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Distribution of surface coat material on fusing neural folds of mouse embryos during neurulation.

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Distribution of Surface Coat Material on Fusing Neural Folds
of Mouse Embryos during Neurulation
Department of Anatomy, School of Medicine, University of Virginia,
Charlottesuille, Virginia 22902
Fusing and non-fusing regions of neural folds from mouse embryos were examined during neurulation for the distribution of extracellular
macromolecules (surface coats) prior to and a t the time of closure. Ruthenium
red staining of 10th day ICR/DUB mouse embryos was used to detect the distribution of surface coat material. Light microscopic examination of fusing and
non-fusing regions in the midbrain, hindbrain, and spinal cord showed a consistent increase in ruthenium red positive material immediately prior to
closure. Heavy deposits of positive staining material were present along apical
neural fold borders and overlying ectoderm cells. This staining pattern was consistent in the three regions examined, but the pattern of initial contact between opposing neural folds differed. In mid- and hindbrain areas contact was
initiated by overlying ectoderm, whereas in spinal cord regions contact was
first established by neuroepithelial cells. Once contact between opposing neural
folds was initiated a decrease in stainable material was observed.
The distribution of carbohydrate-rich surface coat material along prospective zones of
fusion in the palate and nasal processes of rat
and mouse embryos has led to the hypothesis
t h a t these macromolecules are essential for
normal epithelial contact and adherence. During palatogenesis, surface coat material has
been shown to increase dramatically over medial-edge epithelial cells prior to fusion and is
concentrated in prospective contact regions
(Pratt e t al., '73; Greene and Kochhar, '74;
P r a t t and Hassell, '75; Souchon, '75; Greene
and Pratt, '76). Surface coats have also been
observed during fusion of medial and lateral
nasal processes in mouse embryos (Gaare and
Langman, '77; Smuts, '77) and in fusing neuroepithelium of chick (Lee e t al., '76b) and amphibian (Moran and Rice, '75) embryos. In
both the nasal and neuroepithelial areas, increased amounts of surface coat material over
prospective fusion zones was observed immediately prior to contact. These findings suggest t h a t extracellular materials may be
important for normal fusion and adhesion between epithelial surfaces during organogenesis (Greene and Pratt, '76). This view is substantiated by results indicating that inhibitors of surface coat synthesis such as DON
(6-diazo-5-oxonorleucine) prevent adhesion of
(1978)191: 345-350.
epithelial surfaces during palatogenesis in
rats (Greene and Pratt, '76, '77).
To further investigate the role of surface
coat material during epithelial adhesion, fusing neural folds in mouse embryos were examined for the presence, and distribution of saccharide-rich surface macromolecules. Neural
tube closure in mouse embryos was selected
because the fusion process is a continuum,
beginning in the cervical region and proceeding rostrally and caudally, thereby permitting
observations of fusing and non-fusing regions
in the same embryo. Ruthenium red, a stain
used to demonstrate surface coat carbohydrates, was (Luft, '71a,b), used to demonstrate
the presence of surface coat material.
Random-bred ICR/DUB mice (Flow Laboratories, Dublin, Virginia) were used for all investigations and animals were sacrificed a t 10
and 10.5 days of gestation (plug day=day 1).
Following removal from the uterus, embryos
were fixed for one hour in cacodylate-buffered
modified Karnovsky's fixative (2%glutaraldeReceived Aug. 9, '77. Accepted Feb. 16, '78.
This work was supported by NIH Grant 5132 DE07037-02 for
craniofacial development and by Biomedical Research Support
Grant 5S07RR5431-15.
hyde, 2'11 paraformaldehyde) containing 4,500
ppm ruthenium red (Sigma). After fixation,
embryos were rinsed briefly in 0.1 M cacodylate buffer containing 4,500 ppm ruthenium
red and post-fixed for three hours in cacodylate buffered OsO,, also containing 4,500 ppm
ruthenium red (Luft, '71a,b). The tissue was
then dehydrated in alcohol and embedded in
araldite (502). Thick (1 p m ) sections were
made on a n LKB-Huxley ultramicrotome,
stained with toluidine blue, and observed with
the light microscope.
Neural tube closure in ICR mouse embryos
was initiated on t h e ninth day of gestation
and continued to completion over the next 24
hours. Fusion of hind- and midbrain areas was
similar morphologically and in regard to distribution of ruthenium red positive material.
Morphologically, neural folds from these regions elevated from t h e neural plate to a vertical position in which opposing folds were parallel (fig. 1). At this point a bend occurred
near the middle and at t h e apex of each fold
which tended to approximate t h e apices from
either side. Concomitantly, t h e apices, which
were rounded in t h e parallel position, began to
taper a s they approached each other (figs. 2,
3). However, tapering was more pronounced in
hindbrain (figs. 2, 3) than midbrain (fig. 5)
a r e a s which r e t a i n e d a rounded s h a p e
throughout closure. Overlying t h e tips of each
neural fold was a n ectodermal cell layer which
was continuous with ectoderm surrounding
the entire embryo (figs. 1-41, Ectoderm cells
appeared cuboidal except over t h e uppermost
area of approximating neural folds where
these cells were elongated. A t closure, ectoderm cells made initial contact between
opposing neural folds and formed a cellular
plug situated between adjacent neuroepithelial cells from each fold (fig. 4). Finally,
neuroepithelial cells made contact beneath
the ectodermal plug, thereby forming a continuous layer of neuroepithelium around t h e
lumen. Following neuroepithelial fusion, overlying ectoderm cells resumed their cuboidal
Distribution of ruthenium red positive material was similar in hind- and midbrain regions. Approximately 100-120 p m anterior to
a fusion zone, in either of these regions, no ruthenium red stained material was observed
with light microscopy. However, as a n area of
fusion was approached a n accumulation of
stain was observed. Initially, stained material
was present uniformly over the entire luminal
surface of neural folds and overlying ectoderm. This staining pattern was maintained
until approximately 40 p m anterior to fusion
at which point a concentration of positively
staining material accumulated along luminal
surfaces of t h e upper third of opposing neural
folds (figs. 2, 3, 5). The deposition of stain was
heaviest a t the apices of neural folds and occasionally extended over ectoderm cells immediately overlying apical neuroepithelial cells.
Following contact between neuroepithelium
from adjacent folds and a return of ectoderm
to a cuboidal shape, staining decreased until
only a small, evenly distributed deposit of ruthenium red positive material was observed
over the luminal surface and overlying ectoderm.
Neural tube closure in spinal cord regions
differed morphologically from hind- and midbrain fusion, but t h e pattern of ruthenium red
staining remained t h e same. For example,
during spinal cord closure, neural fold apices
were rounded, not tapered as those from hindbrain areas (fig. 6). Furthermore, overlying
ectoderm cells did not completely cover apical
areas and were not involved in initial contact.
However, staining patterns in tail regions
were similar to hind- and midbrain areas and
positively staining material was initially observed 100-120 p m prior to contact in a n even
distribution along l u m i n a l surfaces. T h e
amount of staining then increased along
upper luminal borders and overlying ectoderm
as opposing folds drew closer together (fig. 6).
Neural tube closure in ICR mouse embryos
encompasses a 24-hour period beginning on
t h e ninth and extending into the tenth day of
gestation. Closure begins in the cervical region and proceeds cranially and caudally. I n
cranial regions, increased folding and tapering of neural folds occurs, especially in hindbrain regions, and initial contact appears to be
between overlying ectoderm. Tail regions
show very little folding and no tapering, with
initial contact being accomplished by neuroepithelial cells from opposing neural folds.
This pattern of fusion in cranial areas is in
contrast t o scanning electron microscopic observations of CD-1 mouse and hamster neurulation in which initial contact was observed
between neuroepithelial cells (as we observed
in tail sections) followed by ectodermal fusion
(Waterman, '76).
Although differences occur between cranial
and caudal segments regarding alterations in
neural fold contours, t h e distribution of surface coat material is similar in all regions.
Both cranial and caudal areas show increased
deposition of surface carbohydrates, which a r e
concentrated over prospective zones of fusion
immediately prior to contact. After contact is
initiated, surface coat material diminishes in
t h e fusion zones and returns to levels found in
non-fusing areas of neuroepithelium. These
results a r e similar to those obtained from
lanthanum stained sections of neural folds
during amphibian (Ambystoma maculatum)
(Moran and Rice, '75) and chick (Lee e t al.,
'76) neurulation which also demonstrated t h e
presence of increased surface coat material
along prospective zones of neural fold fusion prior to contact. Lanthanum positive
"bridges" of cell surface material were also observed in amphibians and were thought t o participate in the closure process by pulling t h e
folds together (Moran and Rice, '75). However,
no surface coat "bridges" were observed in
mouse embryos and no evidence was obtained
which would suggest t h a t surface coat material plays a n active role in drawing the neural
folds toget her.
The distribution and location of surface coat
material in t h e mouse neural tube is consiste n t with t h e hypothesis t h a t surface coats
play a role in epithelial fusion and adhesion
during organogenesis. Similar accumulations
of extracellular materials have been observed
in other systems during epithelial fusion including rat and mouse palatogenesis (Pratt e t
al., '73; Greene and Kochhar, '74; Pratt and
Hassell, '75; Souchon, '75; Greene and Pratt,
'76) and fusion of nasal processes in mice
(Gaare and Langman, '77; Smuts, '77). I n each
of these systems increased deposition of surface macromolecules was observed in prospective fusion areas prior t o contact, followed by a
decrease in stainable material after fusion.
The mechanism whereby surface coats media t e epithelial fusion in these systems is
unknown although surface macromolecules
may provide initial adherence until more perm a n e n t cell c o n t a c t s c a n be established
(Greene and P r a t t , '76). I t is known, however,
t h a t interference with existing cell surface
macromolecules during chick neurulation (by
exposure to concanavalin A) (Lee e t al.,
'76a,b), or inhibition of synthetic processes responsible for surface coat production (treatment with DON) (Greene and Pratt, '77) prevents fusion in t h e chick neural tube and r a t
palate respectively. .
Thanks to Robert Cushing for his skillful
technical assistance.
Gaare, J., and J a n Langman 1977 Fusion of nasal swellings
in the mouse embryo. I. Surface coat and initial contact.
Am. J. Anat., 150: 461-476.
Greene, R. M., and D. M. Kochhar 1974 Surface coat on the
epithelium of developing palatine shelves in the mouse as
revealed by electron microscopy. J. Embryol. Exp. Morph.,
31: 683-692.
Greene, R. M., and R. M. P r a t t 1976 Developmental aspects
of secondary palate formation. J . Embryol. Exp. Morph.,
36: 225-245.
1977 Inhibition by diazo-0x0-norleucine (DON)
of r a t palatal glycoprotein synthesis and epithelial cell
adhesion in uitro. Exp. Cell Res., 105: 27-37.
Lee, H., R. G. Nagele, Jr. a n d G . W. K a l m u s
1976a Further studies on neural tube defects caused by
concanavalin A in early chick embryos. Experientia, 32:
Lee, H., J. B. Sheffield, R. G. Nagele and G. W. Kalmus
1976b The role of extracellular material in chick neurulation. I. Effects of concanavalin A, J. Exp. Morphol.,
198: 261-266.
Luft, J. H. 1971a Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy,
and mechanism of action. Anat. Rec., 171: 347-368.
1971b Ruthenium red and violet. 11. Fine structural localization in animal tissues. Anat. Rec., 171:
Moran, D., and R. W. Rice 1975 An ultrastructural examination of the role of cell membrane surface coat material
during neurulation. J. Cell Biol., 64: 172-181.
Pratt, R. M., W. A. Gibson and J. R. Hassell 1973 Concanavalin A binding to the secondary palate of the embryonic rat. J. Dent. Res., 52: 111A.
Pratt, R. M., and R. M. Greene 1975 The effects of diazo0x0-norleucine (DON) on development of the palatal epitheliumin uitro. In: New Approaches to the Evaluation of
Abnormal Development. D. Neubert and H. J. Merker,
eds. Georg Thieme Publishers, Stuttgart, pp. 648-658.
Pratt, R. M., and J. R. Hassell 1975 Appearance and distribution of carbohydrate-rich macromolecules on the
epithelial surface of the developing r a t palatal shelf. Dev.
Biol., 45: 192-198.
Smuts, M. S. 1977 Concanavalin A binding to the epithelial surface of the developing mouse olfactory placode.
Anat. Rec., 188: 29-38.
Souchon, R. 1975 Surface coat of the palatal shelf epithelium during palatogenesis in mouse embryos. Anat.
Embryol., 147: 133-142.
Waterman, R. E. 1976 Topographical changes along the
neural fold associated with neurulation in the hamster
and mouse. Am. J. Anat., 146: 151-172.
1-4 Cross section through the hindbrain region of tenth day embryos showing the parallel position (fig. l),the tapered configuration (figs. 2, 31, and initial contact (fig.
4) between opposing neural folds. The increased distribution of ruthenium red positive material is also demonstrated along the upper luminal borders of neural
folds. Ectoderm (El, mitotic figures (M); neural crest, ( N O ; neural folds, (NF);
pyknotic cell, (PC); pyknotic debris, (PD). One micron ruthenium red-toluidine
blue. Figure 1 X 400; figures 2-4 X 1,100.
5 Midbrain region of a tenth day embryo prior to fusion, showing slight tapering of
neural folds (NF) and a heavy deposit of ruthenium red positive material over
neural fold apices and overlying ectoderm (E). One micron ruthenium red-toluidine blue. X 1,600.
6 Cross section from the spinal cord of a tenth day embryo near the point of fusion
showing increased amounts of ruthenium red positive material over upper neural
fold regions (NF) and overlying ectoderm (E). Mitotic figures (MI; pyknotic cell
(PC). One micron ruthenium red-toluidine blue. x 1,100.
T. W. Sadler
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distributions, fold, embryo, neural, mouse, surface, material, neurulation, coat, fusing
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