J. Embryol. exp. Morph. 96,183-193 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
183
The histogenetic potential of neural plate cells
of early-somite-stage mouse embryos
W. Y. CHAN AND P. P. L. TAM
Department of Anatomy, Faculty of Medicine, The Chinese University of Hong
Kong, Shatin, NT Hong Kong
SUMMARY
The mesencephalic neural plate of early-somite-stage mouse embryos differentiated underneath the renal capsule to form mostly neural tissues together with other tissues some of which
were probably of neural crest cell origin. The capacity to form non-neural tissues such as skeletal
tissues and melanocytes was lost at about the 5-somite stage. The lateral areas of the plate
tended to form non-neural tissues more than the medial areas. The cephalic neural plate of
presomite head-fold-stage embryos differentiated extensively to form both ectodermal and
mesodermal tissues. However, upon completion of neurulation, the mesencephalic neuroepithelium of forelimb-bud-stage embryos gave rise to neural tissues only. Therefore there is a
progressive restriction in the histogenetic capacity of the mesencephalic neural plate during
neurulation and this could be attributed to the cellular commitment for neural differentiation
and the loss of the neural crest cells.
INTRODUCTION
The differentiation of embryonic germ layers is characterized by a progressive
restriction of the developmental capacity and the relocation of cells to their
definitive position in the foetal body. The embryonic ectoderm or epiblast of
mouse and rat egg cylinders is shown to be capable of extensive differentiation to
generate tissues belonging to the three definitive germ layers both in an embryonic
environment and in ectopic sites (Beddington, 1981,1983; Diwan & Stevens, 1976;
Levak-Svajger & Svajger, 1974; Skreb & Svajger, 1975; Skreb, Svajger & LevakSvajger, 1976). This extensive histogenetic potential of the embryonic ectoderm
remains unchanged until the late-primitive-streak stage and there is also no
detectable variation in the potency between the anterior (presumptive cephalic
region) and the posterior regions of the embryonic ectoderm (Beddington, 1983;
Skreb etal. 1976). A restriction in the histogenetic potential seems to have
occurred in presomite head-fold-stage rat embryos and when the cephalic neural
plate is grafted to ectopic sites, definitive endodermal (gut) tissues are absent in
the teratomas (Svajger & Levak-Svajger, 1974). The neural plate of early-somitestage rat embryos is still capable of producing mesodermal tissues in addition to
the neural derivatives but the partially closed neural tube of 10- to 12-somitestage embryos forms primarily neural tissues (Svajger, Levak-Svajger, KostovicKey words: neural plate, neural crest cells, early-somite stage, mouse embryos.
184
W. Y. CHAN AND P. P. L. TAM
Knezevic & Bradamante, 1981). Since the final composition of the graft is likely to
be related to the initial developmental capacities of the tissue (Svajger et al. 1981),
one possible interpretation of the change in histogenetic potential is that neural
plate cells become progressively more committed to the differentiation of neural
tissues during neurulation. However, the other possible factor contributing to this
restriction of potential may be the loss of certain 'mesodermal' cell types from the
neural plate at these stages. During the early stages of cephalic neurulation in the
mouse, groups of cells leave the lateral edges of the neural plate and these cells,
which show a positive staining reaction towards CPC-toluidine blue, are presumed
to be the neural crest cells (Nichols, 1981). The neural crest cells first emigrate
from the mesencephalic portion of the neural plate and are later seen leaving the
rhombencephalic region. A similar pattern of neural crest cell migration has been
described in the cranial region of rat embryos (Tan & Morriss-Kay, 1985). The
common features are the emergence of the neural crest cells from the cranial
neural plate well before the fusion of the neural folds and the early migration
of the mesencephalic crest cells. In the present study, we have examined the
histogenetic potential of the mesencephalic neural plate of early-somite-stage
mouse embryos with a specific attention on the change in the capacity of forming
mesodermal tissues such as cartilage, bone and pigment cells. These tissues are
presumed to be of neural crest cell origin in the craniofacial region of the
embryo (Bee & Thorogood, 1979; Jaenisch, 1985; Morriss & Thorogood, 1978;
Noden, 1983; Rawles, 1947). For a comparison, the cephalic neural plate of presomite head-fold-stage embryos and the neuroepithehum in the mesencephalon of
forelimb-bud-stage embryos were also studied.
MATERIALS AND METHODS
Random bred ICR and inbred C57BL strains female mice were paired with syngeneic males
and the presence of vaginal plugs was checked for occurrence of mating. Embryos were
recovered from the pregnant mice at 8-0 and 9-5 days/j.c. (the afternoon of the plug day = 0-5
dayp.c). According to the somite number, the embryos were placed into five groups: presomite
head-fold stage, 1- to 2-, 3- to 4-, 5- to 6- and 20- to 24-somite stages. At the presomite head-fold
stage, the flattened portion of the ectoderm anterior to the flexure (Fig. 1) was isolated by
dissecting with a pair of fine metallic needles. For the early-somite stages, the head region rostral
to the preotic sulci was taken (Figs 2, 3). In the forelimb-bud-stage embryos, the brain region
that is rostral to the otic capsule was used (Fig. 4). These embryonic parts were then treated with
a mixture of 0-5 % trypsin (Sigma, Type II) and 2-5 % pancreatin (Sigma, Grade III) in calciumand magnesium-free phosphate-buffered saline at room temperature for 10-15 min and the
enzymic digestion was stopped by transferring the tissues to PB1 medium containing 20 % heatinactivated foetal calf serum. The tissues were washed twice with fresh PB1 medium and were
further dissected. The open neural plate of the embryos was bisected sagittally along the neural
groove into two halves. At the presomite stage when the prospective brain areas were not yet
discernible (Fig. 1), the entire half-plate was used, whereas in early-somite-stage embryos that
showed a distinctive subdivision of brain regions, the mesencephalic neural plate was isolated.
The underlying mesoderm was removed by dissecting with electrolytically polished alloy
needles. The surface ectoderm was removed by cutting along the edge of the plate and the cut
was usually made more on the neural plate side (Figs 2, 3). The half-neural plate was then
bisected into medial and lateral portions (Figs 2, 3). The mesencephalon of the forelimb-budstage embryos was divided into dorsal and ventral portions (Fig. 4) which corresponded to the
original lateral and medial parts of the open neural plate.
Differentiation of neural plate cells
185
The tissue fragments were transferred by means of fine glass micropipettes underneath the
renal capsule of syngeneic male mice under light Nembutal (Sigma) anaesthesia. For each
recipient, fragments of neural plate were transferred to one kidney capsule and fragments of the
Fig. 1. The sagittal view of a presomite-stage embryo. The entire portion of the neural
plate rostral to the dotted line was isolated for enzymic treatment. Bar, 100 \im.
Fig. 2. The dorsal view of a 2-somite-stage embryo. The mesencephalic neural plate
was divided into medial portion (M) and lateral portion (L). Bar, 100jwrn.
Fig. 3. The dorsolateral view of a 4-somite-stage embryo, showing the subdivision of
the mesencephalic neural plate into medial portion (M) on the right side and lateral
portion (L) on the left half of the plate. Bar, 100 jum.
Fig. 4. The ventricular aspect of the brain of a 20-somite-stage embryo, showing the
two parts of the mesencephalon that were used for grafting. V, ventral portion; D,
dorsal portion. Bar, 100 fjm.
186
W. Y. C H A N AND P . P. L. T A M
mesoderm and surface ectoderm were transferred to the other capsule. The mice were killed two
weeks after the transfer and the grafts were fixed in Sanfelice fluid and processed for histology.
Paraffin wax sections of 7jum thickness were stained with haematoxylin and eosin. Selected
sections were stained with Bodian's method for nerve fibres and nerve endings (Luna, 1968).
Other grafts were fixed in half-strength Karnovsky fixative followed by 1 % osmium tetroxide
and embedded in Spurr resin. Thick (1-2 ^m) sections were stained with 1 % toluidine blue for
light microscopy. Some of the original preparations of neural plate were fixed in half-strength
Karnovsky fixative and processed for scanning electron microscopy using a JEOL JSM-35CF
electron microscope, while others were fixed in Sanfelice fluid and processed for routine
histology.
RESULTS
Fourteen dissected preparations of neural plates were examined microscopically. The basement membrane that normally associated with neuroepithelium
was lost and large intercellular clefts were seen in the loosened epithelium (Fig. 5).
No adhering mesodermal cells and surface ectoderm were seen in these preparations. For those preparations that were used for grafting experiments, a
preliminary histological examination was not possible but great care was taken to
scrutinize the preparations under the dissecting microscope for contaminating
Fig. 5. A scanning electron micrograph showing the basal aspect of the neuroepithelium of a 4-somite embryo after the enzymic treatment and dissection. The
basement membrane has been removed and no adhering mesodermal cells are seen.
Under light microscope (inset, H & E staining), the neuroepithelium of another
embryo with 4 somites is free of any surface ectodermal and mesodermal cells, bs, basal
surface. Bar, 50jum.
187
Differentiation of neural plate cells
tissues prior to transfer. Between 50 to 75 % of the neural plate fragments grew
into a recognizable mass under the renal capsule after two weeks, but grafts of
mesoderm and surface ectoderm developed poorly and few (10-30 %) of them
formed recognizable tissue masses. Apart from the presence of pigment cells in the
grafts derived from C57BL embryos, there was no other significant difference in
the tissue composition between the two strains used in this study and therefore the
results were pooled. Like the observations made in other studies on the ectoderm
of presomite head-fold-stage rat embryos, the mouse neural plate was capable to
differentiate into a large variety of ectodermal and mesodermal tissues (Table 1).
In the grafts of neural plate of other stages, the most prevalent tissues were those
normally found in the developing nervous system (Table 1). Groups of basophilic
Table 1. The tissue composition of grafts derived from the mesencephalic neural plate
and mesencephalon
Somite no.
No. grafted
No. developed
No. analysed
Presomite
*M:34
L:32
M:17
L:17
M:15
L:17
1-2
3-4
51
54
26
29
18
20
46
41
36
28
18
21
5-6
34
87
18
43
16
37
M:15
L:17
M:ll
L:15
M:3
L:6
M:5
L:ll
M:3
L:6
M:0
L:7
M:l
L:7
M:5
L:4
M:0
L:5
M:5
L:0
15
20
11
17
20
11
16
34
14
13
14
27
7
6
0
3
0
2
3
3
3
4
0
4
2
2
0
0
8
12
0
3
0
1
0
1
2
3
0
4
0
0
0
0
10
13
0
4
0
3
0
0
0
0
0
0
0
0
0
0
20-24
tV:39
D:102
V:24
D:44
V:22
D:31
No. of grafts containing:
Neuroepithelium
Mature grey
matter
Choroid plexus
and ependyma
Keratinized
epithelium
Hair and sebaceous
gland
Cartilage
Bone
Pigment cells
(C57BL only)
Adipose tissue
Striated muscle
* M and L: Medial and lateral portions of the neural plate.
t V and D: Ventral and dorsal portions of the closed neural tube.
V:22
D:31
V:15
D:16
V:ll
D:14
V:0
D:0
V:0
D:0
V:0
D:0
V:0
D:0
V:0
D:0
V:0
D:0
V:0
D:0
188
W. Y. CHAN AND P. P. L. TAM
cells were organized into a thick pseudostratified epithelium which lined the wall
of many cystic and tubular structures. A peripheral layer of cellular processes was
commonly seen on the basal aspect of the epithelium and this tissue arrangement
is similar to that of the ventricular and marginal layers observed in the neuroepithelium of the early neural tube. In other areas, large patches of mature
neuronal cells were found. Typically, these cells had a large soma and they
sprouted out neuronal processes (Fig. 6). The cell bodies were arranged in clusters
surrounded by interwoven meshworks of neuronal processes and this tissue
resembled the grey matter of the central nervous system. Other neural tissues
such as choroid plexuses and ependyma that lined the luminal surface of cystic
structures were also found. There was no significant difference in either the
incidence or the relative amount of neural tissues formed by the cells in the lateral
portion of the neural plate in comparison to those in the medial portion of the
plate (Table 1).
While only neural tissues were seen in grafts of mesencephalon of 5- to 6-somitestage and forelimb-bud-stage embryos, the neural plate of younger embryos also
gave rise to non-neural tissues. Epidermal tissues such as keratinized epithelia,
hairs and sebaceous glands (Fig. 7) were seen in 15-42 % of grafts of presomite
head-fold-stage neural plate (Table 1). The tissues were organized in the proper
topographical relationship seen in adult skin, but the dermal component was
poorly developed. Initially, the skin-forming capacity was found in both portions
of the neural plate but later it was limited to the lateral portion and was eventually
lost by the forelimb-bud stage. Mesodermal derivatives such as bone, cartilage and
adipose tissue (Fig. 8) were also formed by the neural plate cells of early-somitestage embryos. But this mesodermal potency was progressively lost from both the
medial and the lateral portion of the neural plate and was completely absent from
the neural plate cells by the 5-somite stage (Table 1). Pigment cells (Fig. 9) were
present in the neural plate graft of C57BL embryos having one to four pairs of
somites (Table 1).
The grafts that derived from the early-somite-stage neural plate were reexamined with respect to the change in the potency to form presumed neural crest
cell derivatives such as skeletal tissues and pigment cells (Table 2). It is clear from
this analysis that such tissues were formed only in grafts of neural plate of 1- to 4somite-stage embryos. Furthermore, the lateral portion tended to form presumed
Fig. 6. Differentiated neural tissue that resembles grey matter of the central nervous
system and contains silver impregnated processes (arrowhead). Bar, 100jum. Silver
impregnation by Bodian method.
Fig. 7. A cystic structure lined with keratinized stratified epithelium (k), hair follicles
(/) and sebaceous gland (arrowhead). Bar, 100pim. H & E staining.
Fig. 8. The ossification of the cartilage (c) to form bones (b) in the graft derived from
the lateral portion of the neural plate of a presomite head-fold-stage embryo.
a, adipose tissue; m, striated muscle. Bar, 100 jum. H & E staining.
Fig. 9. A cluster of pigment cells (arrowhead) found in the graft of the lateral portion
of the neural plate of an early-somite-stage C57BL embryo. Bar, 100 jum. H & E
staining.
Differentiation of neural plate cells
189
190
W. Y. CHAN AND P. P. L. TAM
neural crest derivatives more frequently than the medial portion (Table 2). The
presumed neural crest derivatives were also often formed in the absence of other
mesodermal (such as adipose and muscles) or epidermal derivatives (Table 2).
Among the 78 grafts involving the lateral areas of the neural plate of early-somitestage embryos, epidermal tissues were found in 10 grafts (Table 1). None of the
medial portions formed any epidermal tissues. The differentiation of epidermal
derivatives in the former case was likely due to the contamination by surface
Table 2. The association ofpresumed neural crest cell derivatives with other epidermal
and mesodermal structures in grafts of early-somite-stage neural plate
1-2
*M L
Tissues contained in grafts
(A) Neural tissues and presumed neural crest cell derivatives
Cartilage or bone
6
Pigment cells
0
Pigment cells +
0
bone or cartilage
Overall:
6
Somite no.
3-4
M
L
5-6
M
L
5
2
1 0
2
0
4
0
3
0
1 0
0
0
0
8
8
0
0
0
0
0
0
0
0
0
0
2
(B) Presumed neural crest cell derivatives + derivatives of surface ectoderm
and cranial mesoderm
Bone or cartilage +
0
0
0
0
epidermal tissue
1
0
Pigment cells +
0
0
epidermal tissue
Bone or cartilage +
0
0
0
0
mesodermal tissue
1
0
0
Overall:
0
*M and L: Medial and lateral portions of the neural plate.
Table 3. The tissue composition of grafts derived from the cranial mesoderm and
surface ectoderm
Somite no.
No. grafted
No. developed
No. analysed
No. of grafts containing:
Keratinized epithelium
Hair and sebaceous gland
Cartilage and bone
Adipose tissue
Connective tissue and
mesenchyme
Presomite
1-2
3-4
5-6
20-24
28
5
5
22
4
4
30
3
3
80
23
23
95
28
23
0
0
4
2
3
0
0
4
1
2
0
0
2
1
2
14
14
22
8
10
13
13
23
7
15
Differentiation of neural plate cells
191
ectoderm in the original grafts. Technically, this is unavoidable because, in early
embryos, the morphological landmarks might not faithfully delineate the neural
and epidermal areas of the ectoderm. When the underlying cranial mesenchyme
and the adjacent surface ectoderm were grafted, mesodermal derivatives such as
cartilage, adipose and connective tissues were commonly formed. Skin derivatives
were only formed in grafts originating from embryos having five or more somites
(Table 3). No neural tissues were ever formed.
DISCUSSION
Embryonic ectodermal cells taken from the anterior region of the mouse egg
cylinder have been shown to colonize the surface ectoderm and neurectoderm in
the craniofacial region of chimaeric embryos (Beddington, 1981). The analysis of
cellular proliferation in jwl8/fw18 mutant embryos similarly suggests that cells in
anterior embryonic ectoderm are topographically predisposed to form cephalic
neurectoderm (Snow & Bennett, 1978). However, when these cells are allowed to
differentiate in an ectopic environment, they express a histogenetic potential far
exceeding their normal ectodermal fate and form both mesodermal and endodermal derivatives (Beddington, 1983).
Our observation on the extensive range of tissue differentiation seen with the
presomite head-fold-stage neural plate agrees with other studies in rat embryos
(Levak-Svajger & Svajger, 1974). A restriction in the capacity to form endodermal
tissues was already apparent in the neural plate at this stage. In the course of
morphogenesis, the neural plate in early-somite-stage embryos lost the capacity to
form mesodermal tissues, particularly those presumed to be neural crest cell
derivatives. From about 5-somite stage onwards, the neural plate differentiated
exclusively into neural tissues. Our results therefore complement those observations made on the neural plate of rat embryos at similar stages of development
(Levak-Svajger & Svajger, 1974; Svajger & Levak-Svajger, 1974; Svajger et al.
1981), where a progressively diminishing capacity to form mesenchymal tissues
has been noted. This progressive restriction in histogenetic potential could be
attributed to the commitment of neural plate ectoderm towards the neurogenic
pathway and this appears to occur concomitantly with morphogenesis of the plate.
When the mesencephalic neural plate was transplanted to the renal capsule, the
graft differentiated predominantly into neural tissues. This is in line with other
studies which demonstrate that neural tissues such as neurones, ganglia and glia
are commonly formed during the in vitro culture of the neural plate (Cohen, 1977;
Ito & Takeuchi, 1984; Kahn & Sieber-Blum, 1983; Norr, 1973; Sieber-Blum &
Cohen, 1980; Skreb & Crnek, 1977; Skreb, Scuknac-Spoljar & Crnek, 1976; Skreb
& Svajger, 1975) or when the neural plate is grafted to renal capsules or the
anterior ocular chamber (Levak-Svajger & Svajger, 1974; Lumsden, 1984; Skreb
etal 1976).
The changes in histogenetic potential may also be related to the ability of the
neural plate to undergo the atypical morphogenetic process of 'neoformation' of
192
W. Y. CHAN AND P. P. L. TAM
mesenchyme in ectopic environment (Svajger etal. 1981). Early differentiation of
ectodermal fragments of early-somite-stage rat embryos proceeds with a sloughing
of cells from the edges of the graft in a manner that is reminiscent of neural crest
cell migration (Svajger etal. 1981). It seems likely that the gradual loss of the
ability to form skeletal tissues and pigment cells by grafts of the mouse mesencephalic neural plate was partly due to the diminishing population of precursor
neural crest cells in plates of advancing developmental stages. Circumstantial
evidence for this possibility was provided by the ability of the lateral portion of the
early-somite-stage neural plate to generate mesodermal tissues rather than the
medial portion. Furthermore, the temporal sequence of changes in the potency
was also coincidental to the emigration of neural crest cells from the mesencephalic neural plate (Nichols, 1981; Tan & Morriss-Kay, 1985). In a recent study,
cartilages and tooth primordia were formed when the lateral portion of the cranial
neural plate of early-somite-stage rat embryos was grafted together with the
mandibular arch epithelium to ectopic sites (Lumsden, 1984). This result has been
taken to indicate that those neural crest cells forming the teeth and the jaw bones
are found in the cranial neural plate. Explants of neural plate from chick embryos
of early-somite stage form cartilage and melanocytes when cultured in vitro or on
chorioallantoic membrane (Bee & Thorogood, 1979; Hall & Tremaine, 1979;
Newsome, 1976). Extensive skeletal differentiation occurs when the lateral areas
of the neural plate of 8-somite-stage chick embryos are recombined with epithelia
from pigmented retina and maxillary process (Bee & Thorogood, 1979). However,
the skeletogenic capacity is lost from explants taken from embryos at the stage
when neural crest cells have left the plate (Hall & Tremaine, 1979). Our interpretation on the mesodermal-tissue-forming capacity of the neural plate therefore
conforms with the morphological observations on the origins and emigration of
neural crest cells at the early stages of neurulation. The apparently greater ability
to form mesodermal derivatives endowed in the lateral portion of the plate may
suggest that the precursors of neural crest cells are strategically located to facilitate
their subsequent migration from the plate.
The authors are grateful to Professor D. J. Riches for his useful comment on the manuscript.
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HALL,
(Accepted 11 March 1986)