Download Regionalisation of the mouse embryonic ectoderm

55
Development 107, 55-67 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Regionalisation of the mouse embryonic ectoderm: allocation of prospective
ectodermal tissues during gastrulation
PATRICK P. L. TAM
Department of Anatomy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong
Summary
The regionalisation of cell fate in the embryonic ectoderm was studied by analyzing the distribution of graftderived cells in the chimaeric embryo following grafting
of wheat germ agglutinin-gold-labelled cells and culturing primitive-streak-stage mouse embryos. Embryonic ectoderm in the anterior region of the egg cylinder
contributes to the neuroectoderm of the prosencephalon
and mesencephalon. Cells in the distal lateral region give
rise to the neuroectoderm of the rhombencephalon and
the spinal cord. Embryonic ectoderm at the archenteron
and adjacent to the middle region of the primitive streak
contributes to the neuroepithelium of the spinal cord.
The proximal-lateral ectoderm and the ectodermal cells
adjacent to the posterior region of the primitive streak
produce the surface ectoderm, the epidermal placodes
and the cranial neural crest cells. Some labelled cells
grafted to the anterior midline are found in the oral
ectodermal lining, whereas cells from the archenteron
are found in the notochord. With respect to mesodermal
tissues, ectoderm at the archenteron and the distallateral region of the egg cylinder gives rise to rhombencephalic somitomeres, and the embryonic ectoderm
adjacent to the primitive streak contributes to the
somitic mesoderm and the lateral mesoderm. Based
upon results of this and other grafting studies, a map of
prospective ectodermal tissues in the embryonic ectoderm of the full-streak-stage mouse embryo is constructed.
Introduction
ectoderm is the elevation of proliferative activity in a
small group of cells in the anterior region of the
embryonic ectoderm near the rostral end of the primitive streak of the gastrulating embryo (Snow, 1977).
The developmental fate of the progeny of this mitotically active population is unknown though neuroectoderm has been suggested (Snow & Bennett, 1978).
Morphological studies in the mouse embryo suggest
that the embryonic ectoderm in the anterior region of
the egg cylinder gives rise to major segments of the
brain on the grounds that this part of the embryonic
ectoderm is topographically related to the first three to
four somitomeres normally underlying the forebrain to
upper hindbrain (Tarn & Meier, 1982; Meier & Tarn,
1982; Jacobson & Tarn, 1982). Orthotopic grafting of
embryonic ectoderm cells to the most anterior region
and the distal tip (the node, the head process or
the archenteron - Theiler, 1972; Poelmann, 1981) of the
primitive-streak-stage mouse embryo results in the
colonization of, respectively, the head and trunk neurectoderm by the graft-derived cells but the precise
segmental distribution of these cells in the cephalic
neural tube is not known (Beddington, 1981). Details of
the normal fate of cells in other anterior and lateral
regions of the embryonic ectoderm are incomplete
because previous mapping studies tended to focus
The embryonic ectoderm (epiblast) of the gastrulating
mouse embryo is believed to be the sole precursor of all
definitive tissues in the fetus. The evidence supporting
this notion is provided by the extensive range of
embryonic and adult tissues produced during the differentiation of the whole epiblast or fragments of it after
transplanting to ectopic sites (Diwan & Stevens, 1976;
Beddington, 1983; Svajger etal. 1986) and the multitude of tissue types colonized by the epiblast cells
grafted orthotopically in the primitive-streak-stage embryo (Beddington, 1981, 1982). It is therefore conceivable that, in order to generate a embryonic body plan
from the histologically homogeneous embryonic ectoderm (Reinius, 1965; Batten & Haar, 1979), an orderly
allocation of cells to various tissue types in specific
regions of the body needs to be accomplished during
gastrulation. The process of pattern generation would
be further facilitated if cells with diversified developmental fate are strategically located within the embryonic ectoderm so that tissues of different lineages but
belonging to specific parts of the body are properly
juxtaposed in preparation for ingression through the
primitive streak.
An example of early regionalization of the embryonic
Key words: embryonic ectoderm, primitive-streak stage,
mouse embryo, microsurgical grafting, lectin-gold labelling.
56
P. P. L. Tarn
mostly on specific groups of cells adjacent to and within
the primitive streak of the gastrulating embryo (Beddington, 1981, 1982; Copp etal. 1986; Tarn & Beddington, 1987). An in vitro study on the morphogenesis
of isolated fragments of primitive-streak-stage embryo
seems to suggest that, within the embryonic ectoderm,
cell populations destined for specific brain parts are
already spatially delineated (Snow, 1981). The present
grafting study was carried out to examine the regionalisation of prospective ectodermal tissues in the anterior
and lateral regions of the embryonic ectoderm. Special
attention is given to (1) the segmental distribution of
the epiblast-derived cells in the neural tube and (2) the
location of cells destined for the surface ectoderm, the
placodes and cephalic neural crest cells of the mouse
embryo.
Materials and methods
Recovery of embryos
Primitive-streak-stage embryos were obtained from a closebred colony of ICR strain mice. At 7-5 days p.c., embryos
were dissected from the uterus in PB1 medium containing
either 4mgml~' bovine serum albumin (Miles Lab) or 10%
fetal calf serum (FCS, Gibco). The parietal yolk sac was
removed microsurgically with a pair of fine glass needles and
the embryos were washed in several changes of fresh PB1
medium. Only late-primitive-streak-stage embryos (Fig. 1)
showing expanded exocoelom and amniotic cavity, a complete amnion and clearly discernible embryonic ectoderm
were used for labelling and grafting. Other features characteristic of embryos at this stage are the early allantoic rudiment
and the anterior-posterior gradient of tissue opacity due to
the presence of the spreading mesodermal wings.
Fig. 1. Scanning electron micrograph of a bisected 7-5-day
primitive-streak-stage embryo showing the exocoelom
completely separated from the amniotic cavity by the
amnion (am), al, allantoic rudiment; ch, chorion; ps,
primitive streak; ect, embryonic ectoderm. Bar= 100/.im.
In vitro culture of embryos
Primitive-streak-stage embryos were cultured in rotating
(30 rev min"') 50 ml glass bottle (Wheaton) containing 3-4 ml
of culture medium. To culture embryos for 22-24 h until they
reached the early-somite stage, a 1:1 (v/v) mixture of rat
serum and Dulbecco's modified Eagle's medium (DMEM,
Gibco) was used as the culture medium. For culturing
embryos for 44-46h until the forelimb-bud stage, a mixture of
mouse serum, rat serum and DMEM (1:2:1 by volume) was
used and the embryos were transferred to fresh medium after
24 h of culture (Hunter et al. 1988). The culture was gassed
with 5% CO 2 , 5% O2 and 90% N2 during the first 24 h of
development to the early-somite stage and then with 5 % CO2
in air for further culture.
Labelling of embryonic ectoderm and preparation of
grafts
Wheat germ agglutinin (WGA)-gold conjugate used for
labelling the embryonic ectoderm was prepared as described
by Tarn & Beddington (1987). About 2-5 nl of the colloidal
WGA-gold label was injected into the amniotic cavity of the
primitive-streak-stage embryo, which was then cultured in rat
serum-DMEM for 3-4 h. After culture, the embryo was
transferred to PB1 medium and rinsed twice in the same
medium. Fig. 2A shows the orientation of embryonic axes of
the egg cylinder with the posterior aspect of the embryo
indicated by the allantoic rudiment and the primitive streak
and the proximal border by the amnion. Using a pair of fine
glass needles, the egg cylinder was transected at the level of
the amnion to remove the extraembryonic region. A longitudinal cut was made down the anterior midline (Fig. 2B) to
unfold the embryonic portion of the egg cylinder. The
dissected embryo was then left in PB1 medium on a warm
stage (30-35°C) for about 5-10min. Often this was sufficient
to cause the spontaneous separation of the more turgid
embryonic ectoderm from the loose mesoderm and the thin
endodermal layer, which started to curl back from the free
edges. Further separation of tissue layers was achieved by
inserting a fine needle underneath the embryonic ectoderm,
which was colored deep red by the gold label, to slice away the
mesoderm and endoderm. A longitudinal cut was then made
on each side of the primitive streak (Fig. 2B) to isolate two
half-fragments of the embryonic ectoderm without the primitive streak (Fig. 2C). The embryonic ectoderm fragments
were transferred to fresh PB1 medium and divided into four
major fragments: two smaller anterior quadrants and two
larger posterior quadrants (fragments A-D; Fig. 2C) from
which smaller clumps of 20-30 cells were isolated for grafting.
Usually 5 to 6 egg cylinders would provide enough tissues for
grafting of 16-20 embryos.
Grafting experiments
The strategy was to graft WGA-gold-labelled cells isolated
from the four major fragments of the embryonic ectoderm to
different sites in the primitive-streak-stage mouse embryos.
Donor cells were grafted to sites in the same quadrant from
which they were isolated. Exact orthotopic grafting, which
was technically much more complicated, was not attempted.
The embryos were then cultured until they developed to the
early-somite stage (22-24 h HI vitro) or to the stage of
formation of forelimb bud and closure of anterior neuropore
(stage 14 - Thieler, 1972; 44-46 h in vitro). The pattern of
tissue colonization by the graft-derived cells was studied in the
chimaeric embryos following histological preparation and
silver enhancement of the colloidal gold particles in the
labelled cells. The manipulation of the egg cylinder and the
Tissue fate of embryonic ectoderm
57
EXT
LAT
Al
fr-
PROXIMAL
,PS
-I
POST
EMB
POST
ANT
LAT
DISTAL
(a)
Ib)
Fig. 2. (A) Anterior (ANT)-posterior (POST) and proximal-distal embryonic axes of the 7-5-day egg cylinder. The
primitive streak (PS) and allantois (AL) mark the posterior side of the egg cylinder and the proximal border is defined by
the amnion attaching to the junction between the extraembryonic (EXT) and the embryonic (EMB) parts of the egg
cylinder. (B) The position of cut I along the anterior midline of the egg cylinder and cuts II on the two sides of the primitive
streak (PS) producing the fragments shown in fig. 2c. LAT, lateral aspect of the egg cylinder, ECT, embryonic ectoderm;
AC, amniotic cavity. (C) The dissection of the half-embryo obtained in B to yield fragments A,B,C and D by cuts III and
IV.
grafting of labelled cells to the embryonic ectoderm followed
that described by Beddington (1987). Briefly, grafting was
done by microinjecting clumps of labelled cells to the embryonic ectoderm after pushing the injection needle through the
primitive endoderm and the mesoderm of the egg cylinder.
The graft was placed well within the ectodermal epithelium
but it was inevitable that, because of the wound produced by
the injection needle, some labelled cells might be lodged in a
mesodermal or even endodermal position after grafting. The
exact location of the donor cells was examined in 40 embryos
which were fixed for histological study of WGA-gold-labelled
tissues at 4-5 h after grafting.
Labelled donor cells were grafted to seven different sites of
the embryonic ectoderm. Because of the variation in the size
of the 7-5-day embryos, it was technically not feasible to
obtain uniform results from different embryos by positioning
the graft on the basis of absolute physical distance from any
morphological landmark. Instead, the seven locations were so
chosen that they represented the Cartesian points of an
imaginary rectangular grid mapped on the lateral aspect of the
embryo using the allantois, the amnion and the distal tip of
Fig. 3. A schematic diagram showing the position in the
embryonic ectoderm where microsurgical grafting of
WGA-gold-labelled embryonic ectoderm cells were made.
Three graftings were made to the midline position (ANTMAR, MID-ANT and ARCH) and others to the lateral
areas with two (PL-PS and ML-PS) adjacent to the
primitive streak (PS) and two midway between the anterior
and posterior midline of the embryo (P-LATand D-LAT).
PL-PS graft was made close to the allantoic rudiment (AL)
and P-LAT close to amnion (AM). MID-ANT, D-LAT and
ML-PS was halfway down the proximal-distal distance in
the embryonic part of the egg cylinder. ARCH was at tip of
the egg cylinder.
the egg cylinder as the reference (Fig. 3). These regions were
separated from one other by a distance of 80-100 ^m along
the orthogonal axes which could be discerned under a
dissecting microscope at 40x magnification. The following
grafts were made:
I Labelled cells from proximal-anterior quadrant (fragment A - Fig. 2C) were grafted to
(1) the anterior midline position near the attachment
of the amnion to the embryonic ectoderm
(anterior-marginal: Ant-Mar).
58
P. P. L. Tarn
11 Labelled cells from the distal-anterior quadrant (fragment
B - Fig. 2C) were grafted to
(2) the region halfway down the anterior midline of the
egg cylinder (mid-anterior: Mid-Ant).
III Labelled cells from the proximal-posterior quadrant
(fragment C - Fig. 2C) were grafted to:
(3) the region lateral to the posterior end of the primitive
streak (posterior-lateral primitive streak: PL-PS), and
(4) the proximal area near to the attachment of the
amnion on the lateral flank of the egg cylinder
(proximal-lateral: P-Lat).
IV Labelled cells from distal-posterior quadrant (fragment D
- Fig. 2C) were grafted to:
(5) the region halfway down the posterior side of the egg
cylinder and lateral to the middle portion of the
primitive streak (mid-lateral primitive streak: MLPS),
(6) the area halfway down on the lateral flank of the egg
cylinder (distal-lateral: D-Lat), and
(7) the distal tip of the egg cylinder, which corresponded
to the site of the archenteron and the head process
(archenteron: Arch).
Embryos were inspected under the dissection microscope
immediately after grafting for the location of the graft. The
labelled cells could easily be discerned by their red coloration.
Embryos containing grafts in the mesodermal or endodermal
germ layers were discarded.
Examination of embryos after culture
Embryos were harvested at 22-24 or 44-46 h of culture and
examined for vitelline circulation and heart activity. The fetal
membranes were then removed for a closer inspection of
developmental features such as the appearance of neural folds
and closure of cephalic neural tube, formation of somites and
forelimb bud and axis rotation. Embryos were fixed with
Carnoy fluid, embedded in paraffin wax and processed for
light microscopy. To visualize the colloidal gold marker,
histological sections were treated with a silver developer and
counterstained with Fast Green (Tarn & Beddington, 1987).
The location of labelled cells in different embryonic tissues
was identified in serial sections of the cultured embryos. The
segmental position of the labelled cells in the neural tube, the
surface ectoderm, the cranial mesenchyme, the somitic mesoderm and the lateral plate mesoderm was determined with
respect to the primary brain parts and the branchial arches in
the cephalic region and to the somite in the trunk region.
Results
The initial location of grafted cells
Forty embryos receiving grafts at different sites of the
embryonic ectoderm were examined at 4-5 h after
grafting. Results in Table 1 show that about 68%
(57-80%) of the experimental embryos have donor
cells confined to the embryonic ectoderm (Fig. 4) and
about 20 % have donor cells in both the ectoderm and
the adjacent mesoderm. About 20-30 WGA-goldlabelled cells were found in these embryos. In about
12 % of cases, labelled cells were found only in the
mesoderm and endoderm of the host embryo. Based
upon this observation, 18 chimaeric embryos (of a total
of 132) that showed tissue colonization by donor cells
only in mesodermal and endodermal tissues and not in
any ectodermal derivatives were excluded from this
I
\
ect
Fig. 4. A primitive-streak-stage embryo examined 4h after
grafting, snowing the proper incorporation of WGA-goldlabelled cells (arrowheads) in the pseudostratified
epithelium of the embryonic ectoderm, ect, embryonic
ectoderm. Silver-enhanced and Fast Green. Bar = 20,um.
study, since they might represent artefacts of improper
graftings.
Embryonic development in vitro
After 22-24 h of culture, over 50% of experimental
embryos developed an actively beating heart, and over
85 % of them formed early neural folds. About 4-8
pairs of somites were formed, which was similar to the
number of somites found in the intact embryos cultured
for the same duration (Table 2). When examined after
44-46h of culture, experimental embryos of the AntMar, Mid-Ant, P-Lat and D-Lat groups developed to
the same extent as the intact embryo with respect to axis
rotation, cephalic neurulation, limb bud formation and
somite number (Table 2). Embryos of Arch, ML-PS
and PL-PS groups were retarded when compared to the
Table 1. The location of
WGA-gold-labelled cells in embryos examined at
4-5 h after grafting
Number of embryos having labelled cells in
Total
Group
Ant-Mar
Mid-Ant
Arch
Lat
Ectoderm
14
5
6
15
40
8
4
4
11
27(68%)
Ectoderm & Mesoderm
mesoderm & endoderm
4
0
1
3
8(20%)
2
1
1
1
5(12%)
Experimental groups: Ant-Mar = anterior margin; MidAnt = mid-anterior midline; Arch = archenteron; Lat = lateral
embryonic ectoderm.
Tissue fate of embryonic ectoderm
59
Table 2. Development of embryos receiving grafts of labelled embryonic ectoderm at the egg cylinder stage and
cultured for 22-24 or 44-46 h in vitro
44-46h
22-241
No. of embryos (%) showing
No. of embryos (%) showing
\Ipnrnl
Groups
No. of
embryos
Neural
folds
Intact embryos
19 (83)
23
Embryos with grafts at:
Ant-Mar
32
30 (94)
Mid-Ant
19 (95)
20
Arch
31(89)
35
ML-PS
9(90)
10
PL-PS
6(100)
6
P-Lat
25 (100)
25
D-Lat
22 (85)
26
oomiic
Somites
Beating
heart
number
(n)
No. of
embryos
Axis
rotation
tube
closure
19 (83)
11 (48)
5-0 ±0-3 (19)
14
12 (86)
10 (71)
12 (86)
7(50)
16-2 ±0-6 (14)
30 (94)
19 (95)
31 (89)
9(90)
6(100)
24 (96)
22 (85)
19 (59)
14(60)
18 (51)
6(60)
3(50)
14 (56)
14 (54)
5-5 ± 0-3 (30)
6-4 ±0-2 (18)"
5-8 ± 0-4 (30)
6-0 ±0-5 (9)
4-5 ±0-7 (6)
5-7 ± 0-3 (24)
5-4 ± 0-4 (24)
16
24
17
16
18
9
25
12 (75)
15 (63)
7 (41)e
9(56)
8(44)c
6(67)
14(64)
10 (63)
13 (54)
5 (29)c
6(38)
6 (33)c
4(44)
11 (44)
15 (93)
22 (91)
15 (88)
15 (94)
15 (83)
7(78)
17 (68)
9(56)
19 (79)
9(53)
4(25)
6(33)
4(44)
15(60)
17-3 ±0-6 (15)
17-1 ±0-4 (21)
14-4 ± 0-8 (14)b
13-5 ± 0-6 (13)b
13-3 ±0-5 (17)b
18-2 + 0-8(6)
16-6 ±0-7 (21)
Yolk sac Forelimb
circulation
bud
number
(n)
Experimental groups: Ant-Mar = anterior margin; Mid-Ant = mid-anterior midline; Arch = archenteron; ML-PS = lateral to the mid-region of
the primitive streak; PL-PS = lateral to the posterior region of the primitive streak; P-Lat = proximal area of lateral embryonic ectoderm; DLat = distal area of lateral embryonic ectoderm.
Somite numbers; a = groups with more somites and b = groups with fewer somites than intact embryos (Duncan's multiple range comparison at
P<0-05).
Embryonic development: c = significantly different from intact embryos at f < 0 0 5 by Chi-squared test.
intact embryo developing under similar culture conditions. Axis rotation and cephalic neural tube closure
were delayed and fewer somites were formed (Table 2).
Tissue distribution of labelled cells
The number of WGA-gold-labelled cells in different
tissues of 36 chimaeric embryos was scored (Table 3).
The total number of labelled cells varied from 28 to 154
per embryo. In embryos of the Ant-Mar, Mid-Ant,
Arch, P-Lat and D-Lat groups, the majority of labelled
cells was found in ectodermal derivatives such as the
neural tube and epithelia of the oral cavity, body
surface and cephalic placodes of chimaeric embryos.
Colonization of the notochord by graft-derived cells
occurred only in the Arch group embryos. More
labelled cells were found in the paraxial and lateral
mesoderm of the PL-PS, ML-PS and D-Lat embryos
than in the other groups (P<0-01, Mann-Whitney
test). About 12 % of the labelled population was found
in the cranial mesenchyme and heart mesoderm of the
Ant-Mar, Mid-Ant and Arch embryos, compared to
33% mesodermal colonization in embryos receiving
graft in the lateral regions. Colonization of the endoderm was found in only 3 chimaeras, with about 10 % of
the labelled cells (2-10 per embryo) present in the gut
epithelium.
Patterns of tissue colonization
Altogether 93 chimaeric embryos, including 15 from the
cell counting study, were analysed in detail for the
spatial distribution of labelled cells in various embryonic tissues (Tables 4-6).
(i) Neural tube
Colonization of the neural tube and placodes occurred
more frequently in embryos receiving grafts along the
anterior midline ectoderm (Ant-Mar, Mid-Ant and
Arch) and the lateral ectoderm (P-Lat and D-Lat), in
contrast to embryos with grafts made to embryonic
ectoderm adjacent to the primitive streak (Table 4).
The segmental distribution of labelled cells in the
neural tube is summarized in Table 5. Most embryos
receiving grafts at Ant-Mar ectoderm had labelled cells
in the prosencephalon, mainly on the floor of the
diencephalon near the invaginating Rathke's pouch
(Fig. 5), the lamina terminalis and the optic evagination. Some labelled cells were also found at the
junction between the forebrain and the midbrain.
Labelled cells of the Mid-Ant graft were found mostly
in the mesencephalic floor and at the junction between
the midbrain and adjacent brain segments. The rhombencephalon and the neural tube at the level of the first
three (occipital) somites (Fig. 6) were colonized mainly
by labelled cells grafted to the D-Lat ectoderm, with a
minor contribution from the P-Lat and PL-PS ectoderm. Colonization of the neural tube at more caudal
somitic levels was observed in embryos with Arch and
ML-PS grafts.
(ii) Surface ectoderm and placode
Colonization of the surface ectoderm was observed in
25-43 % of embryos receiving grafts in the lateral
ectoderm and adjacent to the primitive streak
(Table 4). The surface ectoderm over the branchial
arches was colonized by cells grafted to lateral ectoderm but those over the hindbrain and somitic levels
were derived from grafts in the lateral ectoderm as well
as adjacent to the primitive streak (Table 5). Labelled
cells of the P-Lat and PL-PS grafts were frequently
found in the columnar placode epithelium on the lateral
60
P. P. L. Tarn
Table 3. The distribution of the labelled cell population in the chimaeric embryos examined at 22-24 h after
grafting of WGA-gold labelled embryonic ectoderm cells
Number of labelled cells in
Oral
Total
Grafts
Ant-Mar
Mid-Ant
Arch
PL-PS
ML-PS
Neural
tube
33
50
80
31
27
25
32
23
35
21
42
30
Placode
ectoderm
2
2
4
2
4
2
8
22
3
81
25
25
55
5
4
10
5
57
4
33
55
11
33
26
25
27
21
13
17
35
62
20
24
31
19
5
66
34
32
24
122
97
2
21
Notochord
ll+2ht
6ht
2+2ht
11
18
6
49
59
37
80
110
60
51
105
49
124
79
84
Gut
endoderm
Mesoderm
21
29
19
42
24
72
42
77
102
59
154
121
D-Lat
Surface
5
6
44
67
14
5
28
28
39
134
44
25
64
P-Lat
Neural
crest
10
20
24
6
39
46
6
48
8
25
95
41
106
40
64
17
9
10
8
18
39
20
Tissues containing more than 50 % of the labelled cell population are in italic.
For experimental groups, refer to footnote of Table 2, ht = heart mesoderm.
Table 4. Tissue colonization by WGA-gold-labelled cells in chimaeric embryos
No. of embryos (%) showing colonization in
Groups
No. of
embryos
Neural
tube &
placode
Embryos with grafts at:
Ant-Mar
20
18 (90)
Mid-Ant
13
13 (100)
14(64)
Arch
22
ML-PS
8
4(50)
PL-PS
8
3(38)
P-Lat
14
10 (71)
D-Lat
12
11 (92)
Surface
ectoderm
Oral
ectoderm
1(5)
8(40)
2(15)
0
0
0
3(38)
3(38)
6(43)
3(25)
0
0
0
0
For experimental groups, refer to footnote of Table 2.
Cranial
mesenchyme
1(5)
Neural
crest
cells
6(46)
0
0
2(9)
1(12)
2(25)
4(29)
6(50)
0
0
2(25)
4(29)
0
Somitic
mesoderm
1(5)
1(7)
8(36)
4(50)
1(13)
1(7)
2(16)
Lateral
plate
mesoderm
Heart
Gut
0
2(10)
3(15)
0
0
0
0
0
0
3(38)
5(63)
1(7)
5(41)
0
2(14)
1(8)
4(18)
2(25)
0
0
0
Notochord
0
1(7)
14(64)
0
0
0
0
20
13
15
8
8
10
12
LT
R
OP
15
1
W F
Jen
F-M
2
8
R W F
Midbrain
1
Jen
M-H
Met
Mye
Hindbrain
OT
6
2
5
1
5
2
4
2
1
4
6
3
6
1 1
2
1
3
6
1
1 1 1 1
1
Segmental level by somites
4 5 6 7 8 9 10 11 12 P
Spinal cord
BA1 BA2
2
2
1
3
4
5
3 2 1
1 1
1 1 1 1 1
2 1
3
Segmental level
Mye 1 2
Surface ectoderm
1
1
6
9
5
6
3
9
1
I
1
3
II
3
1
BA1
MBNC
1
III
3
1
BA2
2
1
1
HBNC IV
1
1
1
2
1
V
1
1
2
1
3
2
1
1
1
2
VI VII
1
2
3
2
1
1
4
1
1
2
2
4
3
2
3
1
3
1
1
1
8
Somitic mesoderm
1
1
AP
1
1
MP
2
1
PP
1
1
3
My
1
Lateral mesoderm
Abbreviations
For experimental groups, refer to footnote of Table 2.
Embryonic parts: I—VII = cranial somitomeres; AP, MP, PP = anterior, middle and posterior portions of presomitic mesoderm; BA1, BA2 = first and second branchial arch; F = floor of
brain vesicle; HBNC = neural crest cells in the lateral mesenchymal region of the hindbrain; MBNC = neural crest cells in the lateral mesenchymal region of the midbrain;
My = myelencephalon.
Ant-Mar
Mid-Ant
Arch
MP-PS
PL-PS
P-Lat
D-Lat
N
Cranial mesenchyme
No. of embryos with labelled cells in
Table 6. The distribution of WGA-gold-labelled cells in the cranial mesenchyme, somitic mesoderm and lateral plate mesoderm of the chimaeric
embryos
Abbreviations
For experimental groups, refer to footnote of Table 2.
Embryonic parts: BA1, BA2 = first and second branchial arch; F = floor of brain vesicle; Jen = junction between forebrain and midbrain (F-M) and of midbrain and hindbrain (M-H);
LT= lamina terminalis; Met = metencephalon; Mye = myelencephalon; OP = optic evagination; OT = otic placode or vesicle; P = posterior neuropore and adjacent areas; R = roof of brain
vesicles.
Ant-Mar
Mid-Ant
Arch
ML-PS
PL-PS
P-Lat
D-Lat
N
Forebrain
No. of embryos with labelled cells in
Table 5. The distribution of WGA-gold-labelled cells in the neural tube, placode and surface ectoderm of the chimaeric embryos
o\
8
3
o
3
re
re*
K
re
62
P. P. L. Tarn
aspect side of the head folds of the early-somite embryo
(Fig. 7). The labelled epithelium was located close to
the root of the first two branchial arches and at the
myelencephalic level of the chimaeric PL-PS and P-Lat
embryos. Following further in vitro development,
labelled cells were found in the trigeminal ganglia and
the otic capsule of the advanced embryos, suggesting
that the donor cells might have colonized the trigeminal
and otic placodes after grafting to the PL-PS and P-Lat
ectoderm.
(iii) Cranial mesenchyme and neural crest cells
Labelled cells were found in the cranial mesenchyme of
all groups of experimental embryos especially in those
with grafts in the Mid-Ant and lateral ectoderm
(Table 4). When cells were grafted to Mid-Ant ecto-
' -'
9
10
dm
scl
Fig. 5. A sagittal section of the
diencephalon (di) of an embryo cultured
for 44 h after Ant-Mar grafting showing
the presence of labelled cells in the
neurohypophyseal diverticulum meeting
the invaginating Rathke's pouch (rp) on
the roof of the oral cavity.
Fig. 6. A sagittal section of an embryo
cultured for 24 h after Mid-Ant grafting
showing WGA-gold-labelled cells
(arrowheads) in the rhombencephalon
(rh) and in the mesenchyme adjacent to
the truncus of the heart, fg, foregut
portal; ht, heart tube.
Fig. 7. A frontal section of an embryo
cultured for 24 h after P-LAT grafting
resulting in the colonization of placodal
epithelium at the upper hindbrain level
(arrowheads), fg, foregut; nt, neural
tube.
Fig. 8. A transverse section through the
hindbrain level of an embryo cultured
for 24 h after P-Lat grafting showing the
presence of labelled cells (arrowheads)
in the neuroepithelium (ne) and at the
lateral subectodermal position along the
putative migratory path for neural crest
cells.
Fig. 9. A section along the long axis of
the first branchial arch (I) of an embryo
cultured for 44 h after D-Lat grafting
showing labelled cells (arrowheads) in
the mesenchyme of the branchial arch,
p, first pharnyngeal pouch; II, second
branchial arch.
Fig. 10. A sagittal section through the
trunk of an embryo cultured for 44 h
after ML-PS graft showing the
colonization of somites by WGA-goldlabelled cells (arrowheads), se, surface
ectoderm; scl, sclerotome; dm,
dermamyotome. Bars= 100/im. Silverenhanced and Fast Green.
Tissue fate of embryonic ectoderm
derm, 46% of the embryos showed colonization of
somitomeres II and III (Table 6). Labelled cells grafted
to lateral areas (P-Lat and D-Lat) of the egg cylinder
and to regions adjacent to the primitive streak (ML-PS
and PL-PS) colonized predominantly the rhombencephalic somitomeres (Table 6). Only 3 examples of
colonization of the cranial somitomeres were encountered in 42 embryos receiving grafts at Ant-Mar and
Arch regions.
In embryos receiving grafts at P-Lat and PL-PS
regions, some labelled cells were found subjacent to the
surface ectoderm on the lateral aspect of the cranial
mesenchyme (Fig. 8). These were probably neural crest
cells since they were localized along the migratory path
where such cells are expected (Chan & Tarn, 1988).
Mesencephalic neural crest cells were derived from
grafts in the P-Lat ectoderm and rhombencephalic
neural crest cells came from grafts in the PL-PS
ectoderm (Table 6).
In older embryos receiving P-Lat and PL-PS grafts,
WGA-gold-labelled cells were found in cellular clusters at the base of the branchial arches, which are
reminiscent of primordia of trigeminal and acousticofacial ganglia. Although these cells were likely to be
neural crest cells, a placodal contribution cannot be
ruled out. In embryos with P-Lat and D-Lat grafts,
labelled cells were sometimes found in the mesenchymal core of the branchial arches (Fig. 9) but again it was
not possible in this study to identify their tissue of origin
as either neural crest or cranial somitomere.
(iv) Somitic mesoderm and lateral plate mesoderm
Colonization of the somitic mesoderm (Fig. 10) was
observed predominantly in embryos receiving grafts at
Arch and ML-PS sites and to a lesser extent at D-Lat
sites in the embryonic ectoderm (Table 4). Labelled
cells grafted at the Arch and ML-PS ectoderm colonized the first 8-9 somites and the presomitic mesoderm. Labelled cells derived from the lateral ectoderm
were allocated to the first three somites (Table 6).
Colonization of the lateral plate mesoderm was
observed primarily after grafts were made to the D-Lat
embryonic ectoderm and to the ectoderm adjacent to
the primitive streak (Table 4). The graft-derived cells
colonized both the somatopleure and the splanchnopleure at the level of the lower hindbrain to first 4
somites (Table 6).
(v) Other embryonic tissues
Extensive colonization of the notochord was found in
embryos receiving grafts at the archenteron (Table 4).
In 5 out of the 14 cases, labelled cells were distributed in
the notochord over a length equivalent to 5-6 somites.
Donor cells colonized the ectodermal lining of the
stomadeum, the buccopharyngeal membrane and the
foregut endoderm of embryos receiving Ant-Mar grafts
(Table 4). Two of the 13 Mid-Ant chimaeras also
contained labelled cells in the oral ectoderm, but not in
the foregut endoderm. Colonization of the endoderm of
mid- and hind-gut occurred in 6 cases following grafting
to Arch and ML-PS sites (Table 4). Labelled cells were
63
found in the epimyocardium and pericardium of 4
embryos in the Ant-Mar and P-Lat groups and in 1
D-Lat embryo (Table 4).
Discussion
The developmental fate of cells located in the anterior
and lateral regions of the embryonic ectoderm of intact
late-primitive-streak-stage embryos has been examined. Experimental evidence has been obtained for
regionalisation in the deployment of embryonic ectoderm cells to specific segments of the neural tube and
other ectodermal tissues during gastrulation. The study
of the mesodermal fate of embryonic ectoderm was,
however, fraught with technical problems and proved
less conclusive.
Technical consideration
In the present study, labelled cells were isolated from
four major quadrants of the embryonic ectoderm and
were grafted back to various sites in their quadrant of
origin. Grafting of cells to exactly orthotopic sites was
not attempted because of technical difficulties. There is,
however, no significant variation in the pattern of tissue
colonization among chimaeric embryos of the same
grafting groups. Indeed, it has been shown that when
embryonic ectoderm is grafted to heterotopic sites, the
cells may change their normal fate to conform with the
pattern of tissue differentiation of the new locations in
the embryonic ectoderm (Beddington, 1982). The one
exception is the anterior marginal ectoderm which is
predisposed to produce ectodermal derivatives. Since in
the present study, donor cells were always grafted to
quasi-orthotopic sites, the differentiation of the graftderived cells is likely to reflect the normal fate of cells at
the selected sites in the embryonic ectoderm.
One major drawback of the microsurgical grafting
technique is the possibility that, despite great caution
taken to place the graft within the embryonic ectoderm,
some grafted ectodermal tissue may accidentally be
grafted to the subjacent germ layers. The passage of the
injection micropipette through the tissue layers might
also create artefactual channels through which grafted
cells in the ectoderm could readily migrate to other
germ layers. A survey of 40 embryos examined at 4-5 h
after grafting did reveal that, in about 20% of cases,
grafted cells were distributed to both the ectoderm and
mesoderm which might result in the colonization of
tissues derived from both germ layers in these chimaeric
embryos. In another 12% of cases, the grafted cells
were not found in the embryonic ectoderm and therefore colonization by graft-derived cells would probably
be confined to mesodermal and endodermal tissues. In
both cases, it would be difficult to draw any conclusion
about the normal fate of embryonic ectodermal cells
with respect to mesodermal and endodermal development. In the present study, chimaeric embryos that
showed no colonization of ectodermal derivatives by
WGA-gold-labelled cells were excluded on the assumption that they were the result of misintegration of
64
P. P. L. Tarn
SEct
NTube
1
2
3
4
5
6
7
8
Ecl-M«3
PxMeso
MiscMes
m
H
D
nm
Sme II -IV
Di-Me Me
Sme IV- VI
LPM
Sme V- VII Som
LPM
SpC
Som
LPM
s*c
Som
Me-RhRhSpC
TrSEct
SpC
TiSEct
Rh
Others
OrEct
Pros
n
m
m
GITract
BAr
CrSEct PI
BAr NCC
CrSEct PI
NCC
9
D
11 E3
10
MGN HGN
Noto
Sme IV, V
LPM
SmeV-VH Son
TB
PS
LPM
PS
LPM Ext Emb
PS PGC
Fig. 11. The normal fate of cells in the embryonic ectoderm of the primitive-streak-stage mouse embryo studied by
orthotopic graftings of labelled cells (Data from the present study: grafts 1-4 and 6-8; Tarn & Beddington, 1987: grafts 5
and 9-11; Beddington, 1981, 1982 and Copp et al. 1986: grafts 1,6 and 11). The dotted lines mark the boundary of the
various brain parts and the spinal cord. For results obtained in the present study, only data showing colonization of the
tissue in over 20% of the chimaeras were used for constructing this map. Neural tube (NTube): Pros, prosencephalon;
Di~Me, diencephalic-mesencephalic junction; Me, mesencephalon; Me~Rh, mesencephalic-rhombencephalic junction;
Rh, rhombencephalon; SpC, spinal cord. Surface ectoderm (SEct): CrSEct, head surface ectoderm; TrSEct, trunk surface
ectoderm; PI, trigeminal placode or otic placode/vesicle. Ectomesenchyme (Ect~Mes): BAr, branchial arch mesenchyme;
NCC, presumptive neural crest cells. Paraxial mesoderm (PxMeso): Sme, cranial somitomeres I-VII; Som, somites and
presomitic mesoderm. Other mesodermal tissue (MiscMes): LPM, lateral plate mesoderm including somatopleure and
splanchnopleure; TB, caudal mesenchyme; ExtEmb, extraembryonic mesoderm of the amnion, yolk sac and allantois.
Gastrointestinal tract (GITract): OrEct, ectodermal lining of the oral cavity and buccopharyngeal membrane; MGN, midgut
endoderm; HGN, hindgut endoderm. Others: Noto, notochord; PS, primitive streak; PGC, primordial germ cells.
grafts. Only embryos containing labelled cells in the
ectodermal tissues were studied, with specific emphasis
on external tissues such as the neural tube and the
surface ectoderm. The results for internal (mesodermal
and endodermal) tissues must be viewed with caution
since they might be an artefact of grafting procedure.
The ectodermal derivatives
Results of the present grafting experiments demonstrate clearly that cells in the anterior regions of the
embryonic ectoderm give rise to the neuroectoderm of
the prosencephalon and mesencephalon. Cells destined
for the rhombencephalon come from the ectoderm in
the distal-lateral regions flanking the rostral end of the
primitive streak. The neuroectoderm of the spinal cord
is derived from the ectoderm overlying the archenteron
(alias the node region) and lateral to the anterior and
the middle regions of the primitive streak (sites 4 and 5,
Fig. 11). The various segments of the brain and the
trunk neural tube are represented in the correct craniocaudal order along the anterior-posterior axis of the
embryonic ectoderm of the primitive-streak-stage embryo. Labelled cells grafted to the midline of the
embryo colonize the floor plate and the basal plate
whereas cells grafted to more lateral positions end up
predominantly on the lateral wall of the neural tube. It
seems therefore that not only the craniocaudal pattern
but also the dorsoventral orientation of the neural tube
is established within the embryonic ectoderm at this
stage of gastrulation.
Other ectodermal tissues besides the neuroectoderm
are also derived from cells of the embryonic ectoderm.
Cells in the more proximal areas of the lateral embryonic ectoderm contribute to the cranial surface ectoderm, the epidermal placode and the neural crest cells.
A transplantation study in the mid- to late-primitivestreak-stage chick embryo shows that a crescent-shaped
zone in the anterior and lateral aspects of the epiblast is
destined for neural crest cells and peripheral to this
zone are those cells destined for the epidermis (Rosenquist, 1981). If such a neural crest cell zone could be
taken to demarcate the boundary of the neural primordium then the neural plate of the mouse embryo is
occupying a major portion of the anterior and lateral
regions of the embryonic ectoderm leaving a small area
in the proximal-lateral embryonic ectoderm for the
non-neural ectodermal tissues. Fig. 11 shows the subdivision of the neural primordium into domains for the
Tissue fate of embryonic ectoderm
brain segments and the spinal cord, based on the
distribution of graft-derived cells in major neural tube
segments and particularly at junctions between segments. The forebrain and the midbrain occupy a
relatively small area making up about one third of the
anterior embryonic ectoderm. The hindbrain covers the
distal area of the anterior embryonic ectoderm and
most of the lateral region. The trunk neural tube
occupies the node areas and extends posteriorly and
proximally to embryonic ectoderm lateral to the primitive streak. Taking into account the cup-shaped configuration of the primitive-streak-stage mouse embryo,
there is a remarkable similarity in the spatial pattern of
brain segments in the embryonic ectoderm when compared to that in the epiblast of the stage 4-5 chick
embryo. In the chick, the neural primordium is mapped
in a series of wedges stretching from the prenodal to the
postnodal- areas in the epiblast adjacent to the cranial
end of the primitive streak (Nicolet, 1971; Rosenquist,
1981; Packard, 1986).
Labelled cells derived from grafts in the anteriormarginal embryonic ectoderm also colonize the ectodermal lining of the oral cavity, the buccopharyngeal
membrane and the Rathke's pouch, in addition to
lamina terminalis and the diencephalic (neurohypophyseal) diverticulum of the forebrain. It is interesting to
draw a comparison with the prosencephalic plate of the
early-somite-stage avian embryo. Using chick-quail
chimaeras, the anterior neural primordium of the avian
embryo is found to give rise to the typical neural
structures such as the telencephalon and diencephalon
and also to non-neural tissue including the lining of the
nasal and oral cavities, the prosencephalic neural crest
cells and the hypophysis (Couly & Le Douarin, 1985,
1987; and the morphological studies by Takor Takor &
Pearse, 1975).
The relative size of different parts of the neural tube
as mapped onto the embryonic ectoderm of the primitive-streak-stage embryo is not in proportion to their
ultimate size at subsequent stages of development.
Although the forebrain and midbrain occupy a small
portion of the anterior embryonic ectoderm, by the
early-somite stage they become the most prominent
brain segments and constitute over half the tissue
volume of the head folds (Morriss-Kay, 1981; Jacobson
& Tarn, 1982). This is probably the result of differential
tissue growth in the neural tube. Indeed, it has been
shown in the rat embryo that the most active tissue
growth is encountered in the developing forebrain,
which may account for the rostral and lateral expansion
of this part of the brain during neurulation (Tuckett &
Morriss-Kay, 1985). Concomitant to the morphogenesis
of the neural tube, there is an enormous expansion in
the area of the proximal-lateral embryonic ectoderm
leading to the separation of the hindbrain and the spinal
cord which are originally juxtaposed in the embryonic
ectoderm of the primitive-streak-stage embryo. Analysis of the movement of cells in the proximal regions of
the embryonic ectoderm has revealed a relocation of
cells converging towards the primitive streak during
gastrulation (Lawson etal. 1987) similar to that de-
65
scribed in the chick epiblast (Vakaet, 1984). Such cell
movement is probably linked to the anisotropic growth
of the egg cylinder in the posterior and distal direction
(Tarn & Meier, 1982) and could be achieved by the
active proliferation of cells in the lateral and distal
embryonic ectoderm (Snow, 1977; Poelmann, 1980).
The expansion in tissue areas can also be achieved by
the changes in cell size and shape as exemplified by the
attenuation of the epithelium of the surface ectoderm
accompanying neurulation in the rat embryo (MorrissKay, 1981).
Mesodermal derivatives
Results of tissue colonization by donor cells in this study
suggested that precursor tissues for the paraxial mesoderm and the lateral plate mesoderm are largely confined to the distal-lateral and posterior regions of the
embryonic ectoderm close to the primitive streak.
Colonization of the first three cranial somitomeres
occurs in 1 out of 20 Ant-Mar and 6 out of 13 Ant-Mid
chimaeric embryos. A minor contribution to 'loose
head mesoderm' and even heart mesoderm by grafts of
anterior ectoderm has previously been reported (Beddington, 1981, 1982). As previously discussed, it is
doubtful whether this actually reflects the normal fate
of cells in this regions of the embryonic ectoderm. The
translation of the anterior and lateral ectoderm into the
cephalic neural tube necessitates a forward displacement of a coherent epithelial tissue and a concomitant
expansion of the posterior ectoderm adjacent to the
streak to generate the spinal cord and the paraxial
mesoderm. If the colonization of the rhombencephalic
mesoderm by Ant-Mid graft is real then the generation
of such mesodermal tissues from an anterior sites in the
epithelial sheet would pose a difficult mechanistic
problem. A specific group of prospective mesodermal
cells will have to move towards the streak in a direction
opposite to that of tissue sheet expansion and then to
reverse their course after invagination to reach their
final segmental position. Alternatively, the colonization
of the mesoderm by labelled cells could readily be
explained by a misplaced graft or a local delamination
of the grafted cells from the embryonic ectoderm.
More extensive colonization of the paraxial mesoderm begins with the 4th cranial somitomeres and
extended to all subsequent somites in embryos receiving grafts in the lateral and posterior embryonic ectoderm adjacent to the primitive streak. Previous grafting
experiments carried out for cells in the primitive streak
and adjacent to the streak (Fig. 11: sites 5, 9-11;
Beddington, 1981; Tarn & Beddington, 1987) have
demonstrated a significant contribution to the paraxial
mesoderm and lateral mesoderm during gastrulation of
the mouse embryo. That colonization by graft-derived
cells began with the rhombencephalic somitomeres also
agrees with the morphological finding that the first
three somitomeres are already established in the mesoderm at the mid- to late-primitive-streak stage (Tarn &
Meier, 1982) and with the result of grafting studies that
newly recruited paraxial mesoderm from the embryonic
ectoderm and the primitive streak is allocated to more
66
P. P. L. Tam
caudal somitomeres (Tarn & Beddington, 1986, 1987).
Similar to the situation of the ectodermal derivatives,
there is again striking homology in the mesodermal fate
of cells in the mouse embryonic ectoderm and that of
the chick epiblast at the primitive streak stage of
development. In both cases, the paraxial mesoderm is
located lateral to the anterior region of the primitive
streak and posterior to the Hensen's node. The lateral
plate mesoderm is found adjacent to and within the
middle region of the primitive streak, whereas the
extraembryonic mesoderm is associated with the posterior part of the streak (Nicolet, 1971; Meier &
Jacobson, 1982; Packard, 1986; Copp etal. 1986; Tarn &
Beddington, 1987).
Regionalisation of the embryonic ectoderm
Sufficient information is now available to build a map of
prospective ectodermal and mesodermal tissues for the
embryonic ectoderm of the primitive-streak-stage
mouse embryo. Fig. 11 shows the grafts of embryonic
ectoderm that have been investigated: sites 1-4 and 6-8
from the present study and sites 5 and 9-11 from study
by Tarn & Beddington (1987). Additional information
for sites 1, 6 and 11 is provided by similar grafting
studies by Beddington (1981, 1982) and Copp et al.
(1986). The neural tube occupies most of the anterior
and lateral embryonic ectoderm. The proximal areas of
the embryonic ectoderm contain the cells destined for
the surface ectoderm in the head and oral regions, the
epidermal placodes and the neural crest cells. Paraxial
mesoderm (rhombencephalic somitomeres and
somites) is mapped to the embryonic ectoderm in the
distal-lateral regions and adjacent to the primitive
streak. Within the primitive streak, distinctive regional
diversity of tissue fate is observed: the anterior region is
associated with the trunk neural tube and paraxial
mesoderm, the middle region with the lateral plate
mesoderm and caudal mesoderm and the posterior
region with the extraembryonic mesoderm and primordial germ cells (Copp et al. 1986; Tarn & Beddington,
1987). The present fate map, which is based upon the
result of grafting experiments, is very similar to the map
constructed by Snow (1981) using an entirely different
experimental approach: the prospective fate of the
embryonic ectoderm is deduced from the types of
embryonic tissues formed in embryo fragments containing a full complement of germ layers. Although a spatial
pattern of tissue diversification has been demonstrated
within the embryonic ectoderm by these studies, it does
not imply that the cells are precommitted to specific
lineages prior to gastrulation. Although the consensus
of studies using heterotopic grafts and ectopic teratomas indicates that there is little regional restriction in
the developmental potency of embryonic ectoderm cells
(Beddington, 1983; Svajger et al. 1986), definitive
information on cell potency has to be obtained by a
proper clonal analysis of individual cells of the embryonic ectoderm (Lawson et al. 1987).
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