Download Cell fate, morphogenetic movement and population

Development 101, 627-652 (1987)
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627
Cell fate, morphogenetic movement and population kinetics of
embryonic endoderm at the time of germ layer formation in the mouse
KIRSTIE A. LAWSON1 and ROGER A. PEDERSEN 23
with an appendix by SARA VAN DE GEER4
l
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands,
Laboratory of Radiobtology and Environmental Health and ^Department of Anatomy, University of California, San Francisco, CA 94143,
USA
^Centre for Mathematics and Computer Science, Knuslaan 413, 1098 SJ Amsterdam, The Netherlands
2
Summary
The fate of the embryonic endoderm (generally called
visceral embryonic endoderm) of prestreak and early
primitive streak stages of the mouse embryo was
studied in vitro by microinjecting horseradish peroxidase into single axial endoderm cells of 6 7-day-old
embryos and tracing the labelled descendants either
through gastrulation (1 day of culture) or to early
somite stages (2 days of culture).
Descendants of endoderm cells from the anterior
half of the axis were found at the extreme cranial end
of the embryo after 1 day and in the visceral yolk sac
endoderm after 2 days, i.e. they were displaced
anteriorly and anterolaterally. Descendants of cells
originating over and near the anterior end of the early
primitive streak, i.e. posterior to the distal tip of the
egg cylinder, were found after 1 day over the entire
embryonic axis and after 2 days in the embryonic
endoderm at the anterior intestinal portal, in the
foregut, along the trunk and postnodally, as well as
anteriorly and posteriorly in the visceral yolk sac.
Endoderm covering the posterior half of the early
primitive streak contributed to postnodal endoderm
after 1 day (at the late streak stage) and mainly to
posterior visceral yolk sac endoderm after 2 days.
Clonal descendants of axial endoderm were located
after 2 days either over the embryo or in the yolk sac;
the few exceptions spanned the caudal end of the
embryo and the posterior yolk sac.
The clonal analysis also showed that the endoderm
layer along the posterior half of the axis of prestreak-
Introduction
During late preimplantation development of the
mammal, the inner cell mass differentiates into a
and early-streak-stage embryos is heterogeneous in its
germ layer fate. Whereas the germ layer location of
descendants from anterior sites did not differ after 1
day from that expected from the initial controls
(approx. 90 % exclusively in endoderm), only 62 % of
the successfully injected posterior sites resulted in
labelled cells exclusively in endoderm; the remainder
contributed partially or entirely to ectoderm and
mesoderm. This loss from the endoderm layer was
compensated by posterior-derived cells that remained
in endoderm having more surviving descendants (8-4 h
population doubling time) than did anterior-derived
cells (10-5 h population doubling time). There was no
indication of cell death at the prestreak and early
streak stages; at least 93 % of the cells were proliferating and more than half of the total axial population
were in, or had completed, a third cell cycle after 22 h
culture.
We suggest that the visceral embryonic endoderm,
derived from the primitive endoderm of the late
blastocyst, is displaced onto the yolk sac by a new
population of endoderm inserted from epiblast at the
anterior end of the early primitive streak. This cell
population has colonized the axial endoderm by the
neural plate stage and contributes to the embryonic
endoderm of the early somite embryo.
Key words: cell fate, morphogenetic movement, cell
proliferation, embryonic endoderm, mouse embryo,
postimplantation development, cell lineage, horseradish
peroxidase, microinjection.
layer of primitive endoderm that faces the blastocyst
cavity and a core of primitive ectoderm (Nadijcka &
Hillman, 1974; Enders, Given & Schlafke, 1978). The
fate of the primitive endoderm has been studied in
628
K. A. Lawson, R. A. Pedersen and S. van de Geer
rodent embryos using preimplantation injection
chimaeras and by analysis of tissue potency at early
postimplantation stages. Primitive endoderm cells
isolated from 4-5 day of gestation mouse embryos and
injected into 3-5-day host blastocysts formed only
visceral and parietal yolk sac endoderm (Gardner
& Papaioannou, 1975; Gardner & Rossant, 1979).
Single primitive endoderm cells from 5-5-day donors
and groups of visceral embryonic endoderm cells
from 6-5- or 7-5-day embryos formed both parietal
and visceral endoderm descendants, leading to the
concept that the primitive endoderm lineage contains stem cells capable of forming both cell types
(Gardner, 1982, 1984). Cells isolated from the primitive ectoderm layer at 4-5 days and injected into host
blastocysts were capable of forming all the fetal
tissues, including the embryonic gut, as well as
extraembryonic mesoderm and amniotic ectoderm.
There is no evidence from the blastocyst injection
studies (using glucose phosphate isomerase as a
lineage tracer) that visceral embryonic endoderm
contributes any descendants to the fetal gut at midgestation, although a minor contribution might not
have been detected with this approach (reviewed by
Rossant, 1986).
When visceral embryonic and extraembryonic endoderm are isolated from primitive-streak-stage rat
and mouse embryos, they have extremely limited
tissue potency, as determined by ectopic grafting.
Visceral embryonic or extraembryonic endoderm
either yielded only parietal endoderm-like cells
(Solter & Damjanov, 1973; Diwan & Stevens, 1976)
or did not survive in ectopic grafts (Levak-Svajger &
Svajger, 1971, 1974). By contrast, embryonic ectoderm isolated from mouse and rat embryos before
and during gastrulation and grafted to ectopic sites
has broad tissue potency (Grobstein, 1952; LevakSvajger & Svajger, 1971; Diwan & Stevens, 1976).
Both distal and anterior regions of the mouse embryonic ectoderm isolated at the primitive streak stage
and grafted to ectopic sites formed midgut and
foregut derivatives (Beddington, 1983a). Taken
together with the blastocyst injection studies, these
patterns of postimplantation tissue potency imply
that the primitive ectoderm or its derivatives, rather
than the primitive endoderm, gives rise to the definitive fetal gut tissues.
Little is known about when the definitive endoderm forms (reviewed by Beddington, 1983b, 1986).
When postimplantation mouse chimaeras were
formed by transferring small groups of [3H]thymidine-labelled donor ectoderm cells from the distal
region of the egg cylinder on the 8th day of gestation
to a distal host site, labelled descendants were
detected in the embryonic gut (specifically, midgut)
(Beddington, 1981); orthotopic grafts of the primitive
streak from embryos on the 9th day of gestation also
contributed to gut endoderm (Tarn & Beddington,
1987). No gut derivatives were formed by distal
donors transferred to other host locations or from
other donor sites transferred to a distal host location
(Beddington, 1982). These apparent constraints in
contribution to gut tissues in the postimplantation
chimaeras imply that formation of the definitive
endoderm in the intact mouse embryo is a precisely
organized morphogenetic process.
We have recently described the fate of axial embryonic endoderm cells in midstreak and late streak/
neural plate stage (7-5 day) mouse embryos using
microinjected horseradish peroxidase (HRP) as a
short-term lineage tracer (Lawson, Meneses & Pedersen, 1986). This lineage tracer (Weisblat, Sawyer &
Stent, 1978; Balakier & Pedersen, 1982), while lacking some of the advantages of a cell-autonomous
genetic marker (Rossant, Vijh, Siracusa & Chapman,
1983; Gardner, 1984), nonetheless facilitates labelling
of single cells in situ without disrupting the native cell
relationships of the intact embryo. Our analysis
revealed that the visceral embryonic endoderm (hereafter called embryonic endoderm) is a mixed population at midstreak and late streak stages, consisting
of progenitors of visceral extraembryonic (yolk sac)
endoderm and progenitors of the embryonic gut
endoderm. Based on the relative locations of these
distinct progenitor cell populations, we proposed that
the progenitors of embryonic foregut endoderm
emerge from the epiblast earlier in gastrulation,
replacing the primitive endoderm cells, which contribute to yolk sac endoderm. This proposal emphasizes the similarity between rodent and avian
embryos, as previously noted (Levak-Svajger &
Svajger, 1974; Rossant & Papaioannou, 1977;
Gardner, 1978; Beddington, 19836).
In the current study, we extend this approach to
prestreak- and early-streak-stage mouse embryos (6-7
days) to trace the origin of the axial endoderm of
midstreak- and late-streak-stage embryos and to
examine the extent of the contribution of embryonic
endoderm to the yolk sac. This was accomplished by
marking axial endoderm cells with HRP at prestreak
and early streak stages, culturing embryos for 1 or 2
days, then determining the location of labelled descendants. This approach also provides information
about the morphogenetic movements and population
dynamics of the embryonic endoderm during this
period.
Materials and methods
Embryos
Noninbred Swiss mice of the Dub:(ICR) strain were used.
Gestation was considered to have begun at midnight before
Cell fate in mouse endoderm
the morning on which a copulation plug was found. Females
were killed by cervical dislocation between 15.00 and
16.00 h on the 7th day of pregnancy (6-7-day embryos). Egg
cylinders, including most of the ectoplacental cone, were
dissected from the decidua in Dulbecco's phosphatebuffered saline. Reichert's membrane was removed with
glass needles, and this and all further manipulations were
made in flushing medium II (Spindle, 1980) containing 10 %
fetal calf serum. There was considerable variation in
developmental stage both between and within litters of
nominal 6-7-day embryos. Embryos were classified as 'early
streak' or 'prestreak'. In early-streak-stage embryos mesoderm formation had begun caudally: in the most advanced
embryos of this group the posterior amniotic fold had been
initiated and the thin end of the mesodermal wedge
indicating the anterior end of the primitive streak was
located about one third of the length of the embryonic
ectoderm posterior to the distal tip of the egg cylinder
(fig. 64/1 in Theiler, 1972). The bilateral symmetry of the
prestreak embryos was recognized by the slightly thicker
posterior embryonic ectoderm and slight asymmetry of the
endodermal outline (fig. 52 in Theiler, 1972).
Embryo culture
Embryos that had been microinjected with HRP were
cultured in Dulbecco's modified minimal essential medium
containing 50% rat serum, as previously described (Lawson et al. 1986). Four to six embryos were cultured for
2 2 h ± l - 7 h i n l m l medium. Embryos maintained for 2 days
in culture were transferred to fresh medium (two
embryos/l ml) after thefirstday and incubated for a total of
44h.
Cell labelling with HRP
The conditions for intracellular injection by iontophoresis
have been described previously (Balakier & Pedersen,
1982; Lawson etal. 1986). One axial or near axial endoderm
cell per 6-7-day-old embryo was injected with 4 % HRP
(Sigma Type VI) in 0-05 M-KCI for 15 S with 5 nA continuous
positive current. The site of injection was recorded on a
freehand drawing of the embryo. Control (unincubated)
embryos were injected at one to three sites along the axis.
629
Cells containing HRP were detected by treating the intact
embryos with 0-1% Hanker-Yates reagent (Polysciences)
in OlM-phosphate buffer, pH5-5 (Streit & Reubi, 1977)
plus 5 % (w/v) sucrose and 002 % H2O2 for 15-100 min in
the dark. The position of visible, labelled cells and their
approximate number were recorded on freehand drawings
of the embryos. Embryos were fixed in glutaraldehyde and
embedded in glycol methacrylate as previously described
(Lawson et al. 1986), and exact localization and number of
labelled cells were determined on 10^m serial sections,
followed by photographic reconstruction (Lawson et al.
1986).
Results
(A) Embryos cultured for 1 day
(1) Embryo development
After 1 day of culture, the majority of prestreak-stage
embryos had developed to the midstreak stage
(Table 1). Early-streak-stage embryos had mostly
reached late streak and neural plate stages; the
initially most advanced were forming a head fold and
foregut invagination. The embryos appeared normal
except that, after amnion closure, the amniotic cavity
and the exocoelom overexpanded slightly.
(2) Validity of the labelling technique
In control embryos stained directly after injection,
72 % (58/81) of the sites were successfully injected
(Table 2). Of these, most of the labelled cells were in
endoderm and the remainder in ectoderm/mesoderm. (Ectoderm/mesoderm indicates ectoderm
and/or mesoderm.) In more than half of the successfully injected sites a single cell was stained (Table 3,
series 1; Fig. 1A); almost all of the remaining sites
had two adjacent cells labelled. After 22h culture,
73 % (117/160) of injected embryos had labelled cells
(Table 2), indicating that there was no significant loss
of injected progenitors during culture.
Table 1. Developmental stages reached by 6-7-day-old embryos after culture
Somites
Initial stage
time (h)
n
MS*
LSt
NPt
HF§
1-3
4-6
7-10
Prestreak
Early streak
Prestreak
Early streak
22
22
44
44
67
93
69
93
43
12
0
0
17
37
0
0
7
31
0
0
0
13
26
2
0
0
14
2
0
0
28
44
0
0
1
45
* Midstreak: mesoderm wings extend about two thirds around egg cylinder; posterior amniotic fold with developing exocoelom; head
process not exposed at distal tip of egg cylinder. (The head process is the cranial extension of the primitive streak and is connected to
the streak by the node, visible as a protuberance at the distal tip of the egg cylinder at late streak and later stages (Fig. 1C). For
detailed description see Snell & Stevens (1966), Poelmann, (1981b).)
t Late streak: mesoderm present in midline anteriorly; amnion closing; head process exposed at distal tip of egg cylinder but not
more anteriorly.
t Neural plate' amnion complete; allantois developing; edges of neural plate defined; head process exposed to surface over onequarter length of the anterior half of the egg cylinder.
§ Head-fold: early neural folds and foregut invagination. No somites.
630
K. A. Lawson, R. A. Pedersen and S. van de Geer
Table 2. Incidence of embryos with HRP-labelled cells in various germ layers after injection into a single axial
endoderm cell and 22 h culture
No. with HRP-labelled cells
Endoderm plus
ectoderm/
mesoderm
Culture
time (h)
No.
injected
Total
0
81
58
51 (88 %)
Prestreak
Early streak
22
22
67
93
51
66
30
58
14
5
Total
22
160
117
88(75%)
19(16%)
Stage at
injection
Prestreak + early streak
Endoderm
Ectoderm/
mesoderm
only
7(12%)
0
7
3
10(9%)
Table 3. Cells labelled/injection site(controls)
HRP-labelled sites
Sites
injected
Total
One cell
Two cells
Three cells
Series 1
Series 2
Anterior
Posterior
Total
81
58
34 (58 %)
23 (40 %)
1 (2 %)
47
45
92
34
32
66
16
18
34 (52 %)
16
13
29 (44 %)
2
1
3 (4 %)
Series 3
66
43
27 (63 %)
15 (35 %)
1(2%)
(3) Localization of labelled cells
Of the 117 cultured embryos with HRP-labelled cells,
75% had labelled cells only in endoderm, 16% in
endoderm plus ectoderm/mesoderm and 9 % in ectoderm/mesoderm only (Table 2). For the purpose of
analysis, the position of injection was classified on an
arbitrary scale: the length of the anterior-posterior
axis of the embryo from its anterior limit at the
junction of embryonic and extraembryonic ectoderm
was divided into five imaginary zones (Fig. 2); the
relative position of the zones was the same in all
embryos, but the absolute size of the zones depended
on the size of the embryo.
(a) Embryos with labelled endoderm only. Descendants of cells situated on the axis of the anterior half
(zones I and II), including the distal tip (zone III), of
early-streak-stage embryos were located over the
most anterior and anterolateral part of the embryonic
ectoderm and neighbouring visceral yolk sac at late
streak and neural plate stages (Fig. 3, zones IB, IIB,
and IIIB). They tended to be aligned at right angles to
the embryonic axis. Endoderm descendants of surface cells from, and near, the anterior end of the early
primitive streak (zone IV) were found along the
entire axis of the embryo (Fig. 3, zone IVB), but
mainly in the anterior half (Fig. 1C), including the
distal tip. These groups tended to be aligned along
the axis, skirting the head process and extending into
a horseshoe or crescent in the region of the presumptive foregut invagination (Fig. 3, zone IVB; Fig. 5).
That endoderm cells originating in zone IV~ are
involved in foregut invagination is demonstrated by
the two initially most advanced embryos in the series
that had been injected in zone IV, and in which the
foregut invagination was evident after 22h culture.
Labelled cells were present in the floor of the invaginating foregut of one embryo (Fig. 6A); in the other,
slightly more advanced, embryo, which also had three
labelled cells in the node, labelled cells were present
at the rostral tip of the roof of the invaginating
foregut (Fig. 6B). Cells over the main part of the
primitive streak (zone V) contributed endoderm
descendants to the axis of the posterior half of the
embryo and to the posterior visceral yolk sac (Fig. 4,
zone VB).
The pattern of labelled cells seen in cultured
embryos after labelling at the prestreak stage in zones
I, II and III was broadly similar to that obtained from
early streak stages (Fig. IB; Fig. 3, zones IA, IIA,
and IIIA). However, descendants of zone IV cells
were found in the anterior half only, with considerable anterolateral spread and overlap with those from
zone III (Fig. 3, zone IVA), and zone V contributed
to the entire axis, resembling the behaviour of the
cells of zone IV of the early-streak-stage embryo
(Fig. 4, zone VA). The primitive streak is initiated in
this most posterior region before spreading anteriorly
into zone IV.
Cell fate in mouse endoderm
These results indicate that there is a relative
anterior shift of endoderm during gastrulation that
involves all axial endoderm back to, and including,
that covering the anterior portion of the primitive
streak,
631
(b) Embryos with labelled cells in both endoderm and
ectoderm/mesoderm. The incidence of embryos with
labelled cells in both endoderm and ectoderm/mesoderm was significantly greater in the cultured embryos than in the controls (Table 2) (jr2= 10-94,
v -
y
a
-'•
\
1A
i
•
1
/7 P V
G
Fig. 1. Sections of injected embryos. (A) Longitudinal section of control embryo injected at three sites, showing one
labelled endoderm cell (single arrow), two labelled adjacent endoderm cells (two arrows) and one labelled ectoderm cell
(arrow head), a small part of which extrudes into the endoderm layer. (B) Near-sagittal section of midstreak-stage
embryo that had been injected in zone III 22 h earlier at the prestreak stage, showing 5 of the 23 labelled endoderm
descendants near the anterior limit of the embryo. (C) Sagittal section of neural-plate-stage embryo that had been
injected in zone IV at the early-streak stage: two of the four labelled squamous endoderm cells (large arrows) lie over
and immediately anterior to the head process, the anterior limit of which is indicated by a small arrow, a, anterior limit
of embryo; am, amnion; ec, embryonic ectoderm; hp, head process; m, mesoderm; n, node at anterior end of primitive
streak; ps, primitive streak. Bar, 100^m.
632
K. A. Lawson, R. A. Pedersen and S. van de Geer
IV
IV
Fig. 2. Diagrammatic sagittal sections of egg cylinders
without Reichert's membrane at the prestreak stage (A)
and the early-streak stage (B) to show the position of the
injection zones (I-V). The future cranial-caudal axis
runs from anterior (ant.) to posterior (post.) via the distal
tip of the egg cylinder, dots, embryonic endoderm;
hatching, epiblast; broken line, primitive streak; eec,
extraembryonic ectoderm; vee, visceral extraembryonic
endoderm.
df=2, P<0-005), with a relatively larger contribution by prestreak-stage embryos. However, prestreak-stage embryos had been injected disproportionately often in posterior zones. To determine
whether the presence of descendants in ectoderm/
mesoderm was zone-dependent or stage-dependent,
one axial endoderm cell in either the anterior half
(zone II or junction I—II) or posterior half (zone IV or
junction IV-V) of the embryo was injected. Initial
developmental stage in each litter was equalized over
the two groups and the controls were taken at random
from each group after injection. The number of cells
labelled per injection site in the controls (Table 3,
series 2) and the germ layer distribution of these cells
(Table 4) were similar to those in the previous series.
The incidence of cultured embryos with labelled cells
not found exclusively in endoderm (i.e. endoderm
plus ectoderm/mesoderm or ectoderm/mesoderm
only) was increased after injection into posterior
endoderm compared with anterior endoderm
(Table 4, c and d) and with controls; the frequency
distribution for the anterior endoderm, however, did
not differ from the controls (Table 4, c and a). There
was no significant difference in the behaviour of
posteriorly labelled cells between prestreak- and
early-streak-stage embryos (Table 4, d] and d2).
The localization of labelled cells in the embryos
with label in endoderm plus ectoderm/mesoderm
after injection posteriorly are summarized in Fig. 7:
the results of embryos in this category from the first
series of experiments (Table 2) have been included.
Labelled mesoderm cells were found either at the
anterior edge of the mesoderm (midstreak stage) or
nearby (late streak/neural plate stage), or near the
anterior end of the primitive streak. Labelled ectoderm was found in the distal tip of the egg cylinder at
the anterior end of the primitive streak. The results
from embryos with label.in ectoderm/mesoderm only
are shown in Fig. 8: after injection into early-streakstage embryos, the labelled mesoderm cells tended to
be more posteriorly situated than in embryos in which
the endoderm was also labelled (Fig. 7); this difference was not seen in the embryos injected at the
prestreak stage. The positions of labelled cells in
embryos with label in endoderm, both exclusively
(Fig. 9) and in combination with ectoderm/mesoderm (Fig. 7), supplement the data from the earlier
experiments (Figs 3, 4) and emphasize the anterior
and anterolateral shift of cells from the anterior zone
compared with the axial, mainly anterior, spread of
the posterior cells.
We conclude from these results that the posterior
axial endoderm is heterogeneous during the time
when bilateral symmetry is becoming visible: the
majority of the cells will contribute to the endoderm
of the late gastrula, but a substantial minority will
contribute to ectoderm/mesoderm. Cell pairs were
labelled in 40% of the injections (Table 3); although
many of these pairs must have been sisters connected
by a cytoplasmic bridge, occasional labelling of two
nonsister cells by inaccurate injection cannot be
excluded. In addition, the labelled cells in a' few
embryos will have descended from inadvertently
injected epiblast. Because of these uncertainties, the
proportions of presumptive endoderm and ectoderm/
mesoderm cells in the posterior axial endoderm
cannot be estimated from the germ layer localization
of labelled descendants in the cultured embryos, nor
can the question be answered whether endoderm and
ectoderm/mesoderm can be derived from one cell in
the surface of the streak or only from adjacent, not
necessarily sister, cells.
(c) Embryos with anomalous distribution of labelled
cells. The positions of labelled cells in nine embryos
in the first series of experiments and three in the
second series were anomalous and are not shown in
the figures. Whereas most of these results could be
attributed to mistaken orientation of prestreak-stage
embryos or inadvertent injection into epiblast, two
specimens could not be easily explained. Zone II of
these embryos was injected at the early streak stage,
and mistaken orientation was therefore unlikely;
however, labelled mesoderm as well as labelled
endoderm was found in zone I after culture. If only
endoderm had been injected, the labelled mesoderm
Cell fate in mouse endoderm
cells must have either descended from anterior endoderm or acquired evenly distributed cytoplasmic
HRP by nonlineage transfer: both possibilities are
without precedent. If both epiblast and endoderm
were labelled at injection, the mesoderm could have
descended from epiblast directly, without passing the
primitive streak. Such direct delamination of mesoderm from ectoderm occurs in utero and indicates the
beginning of mesectoderm formation, which has
already started by the neural plate stage in rodents
(Vermeij-Keers & Poelmann, 1980; Smits van
Prooije, 1986).
(4) Cell numbers and population dynamics
In theory, estimates of population doubling time, cell
death and cell cycle distribution can be obtained from
the quantitative data on labelled cells (Fig. 10).
(a) Population growth. The total population increase
(IT) is obtained from the following relation (Lawson
etal. 1986):
r
_ Total labelled cells (22 h) 'Total labelled cells (Oh)
Embryos injected (22 h)
Sites injected (Oh)
1605 /68 + 104+12
173
261
= 5-78.
(Data from Fig. 10; Tables 2, 3 and 4 [series 1 and 2].)
Similarly, the population increase due to descendants in the endoderm layer is given by
IF
Total labelled
, Total labelled
endoderm cells (22 h) 'endoderm cells (Oh)
=
Embryos injected (22 h)/ Sites injected (Oh)
1466/60+104+11
173
261/
= 5-55.
(b) Population doubling time. The population doubling time can be calculated from the number of
labelled cells. Assuming N = A-e b t , where N =
number of labelled cells at t h, and N = 1 when t = 0.
Then the population doubling time (T) is given by
^
1
In2xt
lnN
For the total population of labelled cells:
Ti
In2x22
In 5-78
= 8-7h.
For the population in endoderm:
T
In 2 x 22
In 5-55
= 8-9h.
633
(c) Cell death. The number of cultured embryos with
labelled cells was not less than that expected from the
controls (Tables 2, 4), which implies that, not only
was there no direct toxic effect of HRP, but there was
also no significant cell death in the axial endoderm
population at prestreak and early streak stages.
(d) Cell cycle distribution. The frequency distribution
of labelled cells/embryo indicates heterogeneity in
the division rate of succeeding cell generations
(Fig. 10). In addition, interpretation of the frequency
distribution is complicated by the expectation from
the controls that 42 % of the embryos initially had a
pair of labelled cells and 3 % had a triplet of labelled
cells (Table 3): this will shift the distribution towards
a greater number of descendants compared with a
population derived from single labelled cells. So,
when the data are classified into groups representing
number of cell generations (1, 2, 3-4, 5-8, etc.,
cells/embryo) (Table 5), the groups obtained are
'mixed generation' classes. However, assuming that
single and paired labelled cells will have the same cell
cycle kinetics, the proportions of initial single and
paired labelled cells in controls can be used to
calculate the frequency distribution of the number of
descendants from single labelled cells and, in the
absence of cell death, the distribution of cell generations (Table 6). By this estimate, only 7-6% of the
total population failed to divide; the majority of cells
were in, or had completed, a third cell cycle (Table 6)
after 22 h culture.
(e) Position-dependent variations. Stage- and position-dependent variations in cell cycle characteristics
could also contribute to the spread in the frequency
distribution of labelled cells (Fig. 10). There was no
significant difference between prestreak- and earlystreak-stage embryos (data not shown), but the
number of labelled cells after culture did depend on
the site of injection: posterior cells had more surviving descendants than did anterior cells (Fig. 11).
When the contribution of the total initial population
to endoderm was examined, there was no difference
in the number of descendants from anterior and
posterior sites (Fig. 12), indicating that the surface
layer of endoderm expands uniformly. However,
when those embryos with descendants only in ectoderm/mesoderm were omitted from the analysis and
the contribution of the remaining initial sites to
endoderm was examined, posterior sites contributed
significantly more descendants to the endoderm layer
than did anterior sites (Fig. 13). Therefore, the loss
of surface cells to ectoderm7mesoderm in the streak
is balanced by a relatively large contribution from the
remaining posterior axial endoderm cells to the
endoderm of midstreak-neural plate stages.
634
K. A. Lawson, R. A. Pedersen and S. van de Geer
A
A
ant
post.
post.
ant
Ill
post.
anL
I \ \\\ p°sl
0 1 mm
post.
post.
Cell fate in mouse endoderm
Standard deviations of the means of labelled cells
were large (Figs 11-13) because the population analysed was derived from a mixture of single and paired
post.
635
labelled cells. When the descendants of cells contributing only to endoderm were classified in 'mixed
generation' classes (Table 5), the frequency distribution of the numbers of descendants of anterior cells
(zones I—III) was different from that of descendants
of posterior cells (zones IV and V) (^ = 12-02, df= 5,
P<0-05). The population kinetics were therefore
calculated for these two subpopulations, yielding
population doubling times of 10-5 and 8-4 h for
anterior and posterior regions, respectively (Table 6).
Simplified estimators of population doubling times of
the anterior and posterior regions (10-49 and 8-97 h,
respectively) were used for statistical comparison and
found to be significantly different (P< 0-001) (see
Appendix). The remaining group of 48 embryos with
labelled cells in ectoderm/mesoderm, both exclusively and with endoderm, had a frequency distribution of labelled cells in 'mixed generation' classes
(Table 5) that was different from both anteriorderived endoderm 0^ = 27-16, df=6, P< 0-001)
and posterior-derived endoderm (j2 = 15-45, df=6,
P<0-02) and suggested that this part of the population was dividing more rapidly than the rest of the
posterior endoderm. However, the data did not fit the
Fig. 4. Position of endoderm descendants after injection
into single axial endoderm cells in zone V of (A)
prestreak-stage and (B) early-streak-stage embryos. For
further explanation, see legend to Fig. 2.
Fig. 3. Position of endoderm descendants after injection
into single axial endoderm cells in zones I-IV of (A)
prestreak-stage and (B) early-streak-stage embryos.
Injection position for individual embryos (one
dot/labelled embryo) is indicated on the upper figure for
each group. The location of descendants is projected onto
a sagittal section (middle figure) and on the ventral
surface of the flattened, embryonic part of the egg
cylinder (lower figure). The anterior limit of the
embryonic axis is indicated with a single arrow and the
posterior limit with two arrows in the middle and lower
figures. The anterior boundary of the neural plate and the
wedge-shaped extension of the exposed head process are
also represented in the lowest figure of B. The median
position of the labelled cells of any one embryo is marked
by a dot, the linear spread by a continuous line. Widely
separated clumps of labelled cells in an embryo are
marked by dots and connected by a broken line.
Overlapping positions on and near the midline of sagittal
sections have been displaced outside the section for
clarity. • ' (II) indicates an embryo that was presumably
misoriented at injection.
Fig. 5. Neural-plate-stage embryo that had been injected
in zone IV at the early-streak stage: seven of the eight
labelled descendants (large arrow) are visible in a
crescent shape in the region of the prospective foregut
invagination. The small arrow indicates the anterior
boundary of the neural plate, am, amnion; n, node;
ps, primitive streak. Bar, 100^m.
636
K. A. Lawson, R. A. Pedersen and S. van de Geer
iterative model, possibly because the sample was too
small and the population kinetics could not be calculated.
In conclusion, the axial endoderm displays position-associated heterogeneity in cell cycle kinetics
during the first day of gastrulation.
(B) Embryos cultured for 2 days
(1) Embryo development
Prestreak-stage embryos reached head-fold-6-somite
stages (Table 1) and 19 % had beating hearts: the
majority of early-streak-stage embryos developed
5-8 somites; 80 % had beating hearts.
(2) Localization of labelled cells
The controls did not differ significantly from those
for 22h culture, either in number of labelled cells/
injection site (Table 3, series 3) or in germ layer
localization (Table 7). Of the 165 injected cultured
embryos, 71 (43 %) had labelled cells, 85 % of these
in endoderm only (Table 7).
(a) Embryos with labelled endoderm only. Endoderm
of the anterior half of the axis (zones I—III) contributed to the visceral yolk sac (Fig. 14, zones I—III;
Figs 16A, 17A). The majority of zone IV cells of
prestreak embryos (6/8) also contributed to the
visceral yolk sac; the remainder had descendants
in trunk endoderm (Fig. 15, zone IVA). The proportions were reversed after injection at the early
streak stage (*2 = 6-98, df=2, P<0-05): only a
minority of zone IV cells (6/25) had descendants
exclusively in the visceral yolk sac; the majority
(17/25) contributed only to axial endoderm (Fig. 15,
zone IVB), with descendants at the anterior intestinal
portal, in the ventral and dorsal foregut (Fig. 17B),
and along the trunk and postnodal endoderm
(Fig. 16B). Zone V contributed mainly to the posterior visceral yolk sac, but also to postnodal endoderm, and to axial endoderm after injection at the
prestreak stage (Fig. 15, zone V). Therefore, while
descendants of most of the axial endoderm cells of
prestreak- and early-streak-stage embryos colonize
to.
6A
am
B
Fig. 6. Foregut initiation.
(A) Near-sagittal section of headfold-stage embryo that had been
injected in zone IV 22 h earlier,
showing two of the three labelled
descendants (arrows) in the ventral
endoderm of the very early foregut;
15 additional endoderm
descendants were located posterior
to the node. (B) Sagittal section of
head-fold-stage embryo that had
been injected in zone IV 22 h
earlier, showing two labelled cells
(arrow) in the rostrodorsal
endoderm of the invaginating
foregut. Two additional labelled
cells in adjacent sections were
located in the base of the head
process and five in postnodal
endoderm. am, amnion; h, heart;
hf, head fold; hp, head process;
ps, primitive streak. Bar, 100^m.
Cell fate in mouse endoderm
637
Table 4. Incidence of embryos with HRP-labelled cells in various germ layers after injection into single anterior
or posterior axial endoderm cells and 22 h culture
No. with HRP-labelled cells
Stage at
injection
Region
Anterior
Prestreak +
early streak
Prestreak +
early streak
Prestreak
Early streak
Posterior
Anterior
Posterior
Prestreak
Early streak
Total
Endoderm +
ectoderm/
mesoderm
Ectoderm/
mesoderm
only
Culture
time (h)
No.
injected
Total
0
47
34
33 (97 %)
1(3%)
0
a
0
45
32
31 (97 %)
0
1(3%)
b
22
22
15
36
11
24
10 (91 %)
21 (88 %)
1 (9 %)
2 (8 %)
0
1(4%)
51
35
31(89%)
3 (9 %)
1(2%)
c
14
36
50
11
29
40
8 (73 %)
17 (59 %)
25 (62 %)
2(18%)
5 (17 %)
7 (18 %)
1 (9 %)
7 (24 %)
8 (20 %)
d,
d,
d
22
22
22
Endoderm
only
l
cvd :tf = 7-39, df=2, P<0-01.
cvd .yC ! = 2-04, df=2, P>0-l.
d , v d 2 : ^ z = 0-66,df=2,P>0-25.
A v
ant
ant.
f
0 1 mm
Fig. 7. Position of labelled cells in embryos with label in both endoderm and ectoderm/mesoderm, after injection into
single posterior axial endoderm cells of (A) prestreak-stage and (B) early-streak-stage embryos. The position of zone IV
is indicated by a black strip. • , endoderm; D, ectoderm; • , mesoderm. For further explanation, see legend to Fig. 2.
638
K. A. Lawson, R. A. Pedersen and S. van de Geer
(3) Cell numbers and population dynamics
The frequency distribution is shown in Fig. 18. In
contrast to embryos after 22h culture, a significant
proportion had no labelled cells. From the controls,
the expected number of successfully injected embryos
would be: 43x165/66=107, (Table 7) but only 71
(66%) were found with labelled cells after 44h.
Therefore, either all the descendants of 34% of the
successfully injected cells died during the second half
of the culture period, or the HRP concentration was
below the detection level in these descendants and,
possibly, in some cells of other embryos.
Many axial endoderm cells die between midstreak
and head-fold stages (Poelmann, 1980; Lawson et al.
1986), but it is not known whether cell death is lineage
determined. The equation for population increase
(see section A4a) gives an unbiased estimate even in
the presence of cell death, provided all surviving
descendants are detected. According to the data in
Fig. 17, Table 3 (series 3), and Table 7, the population increase in the endoderm layer during 44 h was
ant.
0 1 mm
Fig. 8. Position of labelled ectoderm and mesoderm cells
in embryos with no label in endoderm, after injection
into single posterior axial endoderm cells of (A)
prestreak-stage and (B) early-streak-stage embryos. The
position of zone IV is indicated by a black strip.
D, ectoderm; • , mesoderm. For further explanation see
legend to Fig. 2.
1041 /16 + 30 + 3
= 7-06.
66
165,
Since the population increase due to descendants
remaining in endoderm during the first 22 h was 5-55,
this implies that the increase during the following 22 h
was only 27 %. The population increase of endoderm
of 7-5-day-old embryos (midstreak to neural plate
stages) was 101 % during 24 h culture to early somite
stages (Lawson etal. 1986); although 6-7 day embryos
may be growing more slowly during their second day
of culture, the discrepancy suggests that at least some
endoderm descendants of labelled cells were not
detected after 44h culture.
Discussion
the visceral yolk sac 44 h later, a relatively short
stretch of endoderm in the region of the anterior end
of the early primitive streak contributes to embryonic
endoderm at early somite stages.
Labelled descendants in the visceral yolk sac were
aligned parallel to the equator of the conceptus
(Figs 14, 15) or as a loosely coherent clump
(Fig. 16A); those in the embryo tended to be aligned
along the embryonic axis (Fig. 15) and did not form a
coherent patch (Fig. 16B).
(b) Embryos with labelled ectoderm/mesoderm. Of
the eleven embryos with labelled cells in ectoderm/
mesoderm, seven had been injected in zone IV at the
early streak stage; four prestreak-stage embryos had
been injected in zones II, IV (two embryos) and V.
The sample was too small to draw conclusions about
the distribution of labelled descendants.
HRP has a distinct but limited usefulness as a lineage
marker in the postimplantation mouse embryo. HRP
was injected intracellularly to analyse endoderm fate
in situ, on the assumption that the technique would
not interfere with the behaviour of the system being
studied. We found no evidence of interference: successfully injected cells did not die within the first cell
cycle, no effect was found on the number of cells in
either S phase or mitosis 10 h after injection (Lawson
et al. 1986), and the majority of injected cells went
through three cell cycles during the first 22 h. The
results from embryos cultured for 44 h, however,
indicate that while the qualitative data from these
embryos are reliable, the quantitative data may be
biased because labelling in some samples was not
resolvable. Up to 64-fold dilutions of intracellularly
injected HRP can be detected reliably (Lawson et al.
1986): it should therefore be possible to follow
Cell fate in mouse endoderm
639
Posterior
Anterior
ant.
0-1 mm
Fig. 9. Position of endoderm descendants after injection into single axial endoderm cells of anterior and posterior
regions of (A) prestreak-stage and (B) early-streak-stage embryos. The positions of zone II (anterior) and zone IV
(posterior) are indicated by a black strip. For further explanation see legend to Fig. 2.
10 12 14 16 18 20 22 24 26 28
HRP-labelled cells/embryo
30
32
34
36
Fig. 10. Frequency distribution of the number of HRP-labelled cells/embryo after 22 h culture {n = 191). Open blocks,
embryos with labelled endoderm only; hatched blocks, embryos with labelled endoderm and ectoderm/mesoderm; crosshatched blocks, embryos with labelled ectoderm/mesoderm only. 0' represents the number of injected embryos without
labelled cells corrected for controls.
640
K. A. Lawson, R. A. Pedersen and S. van de Geer
Table 5. Frequency distribution of the number of labelled cells/embryo after 22 h culture according to site of
injection and germ layer position of descendants
Injection
zones
All
I,II,III
IV,V
IV,V
Germ
layer
descendants
Total
no. of
embryos
All
Endoderm only
Endoderm only
Ectoderm/mesoderm
+ Endoderm
192
66
78
48
HRP-labelled cells/embryo*
1
8
1
5
2
3-4
2
18
11
5
2
(4%)
(2%)
(6%)
(4%)
(9%)
(17%)
(6%)
(4%)
69
24
25
20
27 (14%)
16 (24%)
11 (14%)
0
(36
(36
(32
(42
17-32
9-16
5-8
%)
%)
%)
%)
52 (27 %)
12(18%)
27 (35 %)
13(27%)
17 (9%)
2 (3%)
5 (6%)
10 (21 %)
33-64
1 (1%)
0
0
1 (2%)
*The data have been grouped in classes according to cell generation ('mixed generation' classes). Each class, except 1, contains a
mixture of descendants of initially single cells and cell pairs; e.g. class 5-8 contains third generation descendants of labelled single cells
and second generation descendants of labelled cell pairs.
Table 6. Population kinetics derived from the calculated number of descendants of single labelled cells during 22 h
culture
Percentage distribution over 0-5 cell generations *
zones
Germ
layer
descendants
Total
no. of
embryos
0
1
2
3
4
All
I,II,III
IV,V
All
Endoderm only
Endoderm only
192
66
78
7-6
2-8
11-7
11-3
28-3
2-8
16-6
22-4
22-9
52-2
46-5
40-8
8-5
0
21-9
Injection
5
Population
increase
Population
doubling
time (h)
3-7
0
0
5-98
4-30
610
8-5
10-5
8-4
-
*The fraction with descendants traversing each generation was calculated as follows, assuming that control and incubated
populations had the same initial frequencies of single, double and triple labelled cells, and that the probability of division was the same
regardless of the initial number of labelled cells. The number of undivided single cells was obtained from the cultured embryos with
only one labelled cell. This figure was applied to the proportion of cell pairs to singletons in the initial population to estimate the
number of cultured embryos with two labelled cells that was due to initial cell pairs that had not divided and, hence, by subtraction, to
estimate the number of single labelled cells that had divided once. The process was iterated on the succeeding 'mixed generation"
classes to obtain the entire distribution for single labelled cells and, hence, the population increase, population doubling time and
(assuming no cell death) percentage distribution of cell generations.
descendants through six generations if only dilution is
involved, but additional metabolic degradation or
intracellular segregation of the enzyme would reduce
this sensitivity. The generation time of embryonic
endoderm at the head-fold stage in cultured embryos
is maximally 11-5 h (Lawson et al. 1986), so that at
least five cell cycles will have been completed by the
dividing population of 6-7-day-old embryos after 2
days. Even a small proportion of faster dividing cells,
or an uneven distribution of cytoplasmic HRP between sister cells at this dilution, could result in
failure to detect descendants and cause a bias in the
frequency distribution of labelled cells. The detection
level for injected cells is therefore probably higher
than that expected from dilution alone. This means
that, compared with amphibians (Jacobson & Hirose,
1978; Hirose & Jacobson, 1979; Heasman, Wylie,
Hausen & Smith, 1984; Masho & Kubota, 1986), fish
(Kimmel & Law, 1985; Kimmel & Warga, 1986), a
variety of invertebrates (Weisblat et al. 1978; Kominami, 1983; Nishida & Satoh, 1983; Taghert, Doe &
Goodman, 1984) and murine preimplantation stages
(Balakier & Pedersen, 1982; Cruz & Pedersen, 1985;
Pedersen, Wu & Balakier, 1986), HRP can be
detected for a relatively brief period (1-2 days)
during postimplantation development in the mouse.
It is, however, reliable for short-term lineage studies
and for analysing morphogenetic movement and
population kinetics over three tofivecell generations.
Morphogenetic movement
The position and alignment of endoderm descendants
relative to the site of injection along the embryonic
axis, together with a comparison of embryos injected
at prestreak and early streak stages and the heterogeneity of cell fate in the posterior endoderm, led us
to the following interpretation. At the time the
posterior ectoderm thickens to produce the primitive
streak and overt bilateral symmetry, a subpopulation
of cells appears in the axial endoderm of the posterior
half of the embryo. Some of these surface cells, or
their progeny, move internally, contributing to ectoderm and mesoderm. Others, concentrated near the
anterior end of the early streak, spread anteriorly,
either displacing or replacing the axial endoderm
from the anterior half of the embryo. The displaced
anterior endoderm itself shifts anteriorly and anterolaterally towards, and partly onto, the visceral yolk
Cell fate in mouse endoderm
641
20-
o
&• 10-
1 9-
15
f
=
3H
2-
1
2
3
a-p axis (relative units)
4
5
Fig. 11. Total HRP-labelled cells/embryo after 22 h
plotted against position of the injection site on the
anterior-posterior axis. Bars, mean ± S.D. Statistical
significance of linear regression: F= 9-84, df= 1,188,
P< 0-005.
sac. The posterior-derived endoderm cells, spreading
forward over the distal tip of the egg cylinder, avoid
or are themselves displaced by the head process,
which begins to insert into the endoderm layer at the
node at the late-streak stage and becomes progressively more exposed anteriorly. Endoderm descendants from the posterior half of the streak spread along
the axis but remain posterior to the node; some move
posteriorly onto the visceral yolk sac. Thus, before
foregut invagination has begun, most of the embryonic axis is occupied by endoderm of posterior origin,
partially bisected by the head process.
During the following day, the anterior-derived
endoderm cells are displaced further anteriorly and
anterolaterally onto the yolk sac. The anterior shift
along the axis continues between the midstreak and
neural plate stages (Lawson et al. 1986) and, by the
time somites are forming, the posterior-derived cells
are located at the anterior intestinal portal and have
been incorporated in the foregut, trunk and postnodal endoderm. In addition, midstreak- and latestreak-stage postnodal endoderm shifts posteriorly
towards and onto the visceral yolk sac. The expansion
of endoderm from the anterior end of the primitive
streak after the early-streak stage is clearly illustrated
when the site of injection is classified according to the
2
3
a-p axis (relative units)
4
5
Fig. 12. HRP-labelled endoderm cells/embryo after 22 h
culture, plotted against position of the injection site on
the anterior-posterior axis. To make the log scale
analysis possible, sites contributing exclusively to
ectoderm/mesoderm were assumed to have one
descendant in endoderm. Bars, mean ± S.D. NO
regression.
position of labelled cells 2 ddys later (Fig. 19B): there
is a border in the endoderm between cells that will
populate the anterior and posterior regions of the
yolk sac; the border area contains cells that contribute to the entire axial endoderm of the early somite
embryo.
Although this picture is incomplete, it is strikingly
similar to the detailed map of morphogenetic movement of endoderm in the avian embryo (Spratt &
Haas, I960; Vakaet, 1962, 1970; Nicolet, 1971;
Rosenquist, 1972), where endoderm expands, initially anteriorly, from the anterior end of the early
primitive streak, displacing the hypoblast towards the
margin of the area pellucida and then invaginates to
form the foregut of the early somite embryo (Rosenquist, 1966, 1972). This cell behaviour may be a
morphogenetic phenomenon common to both birds
and mammals, for which the difference between the
flat blastoderm of the chick and the cup-shaped
embryo peculiar to some rodents is a geometric
irrelevance.
Since endoderm expands from the anterior end of
the primitive streak, local differences in cell behaviour could be involved in the expansion. Posterior
642
K. A. Lawson, R. A. Pedersen and 5. van de Geer
endoderm cells have more surviving descendants
after 24 h than do anterior cells, but a proportion of
these contribute to ectoderm/mesodenn and so are
lost to the surface layer. However, the linear regression of endoderm descendants on the position of
ancestors along the axis remained when the part of
the population contributing only to ectoderm/mesodenn was excluded, indicating that posterior endoderm cells contribute more descendants to latestreak-stage endoderm than do anterior cells: the
calculated population doubling time in zones IV and
V was 8-4h, compared with 10-5h in zones I—III.
20'
10.
9
8T
6'
54-
=?. 30.
OS
X
2-
1
2
3
a-p axis (relative units)
4
5
Fig. 13. HRP-labelled endoderm cells/embryo after 22 h
culture, plotted against position of the injection site on
the anterior-posterior axis, excluding embryos with sites
contributing only to ectoderm/mesodenn. Bars,
mean ± S.D. Statistical significance of linear regression:
F= 4-55, df= 1,172, P<0-05.
Since the regression disappeared when the contribution of the total initial population to endoderm
only was considered, the results represent the behaviour of a surface population with a uniform expansion
in the face of a drain posteriorly to ectoderm/mesoderm, i.e. they do not account for the anterior
displacement of endoderm. Even if the exit of cells
from the surface to deeper layers is counteracted by
entry of new cells to the surface, this would allow
expansion from the point of entry but would not
impose directionality on the expanding layer.
The end of the first 22 h culture period of earlystreak-stage embryos coincides with a stage when
there is substantial cell death in the axial endoderm
(Poelmann, 19816) and the number of cells dying is
greater anteriorly than postnodally (Lawson et al.
1986). The head process inserts into the endoderm
layer at this time. Although cell death may create
additional space anteriorly that other posteriorderived cells can occupy, it is unlikely to be the main
source of displacement, since endoderm displacement occurs before the midstreak stage and there is
no cell death at the early-streak stage.
Endoderm displacement could be passive, reflecting morphogenetic movement of the underlying
ectoderm. If so, the position of labelled endoderm
descendants implies anterior displacement of axial
ectoderm in the anterior half of the egg cylinder and
posterolateral shift at the anterior end of the embryo.
In addition, cells contributing to the primitive streak
would elongate the streak part of the axis, initially
anteriorly and, later, both anteriorly and posteriorly.
In the rabbit epiblast, lateral and anterolateral cells
move towards the primitive streak along the rim of
the embryonic shield (Daniel & Olson, 1966) and
similar movements occur in the avian epiblast (Spratt
& Haas, 1965; Vakaet, 1984); the anterolateral alignment of descendants of anterior endoderm in the
mouse is consistent with such a movement. Forward
expansion of axial cells in the epiblast between the
anterior end of the streak and the anterior end of the
embryo could be produced by high proliferative
Table 7. Incidence of embryos with HRP-labelled cells in various germ layers after injection into a single axial
endoderm cell and 44 h culture
No. with HRP-labelled cells
Stage at
injection
Prestreak + early streak
Prestreak
Early streak
Total
Culture
"time (h)
No.
injected
Total
Endoderm
only
Endoderm +
ectoderm/
mesoderm
Ectoderm/
mesoderm
only
0
66
43
42(98%)
0
1(2%)
44
44
44
65
100
165
27
44
71
23
37
60(85%)
2
3
5(7%)
2
4
6(8%)
Cell fate in mouse endoderm
643
II
ant
III
ant.
ant.
0-1 mm
Fig. 14. Position of endoderm
descendants 44 h after injection into single
axial endoderm cells of zones I—III of (A)
prestreak-stage and (B) early-streak-stage
embryos. The location of descendants is
projected onto a sagittal section (middle
figure) and onto the ventral surface of a
flattened, early somite embryo.
O, notochord. For further explanation
see legend to Fig. 2.
644
ant.
K. A. Lawson, R. A. Pedersen and S. van de Geer
activity at or near the cranial end of the primitive
streak (Daniel & Olson, 1966; Snow, 1977, 1978).
The cellular correspondence between the two
layers is, however, less close than would be expected
if they were behaving as a unit. First, injections of
HRP into the distal tip (zone III) of prestreak-stageegg cylinders, made in such a way that both ectoderm
and endoderm were labelled, showed a slight anterior
shift of ectoderm descendants, but this was clearly
less than the anterior shift of endoderm (J. J.
Meneses, personal communication). Second, an indication of the behaviour of ectoderm cells in the streak
compared with the overlying endoderm was given in
the present experiments by embryos with labelled
cells in both ectoderm and endoderm. Of nine such
embryos, injected in zones IV and V (Fig. 7), labelled
ectoderm cells were later found at the distal tip of the
embryo, at the anterior end of the streak, i.e. the
labelled cells had moved forward with the extending
streak but retained their original position relative to
the anterior end of the streak. In contrast, the
labelled endoderm cells of eight of these embryos
were situated far anterior, and those of one embryo
just anterior, to the labelled ectoderm. Third,
although the most proximal endoderm will be freed
from association with embryonic ectoderm when the
latter contributes to forming the anterior and posterior amniotic folds (Snell & Stevens, 1966), the
eventual colonization of the yolk sac by even the
most distal endoderm makes a close cellular correspondence of endoderm and ectoderm movements
unlikely.
Therefore, while the direction of endoderm expansion and the alignment of endoderm descendants may
reflect morphogenetic movement in the underlying
ectoderm in the anterior half of the embryo and
growth of the primitive streak in the posterior half,
the expansion per se appears to be a property of the
endoderm layer.
ant.
Cell fate and cell lineage
Except for a limited stretch of posterior endoderm,
descendants of cells from all zones along the axis
of prestreak and early-streak embryos were found
exclusively in the yolk sac of early-somite-stage embryos 2 days later. Since only descendants of primitive endoderm have been found in yolk sac endoderm
at midgestation (Gardner & Papaioannou, 1975;
Gardner & Rossant, 1979) and visceral embryonic
0 1 mm
Fig. 15. Position of endoderm descendants 44 h after
injection into single axial endoderm cells of zones IV and
V of (A) prestreak-stage and (B) early-streak-stage
embryos. The surfaces of the ventral (v) and dorsal (d)
foregut and dorsal hindgut (d1) are shown separately.
A, blood island. For further explanation, see legends to
Figs 2 and 9.
Cell fate in mouse endoderm
645
\
16A
B
Fig. 16. Embryos cultured for 44 h. (A) 6-somite embryo injected in zone I at the early streak stage has 12 labelled cells
(arrow) in the yolk sac endoderm. (B) 6-somite embryo injected in zone IV at the early streak stage. The distribution of
the 64 labelled cells is indicated by arrows. A fairly coherent strip of 43 cells is located caudally over the embryo and
spreads onto the yolk sac. The remaining 21 cells are scattered along the axis of the trunk, the right side of the anterior
intestinal portal, and in the foregut; aip, anterior intestinal portal; a/, allantois; h, heart; mb, mesencephalon; s, somite;
ys, visceral yolk sac. Bar, 200 jim.
endoderm shows the same behaviour as visceral
extraembryonic endoderm both as donor in blastocyst chimaeras (Rossant, Gardner & Alexandre,
1978; Gardner, 1982) and in vitro (Hogan & Tilly,
1981), it seems likely that most of the axial endoderm
at the onset of gastrulation is visceral embryonic
endoderm derived from primitive endoderm.
The presence of descendants of early-streak stage,
but rarely prestreak stage, posterior endoderm in
embryonic endoderm 2 days later indicates that a new
population emerges in the axial endoderm at the time
of primitive streak formation. The descendants of this
population, which occupies a relatively short stretch
of endoderm over the anterior part of the early
primitive streak, were spread along the entire embryonic axis by the neural plate stage and contributed to
embryonic endoderm, including the foregut, at early
somite stages. The two possible sources for this
population are nonaxial endoderm and epiblast. If
nonaxial visceral embryonic endoderm moved into
the region of the primitive streak at the beginning of
gastrulation and then spread along the axis, the
descendants of these cells would make no permanent
contribution to the developing gut, since they form
part of the primitive endoderm lineage. If, on the
other hand, this new population is inserted into the
endoderm layer from the epiblast, it could contain
the ancestors of the fetal endoderm: the descendants
in the early somite embryo would contribute permanently, and not transitorily, to the developing gut.
The arguments against an epiblast origin of the
posterior-derived endoderm originate in the results of
ectopic transplants. In such experiments (Grobstein,
1952; Levak-Svajger & Svajger, 1971, 1974; Diwan &
Stevens, 1976; Beddington, 1983a), derivatives of
definitive endoderm were formed by epiblast from
prestreak to late-streak stages, while endoderm alone
failed to develop or formed parietal endoderm. Only
at the head-fold stage was the capacity to form
endoderm derivatives no longer present in ectoderm,
but it was present in transplants of mesoderm plus
endoderm (Svajger & Levak-Svajger, 1974). While
these experiments illustrate the potency of the epiblast, they do not necessarily pinpoint the normal fate
of the transplanted cells (Beddington, 1981, 1982,
1983a,fe) or that of the isolated endoderm that failed
to develop in an ectopic site. A second line of
argument comes from histological studies, the
646
K. A. Lawson, R. A. Pedersen and S. van de Geer
-*»zs:?
a/77
•
.->
•'V.
v
,#.»
ys
•
•
*
17A
B
Fig. 17. Sections of embryos cultured for 44h. (A) 9-somite embryo injected in zone II at the early primitive streak
stage, showing 7 of the 19 labelled cells in the visceral yolk sac endoderm. The honey-combed appearance of the cell
apices (arrow) is due to large endocytotic vacuoles that do not contain HRP. (B) Transverse section of the 6-somite
embryo shown in Fig. 15B, which had been injected in zone IV, showing labelled cells in dorsolateral and ventrolateral
foregut (arrows), am, amnion;/g, foregut; h, heart; ne, neurectoderm;>\s, visceral yolk sac. Bar, 100jim.
o 10-
o
0
2
4
6
8
10
12
14
16
18
20 22 24 26 28 30 32
HRP-labelled cells/embryo
34
38
42
45
51
55
64
Fig. 18. Frequency distribution of the number of HRP-labelled cells/embryo after 44h culture, n = 71. For further
explanation, see legend to Fig. 9.
authors of which have concluded that the head
process is the main or sole source of the fetal
endoderm (Jolly & Fe"rester-Tadie\ 1936; Snell &
Stevens, 1966; Poelmann, 1981). However, such morphological studies do not take into account the
heterogeneity of origin and fate of cells in the
endoderm layer at midstreak to late-streak stages
(Lawson et al. 1986).
The arguments for an epiblast origin of the posterior-derived endoderm are as follows. First, the
presence of mesoderm and ectoderm descendants
from posterior endoderm indicates that cells can
Cell fate in mouse endoderm
am.
Yolk sac
post.
ant.
Fig. 19. Site of injection at (A) prestreak stage and (B)
early-streak stage, classified according to position of
endoderm descendants 44 h later. • , anterior and
anterolateral yolk sac; O, posterior and posterolateral
yolk sac; T, anterior intestinal portal; V, ventral
foregut; • , dorsal foregut; D, trunk; A, postnodal
endoderm; brackets, descendants in different embryonic
regions in the same embryo; ', additional descendants in
postnodal endoderm or yolk sac endoderm.
exchange between epiblast and endoderm as the
primitive streak is forming. Although we have not
shown conclusively that labelled descendants in endoderm and ectoderm/mesoderm were derived from
one cell or a pair of sister cells, it is unlikely that the
18 % of embryos in this category were the result of
injection into two neighbouring but unrelated cells.
Second, there is virtually no overlap of clonal descendants colonizing yolk sac and embryonic endoderm; the only exceptions were 4 of the 63 embryos
cultured for 44 h, and these had labelled descendants
spreading from the most caudal or laterocaudal part
of the embryo onto the posterior yolk sac: more
prolonged culture would be necessary to resolve the
fate of these descendants. Third, the expansion of
endoderm from the anterior end of the primitive
streak is greater than can be accounted for by the
morphogenetic movement of the ectoderm alone,
although the generation time in the endoderm is
longer than in the epiblast: 8-4 h in the posteriorderived endoderm compared with 7-6h (Poelmann,
1980), 6-25 h (Solter, Skreb & Damjanov, 1971),
4-4-6-7 h (Snow, 1977) and 3-7-4-2h (Lewis & Rossant, 1982) in vivo and 7-5 h in vitro (K. A. Lawson,
unpublished data) in the epiblast. This argument,
however, is not compelling since (1) the epiblast is
647
also forming the mesoderm layer and (2) the flattened, squamous endoderm will cover a greater
surface area than the columnar epiblast (e.g.
1000 fim2 of basal lamina would be occupied by 10-14
epiblast cells (more if the mitotic cells at the lumen
are included), but only by 2-3 squamous endoderm
cells). Fourth, the behaviour of endoderm at midstreak and late-streak stages strikingly resembles that
in the chick embryo (Lawson et al. 1986); earlier in
avian gastrulation the epiblast-derived definitive endoderm inserts into the hypoblast through the primitive streak (for reviews see Nicolet, 1971; Bellairs,
1982, 1986) and its subsequent behaviour (Rosenquist, 1966, 1971, 1972) is similar, if not identical, to
that of the posterior-derived endoderm in the mouse
described here. Finally, an analysis of the prestreak
epiblast has revealed that the epiblast associated with
zone IV, and a slightly larger region in early-streakstage embryos, does indeed have descendants in
endoderm at midstreak to neural plate stages (K. A.
Lawson, unpublished data).
On the basis of the information available, it seems
most likely that epiblast derivatives are first inserted
into the endoderm layer very early in gastrulation at
the anterior end of the primitive streak. Descendants
of these cells would be incorporated into the ventral
foregut and anterior intestinal portal, whereas the
descendants of cells that emerged slightly later would
maintain a more axial position and colonize the dorsal
foregut. The head process, the cranial extension of
the anterior end of the primitive streak, has begun to
form by the midstreak stage but becomes progressively incorporated into the endoderm layer from the
late-streak stage onwards: the axial part forms notochord and endoderm of the trunk (midgut). Descendants from lateral cells in the head process may be
added later to foregut endoderm, just before the head
folds develop and the foregut invaginates (Jolly &
Ferester-Tadie\ 1936; Snell & Stevens, 1966; Poelmann, 1981).
Heterogeneity of the posterior endoderm
The presence of cells in the posterior axial endoderm
that have descendants in ectoderm/mesoderm indicates that the separation of the epiblast and embryonic endoderm is no longer complete when the
primitive streak begins to form. Formation of the
primitive streak in mammals is associated with (a) an
increase in the frequency of mitotic spindles oriented
perpendicular to the cell sheet in the streak region,
thus producing several cell layers (Snow & Bennett,
1978), (b) extensive disruption of the basal lamina in
the streak region, which is much greater than the
local discontinuities observed before streak formation and in lateral regions thereafter (Poelmann,
1981; Takeuchi & Takeuchi, 1981; Franke, Grund,
648
K. A. Lawson, R. A. Pedersen and S. van de Geer
Jackson & Illmansee, 1983), (c) increase in membrane specialization (adhesive plaques, gap junctions
and nuclear pores) in the epiblast cells (Batten &
Haar, 1979) and (d) acquisition of vimentin and loss
of cytokeratins by emerging primary mesenchyme
cells (Franke, Grund, Kuhn, Jackson & Illmansee,
1982). In addition, it must be supposed that cell
contacts within the endoderm layer (apical tight
junctions) (Batten & Haar, 1979) and desmosomes
(Solter, Damjanov & Skreb, 1970; Franke etal. 1983)
are sufficiently unstable at this stage to allow departure of cells. Our interpretation of the phenomenon is
that afluxof epiblast- derived cells into the endoderm
layer coincides with streak formation and that some
cells, or their descendants, are released back into the
mesoderm or ectoderm via the streak. Alternatively,
disruption of the basal lamina could be a sufficient
condition for visceral embryonic endoderm cells to
leave the surface layer and temporarily contribute to
mesoderm and ectoderm. This seems to be unlikely,
since caudal endoderm cells grafted caudally into the
primitive streak of 7-5-day embryos do not incorporate into embryonic structures (Copp, Roberts &
Polani, 1986).
Population kinetics
The frequency distribution of labelled cells after 22 h
(Fig. 10) indicates lack of synchrony in the cell cycles
of succeeding generations, but could also reflect
attrition due to cell death. There was no indication
that labelled progenitors died at the prestreak or
early streak stage or of loss of all labelled descendants
in some embryos: both phenomena would have led
to an increase in the number of embryos without
labelled cells. It is less easy to assess the loss of
subpopulations of labelled descendants, but important in the present context, since there is significant
cell death in axial endoderm injected at midstreak
and later stages (Lawson et al. 1986). Cell death of
some descendants would not bias the calculated
frequency distribution of descendants of single cells,
assuming that the chance of cell death was the same
for the descendants of singletons and pairs: this
calculation showed that more than 90% of the
population were dividing. Further classification into
cell generations, however, assumes there is no cell
death and the classification will be distorted if half or
more of a generation dies and the effect of cell death
is compounded by heterogeneity in cell cycle length.
The data so classified (Table 6) are compatible with
some cell death in anterior endoderm during the
second cycle after injection, at the earliest, and in the
posterior endoderm during the third cycle. Complete
analysis of population growth requires independent
estimation of cell death and cell cycle length and
variation, as well as of the size of the dividing
population.
Population doubling time and generation time are
only equivalent when all cells are dividing and there is
no cell death. The population doubling time of 8-7 h is
therefore a maximum estimate of generation time. It
is markedly shorter than that of 16-6 h based on cell
counts between 6-5 and 7-5 days in vivo (Snow, 1977).
The discrepancy can be explained by the fact that
Snow counted cells contained within borders set by
the embryonic ectoderm, while we counted the descendants of a sample of cells only initially within
these borders. Since descendants of zone I and some
zone V cells will have moved onto the yolk sac within
24 h, and some from zones IV and V will have passed
into the interior of the embryo, the population
doubling time within the boundaries set by the
embryonic ectoderm will be longer than the generation time of cells initially within these boundaries,
even if loss to the interior is compensated by insertion
of new cells from the epiblast. On the other hand,
HRP injections were limited to the axial endoderm
and may not be representative of the rest of the
endoderm. The calculated population doubling time
for cells that had exclusively endoderm descendants
varied even along the axis, being 10-5 h in the anterior
cells and 8-4 h in the posterior cells. The value for
anterior endoderm agrees with the cycle time of
10-7h in visceral extraembryonic endoderm during
the same period in vivo, using the labelled mitoses
method (Solter & Skreb, 1968) but is longer than that
of combined visceral embryonic and extraembryonic
endoderm calculated from colcemid-blocked mitoses
(6-6h) (Lewis & Rossant, 1982).
The population kinetics of axial endoderm in vitro
during the first day of gastrulation contrast with those
in the following 24h, when the population doubling
time increases to 23-9h (Lawson et al. 1986): about
half the axial endoderm cells die during the time that
the head process inserts into the surface layer; the
generation time of the surviving cells is 11-5 h or less.
Thus, intracellular injection of HRP provides considerable information about cell population kinetics
that would not be obtained from other, non-clonal
approaches.
Appendix by Sara van de Geer
Statistical comparison of population doubling times
The simplified estimator of the population doubling
time is based on the following assumptions:
(i) the distribution of labelled cells is the same for all
embryos within a cultured group or within the control
group,
Cell fate in mouse endoderm
(ii) the distribution of the initial number of labelled
cells is the same for the control group and the
cultured groups,
(iii) at time t (0=£t=£22h) the expected number of
labelled cells per embryo given the initial number is
equal to
where b depends on the culture under consideration
and j = 1, 2, 3,... is the initial number.
Let p and y. denote the expected number of labelled
cells at time t = 0 and t = 22h, respectively. Under
assumption (iii),
Let population 1 and 2 be the embryos with
labelled endoderm descendants after injection into
zones I, II, III and IV, V, respectively, Let ^ denote
the expected number of labelled cells in an embryo of
population i at time t = 22h, and let T, denote the
population doubling time, i = 1,2. Then
=
=
=
=
Ai
var (£,)
T,
var (f,)
6-348
0-312
10-491
0-463
A2
var (fh)
tvar2 (T,)
=
=
=
=
8-115
0-378
8-974
0-192
The control values for series 1 and 2 (Table 3) were
pooled to give one control group with
Thus the doubling time is
T =
649
p = 1-484, var (p) = 0-003.
22 In 2
We use formula (*) to estimate the covariance
between Tj and T2:
In
Let
COV
P=
total number of labelled cells in the control group
number of labelled sites in the control group
The estimated correlation between T^ and T2 is
and
total number of labelled cells in the cultured group
£ = number of labelled embryos in the cultured group
Under assumption (i) p estimates the expected
initial number of labelled cells in a site of the control
group. Assumption (ii) ensures that p is also an
estimator of p, the number of labelled cells in the
cultured group at time t = 0. We estimate T by
T=
22 In 2
The estimated variances of p and fi can be calculated in the usual way:
var (p) =
number of labelled sites in the control group
A Taylor expansion of T around (p, JX) gives
I
P
A*
Hence, the variance of T can be approximated by
22 In 2
\" I van
T
is approximately normally distributed with expectation zero and unit variance. (If two independent
control groups are used, there is no covariance and
V2(l-/&) reduces to V2)
We have
t = -3-86< -1-96.
Thus the hypothesis Tj = T2 can be rejected at the
5% level.
A~^
+ smaller order terms.
var (f) =
If T, = T2, then
For t we find the value
number of labelled embryos in the cultured group
221n2_ f p - p
=niA7
2
average of (number of labelled cells in cultured embryo) ]—fi
t
cover, ,f2)
Vvar (f J var (t 2 )
-1-96 V2JF
[average of (number of labelled cells in control site)2]-f>2
2
var(/j) = "
^
We thank Jenny Narraway for histology, Leen Boom and
Carmen Kroon-Lobo for photographic and art work, and
Mary McKinney for editorial assistance. We are indebted to
Richard Gill of the Centre of Mathematics and Computer
Science, Amsterdam, for advice on the statistical comparison of population doubling times.
This work was partially supported by the Office of Health
and Environmental Research, US Department of Energy,
contract no. DE-AC03-76-SF01012 and by the Hubrecht
Fund.
650
K. A. Lawson, R. A. Pedersen and S. van de Geer
References
H. & PEDERSEN, R. A. (1982). Allocation of
cells to inner cell mass and trophectoderm lineages in
preimplantation mouse embryos. Devi Biol. 90,
352-362.
BATTEN, B. E. & HAAR, J. L. (1979). Fine structural
differentiation of germ layers in the mouse at the time
of mesoderm formation. Anat. Rec. 194, 125-142.
BEDDINGTON, R. S. P. (1981). An autoradiographic
analysis of the potency of embryonic ectoderm in the
8th day postimplantation mouse embryo. J. Embryol.
exp. Morph. 64, 87-104.
BEDDINGTON, R. S. P. (1982). An autoradiographic
analysis of tissue potency in different regions of the
embryonic ectoderm during gastrulation in the mouse.
J. Embryol. exp. Morph. 69, 265-285.
BEDDINGTON, R. S. P. (1983a). Histogenetic and
neoplastic potential of different regions of the mouse
embryonic egg cylinder. J. Embryol. exp. Morph. 75,
189-204.
BEDDINGTON, R. (19836). The origin of the foetal tissues
during gastrulation in the rodent. In Development in
Mammals, vol. 5 (ed. M. H. Johnson), pp. 1-32.
Amsterdam: Elsevier.
BEDDINGTON, R. (1986). Analysis of tissue fate and
prospective potency in the egg cylinder. In
Experimental Approaches to Mammalian Embryonic
Development (ed. J. Rossant & R. A. Pedersen),
pp. 121-147. New York: Cambridge University Press.
BELLAIRS, R. (1982). Gastrulation processes in the chick
embryo. In Cell Behaviour (ed. R. Bellairs, A. Curtis &
G. Dunn), pp. 395-427. Cambridge: Cambridge
University Press.
BELLAIRS, R. (1986). The primitive streak. Anat. Embryol.
174, 1-14.
COPP, A. J., ROBERTS, H. M. & POLANI, P. E. (1986).
Chimaerism of primordial germ cells in the early
postimplantation mouse embryo following
microsurgical grafting of posterior primitive streak cells
in vitro. J. Embryol. exp. Morph. 95, 95-115.
CRUZ, Y. P. & PEDERSEN, R. A. (1985). Cell fate in the
' polar trophectoderm of mouse blastocysts as studied by
microinjection of cell lineage tracers. Devi Biol. 112,
73-83.
DANIEL, J. C , JR & OLSON, J. D. (1966). Cell movement,
proliferation and death in the formation of the
embryonic axis of the rabbit. Anat. Rec. 156, 123-127.
DIWAN, S. B. & STEVENS, L. C. (1976). Development of
teratomas from the ectoderm of mouse egg cylinders. J.
natn. Cancer Inst. 57, 937-942.
ENDERS, A. C , GIVEN, R. L. & SCHLAFKE, S. (1978).
Differentiation and migration of endoderm in the rat
and mouse at implantation. Anat. Rec. 190, 65-78.
BALAKIER,
FRANKE, W. W., GRUND, C , JACKSON, B. W. &
ILLMANSEE, K. (1983). Formation of cytoskeletal
elements during mouse embryogenesis. IV.
Ultrastructure of primary mesenchymal cells and their
cell-cell interactions. Differentiation 25, 121-141.
FRANKE, W. W., GRUND, C , KUHN, C , JACKSON. B. W.
& ILLMANSEE, K. (1982). Formation of cytoskeletal
elements during mouse embryogenesis. III. Primary
mesenchymal cells and the first appearance of vimentin
filaments. Differentiation 23, 43-59.
GARDNER, R. L. (1978). The relation between cell lineage
and differentiation in the early mouse embryo. In
Genetic Mosaics and Cell Differentiation (ed. W. J.
Gehring), pp. 205-241. Heidelberg: Springer-Verlag.
GARDNER, R. L. (1982). Investigation of cell lineage and
differentiation in the extraembryonic endoderm of the
mouse embryo. J. Embryol. exp. Morph. 68, 175-198.
GARDNER, R. L. (1984). An in situ cell marker for clonal
analysis of development of the extraembryonic
endoderm in the mouse. J. Embryol. exp. Morph. 80,
251-288.
GARDNER, R. L. & PAPAIOANNOU, V. E. (1975).
Differentiation in the trophectoderm and inner cell
mass. In The Early Development of Mammals (ed. M.
Balls & A. E. Wild), pp. 107-132. Cambridge:
Cambridge University Press.
GARDNER, R. L. & ROSSANT, J. (1979). Investigation of
the fate of 4-5 day post-coitum mouse inner cell mass
cells by blastocyst injection. J. Embryol. exp. Morph.
52, 141-152.
GROBSTEIN, C. (1952). Intraocular growth and
differentiation of clusters of mouse embryonic shields
cultured with and without primitive endoderm and in
the presence of possible inductors. J. exp. Zool. 119,
355-380.
HEASMAN, J., WYLIE, C. C , HAUSEN, P. & SMITH, J. C.
(1984). Fates and states of determination of single
vegetal pole blastomeres of A", laevis. Cell 37, 185-194.
HIROSE, G. & JACOBSON, M. (1979). Clonal organization
of the central nervous system of the frog. I. Clones
stemming from individual blastomeres of the 16-cell
and earlier stages. Devi Biol. 71, 191-202.
HOGAN, B. L. M. & TILLY, R. (1981). Cell interactions
and endoderm differentiation in cultured mouse
embryos. J. Embryol. exp. Morph. 62, 379-394.
JACOBSON, M. & HIROSE, G. (1978). Origin of the retina
from both sides of the embryonic brain: A contribution
to the problem of crossing at the optic chiasma. Science
VSl, 637-639.
JOLLY, J. & FERESTER-TADIE, M. (1936). Recherches sur
l'oeuf du rat et de la souris. Archs Anat. Microsc. 32,
323-390.
KIMMEL, C. B. & LAW, R. D. (1985). Cell lineage of
zebrafish blastomeres. III. Clonal analyses of the
blastula and gastrula stages. Devi Biol. 108, 94-101.
KIMMEL, C. B. & WARGA, R. M. (1986). Tissue-specific
cell lineages originate in the gastrula of the zebra fish.
Science 231, 365-368.
KOMINAMI, T. (1983). Establishment of embryonic axes in
larvae of the starfish, Asterina pectinifera. J. Embryol.
exp. Morph. 75, 87-100.
LAWSON, K. A., MENESES, J. J. & PEDERSEN, R. A.
(1986). Cell fate and cell lineage in the endoderm of
the presomite mouse embryo, studied with an
intracellular tracer. Devi Biol. 115, 325-339.
LEVAK-SVAJGER, B. & SVAJGER, A. (1971). Differentiation
of endodermal tissues in homografts of primitive
Cell fate in mouse endoderm
ectoderm from two-layered rat embryonic shields.
Experientia 27, 683-684.
LEVAK-SVAJGER, B. & SVAJGER, A. (1974). Investigation
on the origin of the definitive endoderm in the rat
embryo. J. Embryol. exp. Morph. 32, 445-459.
LEWIS, N. E. & ROSSANT, J. (1982). Mechanism of size
regulation in mouse embryo aggregates. J. Embryol.
exp. Morph. 72, 169-181.
MASHO, R. & KUBOTA, H. Y. (1986). Developmental
fates of blastomeres of eight-cell-stage Xenopus laevis
embryos. Develop. Growth Differ. 28, 113-123.
NADIJCKA, M. & HILLMAN, N. (1974). Ultrastructural
studies of the mouse blastocyst substages. J. Embryol.
exp. Morph. 32, 675-695.
NICOLET, G. (1971). Avian gastrulation. Adv. Morphogen.
9, 231-262.
NISHIDA, H. & SATOH, N. (1983). Cell lineage analysis in
ascidian embryos by intracellular injection of a tracer
enzyme. I. Up to the eight-cell stage. Devi Biol. 99,
382-394.
PEDERSEN, R. A., WU, K. & BALAKIER, H. (1986). Origin
of the inner cell mass in mouse embryos: cell lineage
analysis by microinjection. Devi Biol. 117, 581-595.
POELMANN, R. E. (1980). Differential mitosis and
degeneration patterns in relation to the alterations in
the shape of the embryonic ectoderm of early postimplantation mouse embryos. /. Embryol. exp. Morph.
55, 33-51.
POELMANN, R. E. (1981a). The formation of the
embryonic mesoderm in the early post-implantation
mouse embryo. Anat. Embryol. 162, 29-40.
POELMANN, R. E. (1981i>). The head-process and the
formation of the definitive endoderm in the mouse
embryo. Anat. Embryol. 162, 41-49.
ROSENQUIST, G. C. (1966). A radioautogTaphic study of
labeled grafts in the chick blastoderm. Development
from primitive-streak stages to stage 12. Carnegie Inst.
Wash. Contrib. Embryol. 38, 71-110.
ROSENQUIST, G. C. (1971). The location of the pregut
endoderm in the chick embryo at the primitive streak
stage as determined by radioautographic mapping. Devi
Biol. 26, 323-335.
ROSENQUIST, G. C. (1972). Endoderm movements in the
chick embryo between the early short streak and head
process stages. J. exp. Zool. 180, 95-104.
ROSSANT, J. (1986). Development of extraembryonic cell
lineages in the mouse embryo. In Experimental
Approaches to Mammalian Embryonic Development (ed.
J. Rossant & R. A. Pedersen), pp. 97-120. New York:
Cambridge University Press.
651
histological sections of M. miisculus<r->M. caroli
chimaeras. J. Embryol. exp. Morph. 73, 179-191.
A. E. (1986). Processes involved in
normal and abnormal fusion of the neural walls in
murine embryos. Thesis, Leiden University.
SNELL, G. D. & STEVENS, L. C. (1966). Early
embryology. In Biology of the Laboratory Mouse, 2nd
edn (ed. E. L. Green), pp. 205-245. New York:
McGraw-Hill.
SNOW, M. H. L. (1977). Gastrulation in the mouse:
Growth and regionalization of the epiblast. J. Embryol.
exp. Morph. 42, 293-303.
SNOW, M. H. L. (1978). Proliferative centres in
embryonic development. In Development in Mammals,
vol. 3 (ed. M. H. Johnson), pp. 337-362. Amsterdam:
North-Holland.
SNOW, M. H. L. & BENNETT, D. (1978). Gastrulation in
the mouse: assessment of cell populations in the
epiblast of fwi«/fwis embryos. J. Embryol. exp. Morph.
47, 39-52.
SOLTER, D. & DAMJANOV, I. (1973). Explantation of
extraembryonic parts of 7-day-old mouse egg cylinders.
Experientia 29, 701-702.
SOLTER, D., DAMJANOV, I. & SKKEB, N. (1970).
Ultrastructure of mouse egg-cylinder. Z. Anat.
EntwickGesch. 132, 291-298.
SOLTER, D. & SKREB, N. (1968). La dur£e des phases du
cycle mitotique dans diffdrentes regions du cylindreSMITS VAN PROOIJE,
oeuf de la souris. C.r. hebd. S&anc. Acad. Sci., Paris
267,659-661.
D., SKREB, N. & DAMJANOV, I. (1971). Cell cycle
analysis in the mouse egg-cylinder. Expl Cell Res. 64,
331-334.
SPINDLE, A. (1980). An improved culture medium for
mouse blastocysts. In Vitro 16, 669-674.
SPRATT, N. T., JR & HAAS, H. (1960). Morphogenetic
movements in the lower surface of the unincubated and
early chick blastoderm. /. exp. Zool. 144, 139-157.
SPRATT, N. T., JR & HAAS, H. (1965). Germ layer
formation and the role of the primitve streak in the
chick. I. Basic architecture and morphogenetic tissue
movements. J. exp. Zool. 158, 9-38.
SOLTER,
STREIT, P. & REUBI, J. C. (1977). A new and sensitive
(1978). Investigation of the potency of cells from the
postimplantation mouse embryo by blastocyst injection:
a preliminary report. J. Embryol. exp. Morph. 48,
239-247.
ROSSANT, J. & PAPAIOANNOU, V. E. (1977). The biology
of embryogenesis. In Concepts in Mammalian
Embryogenesis (ed. M. I. Sherman), pp. 1-36.
Cambridge, MA: MIT Press.
method for axonally transported horseradish
peroxidase (HRP) in the pigeon visual system. Brain
Res. 126, 530-537.
SVAJGER, A. & LEVAK-SVAJGER, B. (1974). Regional
developmental capacities of the rat embryonic
endoderm at the head-fold stage. J. Embryol. exp.
Morph. 32, 461-467.
TAGHERT, P. H., DOE, C. Q. & GOODMAN, C. S. (1984).
Cell determination and regulation during development
of neuroblasts and neurones in grasshopper embryo.
Nature, Land. 307, 163-165.
TAKEUCHI, I. K. & TAKEUCHI, Y. K. (1981). Intercellular
contacts between the embryonic or extraembryonic
ectoderm and the primitive endoderm in rat egg
cylinders prior to the formation of the primitive streak.
Devi Growth, Differ. 23, 157-164.
ROSSANT, J., VIJH, M., SIRACUSA, L. D. & CHAPMAN, V.
TAM, P. P. L. & BEDDINGTON, R. S. P. (1987). The
ROSSANT, J., GARDNER, R. L. & ALEXANDRE, H. L.
M. (1983). Identification of embryonic cell lineages in
formation of mesodermal tissues in the mouse during
652
K. A. Lawson, R. A. Pedersen and S. van de Geer
gastrulation and early organogenesis. Development 99,
.109-126.
THEILER, K. (1972). The House Mouse. Development and
Normal Stages from Fertilization to 4 weeks of Age.
Berlin: Springer-Verlag.
VERMEIJ-KEERS, C. & POELMANN, R. (1980). The neural
crest: a study on cell degeneration and the
improbability of cell migration in mouse embryos.
Neth. J. Zool. 30, 74-81.
VAKAET, L. (1962). Some new data concerning the
formation of the definitive endoblast in the chick
embryo. J. Embryol. exp. Morph. 10, 38-57.
VAKAET, L. (1970). Cinephotomicrographic investigations
of gastrulation in the chick blastoderm. Archs Biol.
Liege Si, 387-426.
VAKAET, L. (1984). Early development of birds. In
Chimaeras in Developmental Biology (ed. N. LeDouann
& A. McLaren), pp. 71-88. New York: Academic
Press.
WEISBLAT, D. A., SAWYER, R. T. & STENT, G. S. (1978).
Cell lineage analysis by intracellular injection of a
tracer enzyme. Science 202, 1295-1298.
{Accepted 16 July 1987)