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SEM observations of surface alterations associated with neural tube closure in the mouse and hamster.

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BRIEF COMMUNICATION
SEM Observations of Surface Alterations
Associated with Neural Tube Closure
in the Mouse and Hamster
ROBERT EARLE WATERMAN
Department of A n a t o m y , T h e University of N e w Mexico School of
Medicine, Albuquerque, N e w Mexico 871 31
Vertebrate neural tube formation and
differentiation have been extensively examined. Particular interest has been paid
to : ( 1) the induction of a thickened neural
plate by subjacent chorda-mesoderm, (2)
factors responsible for the appearance of
the neural folds and neural grove, ( 3 ) the
migration of neural crest cells from the
neural folds and, ( 4 ) patterns of mitotic
activity and cellular differentiation within
the walls of the neural tube leading to the
complex functional morphology of the central nervous system. Relatively little attention has been given to the details of fusion
between the neural folds resulting in formation of a closed neural tube (MarinPadilla, ’ 7 0 ) .
Small membrane “ruffles” associated with
cells of the neural folds during neural tube
closure in the chick were recently observed
with the scanning electron microscope
(SEM) (Revel, ’74). It was suggested that
these structures may play a role in forming new adhesions between the surfaces of
apposed neural folds. The present study
reports the presence of similar surface
alterations during fusion of the neural
folds in embryonic mice and golden hamsters.
MATERIALS AND METHODS
Timed pregnant CD-1 mice and golden
hamsters were sacrificed by cervical dislocation during day 8 of gestation. The
uterine horns were removed and placed
in physiological saline. Embryos were rapidly dissected free of uterine and extraembryonic tissue and immersed in a 0.2 M
phosphate-buffered 2% paraformaldehyde,
2% glutaraldehyde solution containing
0.01% trinitro phenol (pH 7.3-7.4) (It0
ANAT. REC., 183: 95-98.
and Karnovsky, ’68) for periods from three
hours to three days. Specimens were then
dehydrated in ethanol, dried by the critical
point method using liquid CO,, coated with
gold-palladium in a vacuum evaporator
and viewed with an ETEC autoscan scanning electron microscope operated at 1020 kV. Thirteen mouse and 10 hamster
embryos were observed.
RESULTS
All embryos exhibited partially fused
neural tubes (fig. 1). A narrow band of
altered surface appearance was observed
for a variable distance along the neural
folds in both species. This band is located
between the larger flattened cells of the
body surface and the smaller apical profiles of the cells of the presumptive neural
tube. It occurs both anterior and posterior
to the fused portion of the neural tube,
but is most prominent immediately anterior to the point of fusion.
In the mouse (fig. 2 ) , the altered band
anterior to the area of fusion is characterized by overlapping attenuated cells and
numerous lamellopodial membrane extensions or “ruffles.” The same relative area
in the hamster, however, exhibits more
long cytoplasmic extensions and filipodia
in addition to flattened cell surfaces (fig.
3 ) . Presumably lysing cells and rounded
cell “blebs” associated with the altered
zone are also more numerous in the hamster than in the mouse, Rounded cellular
profiles are likewise more characteristic
of the hamster (fig. 4), and membrane
“ruffles” more prevalant in the mouse (fig.
5 ) , posterior to the area of fusion.
Received May 6, ’75. Accepted May 13, ’75.
95
96
ROBERT EARLE WATERMAN
Figures 1-3
SEM OF NEURAL TUBE CLOSURE
97
Fig. 4 Neural folds of hamster embryo posterior to the fused neural tube. Small blebs,
possible cell lysis and some flattened cells ( * > are seen between the surface cells and cells
of the presumptive neural tube. SEM. X 630.
Fig. 5 Small membrane “ruffles” associated with the posterior point of fusion between
the neural folds in a mouse embryo. SEM. X 2,650.
DISCUSSION
The results of this study reveal the presence of a localized zone of altered surface
appearance along the presumptive area of
fusion between opposed neural folds prior
Fig. 1 Dorsal view of an 8-day old hamster
embryo. The neural folds have fused along the
mid portion of the body and are open anteriorly
and posteriorly. This stage represents the typical
appearance of both hamster and mouse embryos
examined. SEM. x 31.
Fig. 2 Portion of the neural folds anterior to
the area of fusion in an 8.25 day old mouse
embryo similar to that illustrated in figure 1. A
narrow band of surface alteration can be seen
between cells of the body surface and presumptive neural tube. SEM. x 510.
Inset: Area of right neural fold enclosed by
rectangle in figure 2 is seen at higher magnification. Zone of surface alteration is present between
the neural plate and presumptive surface cells.
SEM. x 1,270.
Fig. 3 Stero pair showing slender filipodiumlike cellular extensions and some “blebs” along
the neural folds immediately anterior to the point
of fusion in a hamster embryo. (To be viewed
with stero glasses). SEM. x 2,710.
to their contact and adhesion. Examination of the surface appearance of the neural folds from neural plate to closed neural
tube stages is currently in progress. Preliminary results indicate there is a progressive increase i n the degree of alteration
along the neural fold which is correlated
with the fusion process (Waterman, ’75).
One feature of this alteration appears to
be changes in the membrane properties of
the cells which are expressed as increased
numbers of membranous extensions. I n
this respect, the “ruffles” observed in the
mouse are more similar to those observed
during neurulation in the chick (Revel,
’74) than the filipodium-like processes observed in the hamster. The demonstration
of similar localized membrane activity in
the chick, mouse and hamster suggests
this may represent a common feature of
neural tube closure.
The fact that the zone of alteration is
restricted to the area of the neural fold
involved in fusion strongly suggests that
98
ROBERT EARLE WATERMAN
observed surface changes may be func- is gratefully acknowledged. Supported by
tionally involved in the adhesion process. N.I.D.R. Grant DE 03897-01.
Similar localized epithelial alterations have
LITERATURE CITED
also been observed along the medial edge
of palatal shelves prior to fusion of the Ito, S., and M. .T. Karnovsky 1968 Formaldehyde-glutaraldehyde fixatives containing trinisecondary palate in mouse (Waterman et
tro compounds. J. Cell Biol., 39: 168a-169a.
al., '73) and human embryos (Waterman
Marin-Padilla, M. 1970 The closure of the
and Meller, '74). Because the membrane
neural tube in the golden hamster. Teratology,
ruffles and extensions seen along the neu3: 39-46.
ral fold appear similar to those commonly Nelsen, 0. E. 1953 Comparative Embryology of
exhibited by many migratory cells in vitro,
the Vertebrates. McGraw-Hill, New York.
it has been proposed that they may aid in Revel, J. P. 1974 Scanning electron microscope
establishing initial contacts between the
studies of cell surface morphology and labelling, in situ and in vitro. IITRI, SEM, 1974,
contralateral folds (Revel, '74). It is also
pp. 542-548.
noteworthy that the zone of alteration corresponds in large part to the area between Waterman, R. E. 1975 Scanning electron microscopic observations of neural tube closure in
presumptive surface cells and neural plate
the embryonic mouse and hamster. Anat. Rec.,
from which the neural crest cells arise
181: 506 (abstract).
(Marin-Padilla, '70; Nelsen, '53). Whether Waterman, R. E., L. M. Ross and S. M. Meller
the observed surface features may be pre1973 Alterations in the epithelial surface of
requisite for adhesion between the neural
A/Jax mouse palatal shelves prior to and during palatal fusion: A scanning electron microfolds, and the possibility that they are
scopic study. Anat. Rec., 176: 361-376.
directly related to neural crest formation
Waterman, R. E., and S. M. Meller 1974 Alterneed to be more fully explored.
ACKNOWLEDGMENTS
The technical expertise of Susan Palmer
ations in the epithelial surface of human palatal shelves prior to and during fusion: A scanning electron microscopic study. Anat. Rec.,
180: 111-136.
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