253
Development 120, 253-263 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
A role for CdxA in gut closure and intestinal epithelia differentiation
Ayala Frumkin1,‡, Graciela Pillemer1, Rebecca Haffner1,†, Nora Tarcic2, Yosef Gruenbaum3
and Abraham Fainsod1,*
1Department of Cellular Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem 91010, Israel
2The Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, Jerusalem
91010,
Israel
3Department of Genetics, Hebrew University, Jerusalem 91904, Israel
*Author for correspondence
†Present address: Department of Chemical Immunology, The Weizmann Institute, Rehovot, 76100, Israel
‡Present address: Department of Genetics, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1XB
SUMMARY
CdxA is a homeobox gene of the caudal type that was previously shown to be expressed in the endoderm-derived gut
epithelium during early embryogenesis. Expression of the
CDXA protein was studied during intestine morphogenesis from stage 11 (13 somites) to adulthood in the chicken.
The CDXA protein can be detected during all stages of gut
closure, from stage 11 to 5 days of incubation, and is mainly
localized to the intestinal portals, the region where the
splanchnopleure is undergoing closure. In this region,
which represents the transition between the open and
closed gut, the CDXA protein is restricted to the endodermderived epithelium. At about day 5 of incubation, the
process of formation of the previllous ridges begins, which
marks the beginning of the morphogenesis of the villi.
From this stage to day 11 expression of CDXA is localized
to the epithelial lining of the intestine. In parallel, a gradual
increase in CDXA protein expression begins in the mesenchyme that is close in proximity to the CDXA-positive
endoderm. Maximal CDXA levels in the mesenchyme are
observed at day 9 of incubation. During days 10 and 11
CDXA levels in the mesenchyme remain constant, and by
day 12 CDXA becomes undetectable in these cells and the
epithelium again becomes the main site of expression. From
day 12 of incubation until adulthood the CDXA protein is
present in the intestinal epithelium. Until day 18 of incu-
bation expression can be detected along the whole length
of the villus with a stronger signal at the tip. With hatching
the distribution along the villi changes so that the main site
of CDXA protein expression is at the base of the villi and
in the crypts. The transient expression of CDXA in the mesenchyme between days 5 and 11 may be related to the interactions taking place between the mesenchyme and the
epithelium that ultimately result in the axial specification
of the alimentary canal and the differentiation of its various
epithelia. The main CDXA spatial distribution during morphogenesis suggests a tight linkage to the formation and
differentiation of the intestinal epithelium itself. CDXA
appears to play a role in the morphogenetic events leading
to closure of the alimentary canal. During previllous ridge
formation the CDXA protein is transiently expressed in the
mesenchymal cells thought to provide instructive interactions for the regionalization and differentiation of the gut
epithelium. Finally, CDXA is expressed, from hatching
until adulthood, in the crypts and the base of the villi, in
cells on their way to differentiate and replace those aged
by digestive activity.
INTRODUCTION
and is organized as a cell layer under the mesoderm. Closure
of the gut begins at the rostral end with the formation of the
foregut (Bellairs, 1953a). The foregut begins its formation with
the head fold which introduces a fold in the endodermal layer
(Hamilton, 1952). This endodermal diverticulum is surrounded
by mesoderm, thus forming the splanchnopleure which will
take part in the formation of most of the gut apart from the
stomodeum and the proctodeum (Hamilton, 1952). The
splanchnopleure constitutes the most anterior part of the
foregut (Bellairs, 1953a). The fold in the endoderm forms the
floor of the foregut, which extends caudally resulting in the
elongation of the gut. Any point where the transition between
the closed alimentary tube and the still open splanchnopleure
Following gastrulation and the formation of the three germ
layers, many different morphogenetic events take place simultaneously. One of the morphogenetic processes is the
formation of the gut or alimentary tract. As embryogenesis
proceeds, this tract will give rise to the digestive system
together with the glands associated to it, and in addition, it will
give rise to epithelial elements of other organs such as the respiratory tract. The morphogenesis of the gut, at least in its early
stages, overlaps with the morphogenesis of the endoderm. In
the chicken embryo, during primitive streak regression, the
endoderm has already migrated through the primitive streak
Key words: homeobox, caudal genes, chicken embryos, gut
development, endoderm
254
A. Frumkin and others
occurs, is termed the anterior intestinal portal. Backward continuation of the floor of the gut is brought about as the lateral
endoderm swings ventromedially and both sides meet and fuse
medially (Bellairs, 1953a).
The alimentary canal also undergoes closure from its caudal
end (Hamilton, 1952). At about stage 11 (13 somites;
Hamburger and Hamilton, 1951) the formation of the tail bud
begins, which becomes well established by stages 13-14 (1921 somites; Schoenwolf, 1979). At the same stages, the tail fold
is formed, the posterior intestinal portal is established and it
marks the beginning of the closure of the hindgut. The process
of closure of the hindgut in a rostral direction proceeds in a
manner analogous to the formation of the foregut (Patten,
1951). Closure of the gut continues from both ends towards the
center until the anterior and posterior intestinal portals meet.
At the point where both portals meet the yolk stalk is established. The yolk stalk gradually closes and by day 5 of incubation it remains as a connection between the intestine and the
yolk sac. The yolk stalk together with the veins and arteries of
the yolk sac and the allantoic stalk form the umbilical cord.
The axial differentiation of the alimentary canal initiates
soon after closure begins, as the embryo reaches stages 12-14
(15-20 somites; Bellairs, 1953a). Regional differentiation of
the gut starts in the foregut as it is the first region of the gut
formed. Differentiation of the gut and the organs originating
from it begins with the stomodeum and then continues with the
pharynx, thyroid, lungs, esophagus, stomach, liver, pancreas in
the rostrocaudal direction up to the yolk stalk (Hamilton,
1952). The intestine shows its main divisions even before the
closure of the yolk stalk. By the third day of incubation, the
duodenum is indicated by the hepatic and pancreatic outgrowths. The jejunum extends from the pancreatic outgrowth
to the yolk stalk and the ileum continues up to the caecal
processes.
The endodermal component of the splanchnopleure will
form the epithelial lining of most of the organs derived from
the alimentary canal. The mesenchyme surrounding the epithelium in most organs will derive from the mesodermal layer of
the splanchnopleure. This association between the endoderm
and the mesoderm results in epithelial-mesenchymal interactions which are of great importance in the functional differentiation of distinct regions of the gut. It has been shown that the
surrounding mesenchyme in the digestive tract influences the
morphogenetic pattern of the endodermal epithelial cells with
which they come in contact (Kedinger et al., 1988; Hayashi et
al., 1988; Yasugi et al., 1991).
Once the axial specification of the alimentary canal is
underway, the different regions of the digestive tract develop
their own specialized epithelia. In the chicken embryonic
intestine a number of morphogenetic events take place that lead
to the formation of a fully developed intestinal epithelium.
Formation of the villi in the intestine begins with the formation
of previllous ridges (Hilton, 1902). At about 4.5 days of incubation (stage 24; Hamburger and Hamilton, 1951), in cross
section, the intestinal epithelium appears as a thick-walled
circular tube with a small lumen (Burgess, 1975). Between
days 5 and 8 of incubation (stages 26 to 34 approximately) the
profile of the intestinal epithelium in cross section, changes and
acquires an elliptical shape (Burgess, 1975). The elliptical
shape of the intestinal epithelium can be attributed to the
formation of two longitudinal folds or ridges bulging into the
lumen (Coulombre and Coulombre, 1958). At about day 8
(stage 35) three previllous ridges have formed, giving the
intestinal epithelium a triangular shape on cross section
(Hilton, 1902; Burgess, 1975). Formation of previllous ridges
continues with a new ridge being formed in the valley between
two pre-existing ones, reaching a final number of about 16 previllous ridges (Burgess, 1975; Clarke, 1967; Coulombre and
Coulombre, 1958; Grey, 1972; Hilton, 1902). In the mesodermal component of the intestine, morphogenetic changes also
take place in parallel to the epithelium. From the fourth to the
eighth day of incubation, the mesenchymal layer of the
mesoderm becomes circumferentially oriented (Coulombre
and Coulombre, 1958). On day 8 part of the mesenchyme differentiates into a circularly oriented smooth muscle, the circularis (Burgess, 1975; Coulombre and Coulombre, 1958). On
both sides of the circular muscle layer there remains undifferentiated mesenchyme, which following the eighth day of incubation, becomes longitudinally oriented (Burgess, 1975;
Coulombre and Coulombre, 1958). On day 12-13, longitudinal
smooth muscle appears on both sides of the circularis
(Coulombre and Coulombre, 1958). On day 11 of incubation,
the epithelial ridges become wavy and by day 13 the folds
exhibit a zigzag pattern with very sharp angles (Clarke, 1967;
Grey, 1972; Hilton, 1902). The formation of the zigzag pattern
is the first morphological marker of the breakdown of the longitudinal folds into villi. Single villi develop between every
two successive angles in the previllous ridge, thus giving rise
to two rows of villi (Clarke, 1967; Grey, 1972; Hilton, 1902).
The caudal family of vertebrate homeobox genes appears to
be part of the regulatory gene network active during intestinal
morphogenesis. The murine Cdx1 gene was shown to be
expressed in embryos from day 14 onwards and the transcripts
were restricted to the endoderm-derived epithelial lining of the
intestine (Duprey et al., 1988). The stomach and the duodenum
did not show significant levels of expression of Cdx1 nor did
the smooth muscle layer of the intestine. Cdx1 and a second
member of the murine caudal family, Cdx2, have been studied
in the adult mouse intestine (James and Kazenwadel, 1991). In
these studies it was shown that both genes are expressed in the
adult intestine and colon and they exhibit maximal levels of
expression in different regions along the intestinal rostrocaudal axis. The rat homologue of the Cdx1 gene has been isolated
and shown to be expressed preferentially in the colon during
postnatal development (Freund et al., 1992). The chicken
homeobox gene CdxA (formerly CHox-cad) was shown to be
expressed in the endoderm-derived lining of the embryonic gut
and the yolk sac at stages during which the alimentary canal
undergoes closure and the initial stages of intestinal morphogenesis (Frumkin et al., 1991). Later in embryogenesis CdxA
expression continues predominantly in endoderm-derived
organs (Doll and Niessing, 1993). The syrian hamster Cdx3
was isolated from an insulinoma cell line but it was shown to
be expressed in the adult intestine (German et al., 1992).
In order to study in detail the spatial distribution of the CdxA
protein product (CDXA) we prepared monoclonal antibodies
against it. Here we show that after stage 10, the CDXA protein
is present during gut closure and its main site of expression is
in the intestinal portals. Once the alimentary canal has formed,
the CDXA protein localizes to the intestine and colon. The
anterior boundary of expression is at the junction between the
gizzard and the duodenum. Expression of CDXA is usually
CdxA in gut morphogenesis
restricted to the endoderm-derived epithelia. One exception
takes place during previllous ridge formation where transiently
the CDXA protein localizes to the mesenchyme underlying the
epithelium.
MATERIALS AND METHODS
Embryos and tissues
Fertilized chicken eggs were purchased from local farms. The eggs
were incubated at 37.7°C and rotated every hour. Incubations were
performed for different periods of time until the embryos reached the
desired developmental stages. Embryos were dissected out in ice-cold
phosphate-buffered saline (PBS) and whenever necessary, the
intestine was also isolated in ice-cold PBS. Up to 3 days of development, the embryos were staged according to Hamburger and Hamilton
(1951), and from day 3 of incubation onwards the developmental
stage is shown as E followed by the day of incubation. For 1 day old
chick intestines, embryos were allowed to hatch, and the intestine was
dissected the following day. For adult intestine, laying hens were
decapitated and dissected and the intestine was subdivided into its
different regions according to anatomical characteristics. All tissues
and embryos were fixed in 20% dimethyl sulphoxide (DMSO) in
methanol, overnight at 4°C (Dent et al., 1989). Endogenous peroxidase activity was eliminated by incubating the tissues or embryos in
5% hydrogen peroxide for 4-5 hours at room temperature. The
samples were transferred to 100% methanol and stored at −20°C.
Anti-CDXA monoclonal antibodies and whole-mount
immunohistochemistry
The anti-CDXA monoclonal antibody used is the one made by the
6A4 clone as described by Frumkin et al. (1993). This antibody was
raised against a glutathione-S-transferase-CDXA fusion protein made
in the pGEX-2T plasmid (Smith and Johnson, 1988). The
6A4αCDXA antibody was obtained either from tissue culture supernatant or ascites fluid. Whole-mount immunohistochemical staining
was performed according to Dent et al. (1989) and Davis et al. (1991)
as described by Frumkin et al. (1993).
Histological analysis
The glycerol cleared embryos or tissues were rinsed twice in saline
for 30 minutes and then refixed overnight at 4°C in 4% paraformaldehyde. After dehydration the tissues or embryos were paraffin
embedded, and 7-12 µm serial sections were collected. The sections
were counter stained with fast green and mounted with Entellan
(Merck).
RESULTS
CDXA localization during gut closure
In order to determine the CDXA spatial pattern of expression,
we performed immunohistochemical analysis of embryos and
tissues from stage 11 (13 somites, 2 days of incubation) to the
adult animal. In all instances the embryos or tissues were
processed for whole-mount immunohistochemistry and subsequently sectioned for detailed histological analysis. The antibodies used were monoclonal antibodies raised against a glutathione-S-transferase-CDXA fusion protein prepared in E.
coli in the pGEX vector (Smith and Johnson, 1988) as
described by Frumkin et al. (1993). One monoclonal antibody,
6A4αCDXA, was chosen for the analysis of the CDXA
protein. This antibody was shown to specifically recognize the
CDXA protein (Frumkin et al., 1993).
255
From the spatial localization of the CdxA transcripts
performed by in situ hybridization it was shown that this gene
is expressed in the gut epithelium of stage 19-18 (H & H)
embryos which are about half way through gut closure (3.5-4
days of incubation; Frumkin et al., 1991). In order to define
better the spatial distribution of the CDXA protein during gut
closure, we analyzed embryos from stage 10+-11 to 5 days of
incubation (E5) encompassing most stages during which
closure of the alimentary canal takes place. Earlier in embryogenesis, between stages 6 and 10, formation of the foregut
pocket has already proceeded for a few hours, but the
expression of CDXA remains restricted to the regressing
primitive streak and newly formed mesoderm (Frumkin et al.,
1993). A major change takes place at stage 10 when the CDXA
protein becomes undetectable (Frumkin et al., 1993). At stages
10+-11 (about 2-4 hours of incubation from stage 10), the
CDXA protein can be detected again (data not shown), and its
spatial distribution during gut closure stages is restricted to the
splanchnopleure and the closing alimentary canal (Fig. 1). At
stage 12, expression is strongest in the region of the anterior
intestinal portal (Fig. 1A) and it extends to most of the
splanchnopleure in the region where the gut is still open (Fig.
1A′). Cross sections of embryos at stage 12-15 revealed that
expression of CDXA is restricted to the endodermal epithelium
of the splanchnopleure (Fig. 1A′) and soon after the gut
undergoes closure, the CDXA protein levels decrease.
Negative control embryos of this developmental stage and of
later stages, were carried out. These were stained either with a
second antibody, which showed no staining or monoclonal
antibodies directed against E. coli proteins, which stained in
other regions (data not shown).
At day 3 of incubation (E3) closure of the gut has
proceeded from the rostral and caudal ends towards the
center. CDXA presence can be detected around both intestinal portals, as well as CDXA-specific staining in the
splanchnopleure, in the region where the gut has not yet
closed (Fig. 1B). In the closed alimentary canal, the CDXA
protein could be detected in regions in close proximity to the
intestinal portals (Fig. 1B). Sections of embryos at E3
revealed that the expression of CDXA is restricted to the
epithelium of endodermal origin of the gut and the splanchnopleure (Fig. 1B′). The thickened endodermal epithelium,
which at this stage covers most of the gut and the proximal
regions of the yolk sac, is the one expressing CDXA. The
dorsal-most epithelium, which remains thin, and the thin
epithelium of the yolk sac do not express the CDXA protein
at detectable levels (Fig. 1B′). After 4 days of incubation (E4),
the closure process has advanced so that the majority of the
alimentary tube has already been formed. At this stage,
CDXA localization remains the same, so that high protein
levels are still present in the intestinal portals and in their
vicinity, in the still open splanchnopleure and in the recently
closed gut (Fig. 1C). Regions of the yolk sac near the intestinal portals whose epithelium is continuous with the epithelium of the gut, show some CDXA protein expression as well
(Fig. 1C). Histological analysis of cross sections of embryos
at E4 revealed that the CDXA protein remains localized to the
epithelium of the gut and the yolk sac, as well as the
splanchnopleure (Fig. 1C′). By day 5 of incubation (E5), the
gut has undergone full closure and the yolk stalk has been
formed. At E5, the expression pattern of the CDXA protein
256
A. Frumkin and others
Fig. 1. Expression of the CDXA protein during gut closure. Immunolocalization of the CDXA protein in embryos from 2 days of incubation
(stage 12; H & H) to 5 days of incubation (E5). The embryos at different developmental stages are shown as whole-mount
immunohistochemical preparations (A-D) and representative histological sections (A′-D′). (A) Chicken embryo after 2 days of incubation at
stage 12 stained as a whole mount with the 6A4αCDXA antibody. This embryos exhibits localization of the CDXA protein in the anterior
intestinal portal. (A′) Cross section through an embryo at stage 12 as in A. The section is posterior to the intestinal portal and it shows the
labeled endodermal component of the splanchnopleure. (B) After 3 days of incubation (E3) embryos show gut closure from both ends towards
the middle. At this developmental stage the CDXA protein is expressed in both intestinal portals. (B′) Cross section through a section as in B,
in the region where the gut has not closed showing CDXA expression in the endodermal epithelium of the splanchnopleure. (C) After 4 days of
incubation (E4), the alimentary canal has undergone closure from both ends. The CDXA protein is still expressed in the intestinal portals and in
regions of the closed gut in close proximity to the portals. (C′) Cross section through an embryo at E4 shows that CDXA expression is high in
the epithelium of the splanchnopleure, while expression in the newly closed gut epithelium has already decreased. (D) By day 5 of incubation
(E5) closure of the gut has been finalized and the yolk stalk has been formed. CDXA expression is localized to the yolk stalk and regions of the
gut in close proximity. (D′) Section through an embryo at E5 shows localization of CDXA in the gut epithelium. Abbreviations IP, intestinal
portal; G, gut; N, notochord; S, spinal cord; Y, localization of the yolk. Scale bar, 500 µm (A-D) and 100 µm (A′-D′).
remains basically unchanged taking into account the changes
the alimentary canal has undergone up until now. Wholemount immunohistochemical detection of the CDXA protein
in E5 chicken embryos revealed that most of the protein is
localized in the vicinity of the umbilicus (Fig. 1D). It can be
seen that the ileum and part of the colon are still expressing
the CDXA protein (Fig. 1D). The yolk stalk and the proximal
regions of the yolk sac also express this protein (Fig. 1D). A
cross section through the ileum and the colon reveals that
expression of CDXA remains restricted to the epithelium of
the intestine (Fig. 1D′).
CDXA during intestinal morphogenesis
From about E6 the CDXA protein is expressed throughout the
intestine, and it remains there until adulthood. Following gut
closure, the intestinal epithelium begins its morphogenetic
changes leading to the formation of the villi. The morphogenesis of the intestinal epithelium involves a series of events
that include changes in shape, size, previllous ridge formation,
villi formation and ultimately functional differentiation along
the villi. In order to study the localization of the CDXA
protein during intestinal morphogenesis, we initially
performed whole-mount immunohistochemistry of E6, E7 and
CdxA in gut morphogenesis
257
Fig. 2. Localization of the CDXA protein during intestinal morphogenesis. Immunohistochemical localization of the CDXA protein in the
embryonic intestine between days 6 to 16 of incubation, using the anti-CDXA antibody on whole mounts of isolated embryonic intestines.
(A) After 6 days of incubation (E6) the CDXA protein can be localized to the internal regions of the intestinal duct. The CDXA protein is
expressed in the jejunum, ileum, colon, caeca and the proximal regions of the yolk stalk. (B) At E7, CDXA expression is seen in the jejunum,
ileum and colon. (C) At E8, the CDXA protein remains localized to the intestine as shown by the expression in the caeca. (D) The anterior
boundary of CDXA expression (large arrow) is shown at E9. The junction between the gizzard and the duodenum is shown and it represents the
anterior boundary of expression. (E) Internal aspect of the intestine of a E12 embryo. Localization of the CDXA protein to the previllous
ridges, which at this stage are wavy. (F) External view of an intestine from an E13 embryo. Immunostaining with the anti-CDXA antibody
shows the zigzag organization of the previllous ridges, which contain the CDXA protein. (G) Internal detail of the intestine from an E14
embryo. The CDXA protein remains localized to the previllous ridges. (H) At E16, the CDXA protein remains localized to the ridges as they
break up into villi. Abbreviations: GZ, gizzard; D, duodenum; J, jejunum; I, ileum; C, colon; CC, caeca; YS, yolk stalk. Scale bar, 200 µm.
E8 embryos. The studies were repeated and extended with
isolated embryonic intestines from E5 to E16 (Fig. 2). During
the whole time period between days 5 and 16 of incubation,
the CDXA protein can be detected in the embryonic intestine
(Fig. 2). From E6 onwards, the CDXA protein is localized to
the internal regions of the intestine (Fig. 2A-D). In all cases
the immunostaining is surrounded by a tissue layer that does
not stain with the 6A4αCDXA antibody (Fig. 2A-D). The
duodenum of an E9 embryo exhibits strong CDXA expression
(Fig. 2D). The ileum, colon and caeca also show CDXA
expression as seen at stages E6, E7 and E8 (Fig. 2A-C). At
E6, the yolk sac proximal to the yolk stalk still contains some
CDXA protein (Fig. 2A). Along the anterior-posterior axis,
the rostral boundary of expression of the CDXA protein maps
to the junction between the gizzard or muscular stomach, and
the duodenum (Fig. 2D). This anterior boundary can be
observed as early as E6-E7 and once established, it remains
constant. At the posterior end, the boundary of expression is
not as clearly defined, but it localizes to the caudal regions of
the colon.
CDXA in previllous ridge formation
In the process of differentiation of the intestinal epithelium,
one of the first and major events is the formation of previllous
ridges. From the whole-mount immunohistochemistry pattern
observed, it became evident that the CDXA spatial restriction
correlates with the formation of the previllous ridges. At very
early stages of morphogenesis of the intestine, E6 and E7, the
strongest CDXA staining is observed in the lateral regions,
probably due to geometrical reasons (Fig. 2A,B). If the staining
258
A. Frumkin and others
Fig. 3. Involvement of CDXA in the formation of the previllous ridges. Embryonic intestines from E5 to E16 stained as whole mounts were
serially sectioned for histological analysis. The different panels represent sections from intestines at different developmental stages: (A) E5, (B)
E6, (C) E7, (D) E8, (E) E9, (F) E12, (G) E14 and (H) E16. The arrow points to the epithelium and the arrowhead marks the mesodermally
derived mesenchyme. Scale bar, 100 µm.
is restricted to a single cell layer organized as a circle, then on
the sides of the intestine, the signal is actually the result of
multiple cell layers. By E8 (Fig. 2C), the stripes of staining are
not restricted to the sides of the tube but staining in the middle
of the tube can also be observed which is stronger by E9 (Fig.
2D). This observation suggests that the staining is related to
the formation of the previllous ridges, with a consequent piling
up of CDXA-positive cells in each previllous ridge thus giving
rise to a striped pattern. Later in development, E10-E16, the
number of stripes increases and they change from straight lines
(Fig. 2D) to wavy (Fig. 2E) to a zigzag pattern (Fig. 2F-H).
This observation again suggests a correlation between CDXA
expression and the formation of the previllous ridges and subsequently the villi.
In order to understand better the involvement of the CDXA
protein in the development of the previllous ridges and the
villi it was important to determine the cellular localization of
this protein during intestinal morphogenesis. Embryonic
intestines from E6 to E16 stained as whole mounts, were
serially sectioned. In sections of embryonic intestines from
E6 to E12 the extent of intestinal morphogenesis and the
localization of the CDXA protein were studied in parallel
(Fig. 3). The stage of intestinal development was determined
by the shape of the intestinal epithelium and the number of
previllous ridges (Burgess, 1975; Hilton, 1902). At E5, the
intestinal epithelium is thick walled and circular in cross
section and the CDXA protein is restricted to this endodermderived epithelium (Figs. 3A, 1D′). From E6 to E9 the intestinal epithelium changes from circular to elliptic, triangular and
then six previllous ridges are formed at stages E7, E8 and E9
respectively (Fig. 3B-E). During these same stages,
expression of the CDXA protein is still present in the intestinal epithelium, but concomitantly the CDXA protein appears
in part of the mesenchyme surrounding this epithelium, which
is of mesodermal origin (Fig. 3B-E). The appearance of the
CDXA protein in the mesenchyme is gradual (Figs. 3B-D)
and it reaches maximal levels at E9 (Fig. 3E). The CDXA
protein is restricted to cells in close proximity to the epithelium, and the protein levels decrease as the distance from the
epithelium increases (Fig. 3D). Once the first previllous
ridges are formed the outward margin of the CDXA-positive
mesenchyme tends to be circular, and towards the lumen it
CdxA in gut morphogenesis
fills up all the regions at the base of the epithelium (Fig. 3E).
During E10 and E11 the CDXA distribution remains as in E9
(data not shown). Day 11 of incubation marks the beginning
of the morphogenetic events leading to the formation of the
villi from the previllous ridges (Clarke, 1967; Grey, 1972;
Hilton, 1902). Between the eleventh (E11) and the twelfth
(E12) days of incubation a major change in the distribution
of the CDXA protein takes place. The CDXA protein level in
the mesoderm-derived mesenchyme decreases until it
becomes undetectable, while it remains restricted to the
epithelium (Fig. 3F). Therefore, from E12 onwards the only
site of expression of the CDXA protein is the endodermderived intestinal epithelium. The CDXA-specific staining
remains restricted to the epithelial layer of the intestine as
shown for E14 and E16 (Fig. 3G,H).
259
The main difference between the E18 and the 1 day old staining
is that at this later stage in the differentiation of the intestine,
the CDXA protein is expressed more strongly in the sides of
the villi and decreases towards the tip. The developing crypts
show very low levels of CDXA expression (Fig. 4B’).
In order to complete the description of the CDXA pattern of
expression in the intestine, we studied the spatial distribution
of this protein in the adult intestine. The intestine of adult hens
was subdivided into duodenum, jejunum, ileum, colon and
caeca and fragments of the different sections were processed
for whole-mount immunohistochemistry. From the staining
intensities of the different regions obtained when stained in
parallel, it could be observed that the CDXA protein is
expressed most strongly in the middle region of the intestine,
and decreases towards more anterior and posterior regions
(Fig. 5A-E). Sections of the different regions of the intestine
showed that the CDXA protein remains restricted to the
endoderm-derived epithelium (Fig. 5A′-E′). As in the 1 day old
samples studied, the CDXA protein is not uniformly distributed along the epithelium. In the villus the highest concentration of CDXA protein localizes to the region close to the base
and it decreases towards the tip (Fig. 5A′-E′). The CDXA
protein is also strongly expressed in the crypts.
Expression of CDXA close to hatching and in the
adult intestine
At about E17 another set of morphogenetic changes takes place
in the intestinal epithelium in preparation for hatching and the
subsequent functions of secretion, digestion and absorption.
The morphogenetic changes include the break up of the ridges
into individual villi, elongation of the villi themselves,
formation of the crypts and changes in the connective tissue
within the villi. All the changes take place in the intestinal
DISCUSSION
epithelium from E17 to hatching and point to the importance
in determining the CDXA spatial distribution at these stages.
Closure of the alimentary tube
The CDXA pattern of expression was studied in isolated
intestines from E18 embryos, in chicks 1 day after hatching
The localization of the CDXA protein from the onset of gasand in the adult intestine (Figs 4, 5).
In all instances, sections of intestine
were stained as whole mounts and
then processed for serial sectioning
and histological analysis. At E18, the
CDXA protein is localized along the
whole intestinal epithelium (Fig.
4A,A′). The intestinal epithelium,
which is of endodermal origin,
continues to be one cell thick, and
CDXA is expressed in all these cells
(Fig. 4A′). However the CDXA distribution along the epithelium is not
uniform and it exhibits a slight
gradient, which is stronger at the tip
of the villus and lighter at its base
(Fig. 4A′).
In the 1 day old chick, the intestine
is already capable of performing all
its adult functions and for this reason
we studied the CDXA distribution at
this early stage in the life of the chick.
From the analysis of the wholemount stained intestines of seven
Fig. 4. Spatial localization of CDXA close to hatching. (A) Whole-mount staining of the intestine
chicks we could detect expression of
of a E18 embryo. The internal detail of the intestine suggests that the CDXA protein is localized
the CDXA protein along the sides of
along the whole epithelium of the elongating villi. (A′) Section of an E18 intestine as in A.
the villus, with minimal levels close
CDXA is localized to the epithelium forming the villi with higher protein levels in the tips of the
to the tip of the villus (Fig. 4B). Hisvilli. (B) Whole mount staining of the intestine of a 1 day old chick. The CDXA protein can be
tological analysis of intestines of 1
detected on the sides of the villi with lower levels at the tips. (B′) Section of a 1 day old intestine.
day old chicks shows that the CDXA
The CDXA protein localizes to the sides of the villi with lower levels at the tips and the
protein is still localized to the one cell
connecting valleys between villi. Abbreviations: L, luminal side; M, mesenchyme; E, epithelium;
thick intestinal epithelium (Fig. 4B′).
V, villi. Scale bar, 200 µm (A,B) and 100 µm (A′,B′).
260
A. Frumkin and others
trulation to stage 10 (10 somites)
embryos has already been described
(Frumkin et al., 1993). It was shown
that the CDXA protein appears at
about stage 3 (H and H) when the
primitive streak in the chicken
embryo already assumes its elongated
form. During primitive streak
elongation CDXA is expressed as a
stripe localized about 2/3 length from
the primitive pit or Hensen’s node.
During gastrulation stages, the
CDXA protein is expressed in cells
that migrate and become endoderm.
When formation of the definitive or
gut endoderm during gastrulation
slows down or comes to an end,
expression of the CDXA protein in
this germ layer becomes undetectable
(Frumkin et al., 1993). Between
stages 5-10, as the primitive streak
regresses, the CDXA protein can be
localized along the whole length of
the primitive streak as it shortens. In
parallel to the regression of the
primitive streak, the process of gut
closure initiates at the anterior end,
with the formation of the head fold.
While the chicken embryo still has a
primitive streak, the CDXA protein is
localized to the cells as they migrate
through the streak. At about stage 10
the CDXA protein becomes undetectable (Frumkin et al., 1993) and
the first steps in tail bud formation
take place (Schoenwolf, 1979). From
stage 10 onwards, CDXA reappears
in the endodermal lining of the gut
where it will remain, although it will
become restricted to specific regions
as development progresses. Initially,
expression can be seen in the
endoderm as the splanchnopleure
undergoes closure to form the alimentary canal. CDXA expression at
these stages is mainly localized to the
anterior and posterior intestinal
portals. Expression can also be
detected in the open splanchnopleure
and in the newly closed gut. This
pattern of expression raises the possibility that CdxA plays a role during
gut closure. Gut closure in the
chicken embryo takes place over a
period of 3.5-4 days from the
formation of the head fold (stage 6)
to about day 5 of incubation. The
information regarding the morphogenetic movements that result in
closure of the gut is very fragmentary.
Closure of the foregut has been
Fig. 5. CDXA expression in the adult intestine. Intestines from adult hens were stained as whole
mounts for the localization of the CDXA protein. After immunostaining the intestines were
sectioned for histological analysis. Localization of the CDXA protein was determined in five
regions of the intestine. Whole mount and section pairs from (A,A′) duodenum. (B,B′) jejunum,
(C,C′) ileum, (D,D′) colon and (E,E′) caeca. Epithelial localization of the CDXA along the
whole intestine in the crypts and the base of the villi is shown. Abbreviations L, the luminal side
in the whole mounts and sections. Scale bar, 500 µm (A-E) and 100 µm (A′-E′).
CdxA in gut morphogenesis
studied in some detail and it was concluded that it is brought
about by a series of movements of the endodermal cells
(Bellairs, 1953a,b). The lack of knowledge makes it difficult
to correlate the CDXA spatial localization with known events
during gut closure. However, CdxA represents the first genetic
marker that is probably involved in gut closure and any further
information regarding this gene may provide some insight into
this process.
Morphogenesis of the intestinal epithelium
From about day 4-5 of incubation, the intestinal epithelium
begins a series of morphogenetic events that will bring about
the formation of multiple projections towards the lumen, the
villi. Prior to the differentiation of the intestine, the alimentary
canal undergoes a subdivision along the rostrocaudal axis,
thereby marking the boundaries of the different organs or
regions that are formed from this tube. By E5 the CDXA
protein can be detected in the embryonic intestine. This restriction to the intestine represents the culmination of a process that
took place in parallel to gut closure, where expression of
CDXA was continually restricted to regions close to the intestinal portals as they converged towards the umbilicus. This
restriction from the ends towards the center is probably a result
of the axial specification of the alimentary canal. At E6 the
anterior boundary of CDXA expression has been established
at the gizzard-duodenum junction. This anterior boundary of
expression will remain until adulthood. Simultaneously with
the establishment of the anterior boundary of CDXA localization, the intestinal epithelium begins its morphogenesis.
Formation of the previllous ridges has been studied by a
number of groups (Burgess, 1975; Coulombre and Coulombre,
1958; Grey, 1972; Hilton, 1902). One of the questions raised
has been the morphogenetic events that lead to the formation
of the previllous ridges. Early on, it was suggested that the previllous ridges are formed as a result of continued mitotic
activity in the epithelium, while mechanically restricted by the
mesenchyme, resulting in pressure-induced folding
(Coulombre and Coulombre, 1958; Grey, 1972). In support of
this suggestion is the fact that at the time when the epithelium
changes from the elliptical to the triangular shape, the circular
smooth muscle layer has already formed. In experiments where
most of the mesenchyme was removed from intestinal
fragments leaving the epithelium with 2-6 loosely packed mesenchymal cell layers adjacent to it, when cultured in vitro the
epithelium managed to develop three previllous ridges
(Burgess, 1975). It was concluded from this experiment that
formation of the previllous ridges does not result from
continued mitotic activity of the epithelium in a confined
space. This conclusion was further strengthened by experiments in which the intestine was slit open lengthwise and
cultured as flat fragments. In this case also the epithelium gave
rise to six or more previllous ridges (Burgess, 1975). From
studies like these and others, including cytochalasin B treatments, it was concluded that microfilaments play a major role
in the formation of the ridges (Burgess, 1975). In the context
of this information it is of interest to note that during previllous ridge formation the CDXA protein undergoes an interesting modification in its pattern of expression that suggests its
involvement in the initial stages of intestinal epithelium morphogenesis. Between E6 to E9, the CDXA protein appears in
the proximal mesenchyme that surrounds the epithelium con-
261
comitantly to its expression in the endoderm-derived epithelium. This pattern of expression continues up to E11 and at
E12 the mesenchymal expression disappears and only the
epithelial expression remains. The period between E6 and E12
marks the stages during embryogenesis when the previllous
ridges are being formed. In the manipulations and treatments
described above the possible role of the circular smooth muscle
layer in the formation of the ridges was ruled out. Furthermore
it was suggested that ridge formation is an intrinsic function of
the epithelium, and once formed they are stable (Burgess,
1975). On the other hand, it has to be pointed out that in all
treatments and manipulations, part of the loosely packed mesenchyme was left in close association to the epithelium. This
loosely packed mesenchyme is the one that begins expressing
the CDXA protein during ridge formation, and as shown with
the CDXA expression, it fills all the regions under the epithelium so that the outward margin of the mesenchyme forms a
circle. Therefore, it could be suggested that, as concluded by
Burgess (1975), the epithelium folds by the action of microfilaments and then the projections made by the epithelium are
filled by CDXA expressing mesenchyme. Another possibility
which cannot be ruled out by the Burgess experiments, is that
the mesenchyme in close proximity to the epithelium, which
is CDXA positive, through mitotic activity at specific points,
pushes the epithelium towards the lumen. In this case microfilamental contractions would provide mechanical support for
the folding of the epithelium. In both cases, the fact that the
mesenchyme grows and fills the folds created by the epithelium provides mechanical support for the whole structure.
E17-E18 represent another landmark in the development and
morphogenetic differentiation of the chicken embryonic
intestine. At this stage of development the chick prepares to
hatch, internalization of the yolk sac and its contents into the
body cavity takes place. The contents of the yolk sac are still
being digested mainly by the yolk sac epithelium. This food
store can keep the chick alive for 1-2 days, but soon after
hatching the chicks are capable of eating and digesting solid
food. Also at this stage the villi begin their growth in length to
reach the size needed for full digestive function. At these
developmental stages and onwards through adulthood the
CDXA protein is present in the intestinal epithelium. It can be
suggested that the function at this stage is in the maintenance
of the differentiated state of this epithelium. The correlation
between the CdxA gene expression and specific intestinal
epithelium related events late in embryogenesis and posthatching, can only be established from altered physiological
states and pathological situations.
Expression of the vertebrate caudal family in the
intestine
During primitive streak elongation, the CDXA protein is
expressed as a stripe along the three germ layers, but only in
the endoderm does this labeling extend along the whole width
of the embryo (Frumkin et al., 1993). Localization of this stripe
on the available fate maps (Rawles, 1936; Rosenquist, 1966,
1971; Rudnick, 1952) suggests that the endoderm cells
expressing the CDXA protein correspond to the future ventrolateral intestine (Rosenquist, 1966). These same cells will
again express the CDXA protein once the intestinal morphogenesis gets underway and will continue doing so through
adulthood.
262
A. Frumkin and others
At present the vertebrate caudal gene family is composed of
nine members and five of them have been shown to be
expressed in the intestine of the adult. These genes are the
murine Cdx1 and Cdx2 genes (Duprey et al., 1988; James and
Kazenwadel, 1991), the rat Cdx gene (Freund et al., 1992), the
syrian hamster Cdx3 (German et al., 1992) and the chicken
CdxA gene (Frumkin et al., 1991; Doll and Niessing, 1993; this
work), all of which are expressed in the endoderm-derived
epithelium of the adult intestine. Some of these genes have
been shown to be expressed in the same epithelium during
embryogenesis (Cdx1, Duprey et al., 1988; CdxA, Frumkin et
al., 1991; Doll and Niessing, 1993). Restriction of members of
the caudal homeobox gene family to the intestine from late
embryogenesis and the adult, has been shown for Cdx1, Cdx2
and CdxA. The expression in the intestine exhibits spatial
changes along the anteroposterior axis. The junction between
the stomach and the duodenum appears to be a common
anterior boundary for several members of this homeobox gene
family, as shown for the chicken CDXA protein and the rat
Cdx gene (Freund et al., 1992). Along the intestine, the
expression of the vertebrate caudal type homeobox genes is
not uniform and the different genes exhibit peak levels of
expression in different regions of the intestine (James and
Kazenwadel, 1991; Freund et al., 1992; this work). Cdx1 was
shown to exhibit maximal transcript levels in the distal colon
while Cdx2 was shown to be most active in the proximal colon
(James and Kazenwadel, 1991). The rat Cdx gene also showed
a gradient pattern of expression in the intestine from birth
(Freund et al., 1992). In whole-mount immunohistochemical
localization of the CDXA protein in fragments of adult hen
intestine, the protein was found to be expressed at highest
levels in the middle intestinal regions, jejunum, ileum and
colon,, and the protein levels decrease towards both ends. This
gradient expression of the different caudal type genes in the
intestine and the observation that the peaks of expression of
the different genes do not overlap, suggest that a network of
caudal type genes might be active in establishing axial
positions in the adult intestine.
Interestingly, the Drosophila caudal (cad) gene is also
expressed in the developing gut in the fly and it is restricted to
endodermal derivatives. In addition to the maternal and early
zygotic expression of cad, the fly gene is expressed throughout larval development and in early pupal stages (Mlodzik and
Gehring, 1987). In the third instar larva the cad transcripts can
be detected in the posterior midgut, the Malpighian tubules and
the posterior part of the genital disc, the region that corresponds to the anlagen of the anal plates and the hindgut
(Mlodzik and Gehring, 1987). It is important to point out with
regards to the expression of the caudal type genes in vertebrates, that the posterior midgut is of endodermal origin. In
addition, gut development has been studied in some detail in
the fly embryo and it has been shown that induction across
germ layers and spatial restriction along the gut is mediated by
a network involving homeobox genes and growth factor-like
proteins (Affolter et al., 1993; Immerglück et al., 1990; Reuter
et al., 1990). All this information taken together suggests that
the vertebrate caudal type genes have conserved part of the cad
pattern of expression and these genes might be part of a
network responsible for proper gut morphogenesis, in a
mechanism that resembles the one shown in the Drosophila
embryo.
We thank Drs O. Kahner and M. Ben-Sasson for their comments
on the manuscript. This work was funded in part by a grants from The
Council for Tobacco Research, USA and The Israel Science Foundation to A.F.
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(Accepted 11 November 1993)