Regionalisation of the endoderm progenitors and morphogenesis of the gut portals of the mouse embryo

M E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 7 – 6 0 0

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Regionalisation of the endoderm progenitors andmorphogenesis of the gut portals of the mouse embryo

Vanessa Franklin1, Poh Lynn Khoo1, Heidi Bildsoe, Nicole Wong, Samara Lewis,Patrick P.L. Tam*

Embryology Unit, Children’s Medical Research Institute, University of Sydney, Locked Bag 23, Wentworthville, NSW 2145, Australia

A R T I C L E I N F O

Article history:

Received 10 January 2008

Received in revised form

28 March 2008

Accepted 7 April 2008

Available online 11 April 2008

Keywords:

Fate-maps

Definitive endoderm

Gut

Morphogenesis

Lateral asymmetry

Mouse embryo

0925-4773/$ - see front matter � 2008 Elsevidoi:10.1016/j.mod.2008.04.001

* Corresponding author. Tel.: +61 02 9687 280E-mail address: [email protected]

1 These authors contributed equally to this

A B S T R A C T

This fate-mapping study reveals that the progenitors of all major parts of the embryonic

gut are already present in endoderm of the early-head-fold to early-somite stage (1–9 som-

ites) mouse embryo. The anterior endoderm contributes primarily to the anterior intestinal

portal of the early-organogenesis stage (16–19 somites) embryo. Endoderm cells around and

lateral to the node are allocated to the open ‘‘midgut’’ region of the embryonic gut. The pos-

terior (post-nodal) endoderm contributes not only to the posterior intestinal portal but also

the open ‘‘midgut’’. Descendants of the posterior endoderm span a length of the gut from

the level of the 3rd–5th somites to the posterior end of the embryonic gut. The formation of

the anterior and posterior intestinal portals is accompanied by similar repertoires of mor-

phogenetic tissue movement. We also discovered that cells on contralateral sides of the

anterior endoderm are distributed asymmetrically to the dorsal and ventral sides of the

anterior intestinal portal, heralding the acquisition of laterality by the embryonic foregut.

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1. Introduction

In the mouse embryo, allocation of definitive (gut) endo-

derm commences at early- to mid-streak stage when epi-

blast-derived cells are first incorporated into the pre-

existing layer of visceral endoderm (Lawson and Pedersen

1987; Tam and Beddington, 1992). By the mid-streak stage

the endoderm underneath the primitive streak is populated

by the progenitors of the endoderm in the prechordal part

of the foregut and in the caudal region of the early-somite

stage embryo (Lawson et al., 1986). These progenitors are fol-

lowed shortly by those of the dorsal and ventral populations

of the rest of the foregut (Tam et al., 2007). Precursors of the

mid- and hindgut are recruited to the endoderm later during

gastrulation, and this nascent population is incorporated into

er Ireland Ltd. All rights

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study.

the endoderm layer by intercalation between the foregut and

the caudal-most endoderm (Lawson et al., 1986; Tam et al.,

2004). Recruitment of cells from the epiblast via the primitive

streak to the endoderm continues until the early-head-fold

stage (Tam and Beddington, 1987; Wilson and Beddington,

1996). By then, all the progenitors of the embryonic gut are

presumed to be in place (Wells and Melton, 1999) and are

regionalized into precursors of major rostro-caudal segments

of the gut by the inductive/signalling activity of the adjacent

germ layers (Wells and Melton, 2000).

A fate-mapping study of the endoderm in the anterior re-

gion of the 1–10 somite stage embryo showed that cells con-

tribute to the foregut as well as the rostral part of the

midgut (Tremblay and Zaret, 2005). During the development

of the foregut, cells from the lateral domains of the anterior

reserved.

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588 M E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 7 – 6 0 0

endoderm converge ventrally with those from the anterior

intestinal portal (AIP). This morphogenetic process is reputed

to underpin the assembly of the precursors of the liver from

cells at multiple sites in the lateral regions of the anterior

endoderm and the AIP lip (Tremblay and Zaret, 2005). There

were, however, some gaps in the fate map. The fates of cells

located in the central region of anterior endoderm have not

been examined. In addition, the origin of the endoderm of

the pharyngeal pouches in the embryonic foregut is not

known.

Among the cells that are first recruited to the endoderm at

early gastrulation are the progenitors of the most caudal

endoderm population of the late-bud-stage embryo. Initially

located over the anterior segment of the primitive streak,

these cells are displaced posteriorly during gastrulation

(Tam et al., 2007). Together with the nascent cell population

recruited to the endoderm later, they occupy the node and

post-nodal regions which make up the posterior half of the

endoderm layer of the early-head-fold stage embryo (Tam

et al., 2007). Very little is known about the fates of these cells,

except that some of them will give rise to cells of the posterior

(caudal) intestinal portal (Tam et al., 2004) and the caudal part

of the midgut (Tanaka et al., 2005). In particular, the relative

contribution of the cells of the posterior endoderm, and the

sub-populations therein, to the mid- and hindgut has yet to

be elucidated.

A goal of our study is to completely map the fates of the

endoderm population in the anterior region (the anterior

endoderm) and in the node and post-nodal regions (the pos-

terior endoderm) of the mouse embryo to provide a compre-

hensive view of the pattern of regionalization of progenitor

cells of the embryonic gut. The other objective is to elucidate

the events of morphogenetic movement accompanying the

formation of the anterior and posterior intestinal portals by

tracking the distribution of cells during their formation. This

knowledge of the regionalization and tissue movement of the

endoderm progenitors would be useful for the correlation of

domain-specific gene expression pattern to the prospective

fates of endoderm cells both in wild-type and mutant em-

bryos and for assessing the outcome of experimental manip-

ulations. Our results reveal that the prospective foregut could

be mapped within the anterior endoderm, and the mid- and

hindgut are formed mainly by the expansion of the progenitor

populations in the posterior endoderm.

2. Results

2.1. Experimental strategy

To map the fate of endoderm cells in the early-head-fold to

early-somite stage embryo, whole embryos were electropora-

ted to introduce an expression vector encoding b-galactosi-

dase and EGFP into the endoderm cells or the endoderm

cells were labelled by painting their apical surface with carbo-

cyanine dyes, CM-DiI (chloromethybenzamido-1-1-dioctadec-

yle-3,3,30,3 0-tetramethylindocarbocyanine) and DiO (3-3 0-

dioctadecylozacarbocyanine). The distribution of fluorescent

cells and LacZ-expressing cells in the gut was monitored as

the embryo developed in whole embryo culture. The applica-

tion of these two means of tagging endoderm cells allows an

assessment of the consistency of the fate-mapping results ob-

tained with different techniques.

The prospective fates of cells at 13 sites of the endoderm

were analysed. They included six sites in the endoderm

underneath the head folds and rostral to the first somite:

three in the midline and three in the lateral domains

(Fig. 1A). To ensure that cells at all six sites are readily acces-

sible to labelling or electroporation, experiments were per-

formed on embryos at 1–3 somite stage, before any cells

become hidden underneath the lip of the anterior intestinal

portal (Fig. 1B). Seven sites were tested in the trunk and pos-

terior regions of the embryo (Fig. 1C and D). Two sites in the

trunk region were around the node and in the paraxial region

(near the last formed somites) lateral to the node; and five

sites in the posterior region: three in the midline and two in

the paraxial–lateral region (beneath the presomitic meso-

derm). These seven sites were tested in embryos that had

formed 5–9 somites. At this stage, significant recruitment of

cells through the primitive streak to the endoderm has ceased

(Tam and Beddington, 1987; Wilson and Beddington, 1996).

The full complement of progenitor cells of the embryonic

mid- and hindgut is therefore available for the fate-mapping

analysis.

To validate the efficacy and specificity of the cell marking

techniques, E8.0 embryos labelled with carbocyanine dye

(CM-DiI) or electroporated with a CMV-EGFP-IRES2-lacZ con-

struct were fixed at 0 and 3 h after manipulation, respectively.

Histological analysis of these embryos revealed that, in 8/11

dye-labelled embryos (Fig. 2A and B) and 6/9 electroporated

embryos (Fig. 2C and D), only cells in the endoderm layer were

labelled and expressed the lacZ reporter, respectively. Further-

more, there was no significant disruption of the epithelial

architecture of the endoderm.

2.2. Scoring the distribution of labelled cells

To establish the protocol for scoring the distribution of la-

belled cells in the anterior intestinal portal (AIP), we first

examined the size and shape of this structure in several 16–

19 somite stage embryo. At this stage, the anterior endoderm

of the embryo has been transformed from a shallow invagina-

tion with a crescent-shaped lip (Fig. 2E) into a cul-de-sac with

a tapered rostral end, laterally flanked by extensions as pha-

ryngeal pouches (Fig. 2F) and opened to the posterior via a

narrowing portal. The first two pharyngeal pouches span be-

tween the midbrain–hindbrain junction (marked by En1,

Fig. 2G) to the upper hindbrain (marked by Krox20, Fig. 2H).

A preliminary analysis was performed on several embryos

containing populations of anterior endoderm cells labelled

with CM-DiI and/or DiO (Fig. 2I, J and K) and cultured for

24–28 h in vitro (Fig. 2 I 0, J 0 and K 0). When the embryo was

viewed from the ventral side, labelled cells from the endo-

derm lateral to the A1 site (Fig. 2I) were localized to the ta-

pered region (the oral part) of the AIP and the rostral part of

the first pharyngeal pouch (Fig. 2I 0, with reference to

Fig. 2F). Those from regions lateral to the A2 sites (Fig. 2J) were

found in the wall of the 2nd to about the 4th pharyngeal

pouches (Fig. 2J 0) and those from A4 sites (Fig. 2K) were dis-

tributed widely along the lateral wall of the AIP (Fig. 2K 0). In

Fig. 1 – Fate-mapping of cells at 13 locations in the endoderm of the early-head-fold stage embryo. (A and B) Anterior

endoderm: six sites from rostral to caudal in the midline (A1, A2, and A3), and the lateral region (A4, A5 and A6). Asterisk and

circle mark the cranial and caudal border of the cephalic neural plate, respectively, and the dashed line marks the anterior–

posterior domain of the anterior endoderm. Embryo in (B) shows Hesx1 expression domain in the fore- and midbrain in (i)

ventral and (ii) lateral view to illustrate the position of sites in the anterior endoderm relative to fore/midbrain (fmb: A1 and

A4), upper hindbrain (uhb: A2 and A5) and lower hindbrain (lhb: A3 and A6) regions of the neural plate. (C and D) Posterior

endoderm: two sites over the node (N) and the lateral region of the node (LN), and five sites from caudal to rostral in the

midline (P1, P2 and P3) and the lateral region (P4 and P5). The asterisk and the dot mark the allantoic attachment and the

node, respectively, and the dashed line marks the anterior–posterior domain of the posterior endoderm. The 7-somite stage

embryo [(i) ventral and (ii) lateral view] shows Uncx4.1 expression in the last 5 somites, with the most caudal one

immediately anterior to sites LN and N.

M E C H A N I S M S O F D E V E L O P M E N T 1 2 5 ( 2 0 0 8 ) 5 8 7 – 6 0 0 589

summary, cells derived from the paraxial and lateral popula-

tions in the anterior endoderm decorated the lateral wall of

different regions of the AIP. In contrast, cells from the most

rostral A1 site in the midline (green cells in Fig. 2K) were local-

ized in a medial domain in the lower part of the AIP (Fig. 2K 0,

embryo viewed ventrally), suggesting they have been translo-

cated caudally during the formation of the AIP. When viewed

from the medial side of the bisected embryo, these cells were

found in the ventral wall (the floor) and cells from the A4 site

occupied the lateral wall of the AIP (Fig. 2L). The lateral posi-

tion of the labelled cells is readily discerned after hybridiza-

tion with a Pax9 riboprobe to outline the shape of the AIP

(Fig. 2M). Histological examination of this embryo confirmed

the lacZ-positive cells scored by its position in a lateral view

of the whole embryo were indeed localized on the lateral wall

of the pharynx (Fig. 2M).

Based on these observations, the distribution of the la-

belled cells in the AIP was scored first by examining the whole

embryo in the dorso-ventral view (Fig. 2I 0–K0) and lateral view

(Fig. 2L) to determine the position (dorsal, lateral and ventral)

of the labelled cells in the portal. Their location in the ante-

rior–posterior axis of the AIP was scored with respect to the

oral part, first and second pharyngeal pouches, third and

fourth pharyngeal pouches and the lower part of the AIP adja-

cent to the ventricle of the heart to the entrance of the portal

(Fig. 2N, and markers for orientation in Fig. 2O–R, see legend).

Cells found outside the portal were scored as in the ‘‘midgut’’

region (Table 1), which corresponds to the open region of the

embryonic gut, at the level of 4–5th to 14–15th somites, in the

early-organogenesis stage embryo. Selected specimens were

analysed histologically to confirm the score on whole mount

embryos.

2.3. The anterior endoderm contains the progenitor cells ofthe anterior intestinal portal

2.3.1. Cells in the midline are distributed to the floor and roofof the foregut

Cells at three sites in the midline of the anterior endo-

derm were examined for their contribution to the AIP. The

most anterior site, A1, which is underneath the cardiac

crescent and the rostral margin of the head fold, contains

endoderm cells of the ventral side (the floor), extending

from the entrance to the portal to the level of Ph I/II

(Fig. 3A-A1 and Table 1). Although Site A2 is initially caudal

to A1, A2 cells were distributed to the midline in the oral

and Ph I/II parts to occupy a more rostral position than

the A1 cells (Fig. 3A-A2 and Table 1). Many A2 cells were

also found in the lateral wall of the AIP (Table 1), suggesting

that they have expanded laterally. A3 cells were localized to

the roof of the AIP, overlapping with A2 cells in the oral

part (Fig. 3A-A3). Midline cells that were dye-labelled or

Fig. 2 – Endoderm cells marked by (A and B) dye labelling and (C and D) electroporation with CMV-lacZ-IRES2-EGFP expression

vector at Sites A1 (A), P5 (B), A4 (C) and P5 (D). Histological examination revealed that only the endoderm cells were labelled.

(E) Pax9 expression demarcates the anterior intestinal portal lip of a 5-somite stage embryo, and reveals (F) the shape of the

portal of a 16-somite stage embryo (or: oral part of the anterior intestinal portal, I, II: first and second pharyngeal pouch).

Expression domain of (G) En1 and (H) Krox20 marks the rostral and caudal borders of the oral to second pouch segment of the

portal. (I, I 0) Cells of the site lateral to A1 populate the oral region and the 1st pharyngeal pouch. (J, J 0) Those lateral to A2 are

found in the 2nd to 4th pouches. (K, K 0) Cells of A1 site (green) are localized in the medial domain and flanked by A4 cells in

the caudal segment of the anterior intestinal portal. (L) Bisection of the embryo reveals that A1-derived cells are found on the

floor and A4-derived cells are on the lateral wall. The silhouette of the foregut (dashed line) is revealed by (M) Pax9 expression

in the endoderm. The histological section shows the presence of electroporated cells expressing lacZ at the level of the third

pharyngeal pouch (asterisk). (N) Subdivision of the anterior intestinal portal for scoring the distribution of the descendants of

labelled/electroporated cells (or: oral part, I–II and III–IV: first to second and third to fourth pharyngeal pouch segments; ht:

lower segment of the anterior intestinal portal at the level of the ventricle of the heart. (O–R) Expression domains of Otx2, En1,

Fgf8 and Krox20 are used as landmarks (marked in N) to delineate the segments of the anterior intestinal portal, in

conjunction with pharyngeal morphology and the position of the heart tube.


electroporated displayed similar patterns of distribution (Ta-

ble 1 and Fig. 4A-A1, A2 and A3).

The pattern of floor-tip-roof distribution of A1–A2–A3 cells

in the AIP (Fig. 4A-A1–A3) was further highlighted by compar-

ing the distribution of differently dye-labelled cells from two

of the three midline sites (Supplementary Table S1). Three

examples of the double-labelled embryo were shown

(Fig. 3B): A1 and A2 with floor versus tip distribution, A2

and A3 with tip versus roof, and A1 and A3 with floor versus

roof.

2.3.2. Cells in the lateral domains populate not only thelateral wall of the AIP

Three lateral sites, A4, A5 and A6, located at the same axial

level as A1, A2 and A3, respectively, were analysed. The three

sites contained cells that were distributed widely at all levels

Table 1 – Distribution of painted and electroporated cells from sites in the anterior endoderm to the specific region of theembryonic gut

Site # Embryos analysed # Embryos with labelled cells in

Anterior intestinal portal Open ‘‘midgut’’

Oral Ph I–II Ph III–IV Cardiac level Rostral Caudal

D L V D L V D L V D L V M L M L

A1 DL 8 3 7 4 3

E 5 1 5

Total 13 3 7 5 8

A2 DL 11 8 8 7 3 3 1

E 6 5 3 3 3 4 4 1 2 2 2

Total 17 13 11 10 6 7 5 1 2 2 2

A3 DL 8 3 6 6 6 4

E 4 2 4 3

Total 12 5 10 9 6 4

A4a DL 7 1 3 6 2 3 1 1

E 5 1 1 4 5

Total 12 1 3 6 3 4 5 6

A5a DL 9 1 3 8 5 1 2 1

E 8 1 4 7 3 1 2 2 1

Total 17 1 1 7 15 8 2 4 3 1

A6a DL 10 3 4 2 6 8 4 4 4 2

E 4 2 1 1 2 2 1 1 3 1 1 2

Total 14 1 1 1 5 5 3 8 10 5 5 7 1 1 4

Abbreviations: DL, dye labelling; E, electroporation; Ph, pharyngeal pouch; D, dorsal; L, lateral; V, ventral; M, medial, blank = no cells present.

a Data of lateral sites on the left and right sides of the embryo were pooled.


of the AIP (Fig. 4A-A4, A5 and A6) predominantly in the lateral

wall (Fig. 3A-A5 and A6: dye labelling, 3A-A5: electroporation)

but also in the lateral areas of the floor (Fig. 3A-A4: dye label-

ling and electroporation) and the roof (Fig. 3A-A6: electropor-

ation). A4 cells were found in the lateral areas on the ventral

side of the portal at all levels but in the lateral wall mainly at

the Ph I–II and III–IV level (Table 1). In contrast, A5 cells were

incorporated into the AIP and occupied a domain rostral and

dorsal to A4 cells. The A6 cells extended rostro-caudally

mainly in the lateral region of the AIP, as well as to the lateral

region of the open ‘‘midgut’’ (Table 1).

A comparison of the distribution of anterior endoderm

cells from the most rostral and most caudal midline and

lateral sites at similar axial level revealed that they spanned

across similar anterior–posterior domains in the lateral

regions and the roof/floor of the AIP, respectively (Fig. 3B-

A1 + A4, A3 + A6). In contrast, endoderm cells from the

middle regions (A2 and A5) were distributed to different

axial level within the AIP, with A2 cells occupying more ros-

tral (oral and Ph 1–II) regions than the A5 cells (Fig. 3B-

A2 + A5). In all embryos, cells from the lateral sites (A4,

A5 and A6) always contribute to cells in the lateral wall of

the AIP (Table 2). However, in some embryos, A5 and A6

(but not A4) cells from contralateral sides of the anterior

endoderm were distributed asymmetrically in the wall of

the AIP: A5 and A6 cells from the right hand side were

localized preferentially to the dorsal–lateral region and

those from the left hand side to the ventral–lateral region

(Fig. 4B and Table 2).

2.4. The peri- and lateral nodal endoderm contributes tothe open gut region

The embryonic gut in the mid-region of the early-organo-

genesis (16–19 somite) stage embryo between the AIP and

posterior (caudal) intestinal portal (CIP) opens ventrally to

the parietal yolk sac cavity. Endoderm cells in the medial/ax-

ial region of this open ‘‘midgut’’ were derived from cells

around the node of the early-somite stage embryo (Fig. 5N

and Table 3), whereas those in the lateral/paraxial region

came from cells localized lateral to the node (Fig. 5LN and Ta-

ble 3). Cells from the node and lateral-node regions were also

found in the dorsal and lateral wall of the anterior 2/3 seg-

ment of the CIP (Table 3).

2.5. Endoderm of the posterior intestinal portal is derivedfrom progenitor cells in the post-nodal endoderm

Three sites in the midline (P1, P2 and P3) and two in the

lateral region of the post-nodal endoderm were examined

for their contribution to the open ‘midgut’’ and the CIP. Dye-

labelled cells and electroporated cells from the same site dis-

played similar patterns of distribution (Fig. 5). Descendants of

P1 cells were distributed to the floor of the hindgut portal

(Figs. 5P1 and 6P1) and extended anteriorly to the lateral re-

gion of the caudal part of the open ‘‘midgut’’ (Table 3). P2 cells

were localized to both the floor and roof in the caudal region

of CIP (Figs. 5P2 and 6P2; Table 3). P3 cells were found in the

roof of the CIP and extended anteriorly along the whole

Fig. 3 – (A) Distribution of cells from Sites A1–A6 in the anterior endoderm of neural plate-stage embryo in the anterior

intestinal portal of embryo after 24–28 h of in vitro development. Cells were labelled by applying a dye (dye labelling) or by

electroporation of a CMV-lacZ-IRES2-EGFP vector (electroporation). For each type of experiments, whole mount images were

taken at 1–3 and at 24–28 h in vitro, with histological sections showing the localization of labelled cells (black and white

arrows) in the gut endoderm. Details of distribution pattern are given in Table 1. (B) The relative distribution of cells derived

from two different sites in the midline (A1 + A2, A2 + A3 and A1 + A3), and from midline and lateral regions at similar

anterior–posterior level: (A1 + A4, A2 + A5 and A3 + A6). The embryos were viewed (i) 1–3 and (ii) 24–28 h after labelling.

Abbreviations: we, whole embryo; L and R, left and right hand half of the bisected embryo.


Fig. 4 – (A) Regionalization of the descendants of dye-

labelled or electroporated cells located at six sites (insets) in

the anterior endoderm of the early-head-fold stage embryo

in the anterior intestinal portal of the early-organogenesis-

stage embryo after 24–28 h of in vitro development. Colour

code for the location of the cells in the intestinal portal: red,

dorsal/roof; yellow, lateral wall; green, ventral/floor. (B)

Distribution of labelled cells from contralateral sites in the

anterior endoderm: A5 + A5 and A4,6 + A4,6. Embryos were

images at (i) 1–3 and 24–28 h after labelling as (ii) whole and

(iii) bisected embryos (L and R, left and right half of the

embryo). Cells from the right hand side of the anterior

endoderm were distributed to the dorsolateral region of the

portal. Conversely, endoderm cells from the left hand side

were found in the ventrolateral region. The roof of the portal

is marked by the arrowhead.


length of the medial/axial region of the open ‘‘midgut’’ (Figs.

5P3 and 6P3; Table 3), where they co-localized with the cells

from the node region. Tracing the relative distribution of cells

from different midline sites in the same embryo affirmed the

preferential localization of P1, P2 and P3 cells in the floor, cau-

dal tip and the roof of the CIP, respectively (Fig. 6P1 + P2: floor

and tip, 6P2 + P3: tip and roof, 6-P1 + P3: floor and roof).

Cells from the P4 site were distributed mostly to the lat-

eral–ventral region of the CIP (Figs. 5P4 and 6P4; Table 3). P5

cells were found in the same regions as the P4 cells (Figs.

5P5 and 6P5) but were present more frequently in the rostral

two third part of the CIP (Table 3). A comparison of the distri-

bution of contralateral P5 cells revealed a symmetrical locali-

zation to the lateral wall of the portal and the paraxial region

of the open ‘midgut’’ (Fig. 5P5 + P5), suggesting the lateral cells

in the posterior endoderm, unlike their counterpart in the

anterior endoderm, may not be endowed with any lateral-

ity-related asymmetry.

2.6. Morphogenetic movement of endoderm during portalformation

The pattern of distribution of labelled cells reveals that dur-

ing the morphogenesis of the AIP, cell populations located at

the midline sites (A1, A2 and A3) are extending rostro-caudally

as the portal elongates. Cells from the most rostral (A1) and

caudal (A3) sites are stretched along the floor and the roof of

the portal, respectively, whereas the A2 cells were localized to

and populated the tip of the portal (Fig. 4A-A1, A2 and A3). To

elucidate the movement of the lateral cells during the morpho-

genesis of the AIP, we have tracked in a time-course study the

displacement of cells from the lateral A5 and A6 sites relative

to the A2 cells (Fig. 7A-A2 + A5 and A2 + A6). A2 cells remained

stationary during the first 6 hours and then were displaced ros-

trally to the tip of portal. A5 and A6 cells converged medially

after the crescent-shape AIP lip reached their respective sites

(6 h). The A5 and A6 cells from the contralateral sides were jux-

taposed at the margin of the AIP but they did not intermingle

(14 h). Subsequently, A5 cells extended rostrally within the por-

tal (24 h) to reach the first three pharyngeal pouches (27 h). In

the specimen shown (Fig. 7A-A2 + A6), A6 cells on the right side

appeared to extend more rostrally than their left side counter-

parts (24 and 27 h). The right side A6 cells were localized more

to the dorsal side of the portal than the left side A6 cells which

were found mostly in the lateral area. Similar pattern of move-

ment of A5 and A6 cells was observed in three other embryos

(labelled at A2 + A5, A3 + A5, A2 + A6; data not shown).

The displacement of lateral P5 cells in the CIP was tracked

with reference to P1 and P3 cells, which contributed to endo-

derm over a substantial axial length in the floor and roof,

respectively (Fig. 6P1 and P3). P5 cells remained stationary

for the first 9 hours after labelling (Fig. 7B-P1 + P5, P3 + P5: 0–

9 h). Concurrent with the elongation of the portal, which

was revealed by the rostro-caudal extension of the P1 and

P3 cells, P5 cells extended caudally into the portal (21–23 h).

P5 cells from contralateral side merged medially (24–27 h)

and occupied the lateral sides of the portal. They were juxta-

posed to the P3 cells in the dorsolateral region of the rostral 2/

3 part of the CIP and overlapped with the P3 cells in the lateral

region of the open ‘‘midgut’’ (Fig. 7B-P3 + P5: 28 h) but not with

P1 cells in the floor of the CIP (Fig. 7B-P1 + P5: 28 h). Contrast-

ing to the AIP, the formation of the CIP involves more elabo-

rate rostro-caudal extension of the progenitor cells in all

parts of the hindgut.

3. Discussion

3.1. Progenitors of the AIP are present in the anteriorendoderm by the early-somite stage

Fate-mapping studies of the endoderm of the gastrula-

stage mouse embryo show that the definitive endoderm of

the foregut is allocated during the first 12 h of gastrulation

(Tam et al., 2007). By the late-bud/presomite stage, the ante-

rior endoderm, which lies beneath the head folds and consti-

tutes about half of the total endoderm population of the

embryo, is fated to form the anterior intestinal portal (Lawson

et al., 1986). This progenitor population is broadly sub-divided

Table 2 – Patterns of distribution of dye-labelled cells from lateral sites in the anterior endoderm to specific regions of thegut

Site of labelling: L, left hand side; R, right hand side. #, embryo in which an additional midline site at the same rostro-caudal level was also

labelled.

Regions of the gut: Ph I-II and Ph III-IV: at levels of pharyngeal pouches I to II and III to IV; D, Dorsal; LT, Lateral; V, Ventral; M, Medial.*Cells distributed to different regions of the gut are colour matched with their site of origin, blank, no cells present.


into precursors of the dorsal and lateral/ventral foregut (Tam

et al., 2004). In the early (1–3)-somite stage embryo, high-res-

olution mapping of the anterior endoderm reveals a discrete

regionalization of the progenitor of not only the dorsal and

ventral foregut, but also the multiple sites of origin of the pre-

cursors of the liver bud at the lateral region and the AIP lip

and the presence of endoderm progenitor of the gut adjacent

to the heart and the liver bud (Tremblay and Zaret, 2005). To-

gether, results of this previous study and our present work

have now provided a complete fate map of the anterior endo-

derm of the early-somite stage embryo (Fig. 8A and B).

Generally, cells in the midline of the anterior endoderm

contribute to the formation of the axial endoderm population

of the AIP. Cells that are initially located most rostrally in the

anterior endoderm (A1) go to the floor of the AIP, while those

of the caudal region (A3) populate the roof of the AIP (Tremb-

lay and Zaret, 2005, Fig. 8B). The A1 and A3 populations ex-

pand rostro-caudally presumably in concert with the

elongation of the AIP (Fig. 8C). In contrast, A2 cells spread lat-

erally in the oral part of the AIP (Fig. 8B).

Cells at the rostro-lateral region (the A4 site) spread cau-

dally (Fig. 8C) to occupy the ventral floor on either side of

the A1 cells in the midline (Fig. 8B). Cells of the pharyngeal

pouches and the lateral wall of the prospective oesophagus

are derived from cells in the lateral domains (A5 and A6) of

the anterior endoderm (Fig. 8B). A5 cells, however, are local-

ized in the deeper part of the AIP, rostral to the A6 cells. As

a result, cells from A1, A4 and A6 sites become juxtaposed

to each other in the ventral and lateral endoderm at the en-

trance of the AIP. This is consistent with the previous finding

that liver precursors from these sites are brought together

during the formation of the foregut (Tremblay and Zaret,

2005). A6 cells also extend into the lateral endoderm of the

open ‘‘midgut’’ to the level of the first 3–4 somites. These cells

are found to be incorporated later into the foregut rostral to

the liver bud and the pancreas (Pierreux et al., 2005; Tremblay

and Zaret, 2005).

In contrast to the mouse embryo, not all the progenitors of

the foregut are present in the endoderm layer of the equiva-

lent stage chick embryo (Stage 4, Kimura et al., 2006). Notably

absent are the cells contributing to the ventral foregut, which

after ingression at the primitive streak remain among the

mesoderm cells and are incorporated into the endoderm layer

later at Stage 5 (equivalent to mouse early-head-fold stage).

Fig. 5 – Localization of cells derived from (A) individual sites in the posterior endoderm (P1–P5), lateral node (LN) and node (N)

regions of the early-head-fold stage embryo at 1–3 and 24–28 h of in vitro culture following dye labelling or electroporation.

For embryos imaged 24–28 after dye labelling, black arrows mark the lip of the caudal intestinal portal (CIP). Electroporated

cells expressing the lacZ and EGFP vector are indicated by white arrows on whole embryo images and by red arrows on

histological sections. In (N), labelled cells are also found in the notochord (asterisk) derived from the node. In LN(R + L), cells

derived from lateral node endoderm on two sides are distributed symmetrically in the open part of the embryonic gut. (B)

Embryos showing distribution of cells derived from two sites in the posterior endoderm (P1 + P2, P1 + P3, P2 + P3 and P5 + P5)

to the CIP and the open gut region.


Table 3 – Distribution of dye-labelled and electroporated cells from sites at the level of the node and in the posteriorendoderm to the embryonic gut

Site #Embryosanalysed

Number of embryos with labelled cells in

Anterior intestinalportal

Open ‘‘midgut’’ Posterior intestinal portal

Cardiac level Rostral to 8th somite Caudal to 8th somite Rostral third Middle third Caudal third

D L V M L M L D L V D L V D L V

Node DL 7 5 6 5 3 2

E 8 3 6 5 2 2

Total 15 8 12 10 5 4

LN DL 11 2 4 2 9 6 2 1

E 5 1 2 2 2 2 1 2 1

Total 16 2 4 3 11 2 8 2 3 2 2

P1 DL 12 1 11 11 2 5 12

E 6 3 1 5 1 1 5

Total 18 1 14 1 16 3 6 17

P2 DL 8 4 8 8

E 4 1 1 3 3 1

Total 12 1 1 7 11 9

P3 DL 7 1 4 4 4 1 6 3 2

E 5 1 1 1 1 1 1 5 2

Total 12 2 1 5 5 5 2 11 5 2

P4 DL 9 3 9 4 9 7 5 5

E 6 1 1 1 5 6 5 5 5

Total 15 1 4 10 9 6 14 12 10 5

P5 DL 11 3 10 5 9 5 9 1 3 4

E 4 1 3 1 3 4 1 1 1 1

Total 15 3 10 6 12 1 8 13 2 4 5 1

DL, dye labelling; E, electroporation; D, dorsal; L, lateral; V, ventral; M, medial.

Fig. 6 – Distribution of the descendants of dye-labelled and

electroporated cells at five sites (P1–P5, insets) of the

posterior endoderm of the early-somite stage embryo to the

open part of the embryonic gut and the posterior intestinal

portal after 24–28 h of in vitro development. Colour code –

red: dorsal/roof, yellow: lateral wall, green: ventral/floor.


When these progenitors first emerge, they are localized to the

rostral margin of the anterior endoderm (equivalent to the AIP

lip, Tremblay and Zaret, 2005; Sites A1 and A4, this study).

From Stage 5 to 11, the pattern of distribution of the cells in

the anterior endoderm of the avian embryo is remarkably

similar to that of the mouse. Endoderm cells from the rostral

margin contribute to the ventral foregut and they are joined

by cells from the lateral endoderm. The dorsal foregut is pop-

ulated by cells in the medial domain and the lateral foregut by

those in the lateral domain of the endoderm anterior to the

Hensen’s node (Kimura et al., 2006). Foregut formation in

these two divergent species therefore appears to be accom-

plished by deploying common morphogenetic mechanism.

3.2. Laterality in the AIP endoderm

In the avian embryo, cells in the ventral midline of the

foregut are derived from Hensen’s node (Stage 4) and its inter-

mediate descendants in the prechordal plate (Stage 5, Kirby

et al., 2003). Similarly in the mouse, Gsc-expressing cells of

the prechordal plate (Belo et al., 1998; Nishioka et al., 2005)

and the adjacent definitive endoderm (the A1 cells) are de-

rived from progenitor in the anterior primitive streak (the

mid-gastrula organizer) of the mid-streak stage embryo (Kin-

der et al., 2001). Both the A1 cells and the ventral foregut

endoderm, which contains A1-descendants, express Dkk1

Fig. 7 – Time course imaging of the morphogenetic movement of endoderm cell populations during the formation of (A) the

anterior intestinal portal (Sites A2 + A5, Sites A2 + A6) and (B) the posterior intestinal portal (Sites P1 + P5, Sites P3 + P5)

during the 27- to 28-h period of in vitro development. All images are ventral views unless specified. Left, right: left and right

half of the bisected embryo. White and black arrows mark the lip of the anterior and posterior intestinal portal, respectively.


(Lewis et al., 2007; Nishioka et al., 2005) that codes for a WNT-

antagonist. Loss of Dkk1 function or partial loss of both Dkk1

and Gsc results in the truncation of the head structure

(Mukhopadhyay et al., 2001; Lewis et al., 2007), suggesting

the prechordal plate and/or the descendants of A1 cells in

the pharyngeal endoderm may pattern the head and the oro-

facial structures (Lewis and Tam, 2006). Although cells from

the lateral endoderm converge medially in the foregut floor,

they remain separated in the midline by A1-derived cells. In

the avian embryo, ablation of these lateral endoderm popula-

tion results in randomized directions of the looping of the

heart tube (Kirby et al., 2003), raising the possibility that the

ventral midline and the lateral foregut endoderm may play

a role in the maintenance of the laterality of the body plan.

Consistent with this concept, mapping the distribution of

the A5 and A6 endoderm in our study has revealed an asym-

metric distribution of the contralateral endoderm to the dor-

so-ventral aspects of the pharynx. Cells from the right side of

the anterior endoderm are distributed to right dorsolateral re-

gion and those from the left to the left ventrolateral region of

the AIP. This constitutes a clockwise displacement of cells in

the wall of the pharynx when it is viewed from the anterior

end of the AIP. This asymmetric disposition of cells from con-

tralateral side does not involve any physical rotation of the

AIP but only the distribution of the cells within the epithelium

lining the pharynx. At the 3–4 somite stage shortly after label-

ling, asymmetric expression of Nodal is already evident in the

lateral mesoderm underlying the A5 and A6 sites (Brennan

et al., 2002; and unpublished data). This display of laterality

by the A5 and A6 endoderm seems to herald the direction

of rotation (right to dorsal, left to ventral) of the caudal part

of the foregut, which forms the lower part of the oesophagus

and the stomach. Whether this asymmetric displacement of

pharyngeal endoderm has any impact on the looping of the

heart tube or the body axis is not known. Nevertheless, this

finding highlights that the pharynx may have acquired some

morphogenetic property related to the laterality of the body

plan.

3.3. The mid- and hindgut are formed by the expansion ofthe posterior endoderm

The mapping studies of the anterior endoderm shows that

although this population makes up nearly half of the endo-

derm population present in the early-somite stage embryo,

it contributes primarily to the foregut rostral to the liver bud

(Tremblay and Zaret, 2005; this study), which is less than half

of the length of the embryonic gut. The midgut and the hind-

gut would therefore have to be derived from the endoderm in

the middle (somite and the node level) and the posterior

region of the embryo, as indeed shown by our study.

Endoderm of the open gut region, which spans across the

Fig. 8 – (A) Sites in the anterior endoderm of the early-head-fold and the posterior endoderm of the early-somite stage embryo

analysed by fate-mapping in this study. (B) The fates of the 13 endoderm populations and (C) the morphogenetic tissue

movement accompanying the formation of the anterior and posterior intestinal portals. The anterior and posterior parts of

the gut are opened up along the right hand side to reveal the dorsal, lateral and ventral sides of the portal. The maps are not

joined at the open gut region because of the difference between the initial developmental stages of embryos in the two

mapping experiments.


somite-containing region of the early-organogenesis stage

embryo, is derived from the cells at the node and lateral to

the node, as well as those from the P3 site and P5 sites (see

also Tremblay and Zaret, 2005). In the open gut region, endo-

derm cells in the midline and the lateral region undergo ros-

tro-caudal extension. The midline cells, however, show a

much more substantial extension to cover a longer distance

than the adjacent lateral cells, as also seen in the chick (Kim-

ura et al., 2006).

The formation of the CIP involves the invagination of tissues

such that the caudal-most midline cells (P1) are stretched along

the floor of the portal (Fig. 8B and C). The lateral P4 cells con-

verge medially to the ventral and lateral sides of the portal.

On the dorsal side, P2 and P3 cells in the midline extend ros-

tro-caudally to form the roof of the posterior intestinal portal.

The lateral P5 cells converge medially to the dorsal and lateral

sides of the portal and extend to the lateral regions of the open

‘midgut’’. Morphogenesis of the CIP therefore shares some sim-

ilar features with the AIP in that (a) cells initially located at the

end of the body axis (A1 and P1) populate the floor of the portal

and others in sub-terminal sites (A2, A3, P2 and P3) go to the tip

and roof of the portal, (b) lateral cells undergo rostro-caudal

extension and converge to the lateral side and to the floor (A4

and P4) or the roof (A5, A6 and P5) of the portal. Similar conver-

gence of progenitors of the lateral hindgut is also seen in the

chick embryo at Stage 5–11 (Kimura et al., 2006). Development


of the CIP, however, does differ from that of the AIP. The contra-

lateral endoderm populations intermingle at the ventral mid-

line with each other, and like the dorsal midline cells, the

ventral midline and lateral (P5) populations extendwell beyond

the CIP into the lateral area of the open segment of the embry-

onic gut, which will form the lateral and ventral wall of the fu-

ture midgut when it closes ventrally. The CIP also elongates to

form a longer segment of the gut than the AIP. It is likely that

cellular re-arrangement and cell proliferation are required to

achieve the expansion of the gut endoderm (Kanai-Azuma

et al., 2002).

In essence, the midgut endoderm is mostly derived from

the node and lateral node cells (Wilson and Beddington,

1996), but its caudal segment is also populated by cells de-

rived from the posterior endoderm (P3 and P5) of the early-so-

mite stage embryo. The posterior endoderm (P1–P5), which

harbours the progenitor of the full length of the gut from

the level of forelimb bud to the end of the embryonic gut,

therefore forms both the pre- and the post-umbilical part of

the gut.

4. Materials and method

4.1. Collection and culture of mouse embryos

Embryos were harvested from pregnant ARC/s strain mice

at 8.0–8.5 days post-coitum (E8.0–E8.5). Embryoswere dissected

from the decidua and the Reichert’s membrane was removed.

Embryos were sorted by somite numbers into the 1–3 somites

and 5–9 somites groups. They were kept in 100% rat serum in

a 5% CO2 incubator at 37 �C prior to manipulation. Following

dye labelling or electroporation, embryos were cultured for

24–28 h in a medium made up of 75% heat-inactivated rat ser-

um and 25% Dulbecco’s modified Eagle medium at 37 �C in glass

bottles rotating at 30 RPM with a continuously replenished gas

phase of 5% CO2, 20% O2 and 75% N2 (Sturm and Tam, 1993).

4.2. Dye labelling of the endoderm cells

For dye labelling, cells in the endoderm were painted with

carbocyanine dyes: DiO (D275, Molecular Probes) and CM-DiI

(C-7001, Molecular Probes) (Bildsoe et al., 2007). In each em-

bryo, around 100–150 cells were labelled in order to reveal

the maximal distribution in the embryonic gut of the descen-

dants of the cell population at a particular site. In addition,

double labelling was performed to determine the relative spa-

tial distribution of different cell populations and to track their

morphogenetic movement during gut morphogenesis.

Labelled embryos were imaged by fluorescence micros-

copy within 1 h after labelling to ascertain the site of labelling.

The embryos were re-imaged at specified time intervals

in vitro, in experiments for tracking tissue movement, or at

the end of the culture period to visualise the distribution of la-

belled cells in the embryonic gut. Photographs were taken

using a Leica MZ16 microscope with a SPOT Advanced digital

camera and fluorescent and bright field images were digitally

edited and merged with the SPOT 4.0 software and Adobe

Photoshop 7.0. Embryos containing CM-DiI labelled cells were

then fixed in 4% paraformaldehyde solution and then pro-

cessed for wax histology to determine precisely the location

of the cells in the gut tissue.

4.3. Marking the endoderm by whole embryoelectroporation

In contrast to dye labelling which marks groups of endo-

derm cells, electroporation can be targeted to about 30–50

cells and therefore allows a finer resolution of the fates of

cells at each site. A pCMV-EGFP or pCMV-lacZ-IRES2-EGFP

expression vector was introduced into endoderm cells by

electroporation of the whole embryo (Khoo et al., 2007). The

EGFP-expressing cells were visualised by fluorescence micros-

copy as for the dye-labelled cells at 3 h (to determine the pre-

cision and extent of electroporation) and 24–28 h of in vitro

culture for visualising the distribution of the progeny of the

electroporated cells. After fluorescence imaging, embryos

were fixed briefly in 4% paraformaldehyde for 5 min and

stained overnight with X-gal reagent. The stained embryos

were processed for wax histology to localise the X-gal stained

cells in the embryonic gut. Some X-gal stained embryos were

further processed for in situ hybridisation to reveal the

expression of Pax9 in the foregut (Peters et al., 1998).

4.4. Whole mount in situ hybridization

Embryos were processed for in situ hybridization (ISH)

according to the protocol of Wilkinson and Nieto (1993) with

the following modifications: riboprobes were labelled with

digoxigenin-11-UTP (Roche) using the AmpliScribe kit (Epi-

centre Technologies) or the MAXIscript T7/T3 Kit (Ambion).

SDS was used in place of CHAPS in both prehybridization

and hybridization, no RNA digestion was performed after

hybridization, and formamide was omitted from post-hybrid-

ization washes. The following riboprobes (and the source)

were used: En1 (A. Joyner), Fgf8 (G. Martin), Hesx1 (S. Dunwoo-

die), Krox20 (D. Wilkinson), Otx2 (S.L. Ang), Pax9 (R. Jiang) and

Uncx4.1 (S. Dunwoodie).

4.5. Data collation and analysis

At the end of the 24–28 h of in vitro culture, the early-head-

fold embryo developed to embryos containing 16–19 pairs of

somites. At this stage, the embryonic gut comprises of a fore-

gut portal, an open ‘‘midgut’’ in the trunk (somitic) region and

a hindgut portal. The presence of electroporated or labelled

cells was scored in sub-regions of these three segments of

the embryonic gut: the dorsal, ventral and lateral wall of the

foregut portal, medial and lateral regions of the open ‘‘mid-

gut’’, and the dorsal, ventral and lateral wall of the hindgut

portal. Data were then compiled to show the total number

of embryos electroporated or labelled cells for a specific

site or combinations of sites that had cells in each of these

sub-regions (Tables 1–3).

Acknowledgements

We thank David Loebel for comments on the manuscript and

Kirsten Steiner for assistance with figures. Our work is sup-


ported by the National Health and Medical Research Council

(NHMRC) of Australia and Mr. James Fairfax. P.P.L.T. is a

NHMRC Senior Principal Research Fellow.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.mod.2008.04.001.

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Regionalisation of the endoderm progenitors and morphogenesis of the gut portals of the mouse embryo

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