J. Embryol. exp. Morph. 88, 231-247 (1985)
231
Printed in Great Britain © The Company of Biologists Limited 1985
The ontogenesis of cranial neuromeres in the rat
embryo
II. A transmission electron microscope study
FIONA TUCKETT AND GILLIAN M. MORRISS-KAY
Department of Human Anatomy, University of Oxford, South Parks Road,
Oxford OX1 3QX, U.K.
SUMMARY
The morphogenesis of rhombomeres (neuromeres) caudal to the preotic sulcus during
neurulation in rat embryos is described. A model is proposed to explain the development of the
characteristic neuromeric sulci and interneuromeric gyri based on the cytoskeletal elements and
the kinetic behaviour of the neural epithelium.
Evidence obtained from a study of control, cytochalasin D-treated and colchicine-treated
embryos, at the electron microscopic level, supports the proposed model. The longitudinally
expanding cranial neural epithelium bulges between microtubule blocks present within the
interneuromeric gyri, causing a bulge to develop along the line of least resistance, away from the
microfilament-rich luminal border of the neuromeric sulcus region.
INTRODUCTION
Neuromeres have been observed as a segmental arrangement of sulci and gyri
within the early neural tube of all vertebrate embryos. Their characteristic
morphology is illustrated in Fig. 1. This study addresses the question of how the
sulci and gyri develop from an initially straight neural epithelium, and how their
structure is maintained.
Observations on a variety of vertebrates (chick, fish, urodeles) led Kallen (1956)
to propose that the formation of neuromeric sulci was the result of mitotic
patterning within the neural epithelium. His model is based on transverse sections
of the neural epithelium during the formation of a neuromere, which he termed a
'proliferation centre'. According to Kallen, the furrowing of the luminal border
results from the redistribution of neuroepithelial cells, due to the mitotic
patterning, while the bulging at the basal border is brought about by the formation
of proportionally more cells in the centre of the region, where the mitotic activity
is highest. This interpretation relies strictly on the kinetics of the cells within the
neuromere and their movement across the epithelium between the basal and
luminal borders, and takes no account of the possible migration of cells within the
Key words: rat embryo, neuromeres, neural tube, cytochalasin D, colchicine, cranial
neuromeres.
232
F. TUCKETT AND G. M. MORRISS-KAY
neural epithelium (along the length of the embryo), or of possible regulation of
shape through a periodicity of structural components.
We have suggested that a localized increase in the mitotic index is the initial
means of identifying a neuromere, and once the neuromere is expressed
morphologically as a surface sulcus the mitotic index returns to its basal level
(Tuckett, 1984; Tuckett, Lim & Morriss-Kay, 1985). Kallen's 'proliferation centre'
appears to refer to the localized concentrations of mitotic activity which we have
observed in both transverse and coronal section.
This study investigates the possibility of an alternative hypothesis for the
development of neuromeres which embraces both the mitotic patterning
(orientation of the mitotic spindle axes) and the cytoskeletal components of the
neural epithelium. This hypothesis is illustrated diagrammatically in Fig. 2. It
proposes that growth of the neural tube is generated longitudinally by cell division
in this plane, but that elongation is prevented by the fixed nature of the tube
within the embryo. In consequence, the lengthening epithelium bulges outwards
Fig. 1. A light micrograph of a coronal section, along the neural tube of a 16-somitestage embryo, at the level of the otocysts (o). The lumen of the neural tube is narrowed
caudally (to the right). The neural epithelium (nep) is folded into a series of sulci and
gyri which is characteristic of neuromeres. The centre of each neuromere, the sulcus, is
arrowed; mitotic figures with their darkly staining chromatin are generally localized
within the sulci. The interneuromeric junctions (gyri) are marked with asterisks (*).
Cranial mesenchyme (mes) underlies the neural epithelium. Alcian-blue-stained wax
section (5/mi) from Tuckett (1984). Scale bar represents 40/mi.
Ontogenesis of cranial neuromeres studied by TEM
A
• nep
B
mes
233
C
mes
m
*
b
gyrus
t
t
1™
sulcus
gyrus
sulcus
•
• b
•
•
Fig. 2. Schematic diagram representing coronal sections through a similar part of the
myelencephalon to that shown in Fig. 1. b, 'block'; nep, neural epithelium; mes,
mesenchyme; m, microfilaments. (A) Before generation of a neuromere. Growth of
the neural tube is generated by cell division within the longitudinal plane; however the
neural epithelium is fixed at various points along the tube, thus forming 'blocks' to tube
elongation. The luminal border is rich in microfilament bundles which contract along
the line indicated by the arrows. (B) After generation of a neuromere. The lengthening
epithelium has to bulge either outward, or inward along the line of least resistance. The
microfilament-bound luminal border prevents inward bulging of the neural epithelium,
and thus an outward bulge into the more easily deformable mesenchyme results. Due
to the structural integrity of the neural epithelium and as a consequence of the outward
bulging, a sulcus develops at the luminal border. The blocks to tube elongation are
localized at the interneuromeric junctions (gyri). (C) Modification of the model
following the studies reported in this paper. The gyrus region consists of a fan-shaped
arrangement of cells with microtubule groups aligned perpendicular to the luminal
border.
into the adjacent mesenchyme since the basal border (lacking in microfilaments) is
more easily deformable than the microfilament-bound luminal border. The
repeating pattern of sulci and gyri results from the presence of a series of blocks to
deformation (gyri) between the bulging regions (sulci). This hypothesis is based on
evidence from a previous study (Tuckett & Morriss-Kay, 1985), as follows: (i) cell
division within the neural epithelium at relevant stages is predominantly (98%)
orientated with the mitotic spindle axis parallel to the long axis of the embryo, so
as to increase the length of the neural epithelium; (ii) rostral to the preotic sulcus
cells generated in the midbrain and upper hindbrain appear to flow rostrally to
provide an extrinsic source of cells for the rapidly expanding forebrain, suggesting
the existence of a block to cell movement at or close to the preotic sulcus. Since we
are now suggesting a series of blocks to cell movement between neuromeric sulci,
the present hypothesis relates only to the clearly segmented caudal metencephalon
and myelencephalon, i.e. the region caudal to the preotic sulcus.
234
F. TUCKETT AND G. M. MORRISS-KAY
To examine the feasibility of this model for the development of neuromere
morphology, and to investigate the morphogenetic nature of the proposed blocks
to cell movement between neuromeres, transmission electron microscopy was
employed. The function of microtubules and microfilaments in maintaining the
morphology was examined by treating embryos in vitro with colchicine which binds
to tubulin and prevents microtubule assembly, and cytochalasin D which inhibits
microfilament assembly; each may also disrupt existing microtubules and microfilaments respectively.
MATERIALS AND METHODS
Wistar strain rat embryos were explanted in Tyrode's saline on day 10 of pregnancy (day of
positive vaginal smear = day 0). A total of 39 embryos was used. In the 34 embryos which were
subsequently to be cvltured only Reichert's membrane was opened; in the remaining 5 embryos
the extraembryonic membranes were removed before fixation. At the time of fixation the
embryos had between 12 and 16 somite pairs.
Whole embryo culture
Embryos were cultured at a temperature of 38 °C in 60 ml Pyrex glass bottles containing 2-5 ml
immediately centrifuged, heat-inactivated rat serum (Steele & New, 1974), 2-5 ml Tyrode's
saline and lOjul penicillin/streptomycin (5000 i.u. ml"1 and SOOOjUgmP1). The bottles were
gassed with 5 % CO2:5 % O2:90 % N2 (New, Coppola & Cockroft, 1976a,b) prior to sealing. The
bottles were continuously rotated at 30r.p.m. At the end of the culture period, the embryos
were thoroughly washed in Tyrode's saline and the extraembryonic membranes were removed
before fixation.
Cytochalasin D-treated embryos
A stock solution of l-Omgml"1 cytochalasin D (Sigma, London) was prepared in a 10%
solution of dimethylsulphoxide (DMSO; Sigma, London) and stored at -20°C. 7-5/il of the
stock solution was added to the culture medium (final concentration of 0-15 fig ml"1). Ten
embryos at the 11- to 13-somite stage were cultured in the presence of cytochalasin D for 2h.
Five control embryos were cultured in the presence of 7 • 5 /A of a 10 % solution of DMSO for 2 h.
Colchicine-treated embryos
14 embryos at the 11- to 13-somite stage were treated in culture for 3 h with colchicine (Sigma,
London) which had been added to the culture medium so as to produce afinalconcentration of
0-2 jug ml" 1 . This concentration was found to cause a sufficiently high mitotic arrest without
adversely affecting the gross morphology of the embryo; the duration of colchicine treatment
was less than half the cell-cycle time which we have determined previously (Tuckett & MorrissKay, 1985), and thus only a small number of cells were observed to be arrested at the metaphase
stage. Subsequent treatment was the same as for the untreated, control embryos. Five embryos
were cultured in unsupplemented culture medium for 3 h.
Electron microscopy
After removal of the membranes, embryos were fixed in 2-5 % cacodylate-buffered osmium
tetroxide, washed and dehydrated through graded alcohols before embedding in Spurr resin.
Thin sections of 80-90 nm thickness were collected on copper grids and double stained with
uranyl acetate at 40°C for 45 min and lead citrate for 5 min at room temperature.
Ontogenesis of cranial neuromeres studied by TEM
235
Light microscopy
Semi thin sections of 1 /an thickness were cut immediately adjacent to the thin sections. These
sections were collected on glass slides and stained with either equal parts of 1 % methylene blue
and 1 % azure II in 1 % borax, or 1 % toluidine blue in 1 % borax.
RESULTS
Control embryos
No differences were observed between the control cultured and the noncultured embryos, and thus the subsequent use of the term 'control' will apply to
either of these two states.
In control embryos the neural epithelium displayed a typical pseudostratified
epithelial arrangement. All cells had luminal border attachments and those cells
not in mitosis also had basal border attachments. The characteristic morphology of
the neuromere was clearly seen at the light microscopic level (Fig. 3A). Within the
sulcus the cells had narrow necks and overlapping apices which were more easily
visible at the electron microscopic level (Fig. 4A); the narrow cell apices were
associated with the presence of apical microfilament bundles which were aligned
parallel with the cell surface between the intercellular junctions (Fig. 4B). At the
interneuromeric junction region (gyrus), the luminal border had a somewhat
different morphology. The cell apices were broader than in the sulcus region,
although some overlapping did still occur (Fig. 5A); microfilament bundles were
not continuous along the luminal border but were associated laterally with the
intercellular junctions (Fig. 5B). At the basal border of the outward bulging neural
epithelium, the cell processes formed a broad attachment with the basement membrane (Fig. 4C) whereas at the interneuromeric junction, the basal attachments of
the inward bulging neuroepithelial cells were narrow (Fig. 5C). At high power
these narrow basal cell processes were shown to contain microtubules (Fig. 6A,B)
which were aligned perpendicular to the luminal border. The microtubules had a
diameter of 25 nm and their orientation within the gyrus region as a whole displayed a characteristic fan shape, which was reflected in the arrangement and
orientation of cells within the gyrus. The fan-shaped cellular arrangement can be
seen at the light microscopic level in Fig. 3A. The basement membrane and
mesenchyme formed a short angled cleft between adjacent neuromeres (Fig. 5C).
Cytochalasin D-treated embryos
The shape and organization of the epithelium of cytochalasin D-treated
embryos differed from that of the control embryos as follows. There was a marked
thinning of the epithelium which was noticeable at the light microscopic level (Fig.
3B); this was probably associated with the change in cellular morphology which
was more noticeable at the electron microscopic level (Fig. 7A,B). The cells all
had broad apices which protruded into the lumen; there was no difference in the
apical appearance of sulcus and gyrus cells. The sulci and gyri were more gently
236
F. TUCKETT AND G. M. MORRISS-KAY
B
Ontogenesis of cranial neuromeres studied by TEM
237
curved, with the inward and outward bulges of a similar size; there was no sharply
angled basal cleft formation at the inward bulge.
Within the outward bulging epithelium (sulcus) microtubule groups were
present perpendicular to the cell surface, though not in a clearly organized fanshaped orientation. The microtubule groups were more diffuse in nature, and
could not be resolved at the lowest magnification at which they are discernible in
control embryos. Fig. 7C illustrates the diffuse nature of the microtubule groups
following cytochalasin treatment, and may be compared with Fig. 6A,B which is
taken at the same magnification and illustrates the more ordered arrangement of
microtubules in control embryos. Assuming that their position has not been
affected by the cytochalasin treatment, it would seem that the original sulci (Fig.
3A) have everted and that they now appear as gyri (Fig. 3B); and similarly the
microtubule groups originally present within the gyri (Fig. 3A) are now present
within the sulcus (Fig. 3B).
Two other features which characterized the cytochalasin D-treated embryos
were the increased number of cytoplasmic vacuoles and intercellular spaces
compared with the control embryos.
Colchicine-treated embryos
At the light microscopic level a number of cells was seen to be arrested at the
metaphase stage of mitosis, and those cells which were not arrested were
characteristically beginning to round-up (Fig. 3C). These changes in cellular
morphology resulted in narrowing of the neural epithelium but not to the same
extent as following cytochalasin D treatment. Although the cells were more
rounded than their control counterparts there was a narrowing of cell apices within
the sulci, suggesting that microfilaments were contracting along the luminal border
at the centre of a neuromere (Fig. 8A). The apical border of the gyrus cells (Fig.
8B) was arranged in a similar manner as in the control embryos (Fig. 5A). The
most pronounced effect of colchicine treatment on neuromere structure was the
lack of microtubule groups between adjacent neuromeres; the fan-shaped gyrus
cells lost their elongate form although the unarrested cells maintained their apical
and basal contacts. As a result of the loss of cell elongation, the basal cleft between
Fig. 3. Light micrographs of semi-thin plastic sections showing the morphology of the
neural epithelium and neuromeres in (A) control; (B) cytochalasin D-treated; and (C)
colchicine-treated embryos. Scale bar represents 20jum. (A) The form of sulci and gyri
is clearly seen. Within the sulcus the cells have narrow necks and there is a ruffling of
the apical border which is associated with the overlapping of the cell apices (Fig. 4A).
The fan-shaped arrangement of cells within the interneuromeric junction (gyrus)
region is indicated by the arrows. (B) In this cytochalasin D-treated embryo the pattern
of sulci and gyri is much smoother and more gently curved than in Fig. 3A. The group
of microtubule-rich cells is indicated by the arrows. There is approximately a 50 %
reduction in the thickness of the epithelium as a result of cytochalasin treatment. (C)
The rounded cells with darkly staining chromatin are cells arrested by colchicine at the
metaphase stage of mitosis. A prominent feature of this micrograph is the deep basal
cleft between adjacent neuromeres. Cell shortening has resulted in a thinning of the
epithelium.
238
F. TUCKETT AND G. M. MORRISS-KAY
Fig. 4. For legend see p. 240
Ontogenesis of cranial neuromeres studied by TEM
Fig. 5. For legend see p. 240
239
240
F. TUCKETT AND G. M. MORRISS-KAY
adjacent neuromeres deepened significantly (Figs 3C and 8C; compare with Figs
3A and 5C of the clefts of a control embryo).
As with the cytochalasin D-treated embryos, there were quite large intercellular
spaces but these were more likely to be associated with the rounding-up of the
cells and concomitant loss of junctional contacts rather than a direct effect on
intercellular junctions per se.
DISCUSSION
The electron micrographs of the control embryos illustrated here show that in
the coronal plane there is an alternating pattern of cells with characteristic
cytoskeletal components, correlated with the pattern of sulci and gyri of the
neuromeres. In the sulci, the apical regions of the cells show narrow necks with
overlapping apices, and a continuous line of microfilament bundles in association
with intercellular junctions, parallel to the epithelial surface. This line becomes
discontinuous in the interneuromeric (gyrus) regions, where fan-shaped groups of
cells form the angle between adjacent sulci. These cells have long narrow basal
regions and broaden towards the apical surface, and are rich in microtubules
orientated perpendicular to the surface (i.e. along the length of the cells).
Microtubules and microfilaments have long been recognized as playing an
essential role in cell shape, cell movement, and cytoplasmic organization. The
results of the colchicine and cytochalasin D experiments reported here suggests
that they play important roles in maintaining the shape of neuromeres. Reversal of
neuromere shape from a series of sulci between the microtubule-rich interneuromeric regions to a series of gyri was observed in embryos cultured in
Fig. 4. Electron micrographs from a 16-somite-stage control embryo. (A) At the
luminal border of the neuromeric sulcus the cells have narrow necks and overlapping
cell apices. Microfilament bundles running between the cell junctions are seen at
higher magnification in Fig. 4B. Scale bar represents 5/im. (B) A higher power
micrograph of the neuromeric sulcus showing the apical microfilament bundles (m)
running parallel with the luminal border and between the cell junctions (arrowed).
Scale bar represents ljum. (C) The basal border of the outward bulging neural
epithelium, showing the broad cell attachments which are associated with this region.
Scale bar represents 5jum.
Fig. 5. Electron micrographs from a 16-somite-stage embryo. (A) At the apical border
of the gyrus, the broad cell apices protrude into the lumen. Cell junctions are not so
prominent as in Fig. 4A. Scale bar represents 5 jum. (B) A high-powered micrograph of
the gyrus showing the broadened cell apex, and microfilaments associated laterally
with the cell junctions (arrowed). Scale bar represents ljum. (C) At the interneuromeric junction a short, angled basal cleft develops. The basal cell attachments
become narrow near the angle of the cleft. Scale bar represents 5 jum.
Fig. 6. (A) Control embryo, gyrus region, showing microtubules. These are aligned
perpendicular to the luminal border, close to the cell membrane. This micrograph
represents the lowest magnification at which microtubules were clearly resolvable in
control embryos. Scale bar represents 0-4 pm. (B) Group of microtubules at higher
magnification. Scale bar represents 0-2 ptm.
nesis of cranial neuromeres studied by TEM
Ontogenesis
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Fig. 6
242
F. TUCKETT AND G. M. MORRISS-KAY
•
•
• •. r
Fig. 7. For legend see p. 244
«
Ontogenesis of cranial neuromeres studied by TEM
Fig. 8. For legend see p. 244
243
244
F. TUCKETT AND G. M. MORRISS-KAY
medium containing cytochalasin D. This experiment clearly demonstrates the role
of apical microfllament bundles in maintaining both the shape of the sulcus cells
with their narrow neck regions and overlapping apices, and the shape of the sulcus
region as a whole. Curvature generated and maintained by the microfllament
bundles is expressed as an outward bulging of the neural tube into the adjacent
mesenchyme, which must enable this to occur through its own deformability.
Microfllament bundles orientated parallel to the neuroepithelial surface play an
essential role during neurulation, either by generating curvature, as interpreted by
Baker & Schroeder (1971) or by thickening and contracting the neural plate, as
interpreted by Jacobson & Gordon (1976). They are present in the cranial neural
epithelium of rat embryos at the time of epithelial curvature (Morriss & New,
1979), and their disappearance from the cytoplasm as a result of cytochalasin
treatment corresponds to the loss of neuroepithelial curvature (Morriss-Kay &
Tuckett, 1985).
In the presence of colchicine, the fan-shaped groups of gyrus cells lost their
elongate form but maintained their apical contacts, causing the normally shallow
indentations of the basal neuroepithelial surface to deepen considerably. The cells
lost their polarized shape, becoming rounded. Microtubules have been shown to
play an essential role in the establishment and maintenance of cell polarity and cell
shape in a variety of cell types including the amphibian egg after fertilization
(Kirschner, Gerhart, Hara & Ubbels, 1980; Gerhart et al. 1981) and in the
extension of neurites and maintenance of neurite structure (Seeds, Gilman,
Amano & Nirenberg, 1970; Kirschner, 1982; Fulton, 1984). The present
experiments suggest that while microtubules are not essential for the maintenance
of the periodic neuromere structure, they provide a series of stiffened annular
regions on which the adjacent microfilament-rich sulci can contract; these annular
regions also form a morphological break between the cells of adjacent sulci, and
the fan shape of the cell groups provides the necessary compensatory curvature
between sulci.
Fig. 7. Electron micrographs of a 14-somite-stage cytochalasin D-treated embryo. (A)
At the luminal border of the outward bulging neural epithelium, the cell apices have
expanded and now protrude into the lumen. Microfllament bundles are absent.
Compare with Fig. 4A. Scale bar represents 5/an. (B) At the luminal border of the
gyrus, the cell apices have also broadened; compare with Fig. 5A taken at the same
magnification. Scale bar represents 5 jum. (C) This is the lowest magnification at which
groups of microtubules can be resolved. The microtubules are present within the group
of cells situated in the outward bulging epithelium indicated in Fig. 3B. Compare with
Fig. 5B. Scale bar represents 0-2 /an.
Fig. 8. Electron micrographs of a 16-somite-stage colchicine-treated embryo. (A) At
the apical border of the neuromeric sulcus, the cells have a similar appearance to the
control embryos (Fig. 4A) with narrow necks and overlapping cell apices. Scale bar
represents 5 jum. (B) The gyrus cells have broad apices which protrude into the lumen;
compare with Fig. 5A of a control embryo. Scale bar represents 5 jum. (C) At the deep
basal cleft of the interneuromeric junction, some of the basal attachments have been
lost as a result of cell shortening within the fan-shaped region, and consequently the
remaining basal attachments are broader than in control embryos (Fig. 5C). Scale bar
represents 5
Ontogenesis of cranial neuromeres studied by TEM
245
The mechanism underlying the generation and maintenance of neuromeric
periodicity is not understood. It is likely to involve the adjacent primary
mesenchyme, since this is involved in regionalization of the brain at the time of
neural induction (Spemann, 1938, in amphibian embryos). The cranial
mesenchyme is itself organized into a segmental pattern of somitomeres, before
and during neuromeric development (Anderson & Meier, 1981; Meier & Tarn,
1982). Bernfield, Banerjee, Koda & Rapraeger (1984) have demonstrated that
during glandular morphogenesis, change in shape of the epithelium is governed by
the adjacent mesenchyme, via molecular remodelling of the basement membrane.
We have been unable to find evidence for any longitudinal periodicity of glycosaminoglycans in the neuromeric basement membrane or subsequent mesenchyme
(Tuckett, 1984); the possibility that other extracellular matrix components may
show some neuromere-related periodicity is currently under investigation.
Reflecting their segmental pattern, neuromeres have a close correlation with the
pattern of subsequent development of cranial motor nerve nuclei, in general in a
one-to-one relationship (Adelmann, 1925; Bartelmez & Dekaban, 1962; Vaage,
1969). This observation, taken together with the ultrastructural features reported
here, suggests that they may represent clonal compartments. Jacobson (1983), in a
study of cell lineage in late blastula-stage frog embryos, found groups of
blastomeres whose descendants contributed to specific compartments within the
brain and spinal cord which were determined at, but not before, the 512-cell stage.
In the rhombencephalon there were boundaries along the dorsal and ventral
midlines, a division into dorsal and ventral compartments, and a transverse
boundary separating the rhombencephalon from the mesencephalon (but not from
the spinal cord). Within compartments there was much mixing of cells. Assuming
that similar compartments are also determined in mammalian presumptive neural
plate cells, it would seem likely that mixing within compartments becomes further
restricted as progressively finer aspects of pattern are determined, and that
neuromeres themselves represent clonal compartments (each divided into four
quadrants as defined above). The fan-shaped cell groups of the gyms regions
would, according to this hypothesis, represent intercompartmental boundaries,
across which mixing cannot occur.
CONCLUSION
The experiments reported here provide evidence in support of the hypothesis
for neuromere development and maintenance as proposed in the introduction.
Outward curvature of the neuromeric sulci depends on the presence of a line of
apical microfilament bundles, in the absence of which the direction of curvature is
reversed. The gyrus regions contain cells rich in microtubules which are required
to maintain their fan-shaped organization as viewed in coronal sections. We have
no information on the mechanism underlying the periodic nature of the pattern,
but suggest that neuromeres represent semi-autonomous clonal compartments,
and that the gyrus cells provide a physical barrier to cell movement between
246
F. TUCKETT AND G. M. MORRISS-KAY
compartments as well as a skeletal framework for the support of the contracting
sulcus epithelium.
Thanks are due to Mr Martin Barker for technical assistance, and to Mr Tony Barclay for
photographic assistance. This study was supported by the Medical Research Council and the
University of Oxford.
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SEEDS,
(Accepted 28 February 1985)