J. Embryol. exp. Morph. 78, 211-228 (1983)
211
Printed in Great Britain © The Company of Biologists Limited 1983
Occlusion of the neural lumen in early mouse
embryos analysed by light and electron microscopy
By M. H. KAUFMAN
From the Department of Anatomy, University of Cambridge
SUMMARY
A histological account of neural tube occlusion during early mouse embryogenesis is presented here from an analysis of sections taken from plastic-embedded material. Because the overall
pattern of neuroepithelial apposition and the duration of luminal occlusion appears to vary
slightly from one embryo to another at otherwise similar stages of development, only a general
guide to the events occurring in the mouse embryo between about midday on the 9th to late
on the 10th day of gestation can be given. The earliest evidence of complete luminal occlusion
was seen when the cephalic and caudal extremities of the neural tube were still widely open.
An ultrastructural analysis of the morphological appearance of the closely apposed luminal
cells in zones of partial and complete occlusion has demonstrated that occlusion is brought
about initially by the interdigitation of processes from closely apposed neuroepithelial cells.
This initial event is followed by direct contact over a much more extensive area between the
surfaces of apposed cells with a characteristically flattened luminal border. Apposition and
luminal occlusion is probably facilitated by the presence of viscous extracellular material.
Finally, complete occlusion involving an extensive region of the lumen occurs. The latter
phenomenon is a transient event lasting 1 or at most 2 days in the mouse. At no stage were
junctional complexes observed between closely apposed neuroepithelial cells in regions in
which the neural lumen appeared to be completely occluded, though they were apparent
between adjacent neuroepithelial cells.
Observations on the underlying mechanism(s) of cellular fusion are considered in the light
of the ultrastructural findings. These results are compared with findings from analyses of
various other sites of cellular fusion during embryogenesis. Attention is also drawn to the
similarities and differences observed in the timing and overall pattern of events occurring
during the early development of the neural tube in mouse and human embryos.
INTRODUCTION
A recent study has clearly demonstrated that occlusion of the lumen of the
neural tube - eventually involving up to about 60 % of its length - occurs as a
normal event during early embryogenesis in man (Desmond, 1982). Furthermore, it was proposed that complete occlusion of the neural canal, which was
first apparent at stage 11 (when embryos possessed 13-20 pairs of somites, see
Streeter, 1942; O'Rahilly & Gardner, 1979), plays an important part in facilitating enlargement of the brain (Desmond & Jacobson, 1977). Indeed, these
authors suggested that the latter would only occur once the neural tube had
become a closed compartment filled with cerebrospinal fluid. Evidence from
1
Author's address: Department of Anatomy, University of Cambridge, Downing Street,
Cambridge CB2 3DY, U.K.
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M. H. KAUFMAN
chick embryo studies in which the cerebrospinal fluid pressure within the neural
system was markedly reduced following intubation of the neural tube (Desmond
& Jacobson, 1977), eye (Coulombre, 1956) or myelencephalon (Coulombre &
Coulombre, 1958) has clearly demonstrated that these experimental procedures
invariably lead to the abnormal morphogenesis of both the brain and eye.
Apart from the detailed histological analysis of the human embryonic material
presented by Desmond (1982), observations on neural luminal occlusion in the
chick embryo (Desmond & Jacobson, 1977), and the isolated example of luminal
occlusion in the thoracic region of an early rat embryo illustrated by Freeman
(1972), very little appears to be known about the occurrence and possible significance of this phenomenon.
In the present study, a histological account of neural tube occlusion during early
mouse embryogenesis is given, detailing the period of development during which
this phenomenon may be observed. Unlike the situation in the human embryo
where occlusion occurs 'subsequent to the formation of a closed tube' (Desmond,
1982), in the mouse, at least, occlusion may be observed in embryos with only
10-12 pairs of somites present, when the cephalic and caudal regions of the neural
tube are still widely open. Similarly, while the situation described in the human
embryo suggests that in man the onset and sequential events associated with
neural tube occlusion are remarkably uniform from embryo to embryo, the
present findings tend to indicate that this does not appear to be the case in the
mouse. For the latter reason, the present account can only serve as a very general
guide to the events occurring in the early mouse embryo as development proceeds
between about midday on the 9th to late on the 10th day of gestation.
Only sections from plastic-embedded material are presented here, as
appropriately fixed material embedded in this way shows little in the way of
shrinkage artefacts as are commonly observed in conventional paraffinembedded material. An ultrastructural account of the morphological appearance of the closely apposed luminal cells in zones of partial or complete
occlusion is also presented.
The present findings of occlusion of an extensive, but variable, segment of the
neural lumen in the mouse embryo would seem to confirm the contention that
this phenomenon is probably a normal occurrence in many vertebrate species
(Desmond, 1982). However, the detailed timing of this event, the overall pattern
of neuroepithelial apposition and the duration of luminal occlusion appear to
vary from one species to another, and in the mouse at least, from one embryo
to another at otherwise similar stages of development.
MATERIALS AND METHODS
Female CFLP mice (Hacking and Churchill Ltd) were mated with males of the
same strain and isolated on the morning of finding a vaginal plug (designated the
first day of pregnancy). Between the early afternoon of the 9th day to late on the
Neural luminal occlusion in mouse embryos
213
10th day of gestation individual females were killed by cervical dislocation and
the embryos isolated in phosphate-buffered saline (pH7-3). A total of 30 embryos were selected for this study, being divided in groups according to the
approximate number of somites present, their degree of 'turning', and the
presence of an obvious forelimb bud (the most advanced group). Between five
and ten embryos were assigned to each group, though a small degree of overlap
was inevitable using these somewhat arbitrary morphological criteria. An
additional group of three embryos was isolated early in the morning on the 11th
day of gestation. All of the embryos were dissected free of their membranes and
fixed in 2 % glutaraldehyde in 0-1 M-sodium cacodylate buffer containing 10 gm
sucrose/100 ml. After about 2h the embryos were transferred to 0-1 M-sodium
cacodylate buffer containing 3gm sucrose/100 ml. After l h the material was
postfixed in 1 % osmium tetroxide containing 5gm sucrose/100 ml for 25min.
The material was then dehydrated through a graded ethanol series, and eventually embedded in epoxy resin (Spurr, 1969) and semithin transverse sections
(thickness about 0-5-0-75 jum) taken and stained with methylene blue. Thin
sections at appropriate levels were cut with a Huxley ultramicrotome, double
stained with uranyl acetate and lead citrate and viewed in a Philips EM 300
transmission electron microscope.
An additional group of five embryos was used to determine whether extracellular material was detectable on the neuroepithelial cell surface at sites of
close cell-cell apposition. The cephalic region of embryos in this group was
removed prior to their fixation (for 2 h) in 2 % glutaraldehyde in 0-1 M-sodium
cacodylate buffer containing 3mM-Ca2+ (pH7-2). Decapitation was carried out
in order to allow the ruthenium red access to the neuroepithelial cells lining the
neural lumen in all embryos in this group, whether the rostral neuropore had
closed or not. The embryos were then post-fixed in 1 % osmium tetroxide either
(i) with or (ii) without ruthenium red (2mg/ml) in 0-1 M-sodium cacodylate
buffer. This material was then dehydrated through a graded ethanol series,
embedded, and semithin and thin sections taken as described above. The thin
sections were viewed unstained in the Philips EM 300 transmission electron
microscope orientated at either 20 or 40 kV.
The author's collection of Bouin- and Susa-fixed paraffin-embedded material
covering embryonic development between the 10th-12th days of gestation was
also examined, and the incidence of luminal occlusion in this material is also
briefly reported. The latter information is included here as it confirms the
author's contention that this phenomenon is most clearly seen in appropriately
fixed plastic-embedded material.
RESULTS
1. Histological appearance of neural tube
Because of the considerable degree of variability observed in neural tube
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M. H. KAUFMAN
morphology in mouse embryos that appear, at least externally, to be almost
identical, it will only be possible to provide an approximate guide to the sequence
of events occurring with respect to side-side apposition and occlusion of the
neural lumen. For this reason, an overview of this phenomenon in the mouse will
be presented here, using representative histological sections through the neural
tube at different levels along the neuraxis in a selection of embryos isolated
between the early afternoon of the 9th day to late on the 10th day of gestation.
The earliest embryo in which a moderate degree of occlusion was observed,
had about 10-12 pairs of somites present, and had been isolated in the afternoon
on the 9th day of gestation. This embryo was still 'unturned', but from its overall
appearance was likely to have 'turned' to adopt the characteristic foetal position
within a matter of hours. Only a small region of complete luminal occlusion is
present, though extensive areas of side-side apposition and partial luminal
occlusion are evident at various levels along the spinal axis. Representative
sections through the neural tube at different levels are illustrated in Fig. 1.
The appearance of the neural lumen in an embryo at a slightly later stage of
development, isolated in the evening on the 9th day of gestation, will now be
considered. The cephalic neural folds in this embryo had yet to become apposed
and fused in the regions overlying the presumptive fore-, mid- and hindbrain.
The embryo was partially 'turned' and had approximately 12-14 pairs of somites
present. As in the previous embryo, complete luminal occlusion is only evident
over a relatively short segment, though side-side apposition and partial luminal
occlusion involving extensive regions of the neural tube are clearly seen.
Representative sections through the neural tube at different levels along the
spinal axis in this embryo are illustrated in Fig. 2.
The next embryo in this series was almost completely 'turned' and had also
been isolated in the evening of the 9th day of gestation. The embryo had about
Fig. 1. Representative transverse sections through the neural tube of an 'unturned'
embryo with 10-12 pairs of somites present, isolated in the afternoon on the 9th day
of gestation. Sections stained with methylene blue. (A) Section through the neural
tube at the midcardiac level. Note that the neural lumen is widely patent, and the
notochord (arrowed) clearly seen. (B) Low-magnification view through the 'thoracic' region of this embryo. The section is taken through the caudal one third of the
heart, and through the midtail region. Key: a, atrium; v, ventricle; y, yolk sac; m,
amnion. (C) Higher magnification view through the neural tube in the 'thoracic'
region at a level identical to that illustrated in B. A slight indication of side-side
apposition is apparent at this level. (D) Section through the neural tube at the level
of the sinus venosus, at the caudal extremity of the heart. A considerable degree of
side-side apposition is seen, particularly in the central region of the neural tube,
though the neural lumen is still patent at this level. (E) Section through the neural
tube about half way between the sections illustrated in D above and F below, some
distance proximal to the U-shaped lordotic segment in this 'unturned' embryo. The
middle third of the neural lumen (region between arrows) appears to be completely
occluded. (F) Section through the neural tube just proximal to the lordotic segment.
The dorsal half of the lumen appers to be completely occluded whereas the ventral
segment is still patent. Bar = lOOjton.
Neural luminal occlusion in mouse embryos
215
20 pairs of somites present, and the cephalic neural folds were completely fused.
The region of apposition and occlusion in this embryo was also quite extensive,
involving the neural tube from the hindbrain region just rostral to the otic pits,
at about the level of the middle of the first branchial arch, caudally almost as far
as the caudal neuropore. The appearance of the neural tube in this embryo is
illustrated in Fig. 3.
The next embryo was isolated at about midday on the 10th day, had obvious
forelimb buds and about 25 pairs of somites present. This embryo also had an
extensive region of close side-side apposition with intermittent regions of partial
216
M. H. KAUFMAN
luminal occlusion. At no level, however, did the neural lumen appear to be
completely occluded. The appearance of the neural tube in this embryo is
illustrated in Fig. 4. The transmission electron micrographs which illustrate the
sites of apposition and occlusion in more detail were all taken from this embryo.
The next embryo in this series was also isolated at about midday on the 10th
day of gestation, had about 25 pairs of somites and easily recognisable forelimb
buds present. A moderate degree of luminal apposition was present in this
embryo involving principally the ventral third of the neural canal. The apposed
Fig. 2
Neural luminal occlusion in mouse embryos
Fig. 2. Representative transverse sections through the neural tube of a partially
'turned' embryo with 12-14 pairs of somites present, isolated in the evening on the
9th day of gestation. Sections stained with methylene blue. (A) low-magnification
view at the level of the rostral one-third of the heart. Some degree of side-side
apposition of the walls of the neural tube are apparent. Key: f, foregut; v, ventricle;
w, 'thoracic' wall. (B) Low-magnification view of section though the midcardiac
level. Note the considerable diminution in the volume of the foregut compared to the
situation illustrated in A above, and the almost complete obliteration of the neural
lumen. Key: a, atrium, v, ventricle; f, foregut. (C) Low-magnification view through
the mid-U region of the neural tube. In the proximal (lower) segment the lumen is
virtually occluded, whereas in the distal (upper) segment the ventral half of the
lumen is widely patent. Large blocks of somites (arrowed) are apparent on either side
of the neural tube. (D) Higher magnification view of section through the neural tube
at the level of the rostral one-third of the heart, just distal to the section illustrated
in A above. A degree of side to side apposition is apparent. E. Section through the
neural tube at a similar level to that illustrated in B above. Apart from a short dorsal
segment (above), the majority of the neural lumen at this level appears to be completely occluded. (F) Section through the neural tube just rostral to the lordotic
region. The neural lumen appears to be completely occluded. Bar = 100 (xm.
Fig. 3. Representative transverse sections through the neural tube of an almost
completely 'turned' embryo with about 20 pairs of somites present, isolated in the
evening on the 9th day of gestation. Sections stained with methylene blue. (A)
Section through the cephalic region just rostral to the otic pits and slightly distal to
the origin of the 1st branchial arches (arrowed). Some degree of dorsal and particularly ventral side to side apposition is apparent in the hindbrain region (h). Key:
f, forebrain; g, foregut. (B) Section through the hindbrain at the level of the otic pits
(arrowed). Note that the neural lumen is completely occluded. Key: f, foregut; 1,
first branchial arch; 2, origin of the second branchial arch. Bar = 300 jjm. (C) Section
through the 'thoracic' region at the level of the distal one-third of the heart. The
ventral two-thirds of the neural lumen is occluded at this level. Key: a, atrium, prior
to division into right and left sides; v, ventricle. (D) Section through embryo at the
level of the two horns of the sinus venosus (s). The neural tube is virtually completely
occluded at this level. (E) Slightly oblique section through embryo just above the
forelimb bud (arrow). The lumen in the ventral half of the proximal region of the
neural tube (upper) is almost completely occluded, while in the distal region (lower)
the lumen, though narrow, is completely patent. (F) Section through embryo just
below the forelimb bud. The middle two-thirds of the neural tube shown here
represents the occluded proximal ventral segment of the lumen illustrated in E
above. The paraxial blocks of somites are clearly seen at this level.
Fig. 4. Representative transverse sections through the neural tube of an embryo
with approximately 25 pairs of somites present, isolated at about midday on the 10th
day of gestation. Sections stained with methylene blue. (A) Section through foreand hindbrain regions of embryo at the level of the first branchial arch. Key: f,
forebrain; h, hindbrain; 1, first branchial arch; t, tail region. (B) Section through
'thoracic' region at the outflow of the heart. This section also passes through the
hindbrain at the level of the otocysts. Key: o, otocyst, b, bulbus cordis; v, ventricle.
(C) Slightly oblique section through embryo at the level of the forelimb bud (arrow).
(D) Higher magnification view of neural tube in hindbrain region at the level
illustrated in A above. A considerable degree of side to side apposition is apparent
in the ventral third of the neural tube. Note that the notochord is adherent to the
endodermal lining of the oropharynx at this level (arrow). (E) Neural tube in hindbrain region at level illustrated in B above. The middle segment of the neural lumen
is completely occluded. (F) Neural tube proximal to the forelimb bud at level
illustrated in C above, bar = 200 jum. The majority of the ventral half of the neural
lumen is completely occluded. (G) Neural tube distal to the forelimb bud at level
illustrated in C above. (H) Neural tube in lower trunk region at level illustrated in
A above. (I) Neural tube in lower trunk region just proximal to the caudal
neuropore.
217
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M. H. KAUFMAN
Fig. 3. For legend see p. 217.
Neural luminal occlusion in mouse embryos
h
B
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Fig. 4. For legend see p. 217.
219
220
M. H. KAUFMAN
segment extended for a short distance both rostrally and caudally from the level
of the forelimb bud. This, and the previous embryo, appeared externally to be
at an almost identical stage of development, but the extent and overall pattern
of apposition and luminal occlusion in these two embryos were obviously quite
dissimilar. The pattern seen in this, the last embryo in this series, however,
represents the more typical situation observed at this period of embryogenesis.
The appearance of the neural tube in this embryo is illustrated in Fig. 5.
Of three plastic-embedded embryos examined that had been isolated early on
the 11th day of gestation, no evidence of occlusion, or even of close side-side
apposition was observed.
Reference to the author's collection of paraffin-embedded material covering
the period studied, however, would appear to suggest that the incidence of
luminal occlusion observed in embryos fixed on the 9th and 10th days is extremely low: only 3 out of 58 embryos had evidence of partial luminal occlusion. In all
three cases, the embryos had about 25 pairs of somites present. Only a short
partially occluded segment was evident, being located at the level of the forelimb
buds. No evidence of partial occlusion was observed in the material isolated at
earlier stages of embryogenesis.
Out of 34 paraffin-embedded embryos isolated throughout the 11th day, 3
showed a limited degree of partial luminal occlusion. In all of these embryos,
only a short segment of the neural tube appeared to be involved, being located
either proximal to the level of the hindlimb bud, at the level of the hindlimb bud,
or between the fore- and hindlimb buds.
Somewhat surprisingly, out of five 12th day embryos examined, two had
evidence of partial luminal occlusion with the zone of apposition being located
in one embryo at the level of the hindlimb buds, and in the second embryo,
between the fore- and hindlimb buds. In all but one of the eight paraffinembedded embryos in which a zone of side-side 'fusion' was present, the lumen
was patent dorsal and ventral to the site of apposition. In only one embryo was
the dorsal half of the canal completely occluded. Fixation of embryos for 4h in
Susa produced considerably less evidence of shrinkage artefacts, particularly in
the more advanced groups studied than 12-24 h fixation in full-strength or halfstrength Bouin solution.
It is of interest that a section through the neural tube of a rat embryo with
13-20 pairs of somites present, at approximately the mid- to high-thoracic level,
appears to demonstrate that complete neural tube occlusion also occurs in the rat
embryo at this site (see Figure 1, E, Freeman, 1972). No additional information
on the extent or overall pattern of neural tube occlusion observed in this species
is, however, available.
2. Ultrastructural appearance of neuroepithelial cell surfaces at sites of close
cell-cell apposition
The observations presented in this section are largely derived from an analysis
Neural luminal occlusion in mouse embryos
221
Fig. 5. Representative transverse sections through the neural tube of a second embryo with approximately 25 pairs of somites present, isolated at about midday on the
10th day of gestation. Sections stained with methylene blue. (A) Section through the
'thoracic' region at the level of the distal one-third of the heart. Key: h, distal region
of hindbrain; f, foregut; a, atrium; prior to division into right and left sides; v,
ventricle; t, mid-tail region. (B) Section through embryo at the level of the forelimb
bud. Canalization of the midgut is just visible (between arrows). Bar = 500/mi. (C)
Section through embryo distal to the forelimb bud. (D) Higher magnification view
of neural tube at similar level to that illustrated in A above. (E) Neural tube proximal
to the forelimb bud at level illustrated in B above. (F) Neural tube in the lower trunk
region just proximal to the segment illustrated in C above.
EMB78
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M. H. KAUFMAN
of the electron micrographs taken at various locations along the neural axis from
the embryo illustrated in Fig. 4. Other embryos at slightly different stages of
development (both more and less advanced) were also examined, but the
representative sections from this embryo serve to illustrate the principal
ultrastructural features observed at the neuroepithelial cell surface in embryos
in which occlusion of the neural lumen is taking place.
In locations where cellular contact had been made across the midline, a narrow
luminal slit was still evident. Initial contacts were usually established between
relatively small or occasionally quite large cellular protruberances. Characteristically, the majority of the apposing cell surfaces were either completely flattened or had a few small undulations (see Fig. 6A, B). Even the occasional small
cellular protrusion observed appeared to have a relatively wide cross section (see
Fig. 6A). At locations of very close apposition (e.g. see Fig. 6C), only a few areas
where minimal 'point' contact was established were generally seen. At the cell
surface, the occasional presence of coated pits was noted, while in the subcortical
zone, small numbers of coated vesicles were also apparent.
In areas where extensive cell-cell contact had been established, only small
pockets of luminal fluid remained between these sites of neuroepithelial cell-cell
'fusion'. Despite a detailed search along lengthy stretches where the lumen was
completely occluded (see Fig. 6D-E), no convincing evidence of junctional
complexes linking the two sides was seen, even though the apical complexes
between adjacent neuroepithelial cells were clearly apparent.
Cellular contact was generally established between two non-dividing cells, but
was also not uncommonly observed between.a non-dividing and a dividing cell
(see Fig. 6C-D).
In the ruthenium-red-treated group, a thin layer of positively staining material
was usually apparent on the neuroepithelial cell surface in regions of close
cell-cell apposition, and extended distally until sites of complete neural tube
occlusion were encountered. In those embryos in which there was complete
Fig. 6. Transmission electron micrographs illustrating appearance of
neuroepithelial cell surfaces at sites of cell-cell apposition and luminal occlusion.
These are representative thin sections from various sites along the neural axis of the
embryo illustrated in Fig. 4.
(A) and (B) Apposing neuroepithelial surfaces bridged by relatively large cellular
protrusions. Between these sites of initial contact, the cell surfaces are either completely flattened or only slightly undulating. Both micrographs X2600. (C) Extensive
region of close apposition in which a narrow luminal channel is still visible. Note the
occasional presence of coated pits (arrowheads) and coated vesicles (arrows). X3700.
(D) Extensive regions in which the neural lumen is completely obliterated, interspersed with small lacunae containing 'spinal' fluid. x3100. (E) Higher magnification
view of region in which the neural lumen is completely obliterated. Note the presence
of coated pits and vesicles with granular contents (arrowed) in close proximity to the
neuroepithelial cell surfaces, and the absence of junctional complexes between the
apposed neuroepithelial cell surfaces. In this, as in A-D above, apical junctional
complexes are observed between adjacent neuroepithelial cells, x 12 500.
Neural luminal occlusion in mouse embryos
223
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r
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•
224
7A
M. H. KAUFMAN
Neural luminal occlusion in mouse embryos
225
occlusion of the neural lumen, however, the neuroepithelial cell surfaces in nonoccluded regions just distal to the initial sites of occlusion generally remained
unstained, presumably because the ruthenium red failed to penetrate beyond the
occluded segment. Examples of electron micrographs of the neuroepithelial cell
surface in ruthenium-red-stained and unstained control material are presented
in Fig. 7.
DISCUSSION
It seems evident from this and previous experimental and descriptive studies
on neural tube occlusion (chick: Desmond & Jacobson, 1977; man: Desmond,
1982) that there are considerable species differences in the overall pattern of
neuroepithelial cell apposition and luminal occlusion along the spinal axis. The
underlying morphogenetic processes which eventually bring about this
phenomenon are obviously rather complex, and presumably involve an interplay
between a series of morphological changes which must occur within the cellular
components of the neural tube, and extrinsic cellular and extracellular 'forces'
which act on the neural tube from without (see, for example, Schroeder, 1971;
Karfunkel, 1974). Once medial migration of the walls of the neural tube has been
initiated, close apposition and eventual, albeit transient, 'fusion' may be the
inevitable consequence. It is possible that the first contact may be established
across the midline by the interdigitation of relatively small diameter projections
which protrude from the surfaces of the many neuroepithelial cells whose apices
abut on the spinal lumen. The initial contact and adhesion may in addition be
facilitated by the presence of viscous ruthenium-red-positive extracellular
material at the luminal surface of these cells.
An increase in ruthenium-red-positive material has been demonstrated along
apical neural fold borders and on the overlying ectoderm cells in regions immediately prior to and at the time of neurulation (Moran & Rice 1975; Sadler,
1978), and its removal at this time can interfere with neural tube closure (Lee,
Sheffield, Nagele & Kalmus, 1976; O'Shea & Kaufman, 1980). The presence of
carbohydrate-rich surface coat material is also observed along prospective zones
of fusion in the palate (Greene & Kochhar, 1974; Pratt & Hassell, 1975) and
during fusion of the medial and lateral nasal processes (Gaare & Langman, 1977;
Fig. 7. Unstained transmission electronmicrographs from two embryos with about
15-20 pairs of somites present to illustrate the presence of extracellular material at
the surface of neuroepithelial cells in sites of close cell-cell apposition. Both embryos were isolated at about 6 p.m. in the evening on the 9th day of gestation. (A)
Ruthenium-red-stained section showing the presence of a thin coating of positively
stained material at the neuroepithelial cell surface which apparently fails to
penetrate beyond the apical junctional complexes between adjacent cells. x6100.
(B) Higher magnification view of ruthenium-red-positively staining material at the
neuroepithelial cell surface, x 14 250. (C) Appearance of neuroepithelial cell surface
in unstained control embryo, x 14 500.
226
M. H. KAUFMAN
Smuts, 1977). In all of these locations, a decrease in the distribution of surface
macromolecules is observed after fusion.
Possibly slightly later, after initial side-side contact has taken place, the cells
with mound-like and flattened surfaces become more closely apposed, and allow
contact to be established over much more extensive, though still localized, areas.
Concomitant with the progressive increase which occurs in the surface area of
contact, the luminal volume necessarily decreases.
The underlying mechanism(s) which eventually lead to the complete occlusion
of the spinal lumen is still far from clear, though the absence of large numbers
of pinocytic vesicles in the subcortical region of the neuroepithelial cells tends
to suggest that absorption of the luminal fluid probably plays only a minor role
in its removal from this site. The presence of moderate numbers of coated
vesicles with granular contents in the subcortical zone and coated pits, which may
well be their precursors (see Pratten, Duncan & Lloyd, 1980), at the cell surface,
may be indicative of their role — possibly facilitating the removal of excess extracellular matrix material at the fusion site - during the final stages of apposition
and cell-cell adhesion.
Curiously, despite the presence of obvious junctional complexes between the
apical zones of adjacent neuroepithelial cells, no convincing complexes of any
type could be discerned at the fusion interface. While this does not unequivocally
exclude the possibility that some type(s) of specialized complexes are in fact
formed, clearly other techniques e.g. freeze fracture analysis, would be required
to demonstrate them.
In the mouse, unlike the situation in the human embryo (see Desmond, 1982),
since apposition and fusion is first apparent in embryos with about 10 pairs of
somites when both the rostral and caudal neuropores are still widely open, this
would seem to be evidence in favour of the hypothesis that at least at this
relatively early stage of embryogenesis most of the luminal fluid is probably
displaced caudally or cranially (into the amniotic cavity) rather than resorbed
locally. However, once the rostral neuropore, in particular, has closed (for
timing, see Geelen & Langman, 1977; Kaufman, 1979), and complete luminal
occlusion occurred some distance caudally, it seems likely that the production
and only limited resorption of cerebrospinal fluid would facilitate the dilation of
the brain and optic vesicles. While a situation similar to this appears to occur in
man and in the chick embryo, this is obviously a considerable oversimplification,
as in the majority of mouse embryos, for example, the neural lumen becomes
patent along its entire length just before the caudal neuropore eventually closes
(for timing, see Copp, Seller & Polani, 1982). Presumably, once 'cerebral' dilation has been initiated, the equalization between the CSF pressure and the
amniotic fluid pressure - which occurs after the lumen becomes patent along its
entire length, and remains thus at least until the caudal neuropore closes - does
not appear to be detrimental in any way, at least in the mouse.
While the above account is undeniably somewhat speculative, at least as far
Neural luminal occlusion in mouse embryos
227
as providing a detailed picture of the underlying mechanism of cell-cell fusion
in this location, it does appear to confirm the contention that neural luminal
occlusion probably plays an important morphogenetic role in the development
of the vertebrate nervous system.
Apart from answering certain questions regarding the timing and overall pattern of events in the mouse, this study raises other important questions. For
example, it would be of considerable interest to know whether chemical messengers play a role at any stage in guiding the two morphologically indistinguishable
neuroepithelial cell surfaces together. Equally, an analysis of the events occurring when the two sides separate once more late on the 10th or early on the 11th
day (in the mouse) would be instructive. Similarly, descriptive and experimental
observations on neuroepithelial cell-cell apposition and luminal occlusion in
other vertebrate species might enable both the full significance of this
phenomenon and its evolutionary history to be established.
This work was supported by a grant from the National Fund for Research into Crippling
Diseases. I thank Mr. J. Skepper and Mr. M. Wombwell for their expert technical assistance,
and Mr. J. Skepper additionally for his invaluable advice on the interpretation of the electronmicrographs.
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{Accepted 24 August 1983)