tube closure and
supports the idea that this periodicity is imposed by the
somites.
Axial Curvature M a y Affect Closure of the
Rhombencephalic Neuropore
The independent closure events at mesencephalic
and rhombocervical levels lead to the formation of the
RNP. On many occasions, we observed that removal of
the vitelline membrane resulted in simultaneous upward lifting of the head and widening of the RNP (see
Fig. 11, suggesting that RNP closure delay may be
aided by progressive dorsal flexion of the axis. This is
similar to the mechanism proposed for the role of axial
curvature in closure of the caudal neural tube in both
chick and mouse embryos (Brook et al., 1991; Van
Straaten et al., 1993a; Peeters et al., 1996).
Closure of the PNP is Dependent on
Convergent Extension
Following the rapid rhombocervical closure, the PNP
closure point gradually progresses caudally beyond the
last somite formed until it is flanked by the presomitic
mesoderm. Clearly, closure factors other than the
somites must become gradually more important in
achieving closure. These could include convergent extension, apical constriction, and ectodermal expansion.
The process of convergent extension transforms the
initially short and broad caudal neural plate into an
elongated and slender structure. In amphibia, this reshaping is based mainly on directed cell rearrangement along the midline of the neural plate probably
driven by changes in the notoplate (Jacobson and Gardon, 1976; Jacobson, 1978, 1991; Keller et al., 1985;
316
VAN STRAATEN ET AL.
Jacobson et al., 1986; Keller and Tibbetts, 1989). In the
chick embryo, a marked eightfold elongation and a
twofold narrowing occurs throughout HH stages 4-1 1,
which appear to involve not only cell rearrangement
but also changes in cell shape and cell number
(Schoenwolf, 1986, 1994; Schoenwolf and Alvarez,
1989; Schoenwolf and Sheard, 1989). Elongation of the
neural plate appears to be especially associated with
the phase of neural tube closure in the amphibian and
chick embryo (Jacobson and Gordon, 1976; Jacobson,
1984; Schoenwolf and Alvarez, 1989). Thus, the rate of
elongation was seven times higher in the portion of the
PNP cranial to Hensen’s node than in the closed neural
tube of the chick (Jacobson, 1981). The enhanced
neuraxial elongation between sst 7 and 11 in the
present study indicates that convergent extension is
occurring and may play an important role in the mechanism of PNP closure between sst 7 and 11.
Rapid, Two-step Closure of the PNP
From sst 10 onward, the rate of caudal progression of
the PNP closure point accelerates to 480 pdsomite
stage, and the length of the PNP rapidly diminishes,
resulting in its final closure (except for a small remnant). This new phase of neurulation seems to be
unique in its sudden enhancement, but it is the likely
continuation of the gradual reduction in width of the
PNP, which, at this time, becomes sufficiently narrowed to allow fast closure.
Following rhombocervical closure, the PNP exhibits
a pear-like shape, with cranial tapered and caudal wide
portions. The pear shape can be explained by assuming
that movements of convergent extension (and, thus,
narrowing of the neural plate) progress in a craniocaudal direction along the body axis that are active in the
tapered portion and that are becoming active in the
wide portion of the PNP (Fig. 8).This peculiar shape of
the PNP coincides with a two-step closure. Due to integral narrowing of the PNP, its tapered portion is the
first to close. We hardly observed button-like closure
points during closure of this PNP portion, which suggests that the folds close in a craniocaudal, zipper-like
manner rather than simultaneously over their full
length. Narrowing continues in the wide portion of the
PNP, which transforms into a slit. From sst 12 onward,
this slit appears to close almost instantaneously over its
entire length (except for the caudal region). Numerous
button-like contacts were observed during closure in
this region, suggesting a fundamentally different method of closure from that of the tapered region of the PNP.
The slit-like appearance of the PNP has also been
noted by others (Portch and Barson, 1974; Schoenwolf,
1979).Convergent extension may assist in the creation
of this shape: elongation of the midline has been postulated to generate transverse buckling tension, which
forces the neural walls to elevate and the neural folds
to converge (Jacobson, 1978). We suggest that the enhanced elongation preceding and during the final
phase of PNP closure causes transverse buckling of the
Fig. 9 Scanning electron micrograph of the PNP of an sst 11 chick
embryo. The PNP is slit like, with the neural folds extending dorsally and
undergoing convergence. The wide portion (w) of the PNP is open,
whereas the tapered portion (t) shows a suture line that indicates apposition or adhesion contact between the neural folds. More cranially, the
folds are fused (f). The lengthof both portions of the PNP amountedto 750
pm, as measured in dorsal view, although, in this oblique scanning electron micrograph view, the PNP seems shortened. Scale bar = 100 pm.
neural folds and results in the slit-like appearance of
the PNP (Fig. 91, leading to rapid completion of neural
tube closure.
EXPERIMENTAL PROCEDURES
Preparation, Manipulation, and
Measurement of Embryos
Eggs of White Leghorn chicks were incubated at
37°C and 55% humidity in a roller incubator (Poly-
317
MULTIPHASIC NEURAL TUBE CLOSURE
hatch; Brinsea Products, Sandford, United Kingdom)
for 35-50 hr to obtain embryos ranging from HH
stages 7 to 15. After cracking the egg shell very gently,
the contents were floated into a bowl with excess warm
saline (Locke’s solution: 154 mM NaC1, 6 mM KC1, 2
mM CaC12, 10 mM D-glucose), and the embryo was
viewed with a WILD M5 dissecting microscope. By using side illumination, the number of somite pairs could
be visualized and subsequently counted.
The egg white above the embryo was removed. A
droplet of stain (1%toluidine blue in 1%borax diluted
10 times with saline) was floated over the vitelline
membrane, and, within 2 min, areas of the embryo contacting the vitelline membrane (specifically the neural
folds) were sufficiently stained. A photograph was
taken.
Several morphometrical parameters were determined on the embryo in dorsal view: length of the
neuraxis, length of all neuropores, and width of the
PNP, by using a scaled eyepiece graticule at a magnification of x 25. Data were depicted on a scale drawing
of each embryo. The anterior extremity of the neuraxis
was defined by the anterior end of the embryo. The
caudal extremity of the neuraxis, the length of the PNP
and of its portions, and the most caudal point of neural
fold closure (the PNP closure point) were defined as
indicated in Figure 8.
To expose the embryo, saline was injected gently underneath the vitelline membrane, and the membrane
was torn away over an area that exceeded only slightly
the size of the embryo. A second photograph was taken.
The length of the neuraxis and of the PNP were measured again and were compared with the previous data.
Embryos in which these lengths differed by more than
5% (less than 10%of the total studied) were excluded
from the next part of the study. The locations and types
of neural fold contacts were determined by opening the
neural tube step by step with two tungsten needles; the
smallest step was about 50 pm. Fusion was noted when
the folds could not be separated undamaged, adhesion
was noted when the folds could be separated with adhesion bridges temporarily present, and apposition was
noted when folds could be separated without any adhesive contacts (Van Straaten et al., 1993b). The positions and lengths of these contacts were depicted on the
drawing of each embryo.
Morphometric Analysis and Reconstruction of
the Neurulation Process
A total of 117 embryos were used between sst 4 and
18. It was not possible to measure all parameters in
every embryo, so the data presented in Table 1and in
Figures 3-6 are based on different numbers of embryos
for each parameter. In 70 of the embryos, the distance
between the anterior extremity of the embryo and the
first somite and the craniocaudal length of the somites
were determined. Although individual somites change
in size during development, their average length ap-
peared uniform throughout the stages in this study and
was assumed to be 140 Fm at all stages.
The morphometrical data were plotted graphically.
For the data on neuraxis elongation, a polynomial
function was found to fit the data better than a linear
regression function (Fig. 3). When computing progression rates of the PNP closure, partial regression lines
were constructed.
A reconstruction of the entire neural tube closure
process was performed by using the position of the first
somite as a reference (Fig. 7). The lines indicating the
anterior and posterior extremities of the embryo were
deduced from the distance between the anterior extremity and the first somite and from the polynomial
relationship in Figure 3. The length of the neuropores
and the width of the PNP were drawn according to
their linear regression formulae. The PNP closure
points at each stage were connected and drawn as a
continuous, curved line; its irregularities at sst 6-8 as
well as the shape of the neuropores and of the neural
folds were deduced from the photographs. The lengths
of neural fold contacts were based on Figure 4, and
their locations were based on the drawings,
The position of the PNP closure point a t a given
somite stage was calculated as follows: [length of the
neuraxis] - [length (anterior extremity to somite l ) ] [length of the PNP]. The rate of progression of the closure point, relative t~ somite 1,was calculated subsequently for several phases of neurulation.
ACKNOWLEDGMENTS
We acknowledge the technical assistance of Paul van
Dijk. This work was supported in part by a grant from
the Netherlands Organization for Scientific Research
(431241).
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