RESEARCH ARTICLE
251
Development 134, 251-260 (2007) doi:10.1242/dev.02724
Sequential allocation and global pattern of movement of
the definitive endoderm in the mouse embryo during
gastrulation
Patrick P. L. Tam1,2,*, Poh-Lynn Khoo1, Samara L. Lewis1, Heidi Bildsoe1, Nicole Wong1, Tania E. Tsang1,
Jacqueline M. Gad1,‡ and Lorraine Robb3
During mouse gastrulation, endoderm cells of the dorsal foregut are recruited ahead of the ventral foregut and move to the
anterior region of the embryo via different routes. Precursors of the anterior-most part of the foregut and those of the mid- and
hind-gut are allocated to the endoderm of the mid-streak-stage embryo, whereas the precursors of the rest of the foregut are
recruited at later stages of gastrulation. Loss of Mixl1 function results in reduced recruitment of the definitive endoderm, and
causes cells in the endoderm to remain stationary during gastrulation. The observation that the endoderm cells are inherently
unable to move despite the expansion of the mesoderm in the Mixl1-null mutant suggests that the movement of the endoderm and
the mesoderm is driven independently of one another.
INTRODUCTION
Gastrulation of the mouse embryo culminates in the formation of
three primary germ layers: ectoderm, mesoderm and endoderm.
These layers contain the precursors of all the tissues of the fetal body
(Tam and Behringer, 1997; Lawson, 1999; Tam and Gad, 2004).
Fate-mapping studies have revealed a body plan of the embryo that
can be discerned from the regionalization of the germ-layer
progenitors in the epiblast (Lawson et al., 1991; Lawson and
Pedersen, 1992; Quinlan et al., 1995; Parameswaran and Tam, 1995)
and the primitive streak (Tam and Beddington, 1987; Kinder et al.,
1999; Kinder et al., 2001). Subsequently, the precursors of major
tissue types of the body can be discerned at specific locations, with
reference to the orientation and polarity of the primary embryonic
axes, in the ectoderm (Beddington, 1981; Beddington, 1982; Tam,
1989), the mesoderm (Lawson and Pedersen, 1992; Parameswaran
and Tam, 1995; Tam et al., 1997) and the endoderm (Lawson and
Pederson, 1987; Lawson et al., 1986; Tam and Beddington, 1992;
Tam et al., 2004). The endoderm, which is the focus of the present
study, contains the precursors that give rise to the epithelium of both
the gut and the associated organs, such as the liver, pancreas and the
respiratory tract.
Analysis of the developmental fates of cells in the endoderm
layer of the pre-streak mouse embryo has shown that they
contribute almost exclusively to the extraembryonic endoderm.
However, after gastrulation commences, some cells in the
endoderm in the vicinity of the newly formed primitive streak are
fated to become the gut endoderm of the early-somite embryo
(Lawson and Pedersen, 1987). By quantifying the epiblast-derived
1
Embryology Unit, Children’s Medical Research Institute and 2Faculty of Medicine,
University of Sydney, Locked bag 23, Wentworthville, New South Wales 2145,
Australia. 3The Walter and Eliza Hall Institute of Medical Research, 1G, Royal Parade,
Parkville, Victoria 3050, Australia.
*Author for correspondence (e-mail: [email protected])
‡
Present address: Kolling Institute of Medical Research, Royal North Shore Hospital,
St Leonards, NSW 2065, Australia
Accepted 1 November 2006
cells in the endoderm, it was estimated that approximately 5%
(about 400 cells) of the total population of the endoderm is
recruited in the first 4 hours after the onset of gastrulation and that
this incoming population rises to 10% of the total population by
the mid-streak (MS) stage (Tam and Beddington, 1992). The
majority of the newly recruited cells were localized in the
endoderm in the vicinity of the anterior end of the primitive streak
where trafficking of epiblast-derived cells to the endoderm takes
place (Tam and Beddington, 1992). At the MS stage, endoderm
cells in the distal region of the embryo, as well those overlying the
primitive streak, are fated to become foregut and ‘posterior’
endoderm (Lawson et al., 1986), but there seems to be no
precursors for the ‘mid-gut’ endoderm. This raises the issue of
whether the allocation of the endoderm to different segments of the
gut follows the same anterior-posterior order of allocation as that
of mesodermal derivatives (Tam and Tan, 1992; Kinder et al.,
1999).
In the present study, the contribution of cells from different
regions of the endoderm of mid-gastrula-stage embryos to different
segments of the embryonic gut of early-somite-stage embryos was
mapped. Previous analyses on gastrula embryos mainly focused on
endodermal cells along the anterior-posterior body axis (Lawson et
al., 1986; Lawson and Pedersen, 1987) and did not encompass cells
in the non-axial regions of the endoderm layer. Our goal is to
elucidate the developmental fates of the entire endoderm population
of the MS embryo to achieve a comprehensive fate map of the
endoderm that is comparable in its coverage to that of the no- to
early-bud-stage embryo (Tam et al., 2004). We have tracked the
localization of the descendants of a group of cells that can be
visualized by the expression of fluorescent protein tags (achieved by
electroporation of expression vectors) or by the emission signal of
lipophilic flurochrome (by painting the surface of the cells). The use
of these vital cell markers also enables the elucidation of the overall
pattern of cell movement in the endoderm of live embryos. Our
results reveal that the allocation of definitive endoderm begins with
precursors of the rostral-most foregut and the most-posterior
segment of the embryonic gut, followed by the rest of the foregut and
the mid- and hind-gut. The newly recruited definitive endoderm
DEVELOPMENT
KEY WORDS: Definitive endoderm, Allocation, Movement, Mixl1, Gastrulation, Mouse embryo
252
RESEARCH ARTICLE
Development 134 (2)
rapidly expands to displace the pre-existing visceral endoderm to
extraembryonic sites. In addition, we studied the movement of cells
in the Mixl1-null embryo, which is deficient of definitive endoderm.
Based on the results of this analysis, we postulate that the accretion
of cells via recruitment from the epiblast and the primitive streak
may produce the propulsive force that drives the anterior expansion
of the definitive endoderm during gastrulation.
MATERIALS AND METHODS
Mice
Embryo culture
Embryos were harvested from pregnant mice at 7.0 days post coitum (E7.0).
They were dissected from the uterus and the decidua. Following the removal
of the Reichert’s membrane, embryos were sorted into the MS and mid- to
late-streak (M-LS) stages based on their morphology (Downs and Davies,
1993). Prior to experimental manipulation, embryos were kept in DR75
culture medium (Sturm and Tam, 1993) comprised of 75% heat-inactivated
rat serum and 25% Dulbecco’s modified Eagle medium. The culture medium
was kept at 37°C under 5% CO2, 5%O2, 90% N2 in a bottle rotating at 30
RPM. After manipulation (electroporation, painting or cell transplantation),
embryos were cultured in the DR75 medium for up to 48 hours.
Fate mapping by whole-embryo electroporation
The fates of the endoderm of MS and M-LS embryos were mapped by
tracing the distribution of EGFP-tagged cells after development to the
early-head-fold (EHF) stage (24 hours of culture) and early-somite stage
(40-46 hours of culture). Cells in the endoderm of gastrula-stage mouse
embryos were marked by introducing either a CMV-EGFP or CMV-lacZIRES2-EGFP expression vector into the embryos via electroporation. The
embryos were soaked for 5 minutes in an aqueous plasmid solution (1-1.5
␮g/␮l DNA) and were then washed in pH 7.5 Tyrode Ringer solution to
remove the excess DNA that had not been adsorbed on the apical surface
of the endoderm cells. The embryos were then positioned between a plate
and a needle platinum electrode (Davidson et al., 2003). Electroporation
was performed using a BTX Electro Square Porator T820 that delivers
with a 15 V charging voltage 5 square-wave pulses for 50 milliseconds
each with a 1-second inter-pulse gap at a low voltage mode. The
fluorescent EGFP-expressing cells were visualized using a Leica MZ16
FA fluorescence stereomicroscope. The image data were captured by a
SPOT 2 Slider digital camera and editing was performed using SPOT 32
application software and Adobe Photoshop CS. The lacZ-expressing cells
were visualized by X-gal staining of specimens fixed in 4%
paraformaldehyde for 5 minutes at the end of the culture experiment. The
stained embryos were processed for histology to localize the X-gal-stained
cells in the embryonic tissue.
Cells at seven sites of the endoderm were studied (Fig. 1A). A total of six
sites were localized along the anterior-posterior axis: two in the anterior
region (anterior-proximal and anterior-distal domains), one in the distal
region and three in the posterior endoderm (posterior-distal, posterior-middle
and posterior-proximal domains). The area between the anterior and
Fig. 1. Sites of electroporation and domains in the endoderm for
fate mapping experiments. (A) Regions of the endoderm for testing
cell fates by electroporation: anterior-proximal (AP), anterior-distal (AD),
distal (Dist), lateral (Lat), posterior-distal (PD), posterior-middle (PM) and
posterior-proximal (PP). (B) Regions of the endoderm for scoring the
distribution of GFP-expressing cells in early-head-fold-stage embryos.
(C-C⬙) Early-somite-stage embryo. The sub-division of the endoderm is
shown as a schematic lateral view (C), an oblique view (C⬘) and a
ventral view (C⬙). In the early-somite-stage embryo, although the
domain of embryonic foregut may be approximately delineated by the
foregut portal, the precise sub-division of gut segments is not
morphologically evident, and a substantial length of the mid- and hindgut is yet to be formed. Embryonic endoderm of the anterior (A, rostral
segment of the foregut), middle (M, posterior segment of the foregut
and endoderm at the somitic level) and posterior (P, endoderm
associated with the presomitic mesoderm) regions. AYS, anterior yolk
sac; LYS, lateral yolk sac; PYS, posterior yolk sac.
posterior regions was designated as the lateral site. The site of
electroporation was ascertained by the localization of EGFP-expressing cells
after 3 hours of in vitro development. The distribution of EGFP-expressing
endoderm cells was monitored after 24 hours of in vitro culture when the
embryo had developed to the EHF (equivalent to E8.0) stage (Fig. 1B). For
scoring the location and number of EGFP-expressing cells, the yolk sac was
partitioned into the anterior and posterior halves, and the embryonic
endoderm was subdivided into anterior (underneath the anterior half of the
head folds), middle (the posterior half of the head folds) and posterior (from
the posterior margin of the head folds to the posterior end of the primitive
streak) regions (Fig. 1B). After fluorescence imaging and digital
photography, embryos were cultured for another 24 hours, during which
time they developed to the early-somite stage, which is equivalent to about
E8.75 in vivo. The distribution of EGFP-expressing cells was scored in three
regions (anterior-, lateral- and posterior-third) of the yolk sac, and in the
anterior (in the foregut portal and ventral to the heart), middle (the somites)
and posterior (the presomitic mesoderm) regions of the embryo (Fig. 1C⬘C⬙). The number of EGFP-expressing cells in these regions was scored (for
details, see Tam et al., 2004). For estimating the relative contribution of cells
that were derived from the endoderm in different regions of the MS and MLS embryo, the data were presented as percentage of the total population
(Table 1).
DEVELOPMENT
Two strains of mice were used for gastrula-stage endoderm-fate analysis:
out-bred albino ARC/s mice, which provided the embryos for the fatemapping studies, and Mixl1+/GFP mice generated by targeting an inframe
GFP transgene to the Mixl1 locus, which resulted in the deletion of exon 1
of the gene (Hart et al., 2002). A similar strategy was used to generate a lacZ
knock-in Mixl1 allele (L.R., unpublished), which provides another reporter
of Mixl1 activity to compare with the GFP reporter. Mixl1+/GFP mice were
inter-crossed to obtain embryos of Mixl1+/GFP and Mixl1GFP/GFP genotypes.
To obtain donor cells for the fate-mapping study, a new stock of
Mixl1+/GFP;lacZ mice was produced by crossing Mixl1+/GFP mice with
transgenic mice that express the Hmgcr-lacZ transgene (Tam and Tan, 1992).
Mixl1+/GFP;lacZ mice were inter-crossed to produce Mixl1+/+, Mixl1+/GFP
and Mixl1GFP/GFP embryos that also widely express the lacZ transgene in all
embryonic cell types. The Mixl1+/GFP and Mixl1GFP/GFP embryos were the
donors of cells for the transplantation experiment for testing the endodermal
potential of primitive-streak cells. ARC/s strain embryos were used as
recipients of cell transplantation.
Endoderm formation during mouse gastrulation
RESEARCH ARTICLE
253
Table 1. Distribution of GFP-expressing cells in the endoderm of embryos cultured for 24 hours or 40-44 hours after
electroporation
Distribution of descendants of EGFP-expressing cells in the endoderm (% of cell population)
At 24 hours in vitro
Extraembryonic
Site*
Stage†
N
A
AYS
Anterior-proximal
MS
M-LS
12
2
96
100
Anterior-distal
MS
M-LS
6
7
35
39
Distal
MS
M-LS
7
8
5
28
MS
M-LS
5
4
1
4
MS
M-LS
7
6
1
MS
M-LS
5
6
4
MS
M-LS
7
7
38
42
Posterior-distal
Posterior-middle
Posterior-proximal
Lateral
B
PYS
At 48 hours in vitro
Embryonic
C
Ant
D
Mid
Extraembryonic
E
Post
4
10
11
N
F
AYS
G
LYS
12
4
91
90
3
27
36
26
11
2
3
5
3
46
53
41
48
43
24
11
5
9
16
22
2
8
18
5
64
45
17
38
6
4
11
5
4
32
11
8
21
49
47
31
8
6
12
16
28
4
3
12
12
15
9
10
6
80
60
34
22
1
15
12
5
35
32
7
34
H
PYS
Embryonic
I
Ant
J
Mid
K
Post
9
7
2
10
28
27
11
10
1
1
41
42
16
18
26
16
2
2
17
24
39
27
37
32
19
21
5
1
7
29
69
49
95
37
2
6
55
32
9
11
12
6
3
9
10
*Refer to Fig. 1A for sites of electroporation.
†
Stage of development. For each stage, % values in columns A-E or F-K add up to 100%.
For site of labeled cells see Fig. 1C.
Bold font indicates the region where noticeable changes in the relative contribution of cells were found between MS- and M-LS-stage embryos.
MS, mid-streak (E7.0); M-LS, mid- to late-streak (E7.25); N, number of specimens analyzed; AYS/LYS/PYS, anterior, lateral and posterior region of the yolk sac, respectively;
Ant/Mid/Post, definitive endoderm of the anterior (foregut) region, middle (somite level) region and posterior (presomitic mesoderm) region of anterior-posterior body axis.
Cells in the endoderm of MS- and M-LS-stage ARC/s, Mixl1+/GFP and
Mixl1GFP/GFP embryos were labeled by painting the with carbocyanine dyes:
DiO (D275), CM-DiI (C-7001) or DiI (D282, D3911, Molecular Probes).
While holding the embryo by suction against a wide-bored pipette with
polished tip, the endoderm was painted by touching the cells with a bolus of
dye that was partially extruded from the tip of another micropipette. A broad
area of the endoderm of the ARC/s embryos was painted in single color with
either DiI (red) or DiO (green), or in both of these colors by painting
consecutively with DiI and DiO. Painting was performed using DiI on the
Mixl1+/GFP and Mixl1GFP/GFP embryos to contrast labeled cells with the
green fluorescent host tissues. Painted embryos were imaged by
fluorescence microscopy and digital photography 1 hour after labeling to
determine the paint pattern; and at 6-, 12- or 24-hours in vitro to visualize
the distribution of labeled endoderm cells in the embryonic gut and the yolk
sac.
Testing endoderm potential by cell transplantation
Pregnant Mixl1+/GFP;lacZ mice were euthanized at E7.0 to harvest MS-stage
embryos, which provided the cells for the transplantation experiment. The
embryos were examined under the fluorescence microscope and genotyped
based on the intensity of GFP fluorescence: Mixl1+/+, no green signals;
Mixl1+/GFP, moderate to weak green fluorescence; Mixl1GFP/GFP, strong
green fluorescence. In addition, because Mixl1 is expressed specifically in
the primitive streak (Robb et al., 2000; Hart et al., 2002), the relative size of
the GFP or lacZ-expression domain (see Fig. S1A,B in the supplementary
material) enables the staging of gastrulation and guides the identification of
the anterior part of the primitive streak for harvesting cells (see Fig. S1C in
the supplementary material). Donor embryos were dissected by polished
alloy metal needles to isolate small fragments of tissues from the anterior
segment of the primitive streak and the adjacent epiblast, from which any
adherent mesoderm and endoderm were removed as completely as possible.
The fragments were then dissociated into clumps of 10-15 cells using glass
needles. These cell clumps were transplanted using Leica mechanical
micromanipulators to the anterior segment of the primitive streak of stagematched MS ARC/s embryos. Within 1 hour after transplantation, the
recipient embryo was examined to ascertain the presence and proper location
of the GFP-expressing grafted cells (see Fig. S1D in the supplementary
material).
After 24 hours of in vitro development to the early-somite stage, the
recipient embryos were imaged by fluorescence microscopy to visualize the
distribution of the GFP-expressing cells (see Fig. S1E in the supplementary
material). They were then fixed in 4% paraformaldehyde and stained with
X-gal reagent to visualize the Hmgcr-lacZ-expressing cells (see Fig. S1E in
the supplementary material). Embryos containing positively stained graftderived cells were processed and examined by histology. The number and
distribution of the graft-derived cells in the tissues of the host embryo,
especially in the endodermal derivatives, were scored in serial histological
sections of the specimen.
RESULTS
Precursors of definitive endoderm are localized to
the posterior and distal sites
To map the fate of endodermal cells in the MS embryo, whole
embryos were electroporated with a plasmid encoding EGFP and the
distribution of the fluorescent cells was monitored after development
to EHF stage and early-somite stage (Table 1). In the MS embryo,
precursors of the definitive endoderm of the early-somite embryo (48
hours in vitro) constituted approximately 47% of the endodermal
population. Precursors of anterior (foregut) endoderm were found in
the distal (anterior-distal, distal and posterior-distal) domains (Fig.
2C,F,G; Table 1, column I of MS embryo). Precursors of middle
endoderm (at the somite level) were also localized to distal sites
(anterior-distal, distal and posterior-distal; Fig. 2F; Table 1, column
J of MS embryo). Precursors of the posterior endoderm of earlysomite-stage embryos were found in the distal-to-posterior region
(distal, posterior-distal and posterior-middle; Fig. 2E; Table 1,
column K of MS embryo). Therefore, precursors of the different parts
of the embryonic gut endoderm at the early-somite stage are found
predominantly in the anterior-distal, distal, posterior-distal and
DEVELOPMENT
Tracking the endoderm by painting cells
RESEARCH ARTICLE
posterior-middle regions of the MS embryo (Fig. 3). These regions
are within the Cer1-expression domain and partially overlap the
expression domain of Sox17, a definitive endoderm marker (Fig. 3A).
In the MS embryo, precursors of the anterior yolk sac (AYS)
endoderm were derived from the anterior-to-distal sites (anteriorproximal, Fig. 2B; anterior-distal and distal, Table 1, column F of
MS embryo). The precursor of the lateral yolk sac (LYS) endoderm
was in the anterior-distal and lateral sites (Table 1, column G of MS
embryo). The posterior yolk sac (PYS) endoderm was derived from
lateral (Fig. 2A), posterior-middle and posterior-proximal (Fig. 2D)
sites (Table 1, column H of MS embryo). Precursors of the
extraembryonic (yolk sac) endoderm are therefore localized mainly
in the anterior (proximal to distal) and posterior proximal regions of
the endoderm (Fig. 3B⬙). There are, however, overlaps in the
domains of precursors of the extraembryonic endoderm and the
definitive endoderm. Specifically, precursors of AYS and anterior
gut endoderm are co-localized in the anterior-distal and distal
regions, as are those of the PYS and posterior gut endoderm in the
posterior-middle region (Fig. 3B⬘,B⬙; Table 1, columns F-K for the
anterior-distal, distal and posterior-middle sites).
Development 134 (2)
Histological examination of the MS embryo (n=8) electroporated
with the CMV-EGFP-IRES2-lacZ expression vector (Fig. 2H)
confirmed that only endodermal cells were labeled (Fig. 2I). At the
end of in vitro culture, lacZ-expressing cells (Fig. 2J) were localized
in only the endoderm in the early-somite-stage embryo (n=8;
foregut: Fig. 2K; midgut: Fig. 2L).
Expanding occupancy of the gut-endoderm
precursors during gastrulation
To test whether the localization of the precursor population changes
during gastrulation, another snapshot of the regionalization of cell
fates in the endoderm was taken at the M-LS stage, which is around
6 hours more advanced than the MS stage. The most noticeable
difference was the relative contribution of cells in the posteriorproximal and lateral endoderm to the yolk sac. There was a reduced
contribution to the PYS of cells at these two sites in the M-LS
embryo (Table 1, column H, bold font), whereas the contribution to
the LYS from the lateral site was increased (Table 1, column G, bold
font). These changes in the relative abundance of the yolk sac
precursors at specific endoderm sites suggest that, during
Fig. 2. Localization of EGFP and lacZexpressing cells in the endoderm
following electroporation of expression
vectors. Distribution of EGFP-expressing cells
at the mid-streak (MS) stage (3 hours), at the
early-head-fold-stage (24 hours) and in the
early-somite-stage embryo (46-48 hours) after
electroporation of the endoderm of midstreak-stage (E7.0) embryos at seven sites: (A)
lateral, (B) anterior-proximal, (C) anterior-distal,
(D) posterior-proximal, (E) posterior-middle, (F)
posterior-distal and (G) distal. Examples of the
pattern of distribution of EGFP-fluorescent
cells after electroporation and in vitro culture
are shown as a set of three (at 3 hours, 24
hours and 46-48 hours) for each site. All
figures are lateral views with anterior to the
left, except for anterior views of anteriorproximal (24 hours and 46-48 hours) and
anterior-distal (46-48 hours), and posterior
views of posterior-proximal (24 hours and 4648 hours) and posterior-middle (46-48 hours)
sites. (H-L) Localization of the electroporated
lacZ-expressing cells in (H,J) whole-mount and
(I,K,L) histological sections of the embryo.
(H,I) Endoderm of an E7.0 MS-stage embryo
after 3 hours of in vitro culture. (J-L) endoderm
of the dorsal foregut (K) and the middle region
(L) of an early-somite-stage embryo (J) after 24
hours of in vitro culture. The faint staining in I
at the basal region of the epilast is probably
due to the spillage of the product of the X-galstaining reaction, as opposed to genuine
staining, which would be found in the whole
cell.
DEVELOPMENT
254
gastrulation from the MS to the M-LS stage, the PYS precursors exit
the posterior-proximal site, and that the AYS and PYS precursors
that previously occupied the lateral site have departed and their place
has been taken up by the precursor of the LYS.
The departure of the yolk sac-endoderm precursors led to a slight
increase (by 5% to 52.7%) in the relative amount of gut-endoderm
precursors in the M-LS embryo. The most obvious changes were the
increase in the abundance of gut endoderm precursors in the posteriorproximal site (Table 1, column I-K of M-LS embryo, bold font). This
was accompanied by a decrease in the precursor of the posterior
endoderm and an increase in the middle (somite-level) endoderm in
the posterior-middle site (Table 1, column J, K of MS and M-LS
embryo, bold font). This pattern of cell movement was already evident
from the changes in the relative distribution of the embryonic-gutendoderm precursors in the posterior-middle and posterior-proximal
regions of the embryos examined after 24 hours of in vitro
development (Table 1, column D, E of MS and M-LS embryos, bold
font). Based on the changes in the relative abundance of precursor
types (Table 1, columns F-K), a pattern of displacement can be
constructed to reflect the movement and regional expansion of the
precursor populations from the seven sites (Fig. 3C) to the
extraembryonic endoderm during the development from the MS to the
M-LS stage (Fig. 3D), and to the EHF stage (Fig. 3E,E⬘). Similarly,
RESEARCH ARTICLE
255
corresponding maps of the morphogenetic movement of precursors of
the gut endoderm are shown in Fig. 3F-G⬘. During gastrulation, the
pre-existing cell populations moved from the anterior, lateral and
posterior proximal sites to the extraembryonic endoderm. This was
accompanied by an expansion of the abundance of precursors of the
definitive endoderm, predominantly towards the anterior and distal
regions. This coincides with the distal extension of the primitive streak
(see Fig. S1A,B in the supplementary material).
Cellular recruitment to the endoderm may drive
cell movement
The re-construction of the pattern of cell movement in the endoderm
based on the regionalization of cell fate implies that cell movement
may be associated with the continuous accretion of cells to the
endoderm from the primitive streak during gastrulation. To test this
hypothesis, we studied the Mixl1-null-mutant embryos, which were
shown to be deficient of Sox17- and Cer1-expressing gut endoderm,
presumably resulting from an inability to direct the allocation of
mesendodermal progenitors to the definitive endoderm (Hart et al.,
2002). We first tested whether Mixl1-null-mutant cells may have an
impaired potential to form endoderm by cell-transplantation
experiments. Cells from the anterior region of the primitive streak
(see Fig. S1C in the supplementary material) of Mixl1+/GFP
Fig. 3. Fate maps of the precursors of
embryonic and extraembryonic
endoderm and the pattern of cell
movement in the endoderm of the midstreak stage embryo. (A) The expression
domain of Cer1 and Sox17 in the
endoderm of a mid-streak embryo (anterior
to the left). Cer1 expression overlaps in the
distal, posterior-distal and posterior-middle
regions, which contribute to the gut
endoderm, but is also expressed in the
endoderm immediately distal and lateral to
the primitive streak, and in the anterior
proximal endoderm. Sox17 expression
overlaps in the posterior-middle region and
is also expressed in the extraembryonic
visceral endoderm. Both genes are
expressed in the anterior visceral endoderm
(asterisk, anterior proximal region). (B) Fate
maps of the endoderm of mid-streak-stage
gastrula (left figures in B⬘ and B⬙) showing
the localization of the precursors of
embryonic (gut) endoderm (B⬘) and
extraembryonic (yolk sac) endoderm (B⬙) of
the early-somite-stage embryo (right figures
in B⬘ and B⬙). (C) Color coding of the seven
sites in the mid-streak-stage embryo for
testing endodermal cell fates (see Fig. 1A).
(D-G’) The trajectories of the progenitors of
the yolk sac endoderm (D-E’) and
embryonic endoderm (F-G’) during
development from the mid-streak to the
mid- to late-streak stage (D,F) and then to
the early-head-fold stage (E,E’,G,G’),
showing the distribution of cells arising
from (E,G) anterior and distal sites and
(E’,G’) posterior sites separately. Cells
originated from each of the seven sites are
color-coded. In D and F, the origin of the arrow shows the location of the cells at the mid-streak stage and the head of the arrow marks the
predicted position of these cells at the mid- to late-streak stage, based on the cell-fate data. Double-headed arrows indicate the expansion of the
distal and posterior-distal endoderm. Red line in the posterior region of the embryo (B,C) marks the primitive streak.
DEVELOPMENT
Endoderm formation during mouse gastrulation
256
RESEARCH ARTICLE
Development 134 (2)
Table 2. Contribution of the anterior primitive-streak cells of mid- to late-streak stage Mixl1+/– and Mixl–/– mutant embryos to
the germ layer derivatives of the wild-type host embryo
Donor cells (genotype)
Mixl1+/– (Mixl1+/GFP;Hmgcr-lacZ)
Mixl1–/– (Mixl1GFP/GFP;Hmgcr-lacZ)
APS / M-LS
APS
14
APS / M-LS
APS
32
Donor cells: tissue of origin / stage
Site of transplantation
Number of recipient embryos analyzed
Tissue distribution
Cell count
% total
Cell count
% total
48
48
6
8.3
8.3
1.0
45
14
0
2.8
0.9
0
5
45
0.9
7.8
0
122
0
7.5
0
0
0
0
35
58
2.2
3.6
Cranial
Somite
Presomitic
83
78
153
14.3
13.4
26.4
671
386
60
41.5
23.8
3.7
Lateral mesoderm
Primitive streak
12
102
2.1
17.6
132
93
8.3
5.7
Total number of cells scored
Average number of graft-derived cells/embryo
580
41.4
Gut endoderm
Anterior
Middle
Posterior
Axial mesoderm
Head process
Notochord
Neural tissues of the trunk
Neural tube
Floor plate
Paraxial mesoderm
1616
50.5
Bold font indicates noticeable differences between the contribution of the Mixl–/– cells to specific types of host tissues and that of Mixl1+/– cells.
M-LS, mid to late-streak; APS, anterior primitive streak.
movement of GFP-positive mesoderm and DiI-labeled endoderm
in the embryos at different time points of in vitro development,
the endoderm cells in the Mixl1GFP/GFP were found to remain
stagnant while the mesoderm spread anteriorly (Fig. 4A,B). The
impaired movement was not limited to the endoderm associated
with the anterior half of the primitive streak, because cells
overlying the posterior half of the primitive streak of Mixl1GFP/GFP
embryos also remained stationary (Fig. 4C). Therefore, the
endoderm in the Mixl1-null mutant is inherently unable to move
and could not be mobilized by the expansion of the underlying
mesoderm.
Allocation of the lateral and medial gut
endoderm
The transplantation experiment showed that cells derived from the
primitive streak contribute to the gut endoderm in the anterior and
middle region of the EHF-stage embryo (Table 2). To elucidate
whether there was a sequential order of allocation of medial and
lateral populations of the gut endoderm in these regions, the fate of
cells in the endoderm at the anterior region of the primitive streak
of embryos at the MS and M-LS stages was examined. Endoderm
at the anterior region of the primitive streak of Mixl1+/GFP MS (Fig.
5A, 0 hour) and M-LS embryos (Fig. 5B, 0 hour) was painted with
DiI, and the distribution of the labeled cells was examined at 12
hours (late-streak stage) and 24 hours (EHF stage) of in vitro
culture. Labeled endoderm cells of MS-stage embryos were first
present in the lateral proximal region of the embryo (Fig. 5A, 12
hours) and finally in the lateral region of anterior and middle
endoderm (Fig. 5A, 24 hours). By contrast, the endoderm cells from
a similar site in the M-LS-stage embryo (Fig. 5B, 0 hour) were
DEVELOPMENT
(Mixl1+/–, n=15) and Mixl1GFP/GFP (Mixl1–/–, n=47) embryos were
transplanted orthotopically to the ARC/s (wild type) embryos (see
Fig. S1C in the supplementary material). Mixl1+/– and Mixl1–/– cells
were similar in their ability to multiply and colonize the host tissues
(Table 2, and see Fig. S1F,G in the supplementary material).
However, histological examination and cell-count analysis revealed
that Mixl1–/– primitive-streak cells contributed less to the endoderm
than Mixl1+/– cells and more to the mesoderm of the host embryo
than Mixl1+/– cells (Table 2). This strongly suggests that the loss of
Mixl1 function has an impact on the endoderm potential of the
mesendoderm progenitors, which are presumably found in the
primitive streak (D’Amour et al., 2005; Yasunaga et al., 2005; Tada
et al., 2005; Ng et al., 2005; Loebel and Tam, 2005). The reduced
contribution to the endoderm was apparent even when the Mixl1deficient cells had been in an environment conducive to endoderm
formation. The deficiency of definitive endoderm is therefore likely
to be caused by the impaired recruitment of cells to the endoderm
during gastrulation (Hart et al., 2002).
In Mixl1GFP/GFP embryos, the allocation and differentiation of
the mesoderm were unaffected. Although the anterior expansion
of the mesoderm appeared to be delayed, a complete mesodermal
layer was formed eventually (see Fig. S2A,B in the supplementary
material). To test whether the movement of the endoderm was also
retarded in the Mixl1GFP/GFP embryo, endoderm cells overlying
the anterior half of the primitive streak were painted with DiI and
their position was determined after 24 hours of in vitro culture. In
the Mixl1+/GFP embryo (n=14), labeled cells were distributed
along the anterior-posterior axis whereas, in the Mixl1GFP/GFP
embryo (n=11), labeled cells stayed in the posterior region (see
Fig. S2C,D in the supplementary material). By tracking the
Fig. 4. Impaired cell movement in the endoderm of Mixl1GFP/GFP
mutant embryos. (A,B) The displacement of DiI-labeled endoderm
(yellow because of overlaying of red dye color on the green
fluorescence) in Mixl1+/GFP (A) and Mixl1GFP/GFP (B) mutant embryos after
1-, 6-, 12- and 24-hours of in vitro culture. (C,C⬘) Summary of the
distribution of DiI-labeled endodermal cells along the anterior-posterior
axis of the embryos and in the posterior yolk sac 24 hours after
labeling. Each bar represents the pattern of distribution of labeled cells
in a single representative embryo after the endoderm overlying the
anterior (C) or the posterior (C⬘) region of the primitive streak was
painted.
distributed initially along the axial and paraxial region (Fig. 5B, 12
hours) and subsequently moved to the medial region of the anterior
and middle endoderm (Fig. 5B, 24 hours). In summary, precursors
of the lateral-anterior and middle endoderm, which constitute most
of the foregut endoderm (Tremblay and Zaret, 2005), may be
allocated separately from those of the medial endoderm, and these
two endoderm populations reach their destination via different
paths (Fig. 5C). During the morphogenesis of the foregut portal, the
lateral and medial population will become the ventral and dorsal
endoderm of embryonic gut, respectively (Fig. 5D). These results
RESEARCH ARTICLE
257
Fig. 5. Allocation of the endoderm to the dorsal and ventral cell
population of the embryonic foregut. (A,B) DiI labeling of the
endoderm overlying the anterior end of the primitive streak in midstreak (A) and mid- to late-streak (B) embryos (as staged by the distal
extension of the GFP-expression domain), followed by visualization of
the labeled cells at 0 hour (merged GFP and DiI, left images), 12 hours
(DiI only, middle images) and 24 hours (top right images, DiI only;
bottom right images, merged GFP and DiI) of in vitro development.
(C) The allocation and the path of displacement of lateral (green) and
medial (dark blue and light blue) populations of the anterior endoderm
at the mid-streak and mid- to late-streak stages of gastrulation. The
diagrams show the trajectories of these two populations of anterior
endoderm in the normal (left panels) and the flattened-disc (right
panels) configurations of the embryo (Tam and Gad, 2004). (D,D⬘) The
spatial correlation of medial and lateral divisions of the anterior
endoderm of the late-bud-stage embryo (D, lateral view showing Sox17
expression in the anterior endoderm) and the resultant dorsal (roof) and
prospective ventral (floor) regions of the foregut portal of the earlysomite-stage embryo (D⬘, transverse histological section).
reveal that the timing of recruitment to the endoderm influences the
allocation of cells to the medial (dorsal) and lateral (ventral)
endoderm of the gut.
Trajectories of gut-endoderm precursors to the
embryonic gut
The trajectory of gut-endoderm precursors of MS-stage embryos to
the endoderm of different parts of the embryonic gut of EHF-stage
embryos was tracked by mapping the distribution of cells originated
from five areas (Fig. 6A) covering the whole of the posterior and
DEVELOPMENT
Endoderm formation during mouse gastrulation
258
RESEARCH ARTICLE
Development 134 (2)
distal endoderm. Cells anterior to the primitive streak (distal and
posterior distal) and those at anterior end of the primitive streak
(APS) contributed to the endoderm from the prechordal to the
somitic levels, but rarely to the posterior (presomitic mesoderm)
endoderm (Fig. 6B,C, and see Table S1 in the supplementary
material). Cells in the middle segment of the primitive streak
(posterior middle) contributed exclusively to the endoderm from the
hindbrain to the presomitic mesoderm level, but not to that in the
prechordal to midbrain level. The endoderm in the posterior
segment of the primitive streak (posterior distal) colonized only the
posterior-most endoderm (see Table S1 in the supplementary
material).
Although the endoderm associated with the primitive streak can
contribute to the embryonic endoderm along the whole anteriorposterior body axis of the EHF embryo (see Table S1 in the
supplementary material), the most preponderant contribution is to
the anterior endoderm and the most-posterior endoderm.
Furthermore, within the anterior endoderm, most labeled cells were
found in the anterior-lateral and posterior endoderm, and fewer
were found the middle endoderm (Fig. 6D). To reveal the source of
anterior medial endoderm, double-color painting of the posteriordistal to the posterior-proximal endoderm was performed, with an
overlapping area in the APS (yellow area in Fig. 6E). The result
showed that the medial population of the anterior and middle
endoderm was derived from cells in the posterior-distal (red cells
in Fig. 6E) and APS (yellow cells in Fig. 6E), whereas the lateral
anterior endoderm and the posterior endoderm were derived from
the APS and posterior-proximal cells (green cells in Fig. 6E). APS
cells contributed to the medial population of both the anterior and
middle endoderm of the embryo. Endoderm cells in a large area of
the middle and posterior regions were not labeled (Fig. 6E),
suggesting that they may be recruited to the endoderm after the
labeling at the MS stage. The allocation of precursors of definitive
endoderm during gastrulation therefore commences with the
anterior and the most-posterior endoderm, followed by those of
middle endoderm and the rest of posterior endoderm. Histological
examination of the dye-labeled embryos revealed that labeling was
limited to the superficial endoderm layer (Fig. 6F, three embryos
examined 1 hour after labeling) and the gut endoderm (Fig. 6G-I,
four out of six embryos examined after 24 hours of culture).
However, in two of the six embryos examined at 24 hours, although
the majority of labeled cells was in the endoderm, a few were also
found in the mesoderm, as previously reported by Lawson and
Pedersen (Lawson and Pedersen, 1987).
DISCUSSION
Progressive allocation of the definitive endoderm
during gastrulation
Fate mapping of the endoderm of MS embryos revealed that the fate
of cells in the area distal to the anterior region of the primitive streak
is to become the medial population of the anterior endoderm (the
dorsal foregut). The location of some dorsal-foregut endoderm
DEVELOPMENT
Fig. 6. Allocation of the embryonic gut
endoderm during gastrulation. (A) Five separate
sites in the endoderm were painted with DiI (red) or
DiO (green) and the movement of these cells
tracked at 3 hours and 28 hours post-labelling.
(B) Cells from the distal site (Dist, red) were found
mostly in the prechordal region, whereas those from
the posterior-distal site (PD, green) were distributed
to the middle (somitic) region of the embryo. Dotted
circle (red in A, blue in B-E) marks the anterior
region of the primitive streak (APS) (C) Cells from the
PD to posterior-middle (PM) region (red) were
distributed to the medial regions from the forebrain
to the posterior (presomitic mesoderm) level (dashed
line), whereas the APS cells (green cells from the
yellow region) were found mainly in the middle
endoderm. (D) Cells from the painted (green) region
covering the whole primitive streak from APS to
posterior-proximal (PP) region were distributed
mostly to the lateral and intermediate regions of the
anterior endoderm and to the most posterior
endoderm and posterior yolk sac. Some labelled
cells were present in the middle medial endoderm.
(E) From the posterior endoderm, Dist, PD and APS
cells (red) were distributed to the medial endoderm
spanning from the forebrain to the somitic level.
APS, PM and PD cells (green) were distributed to the
lateral endoderm at the forebrain to hindbrain level
and to the posterior endoderm. Overlapping
contribution from the APS cells is observed in the
medial middle endoderm (yellow domain). Few
labelled cells are found in the lateral posterior
endoderm. (F-I) Histological examination of (F) E7.0
MS embryo (inset: whole embryo) showing DiIlabelled cells in the endoderm and (G) labelled
embryo after 24 hours of in vitro culture containing
labelled cells in the foregut portal (H) and in the
endoderm (I) at the somite level.
precursors distal to the anterior primitive streak (Lawson and
Pedersen, 1987) (this study) may be due to the displacement of
precursors that had been recruited since the early-streak stage. The
preponderance of precursors of the lateral population of the anterior
endoderm (the ventral foregut) in the vicinity of the primitive streak
of the mid- to late-streak embryo suggests that these precursors are
recruited after the medial population. Recruitment of the endoderm
of the foregut therefore follows a temporal schedule of early
recruitment of the medial (dorsal) endoderm and later recruitment
of the lateral (ventral) endoderm.
Previous analyses of cell fates in the primitive streak of the chick
embryo showed that, at Stage 3, cells emerging from the rostral
region of the streak contribute to a wide mediolateral domain of the
anterior endoderm, whereas those emerging later at Stage 3+ and
Stage 4 contribute to the dorsal and ventral midline of the foregut
(Lawson and Schoenwolf, 2003; Kirby et al., 2003). A slightly
different order of appearance of endoderm types was revealed by
mapping the fate of cells in the lower layer of the chick gastrula
embryo. In Stage 2 to Stage 3+ (early- to mid-primitive-streak stage)
gastrula, the lower layer contains the precursors of the mid-hindgut
and dorsal foregut, and they are clustered around the anterior region
of the primitive streak (Kimura et al., 2006). As gastrulation
proceeds to Stage 4 (definitive-streak stage), precursors of the lateral
foregut and more of those for the mid- and hind-gut appear in the
lower layer. In the chick gastrula, in contrast to the mouse,
precursors of the ventral foregut appear in the lower layer as late as
Stage 5, presumably due to the extended retention of these
endodermal cells in the mesodermal layer after their ingression at
the primitive streak (Kimura et al., 2006).
Of particular interest is that the fate-mapping study in the chick
revealed the presence of a small population of mid- and hind-gut
precursors in the lower layer of the Stage 2-3 chick gastrula
(Kimura et al., 2006). Similarly, in the endodermal layer of the
early-streak mouse embryo, a small number of cells that contribute
to trunk and posterior endoderm are found adjacent to the primitive
streak (Lawson and Pedersen, 1987). Presently, the precise fate of
these cells of the early-streak embryo is not known. In the MS
embryo, in addition to precursors of the foregut endoderm, the
endodermal layer also contains cells that contribute to the endoderm
in the most-posterior region of the early-somite embryo. It has been
shown that the descendants of these cells can contribute extensively
to the gut from the upper-trunk level to the caudal end in 20- to 23somite embryos (Tanaka et al., 2005) (Lewis and P.P.L.T.,
unpublished). If these cells are the descendants of the posterior
endoderm precursors of the early- to mid-streak embryo, it would
suggest that the endoderm of the mid- and hind-gut is allocated very
early in gastrulation and ahead of the bulk of foregut precursors.
Because the recruitment of cells to the endoderm appears to have
ceased by the early-somite stage (Tam and Beddington, 1987) and
the precursors for major segments of the gut are present in the noto early-bud embryo (Lawson et al., 1986; Tam and Beddington,
1992; Tam et al., 2004), allocation of the full complement of
definitive-endoderm precursor has to be accomplished before the
morphogenesis of major parts of the mid- and hind-gut of the
embryo takes place.
Together, these observations point to a probable sequence of
allocation of the definitive endoderm proceeding with: (a) the mostposterior endoderm and the dorsal endoderm of the rostral segment
of the foregut at early-streak stage; (b) the ventral endoderm of the
rostral foregut and additional posterior endoderm at the MS stage;
(c) the dorsal and then the ventral endoderm of the posterior segment
of the foregut at the late-streak to late-bud stage; and, finally, (d) the
RESEARCH ARTICLE
259
endoderm of the embryonic mid- and hind-gut at the late-bud- to
EHF-stage. If no further recruitment of definitive endoderm takes
place after the presomite stage (Tam and Beddington, 1987), the
mid- and hind-gut (and presumably also the tail gut) would have to
be generated by the expansion of all of the precursors that have been
allocated to the definitive endoderm shortly after the completion of
gastrulation.
Accretion of cells may drive cell movement in the
endoderm
During the development from the MS stage to the M-LS stage,
precursors of extraembryonic endoderm move proximally towards
the ectoplacental pole of the conceptus from the anterior-proximal
and posterior-proximal sites (Fig. 3D-E⬘) The precursors of
embryonic endoderm display a concerted movement: cells in the
posterior-middle to distal region of the MS-stage embryo are
displaced anteriorly and proximally to occupy a wider domain in the
lateral and posterior regions of the embryo (Fig. 3F-G⬘). It is worth
noticing that, whereas the precursors of the anterior definitive
endoderm appear to move in step with the mesodermal layer in the
chick gastrula (Lawson and Schoenwolf, 2003; Kimura et al., 2006),
the dorsal-foregut endoderm in the mouse embryo may have moved
more anteriorly than that of the anterior mesoderm by the MS stage
(Parameswaran and Tam, 1995; Kinder et al., 1999). This may
suggest that the endoderm could move independently of the
mesoderm.
The overall pattern of anterior and proximal displacement of the
definitive endoderm is reminiscent of that of the visceral endoderm
in the pre-gastrulation embryo (Thomas and Beddington, 1996;
Rivera-Perez et al., 2003). Movement of the visceral endoderm has
been attributed to active cell migration (Srinivas et al., 2004), to the
propulsion generated by differential cell proliferation (Yamamoto et
al., 2004) or to guidance-mediated Wnt signaling (Kimura-Yoshida
et al., 2005). Results of the present study on Mixl1-mutant embryos
reveal that a loss of Mixl1 function reduces the endoderm potential
of primitive-streak cells. This finding is consistent with the lack of
contribution by Mixl1–/– ES cells to the gut endoderm of the
chimaeric embryo, and the reduced population of Sox17 and Cer1expressing cells in the mutant embryo (Hart et al., 2002). The
inefficient recruitment of cells to the endoderm may lead to the
lessening of the flow of cells into the endoderm immediately
adjacent to the primitive streak. The finding that the endoderm cells
remain stationary in Mixl1–/–-mutant embryos suggests that one of
the forces driving endoderm movement might be the propulsion
generated by the accretion of cells in the posterior region of the
endoderm during gastrulation. A similar mechanism of driving cell
movement by differential accretion of cells has been proposed for
the visceral endoderm of the mouse embryo before gastrulation
(Yamamoto et al., 2004). In Mixl1–/– embryos, endoderm cells
overlying the primitive streak remain stagnant while the mesoderm
expands. This finding further highlights the independence of the
movement of the mesoderm and the endoderm, and that any traction
force that might be exerted by the expanding mesoderm is
insufficient to mobilize the endoderm cells.
We thank Peter Rowe and David Loebel for comments on the manuscript; and
Kirsten Steiner for contributing Fig. 5D. Our work is supported by the National
Health and Medical Research Council (NHMRC) of Australia and by James
Fairfax. P.P.L.T. is a Senior Principal Research Fellow and L.R. is a Principal
Research Fellow of the NHMRC.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/2/251/DC1
DEVELOPMENT
Endoderm formation during mouse gastrulation
RESEARCH ARTICLE
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