A role for CdxA in gut closure and intestinal epithelia differentiation

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 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 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 (19 – 21 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 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.

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.

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).

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).

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).

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 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′).

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 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.

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 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 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).

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 epithelium from E17 to hatching and point to the importance in determining the CDXA spatial distribution at these stages. The CDXA pattern of expression was studied in isolated intestines from E18 embryos, in chicks 1 day after hatching and 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 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.

The localization of the CDXA protein from the onset of gaschicks we could detect expression of the CDXA protein along the sides of the villus, with minimal levels close to the tip of the villus (Fig. 4B). Histological analysis of intestines of 1 day old chicks shows that the CDXA protein is still localized to the one cell thick intestinal epithelium (Fig. 4B′). 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 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.

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 mor-phogenesis. Between E6 to E9, the CDXA protein appears in the proximal mesenchyme that surrounds the epithelium concomitantly 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.

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.

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.