/. Embryo!, exp. Morph. Vol. 55, pp. 279-290, 1980
Printed in Great Britain © Company of Biologists Limited 1980
279
Fine structure of the lumbosacral neural folds
in the mouse embryo
By DORIS B.WILSON 1 AND LAUREL A. FINTA 1
From the Division of Anatomy, Department of Surgery,
University of California
SUMMARY
The neural folds in the lumbosacral region of the normal 8-day and 9-day mouse embryo
were studied by means of transmission electron microscopy with and without lanthanum
treatment. The cells showed an abundance of ribosomes, microtubules arranged parallel
to the long axes of the cells, and microfilaments extending across the apices. At the luminal
border junctional complexes were common, and an occasional midbody was seen stretching
between adjacent cells nearing the end of telophase. In the 8-day embryos, gap junctional
vesicles (annular nexuses) bounded by layered membranes and containing cytoplasm with
ribosome-like material were commonly observed; at 9 days the vesicles were relatively
rare. The lanthanum-treated material demonstrated that the tracer was able to pass through
the subluminal junctional complexes and throughout the intercellular spaces. However, the
space between the membranes of the gap junctional vesicles lacked lanthanum and thus
apparently did not communicate with the intercellular space.
INTRODUCTION
The formation of the neural tube has been the subject of numerous transmission electron microscopic (TEM) studies on normal embryos of the amphibian
(Schroeder, 1970; Burnside, 1971, 1973; Karfunkel, 1974; Decker & Friend,
1974; Mak, 1978) and chick (Bellairs, 1959; Fujita & Fujita, 1963; Handel &
Roth, 1971; Karfunkel, 1972; Revel, 1974; Bancroft & Bellairs, 1975; Revel
& Brown, 1976; Camatini & Ranzi, 1976). In contrast, comparable TEM
studies on early rodent embryos are relatively sparse (Hinds & Ruffett, 1971;
Freeman, 1972; Sadler, 1978). In the mammal, the early stages of neural tube
closure, particularly the formation and elevation of the neural folds, are of
special importance in the lumbosacral region, since this is a site frequently
affected by non-closure malformations (dysraphism) (Auerbach, 1954; Wilson,
1974; Lemire, Loeser, Leech & Ellsworth, 1975). In view of the paucity of
TEM information on the lumbosacral neural folds in normal mammalian
embryos, the current study was undertaken on normal 8-day and 9-day mice
so as to establish a basis for future fine structural analyses of this region in
abnormal mouse embryos at comparable stages of development.
1
Authors'1 address: Division of Anatomy, M-004, Department of Surgery, School of
Medicine, University of California, San Diego, La Jolla, California 92093, U.SA.
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D. B. WILSON AND L. A. FINTA
MATERIALS AND METHODS
C57BL/6J mice were bred, females were checked daily for vaginal plugs
and killed by cervical dislocation early on the eighth or ninth day post-plug
(day of plug = day 0), and the embryos were removed in saline. In addition,
8- and 9-day embryos were obtained from matings of C57BL/6J and normal
( + / + ) individuals of the splotch (Sp) mutant mouse maintained on a C57BL/6J
background. Six embryos corresponding to developmental stage 12 (Theiler,
1972) were selected from three litters, and four embryos at developmental
stage 14 were selected from three litters. The lumbosacral region of each
embryo was fixed for 1 h in cold (4 °C) half-strength Karnovsky's solution
(Karnovsky, 1965), rinsed in 0-1 M cacodylate buffer (pH 7-2) and postfixed
in cold 1 % osmium tetroxide-Ol M cacodylate buffer for 1 h. The specimens
were then dehydrated in graded ethanols and propylene oxide and flat-embedded
in Epon-Araldite. Thick sections for orientation with light microscopy were
stained with methylene blue-azure II. Thin sections were placed on naked
200 mesh copper grids and stained with uranyl acetate (Watson, 1958) for
20 min and lead citrate (Reynolds, 1963) for 10 min.
For the lanthanum studies, the technique of Revel & Karnovsky (1967) was
used. A 4 % solution of lanthanum nitrate was brought to pH 7-8 by means
of vigorous stirring with 0-02 N-NaOH and was added to the above formaldehyde-glutaraldehyde mixture to give a final concentration of 1 % lanthanum.
The colloidal lanthanum was not used in the buffered rinses, in the osmium
tetroxide, or during dehydration.
Observations were made with a Zeiss 9S-2 electron microscope at direct
magnifications up to x 28000.
FIGURES 1-6
Fig. 1. Cross section of lumbosacral region of neural groove at 8 days' gestation.
L, presumptive lumen, x 180.
Fig. 2. Higher magnification of neuroepithelium at 8 days' gestation. Arrow indicates
internal cellular process, x 600.
Fig. 3. Cilium in apical portion of neuroepithelial cell in lateral region of lumbosacral neural groove at 8 days' gestation, x 28 500.
Fig. 4. Microvillous projection (small arrow) at margin of neuroepithelial cell
at 8 days' gestation. Large arrow, junctional complex, x 28 500.
Fig. 5. Lanthanum in intercellular space (small arrows) between two neuroepithelial cells at 8 days' gestation. Large arrow, possible gap junction, x 84000.
Fig. 6. Midbody at the end of a mitotic division. Small arrows, microtubules. L,
presumptive lumen. Large arrow, dense band, x 28 500.
Neural folds in mouse embryos
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D. B. WILSON AND L. A. FINTA
RESULTS
No differences were noted at the light microscopic or electron microscopic
level between offspring of C57BL x C57BL and those of C57BL x normal (4- / +)
individuals of Sp/ + parentage. Hence the following description applies to
all embryos analyzed in the current study.
Light microscopy
Epon-Araldite thick sections of both the upper and lower lumbosacral
regions of 8-day and 9-day embryos were studied. At 8 days the sections
demonstrate a neural groove that is relatively wide and V-shaped (Fig. 1). The
neurectodermal cells of the groove are tall and columnar. At the lateral edges
of the groove the cells change to cuboidal. Mitotic figures are scattered along
the presumptive luminal border, and deeper lying nonmitotic cells maintain
contact with the lumen by means of a slender internal cellular process (Fig. 2).
In sections taken from the upper lumbosacral region the cell surfaces tend to
bulge slightly upwards into the lumen, whereas sections removed from the
lower lumbosacral region show more flattened luminal cell surfaces. In both
regions of the groove the cells appear to be separated from one another subluminally by extracellular spaces of varying size, except for points of contact
by means of small lateral projections. These contact points and extracellular
spaces may be exaggerated by shrinkage due to tissue preparation.
In the upper lumbar region of 9-day embryos the neural folds have fused to
form a neural tube. Lower lumbar and sacral regions show varying degrees of
closure. In open regions the neural groove is horseshoe-shaped and the medial
aspects of the folds are concave.
Electron microscopy
8 days. At the electron microscopic level low magnifications of the 8-day
lumbosacral region reveal a neuroepithelium with nuclei at varying levels and
mitotic nuclei interspersed between the columnar cells at the presumptive
luminal border. Short microvilli are scattered over the luminal surface, and
centrally located, single apical cilia are occasionally present (Fig. 3). Junctional
complexes occur between adjacent cells at the luminal border, and microfilaments
can sometimes be seen extending outward from the junctions across the apices
of the cells. In most cases a long microvillous projection also extends into the
lumen at the cell margin adjacent to the junctional complex (Fig. 4).
The intercellular junctional complexes are permeable to lanthanum, as
evidenced by the presence of the tracer throughout the intercellular spaces. In
some instances the lanthanum-filled space is narrowed and suggestive of a gap
junction (Fig. 5).
Midbodies are common at the luminal surface and consist of a narrow
cytoplasmic bridge between two cells in the process of completing a mitotic
Neural folds in mouse embryos
283
Fig. 7. Internal cellular process of neuroepithelial cell at 8 days' gestation. Note
numerous microtubules (small arrows). Large arrow, gap junctional vesicle,
x 28 500.
Fig. 8. Gap junctional vesicle (arrow) in mitotic cell at 8 days' gestation, x 28500.
Fig. 9. Gap junctional vesicle in lanthanum-treated embryo at 8 days' gestation.
Note presence of lanthanum in adjacent intercellular spaces but not between
membranes of the vesicle, x 38000.
division. Within the cytoplasmic bridge are parallel stacks of microtubules
with a csntrally located dense band (Fig. 6). A few cells also show large
irregular blebs on the luminal surface.
The neuroepithelial cells of the upper and lower lumbosacral neural groove
contain a variety of cytoplasmic organelles including free ribosomes, polyribosomes, rough endoplasmic reticulum (RER), and small, dense mitochondria.
In some cells the rough endoplasmic reticulum exhibits circular or whorled
zones. Pinocytotic invaginations are frequently found at the luminal surface
of the internal processes of the nonmitotic cells. These processes also show
numerous microtubules running parallel to the long axes of the cells (Fig. 7).
A notable feature of both upper and lower lumbosacral neural groove is the
presence of gap junctional vesicles (annular gap junctions or annular nexuses)
(Figs. 7-9). The vesicles are bounded by a dense, layered membrane and
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D. B. WILSON AND L. A. FINTA
contain ribosome-like material. Most vesicles have an electron-lucent zone
located within the vesicle subjacent to its membrane.
The gap junctional vesicles are most frequently found near the luminal
surface in the internal cellular processes of nonmitotic cells, but they have also
been observed at deeper levels in the cellular processes, near the basal lamina
of the neuroepithelium, and in mitotic cells. The occurrence of the vesicles is
similar in both upper and lower lumbosacral regions, and each section of
neural groove examined has at least one gap junctional vesicle. Some sections
have as many as seven individual vesicles, with an occasional cell having two
vesicles. Examination of adjacent sections confirms that the vesicles are distinct
and separate from one another and not sections of the same vesicle. The
lanthanum preparations show that the space between the membranes of the
vesicles lacks lanthanum and that the vesicles are not in communication with
the intercellular space (Fig. 9).
9 days. At 9 days of gestation the mitotic nuclei are situated at the luminal
border and are interspersed among the internal cellular processes extending
from the deeper nonmitotic cells (Fig. 10). The luminal borders of many of
the ventricular cells and of the internal cellular processes bulge prominently.
At higher magnifications, thick bundles of microfilaments are commonly seen
spanning the apices of these cells (Fig. 11). Midbodies are also common. Cilia,
cytoplasmic blebs and microvilli project into the lumen and long microvillous
projections at the cell margins adjacent to junctional complexes are similar in
size and location to those noted in the 8-day embryos. The appearance and
distribution of intracellular organelles are also similar to those observed at
8 days.
In regions where the neural folds have begun to fuse dorsally, the cells at
the apices of the folds approach one another and become apposed. Long
undulating cytoplasmic processes extend between the dorsal aspects of the
apposing cells, forming an interdigitating tangled web (Fig. 12).
In the 9-day lanthanum preparations, the tracer penetrated the junctional
complexes at the luminal borders of the neuroepithelial cells and was present
in the intercellular spaces. However, in rare instances the lanthanum failed to
penetrate into the intercellular space subjacent to an apical junction.
FIGURES
10-12
Fig. 10. Luminal aspect of lumbosacral groove at 9 days' gestation. The surfaces
show prominent bulges and projections into the lumen. M, mitotic cell. Arrow
indicates a rare gap junctional vesicle, x 3900.
Fig. 11. Higher magnification of apex of a ventricular cell at 9 days' gestation
showing apical bulging into the lumen (L). Arrows indicate bundles of microfilaments, x 24000.
Fig. 12. Low magnification of dorsal neural folds in the process of fusing with
each other at 9 days' gestation. L, Lumen, x 5400.
Neural folds in mouse embryos
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D. B. WILSON AND L. A. FINTA
With respect to gap junctional vesicles, these structures are relatively rare
at 9 days (Fig. 10). In a total of 20 sections, only four vesicles could be found.
The structure of these did not show any apparent differences from that in the
8-day embryos, and their membranes did not contain lanthanum. The locations
of the vesicles were also similar to those seen at 8 days, i.e. near the lumen,
within an internal cellular process, and in a mitotic cell.
DISCUSSION
The C57BL mouse was chosen for this study primarily because it has been
commonly used as a normal base line in neurological investigations (Sidman,
Angevine & Pierce, 1971). Moreover, it also represents a genetic background
upon which several neurological mutants of the mouse occur (Sidman, Green
& Appel, 1965).
The neuroepithelial cells in the lumbosacral folds in the normal 8-day and
9-day mouse embryos used in the current study show features similar to those
described in various other regions of the chick and rodent neural folds during
early development (Freeman, 1972; Karfunkel, 1972; Bancroft & Bellairs,
1975; Camatini & Ranzi, 1976; Revel & Brown, 1976). These features include
microtubules arranged parallel to the long axes of the cells, especially in the
internal cellular processes, and an abundance of free ribosomes and polyribosomes. Apical cilia are also present but are not as frequent or well developed
as during later stages of neural development (Sotelo & Trujillo-Cenoz, 1958;
Bancroft & Bellairs, 1975; Wilson, 1978).
The bundles of microfilaments extending across the apical regions of the
cells are not as prominent or densely arranged in the 8-day mouse as at 9 days
when the medial aspects of the neural folds become concave. The arrangement
of these microfilaments is similar to that seen in the amphibian and chick, and
their role has been postulated as producing the apical constriction necessary
for changes in the shape of the cells during neurulation (Karfunkel, 1972, 1974;
Burnside, 1973).
The observation that the luminal surfaces of the 8-day neuroepithelial cells
bulge somewhat in the upper lumbosacral region, whereas those in the lower
region tend to be more flat, most likely reflects the fact that the upper region
is slightly more advanced than the lower region at any given stage of development. This bulging, as well as the formation of apical blebs, becomes more
prominent as the folds elevate and begin to approach one another at 9 days;
similar protrusions were observed during elevation of rat neural folds (Freeman,
1972).
The luminal surfaces of the neuroepithelial cells at 8 and 9 days also
exhibit an occasional cytoplasmic bridge between two cells nearing the end of
telophase. Once the cells separate completely the cytoplasmic bridge is pinched
off and portions remain as debris at the lumen. These bridges have been
Neural folds in mouse embryos
287
termed midbodies (Allenspach & Roth, 1967), although the term has also been
used more restrictively to designate remnants of the mitotic spindle or the
dense band in the center of the bundles of spindle microtubules (Buck, 1963;
Krystal, Rattner & Hamkalo, 1978). The presence of midbodies along the
luminal aspects of the 8- and 9-day neuroepithelium reflects the mitotic activity
of these cells, and these structures are particularly common after closure of
the neural tube (Allenspach & Roth, 1967; Bancroft & Bellairs, 1975;
Wilson, 1978).
An impressive array of long finger-like interdigitations was observed at 9 days
in those regions where the neural folds had approximated one another and
were beginning to fuse. These cytoplasmic processes appear to be similar to
those observed by means of scanning electron microscopy in the mouse and
hamster (Waterman, 1976) and in the chick (Revel & Brown, 1976). Transmission electron microscopy in the chick (Bancroft & Bellairs, 1975) and in
the amphibian (Moran & Rice, 1975) has also demonstrated these structures,
and it is possible that they provide a means of initial contact and/or maintenance
of fusion of the folds.
In the 8- and 9-day unfused neural folds, the apical regions of the neuroepithelial cells are bound to one another by means of junctional complexes.
However, there is a relatively large amount of extracellular space subapically
in the 8-day mouse neural tube, and this is similar to that observed in the
chick at a comparable stage of development (Bancroft & Bellairs, 1975). While
this may be an artifact produced by the aldehyde fixative, there is some evidence
that the extracellular space may well be extensive in immature neural tissue
(Sumi, 1969; Hinds & Ruffett, 1971), and this may allow for the relatively
rapid changes in cell shape and movement which occur during the early
stages.
Although the exact nature of the junctional complexes could not be confirmed
in the current study without special techniques such as freeze-fracture, the
junctions at the luminal surface appear to be gap junctional in nature, since
lanthanum passed freely through the intercellular spaces to deeper levels. In
the amphibian, Decker & Friend (1974) noted that gap junctions become
widely distributed in the neural folds during closure. Likewise in the chick,
Revel & Brown (1976) describe small gap junctions or gap junction-like
structures in the gutter stage of the neural groove. An occasional juxtaluminal
zonula occludens was observed in more mature regions of the developing
neural tube in the chick (Revel & Brown, 1976); this agrees with our observation
of an occasional junction which was not permeable to lanthanum in our 9-day
mouse material. Although gap junctions have been cited as a means of cell to
cell communication and are particularly common during embryonic differentiation (Decker & Friend, 1974; Fisher & Linberg, 1975; Hayes, 1977), tight
junctions and gap junctions are often closely associated with one another in
developing tissue, and the complex changing patterns of these junctions in the
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neural folds and neural tube preclude a clear definition of their nature and
function (Revel & Brown, 1976).
Of special interest in the current study is the presence of gap junctional
vesicles (annular nexuses). Although the role and significance of the vesicles
are unknown, these unusual structures have been shown by means of freezefracture and tracer techniques to bud off from finger-like projections of gap
junctions into the cytoplasm, and it has been postulated that the vesicles may
represent a means of interiorizing and disposing of gap junctions (Albertini,
Fawcett & Olds, 1975). Gap junctional vesicles have been noted in a variety
of cells including ovarian granulosa cells (Espey & Stutts, 1972; Merk, Albright
& Botticelli, 1973; Albertini et al. 1975; Coons & Espey, 1977; Tung & Larsen,
1979) and various adenocarcinoma cells, particularly during dissociation experiments (Leibovitz et al. 1973; Letourneau, Li, Rosen & Ville, 1975; Murray,
Larsen & O'Donnell, 1978; Murray, 1979). In the current study at least one
of these vesicles was found per section of lumbosacral neural folds in the
normal 8-day mouse embryo; in contrast, gap junctional vesicles were rarely
found in the normal 9-day lumbosacral folds, suggesting that they may play
a role in mediating a normal loss of cell to cell contact and/or communication
at this critical stage of neural tube closure.
In the loop-tail (Lp) and splotch (Sp) mutant mouse, homozygous individuals
show closure defects of the neural tube. Although fine structural characteristics
of microtubules, microfilaments, midbodies, and junctional complexes in the
9-day abnormal embryos are similar to those seen in their normal litter-mates
and in the normal 9-day C57BL individuals of the current study, one striking
difference is the increased number of gap junctional vesicles in the abnormal
neural tubes (Wilson & Finta, 1979; Wilson, 1979). Whether this represents
a cause or an effect of the abnormality remains to be explored in these mutants,
particularly during the eighth day of development. The relationship of gap
junctional vesicles to gap junctions and tight junctions during normal as well
as abnormal neural development would also seem to warrant further attention.
This research was supported by National Institutes of Health grant no. HD09562 from
the National Institute of Child Health and Human Development.
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