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To study the developmental origin of the pancreas we used DiI crystals to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. Endodermal precursor cells for the stomach, pancreas and intestine were found to segregate immediately after completion of gastrulation. Transplantation experiments showed that region-specific endodermal fates are determined sequentially in the order stomach, intestine, and then pancreas. Non-pancreatic endoderm transplanted to the stomach region generated ectopic pancreas expressing both insulin and glucagon. These results imply that a pancreas-inducing signal is emitted from somitic mesoderm underlying the pre-pancreatic region, and this extends rostrally beyond the stomach endoderm region at the early somite stage. Transplantation experiments revealed that the endoderm responding to these pancreatic-inducing signals lies within the pre-pancreatic region and extends caudally beyond the region of the intestinal endoderm. The results indicate that pancreatic fate is determined in the area of overlap between these two regions.

In this study, the initial specification of foregut endoderm in the chick embryo was analyzed. A fate map constructed for the area pellucida endoderm at definitive streak-stage showed centrally-located presumptive cells of foregut-derived organs around Hensen's node. Intracoelomic cultivation of the area pellucida endoderm at this stage combined with somatic mesoderm resulted in the differentiation predominantly into intestinal epithelium, suggesting that this endoderm may not yet be regionally specified. In vitro cultivation of this endoderm for 1-1.5 day combined with Hensen's node or its derivatives but not with other embryonic structures/tissues elicited endodermal expression of cSox2 but not of cHoxb9, which is characteristic of specified foregut endoderm. When the anteriormost or posteriormost part of the area pellucida endoderm at this stage, whose fate is extraembryonic, was combined with Hensen's node or its derivatives for 1 day, then enwrapped with somatic mesoderm and cultivated for a long period intracoelomically, differentiation of various foregut organ epithelia was observed. Such epithelia never appeared in the endoderm associated with other embryonic structures/tissues and cultured similarly. Thus, Hensen's node and its derivatives that lie centrally in the presumptive endodermal area of the foregut are likely to play an important role in the initial specification of the foregut. Chordin-expressing COS cells or noggin-producing CHO cells transplanted into the anteriormost area pellucida of the definitve streak-stage embryo could induce endodermal expression of cSox2 but not of cHoxb9, suggesting that chordin and noggin that emanate from Hensen's node and its derivatives, may be involved in this process.

We have engaged in a number of studies in our laboratory that have focused on the molecular mechanisms underlying gut formation, with particular attention being paid to the establishment of regional differences found in the entire gut and within each digestive organ. We have found from our analyses that the presumptive fate of the endoderm in the embryos of vertebrates is determined quite early during development, but the realization of this fate often requires molecular cues from the neighboring tissues such as the lateral plate mesoderm and the mesenchyme derived from it. The mesenchyme seems often to exert instructive or supportive induction effects and, in some cases, a completely inhibitory role during the differentiation of the endodermal epithelium. In addition, many reports on the formation of the stomach, intestine, liver and salivary gland in vertebrates, and of Drosophila gut, all indicate that the morphogenesis and cytodifferentiation of these organs are regulated by the regulated expression of genes encoding growth factors and transcription factors. We have further shown that the epithelium can regulate the differentiation of the mesenchyme into the connective tissue and the smooth muscle layers, thus demonstrating the occurrence of literally interactive processes in the development of the digestive organs.

Nude mice exhibit athymia and hairlessness by a loss-of-function mutation in the transcription factor Foxn1 gene. Although the immunological functions of Foxn1 have been studied intensively, there ...

In the avian embryo, the endoderm, which forms a simple flat-sheet structure after gastrulation, is regionally specified in a gradual manner along the antero-posterior and dorso-ventral axes, and eventually differentiates into specific organs with defined morphologies and gene expression profiles. In our study, we carried out transplantation experiments using early chick embryos to elucidate the timing of fate establishment in the endoderm. We showed that at stage 5, posteriorly grafted presumptive foregut endoderm expressed CdxA, a posterior endoderm marker, but not cSox2, an anterior endoderm marker. Conversely, anteriorly grafted presumptive mid-hindgut endoderm expressed cSox2 but not CdxA. At stage 8, posteriorly grafted presumptive foregut endoderm also expressed CdxA and not cSox2, but anteriorly grafted presumptive mid-hindgut endoderm showed no changes in its posterior-specific gene expression pattern. At stage 10, both posteriorly grafted foregut endoderm and anteriorly grafted mid-hindgut endoderm maintain their original gene expression patterns. These results suggest that the regional specification of the endoderm occurs between stages 8 and 10 in the foregut, and between stages 5 and 8 in the mid-hindgut.

The light-sensitive neural retina (NR) and the retinal pigmented epithelium (RPE) develop from a common primordium, the optic vesicle, raising the question of how they acquire and maintain distinct identities. Here, we demonstrate that sustained misexpression of the Chx10 homeobox gene in the presumptive RPE in chick suppresses accumulation of melanin pigments and promotes ectopic NR-like neural differentiation. This phenotypic change involved ectopic expression of NR transcription factor genes, Sox2, Six3, Rx1 and Optx2, which, when misexpressed, counteracted RPE development without upregulating Chx10. These results suggest that Chx10 can function as a cell autonomous regulator of the regional identity in the primordial retina, presumably through a downstream transcriptional cascade.

The development of digestive organs in vertebrates involves active epithelial–mesenchymal interactions. In the chicken proventriculus (glandular stomach), the morphogenesis and cytodifferentiation of the epithelium are controlled by the inductive signaling factors that are secreted from the underlying mesenchyme. Previous studies have shown that Fgf10 is expressed in the developing chicken proventricular mesenchyme, whereas its receptors are present in the epithelium. In our present study, we show that FGF10 is an early mesenchymal signal that is critically associated with the developmental processes in the proventricular epithelium. Furthermore, virus-mediated Fgf10 overexpression in ovo results in a hypermorphic epithelial structure and an increase in epithelial cell number. In contrast, the overexpression of a secreted FGFR2b (sFGFR2b), an FGF10 antagonist, blocks cell proliferation and gland formation in the proventricular epithelium in ovo. This downregulation of proliferative activity was subsequently found to retard gland formation and also to delay differentiation of the epithelium. These results demonstrate that FGF10 signaling, mediated by FGFR1b and/or FGFR2b, is required for proliferation and gland formation in the epithelium in the developing chick embryo.

The epithelium of the chicken embryonic glandular stomach (proventriculus) differentiates into both a glandular and a luminal epithelium, the cells of which express specific marker genes. The subsequent formation and differentiation of the glands then proceed under the influence of the mesenchyme. To search for possible candidates for the mesenchymal factors involved, we have now investigated the expression and function of Wnt5a in this process. Our current results show that Wnt5a is expressed in the mesenchyme during active gland formation and that overexpression of this gene in ovo results in the increased and ectopic expression of some of the marker genes of the luminal and glandular epithelia. In particular, the overexpression of Wnt5a markedly enhances the expression of the embryonic chicken pepsinogen gene, a marker of the glandular epithelium, indicating its role as a mesenchymal factor that regulates the differentiation of the proventricular epithelium.

Peroxisome proliferator-activated receptors (PPARs) play very important roles in various biological phenomena such as regulation of lipid metabolism, homeostasis, cell differentiation and proliferation, in a variety of organs and tissues. However, their functions in the development of the digestive organs have not been studied yet, although it has been supposed that they are involved in the tumor development and regression of digestive organs. To provide fundamental data to analyze functions of PPARs in the developing digestive organs in the chicken embryos, we performed thorough analysis of expression of PPARalpha, beta (delta) and gamma in the esophagus, proventriculus (glandular stomach), gizzard (muscular stomach), small and large intestines from early developmental stages to post hatch stages. The results showed that each PPAR is expressed in spatio-temporally regulated manner. In general, PPARbeta is widely expressed among digestive organs whereas PPARalpha and gamma showed restricted expression. In the intestine, all PPARs are expressed after hatch, indicating that they play important roles in the physiology of the adult intestine.

Musashi-1 (Msi-1) is an RNA-binding protein that plays key roles in the maintenance of neural stem cell states and in their differentiation into neural cells. Msi-1 has also been proposed as a candidate marker gene of mammalian intestinal stem cells and their immediate lineages. In this study, we examined Msi-1 expression in the small intestine and the stomach of both chicken and mouse during embryonic, fetal and postnatal development. In addition, we analyzed the expression of c-hairy-1, a chicken homologue of mouse Hes1, and assessed the proliferative activity of the cells expressing both of these factors. Significantly, during the development of these digestive organs in both species Msi-1 expression showed dynamic changes, suggesting that it is important for digestive organ development, particularly for epithelial differentiation. Based on our observations of the expression patterns of Msi-1 and c-hairy-1 in the adult small intestine, we speculate that Msi-1 is also a stem cell marker of the chicken small intestinal epithelium.

Fibroblast growth factor 10 (FGF10) is involved in numerous different aspects of embryonic development and especially in active epithelial-mesenchymal interactions during morphogenesis of many organs as a mesenchymal regulator by activating its receptors (FGFR1b and FGFR2b) expressed in the epithelial tissue. FGFR2b is also activated by FGF7 although FGF7 does not bind to FGFR1b. To provide basic data to analyze function of FGFs in the developing gut, here we cloned Fgf7 and studied expression patterns of Fgf7, Fgf10 and Fgfr1-4 during the development of chicken stomach (glandular stomach; proventriculus and muscular stomach; gizzard). Fgf10 is expressed both in the proventricular and gizzard mesenchyme while Fgf7 is expressed only in gizzard mesenchyme. Fgfr1-4 are expressed both in the epithelium and mesenchyme with a different spatial expression patterns. Furthermore, RT-PCR analysis reveals that Fgfr1b and Fgfr2b are expressed only in epithelia of both organs.

In an attempt to clone genes expressed in the gizzard of the chicken embryo by differential display, we obtained a cDNA of a gene encoding a protein with a putative nuclear localization signal and a DNA-binding motif and designated it DDSG1 (differential display-screened gene expressed in the gizzard-1). Besides its expression in the gizzard, the gene is expressed in central and peripheral nervous systems such as brain, spinal cord and dorsal root ganglia in specific patterns.

In this study, we set out to test the ability of endoderm from 1.5-day-old chick embryos (just before digestive tube formation) to develop region-specific characteristics when cultured heterotopically. Various parts of the 1.5-day endoderm were cultured in combination with the flank somatic mesoderm of 3- to 3.5-day chick embryos, and these cultures were analyzed for the expression of several transcription factors and the differentiation of the endoderm. By 1.5 days of normal development, the transcription factors, which are expressed in specific digestive organs, cSox2, CdxA, and cHoxb9/a13 were already expressed in the endodermal cells of the presumptive areas of their later expression domains. When 1.5-day pre-gut endoderm was cultured for 14-15 days, it showed specific differentiation into appropriate organ structures. In general, the more anterior part of the pre-gut endoderm formed the more rostral digestive organ structures while the posterior part became the caudal gut. The differentiation of these regions of endoderm matches their normal fate as recently elucidated (Matsushita [1996a] Rouxs Arch. Dev. Biol. 205:225-231; Matsushita [1999] Dev. Growth Differ. 41:313-319). Expression of cSox2, CdxA, and cHoxb9/a13 in endoderm cultured for 4-5 days is also consistent with their normal fate. Thus, each part of the pre-gut endoderm appears to be already regionally committed to some extent, in accordance with its fate by 1.5 days of development.

Dolichyl monophosphate (Dol-P) is involved in the attachment of carbohydrate chains to proteins in the formation of N-linked glycoprotein. We found that this compound induces apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5-20 min), reduction in mitochondrial transmembrane potential (delta psi m) and translocation of apoptosis-inducing factor (1-3 hr), caspase-3-like protease activation (2-4 hr), chromatin condensation and DNA ladder formation (3-4 hr) were observed successively. In this study, we examined mitochondrial morphological changes by electron microscopy and delta psi m by JC-1 from immediately after treatment of Dol-P. After 5 min of treatment, we observed clearly that mitochondrial cristae began to be disrupted ultrastructurally and almost all the cristae were disintegrated after 1 hr of treatment. The delta psi m of Dol-P treated cells was reduced to 34% as compared with that of control cells immediately after treatment and was quartered within 1 hr. The reduction in delta psi m was not inhibited by cyclosporin A, N-acetyl-L-cysteine and vitamin E. These results indicate that mitochondrial disruption is one of the first triggering events of Dol-P-induced apoptosis.

A gene encoding embryonic chicken pepsinogen (ECPg), a zymogen of the digestive enzyme pepsin, is expressed specifically in epithelial cells of glands of embryonic stage proventriculus (glandular stomach) under the influence of mesenchyme. We found four GATA and one Sox binding motifs in 1.1 kb of the 5′ flanking region of the ECPg gene which are essential to the organ-specific expression of the gene. The expression of cGATA-5 and cSox2 in the proventriculus from day 6 to day 12 of incubation was therefore analyzed. cGATA-5 was more strongly expressed in glandular epithelial cells than in luminal epithelial cells, while cSox2 gene expression was weaker in glandular epithelial cells. Using heterologous recombination explants we also discovered that the expression of cGATA-5 and cSox2 in epithelial cells was affected by mesenchyme when the latter induced ECPg gene expression in epithelial cells. Introduction of expression constructs into epithelial cells by electroporation demonstrated that cGATA-5 upregulated transcription of a reporter luciferase gene via a cis element in the 5′ flanking region of the ECPg gene. The gel mobility shift assay revealed that the cGATA-5 protein specifically binds to the GATA binding sites. cSox2 downregulated the activity of luciferase but it was not through the Sox binding motif. These results suggest that cGATA-5 positively regulates transcription of the ECPg gene and is involved in spatial regulation of the pepsinogen gene during development.

Epithelial-mesenchymal interactions are very important in the development of the vertebrate gut. In the avian embryonic stomach (proventriculus), expression of embryonic chick pepsinogen (ECPg) gene, which is specific to developing glandular cells in stomach epithelium, is regulated by mesenchymal influence. Molecular mechanisms of tissue-specific transcriptional regulation of the ECPg gene and the molecular nature of the mesenchymal signals were analyzed using a combination of the classic organ culture system and gene transfer strategies. In the present review, three methods for the introduction of DNA into tissues are described: lipofection, electroporation and retroviral infection, and characteristics of each system are discussed.

The embryonic gut of vertebrates consists of endodermal epithelium, surrounding mesenchyme derived from splanchnic mesoderm and enteric neuronal components derived from neural crest cells. During gut organogenesis, the mesenchyme differentiates into distinct concentric layers around the endodermal epithelium forming the lamina propria, muscularis mucosae, submucosa and lamina muscularis (the smooth muscle layer). The smooth muscle layer and enteric plexus are formed at the outermost part of the gut, always some distance away from the epithelium. How this topographical organization of gut mesenchyme is established is largely unknown. Here we show the following: (1) Endodermal epithelium inhibits differentiation of smooth muscle and enteric neurons in adjacent mesenchyme. (2) Endodermal epithelium activates expression of patched and BMP4 in adjacent non-smooth muscle mesenchyme, which later differentiates into the lamina propria and submucosa. (3) Sonic hedgehog (Shh) is expressed in endodermal epithelium and disruption of Shh-signaling by cyclopamine induces differentiation of smooth muscle and a large number of neurons even in the area adjacent to epithelium. (4) Shh can mimic the effect of endodermal epithelium on the concentric stratification of the gut. Taken together, these data suggest that endoderm-derived Shh is responsible for the patterning across the radial axis of the gut through induction of inner components and inhibition of outer components, such as smooth muscle and enteric neurons.

In situ analysis of the chicken cSox2 gene, a member of the transcription factor family containing an Sry-like high-mobility group (HMG) box, demonstrated localized expression in the embryonic endoderm. Transcripts of cSox2 appeared before commencement of morphogenesis and cytodifferentiation in the rostral gut epithelium from the pharynx to the stomach. The caudal limit of cSox2 expression coincided with that of the region competent for proventricular differentiation and to the rostral limit of the domain of CdxA, a homologue of Drosophila caudal. During morphogenesis, the level of transcripts of cSox2 decreased in epithelia invaginating into surrounding mesenchyme to form glandular or tubular structures, such as the primordia of the thyroid and lung, glandular epithelium of the proventriculus, and secondary bronchus of the lung. Tissue recombination experiments demonstrated that cSox2 expression is regulated by the underlying mesenchyme as well as morphogenesis and cytodifferentiation. The results suggest that cSox2 plays pivotal roles in generating morphologically and physiologically distinct types of epithelial cells in the gut.

The mucosubstances in the epithelium lining the segment from gizzard to duodenum during development of the chick embryo was studied histochemically using monoclonal antibodies against gizzard mucus and lectins, with attention to the regional differentiation of the epithelium in this segment. The anterior limit of epithelial CdxA mRNA expression detected by in situ hybridisation, which served as the position of the gizzard-duodenal boundary, was clearly found from d 3. Granules positive for some antibodies or lectins were found in the region ranging from the posterior part of the gizzard to the duodenum at d 3, which was followed by an increase in the number of granules and a gradual enlargement of the granule-positive area to the anterior part of the gizzard over 4-6 d. From d 4, the epithelia of the gizzard body and of the pyloric or duodenal region came to be differently stained with some antibodies or lectins. From d 10, each region showed a specific pattern of staining. The epithelia of the gizzard body and pyloric region contained abundant mucus granules with a different staining pattern. In the duodenum the number of stained granules was low except in occasional goblet cells. Thus the epithelia of the gizzard body, pyloric region and duodenum may produce different mucosubstances and the regional differentiation in these epithelia may start at rather early stages soon after the formation of digestive tube.

The primitive epithelium of embryonic chicken proventriculus (glandular stomach) differentiates, after day 6 of incubation, into luminal epithelium, which faces the lumen and abundantly secretes mucus, and glandular epithelium, which invaginates into mesenchyme and later expresses embryonic chicken pepsinogen (ECPg). So far it is not well understood how undifferentiated epithelial cells differentiate into these two distinct cell populations. Spasmolytic polypeptide (SP) is known to be expressed in surface mucous cells of mammalian stomach. In order to obtain the differentiation marker for proventricular luminal epithelial cells, we cloned a cDNA encoding chicken SP (cSP). Sequence analysis indicated that cSP has the duplicated cysteine-rich domain characteristic of SP. Examination of the spatial and temporal expression pattern of cSP gene revealed that, during embryogenesis, cSP was expressed in lumina epithelial cells of the proventriculus, gizzard, small intestine, and lung, but not the esophagus. In the proventriculus, cSP mRNA was first detected on day 8 of incubation and was localized to differentiated luminal epithelial cells. By using cSP as a molecular marker, the effects of mesenchyme on the differentiation of epithelium were analyzed in vitro. On the basis of these data, a model is presented concerning the differentiation of proventricular epithelium.

During embryogenesis, smooth muscle cells of the gut differentiate from mesenchymal cells derived from splanchnic mesoderm. We have isolated a gene involved in the differentiation of smooth muscle cells in the gut using differential display between the chicken proventriculus in which the smooth muscle layer develops poorly and the gizzard in which smooth muscles develop abundantly. The protein encoded by this gene showed highest similarity to mouse FK506 binding protein, FKBP65, and from the function of this protein it was designated chicken FKBP/smooth muscle activating protein (cFKBP/SMAP). cFKBP/SMAP was first expressed in smooth muscle precursor cells of the gut and, after smooth muscles differentiate, expression was restricted to smooth muscle cells. In organ culture of the gizzard, the differentiation of smooth muscle cells was inhibited by the addition of FK506, the inhibitor of FKBPs. Moreover, overexpression of cFKBP/SMAP in lung and gizzard mesenchymal cells induced smooth muscle differentiation. In addition, cFKBP/SMAP-induced smooth muscle differentiation was inhibited by FK506. We postulate therefore that cFKBP/SMAP plays a crucial role in smooth muscle differentiation in the gut and provides a powerful tool to study smooth muscle differentiation mechanisms, which have been poorly analyzed so far.

Three groups of pepsinogens exist in vertebrates, namely, pepsinogen A, pepsinogen C, and prochymosin, which are produced at different developmental stages. In the chicken, prochymosin is expressed only in the embryonic stage, while pepsinogens A and C are secreted from adult chicken proventricular (glandular stomach) mucosa. In order to understand the mechanism of transcriptional regulation of these genes, we have cloned the genes encoding chicken pepsinogens A and C and analyzed the sequences possibly involved in their regulation. 5'-Upstream sequences of both genes contain putative binding motifs for transcription factors such as GATA, Sox, and HNF-3 beta, which are expressed in the chicken gut epithelium. Moreover, we found seven putative binding motifs for human MZF-1 in intron 8 of pepsinogen A gene. These transcription factors may act as regulators of expression of chicken pepsinogen genes.

Sonic hedgehog (Shh) gene encodes a secreted protein that acts as an important mediator of cell-cell interactions. A detailed analysis of Shh expression in the digestive organs of the chicken embryo was carried out. Shh expression in the endoderm begins at stage 7, when the formation of the foregut commences, and is found as narrow bands in the midgut. Shh expression around the anterior intestinal portal at stage 15 is restricted to the columnar endoderm lined by the thick splanchnic mesoderm, suggesting that the existence of thick splanchnic mesoderm might be necessary for Shh expression in the columnar endoderm. After the gut is closed, Shh expression is found universally in digestive epithelia, including the cecal epithelium. However, its expression ceases in the epithelium of the proventricular glands, the ductus choledochus and ductus pancreaticus that protrude from the main digestive duct. When the gizzard epithelium differentiated into glands under the influence of the proventricular mesenchyme, the glandular epithelium lost the ability to express Shh. These findings suggest that Shh expression in the epithelium may be regulated by surrounding mesenchyme throughout organogenesis of the digestive organs and is closely involved in epithelial-mesenchymal interactions in developing digestive organs.

From many recent studies, it has been argued that keratins (cytokeratins) play important roles in the morphogenesis and differentiation of organ development. To learn the role of keratin in digestive tract development, a cDNA of the chicken homolog of keratin-19 (GK-19) was cloned and its expression pattern was analyzed in the digestive tract of chicken embryos. The GK-19 full-length sequence was approximately 1.6 kb and showed more than 80% similarity to human and mouse keratin-19. The result of in situ hybridization with the proventriculus (glandular stomach) of different developmental stages showed that GK-19 expression disappeared specifically in the glandular epithelium from day 6 to day 9 of incubation. Furthermore, GK-19 was localized in the notochord, floor plate, anterior lobe of the pituitary gland and mesonephros. These results suggest the possibility that GK-19 may have multiple roles in organogenesis during embryogenesis.

CdxA, a chicken homeobox-containing gene related to caudal in Drosophila, has been implicated in the regionalization of endoderm. It is reported here that, in the development of the chicken embryo, CdxA expression appears in the endoderm at day 1.5 of development as bilateral bands on either side of the splanchnopleure which later contribute to intestinal epithelium. The CdxA-expressing area extends medially and caudally as formation of the gut tube progresses. It is also shown that the rostral limit of CdxA expression demarcates the boundary between stomach and duodenum after day 3 of development. CdxA is not expressed in digestive tract appendages which open into the intestine, such as pancreas, liver and allantois. Early restriction of CdxA expression in intestinal lineage suggests that the intestinal specification involving CdxA expression commences before the gut tube is formed. The expression of CdxA in epithelial-mesenchymal tissue recombinants suggests that mesenchymal influence regulating CdxA expression plays an important role in confirming the boundary between the stomach and intestine. Chronological change in the spatial distribution of CdxA transcripts and the results of tissue recombination experiments, together with precise fate maps of early endoderm and splanchnic mesoderm, lead to a model of mechanisms by which intestinal specification is brought about.

Ontogeny and cellular localization of the lutropin receptor (LH-R) mRNA were studied in fowl gonads between day 5 of incubation and day 1 of posthatch. In situ hybridization using an antisense cRNA probe demonstrated that LH-R gene expression began at embryonic day 7 in females and day 14 in males. In female embryos, the specific hybridization was detected in not only developing left ovaries but also regressing right ovaries. The LH-R mRNA expression in the right ovary was detectable until hatch. LH-R mRNA was localized in the medullary cords in ovaries and interstitial tissues in testes. These results are consistent with previous reports on ontogenic analysis of LH-dependent steroidogenesis in chicken embryo gonads.

Epithelial cells of chicken proventriculus (glandular stomach) differentiate into two types; luminal and glandular epithelial cells. The molecules regulating the differentiation of proventricular epithelial cells are not well understood. As the first step in screening the molecular determinants involved in the cell differentiation process, we tried to establish an in vitro culture system for isolated proventricular epithelial cells. Various basal media, growth factors and sera were tested. The medium that supports well the proliferation of epithelial cells was composed of Ham's F12 as the basal medium with epidermal growth factor (10 μg/mL), insulin (10 μg/mL), cholera toxin (1 μg/mL) and bovine pituitary extract (100 μg/mL). Fetal calf serum stimulated cell proliferation 1.7-fold, while horse serum was rather toxic. Proventricular epithelial cells proliferated for 3 days, but began to die out within 1 week of culture. Cultured epithelial cells never expressed embryonic chicken pepsinogen (ECPg), a marker gene of glandular epithelial cells, or maintained ECPg expression. The capacity for ECPg expression in cultured epithelial cells was analyzed by recombination with the proventricular mesenchyme and ECPg was detected in epithelial cells cultured up to 3 days. We concluded therefore, that epithelial cells keep the capacity for ECPg expression for 3 days of cultivation and proventricular mesenchymal cells are required for the actual expression of the ECPg gene.

Regulatory mechanisms of cytokeratin expression in the stomach (the proventriculus and the gizzard) epithelia of chicken embryos were examined by epithelial-mesenchymal recombination experiments. In the proventriculus, antigens detected by monoclonal antibodies (PKK1 and AE3) against cytokeratins invariably decreased in gland cells which synthesize pepsinogen, while luminal and duct epithelial cells were positive to these antibodies. When recombined and cultured with the proventricular mesenchyme, the esophageal, proventricular and gizzard epithelia formed proventriculus-type, pepsinogen-expressing glands and the expression patterns of PKK1 and AE3 antigens were identical to that in the normal proventriculus. Under the same experimental conditions, the small-intestinal and allantoic epithelia also formed complex glands but did not express pepsinogen, and PKK1 and AE3 antigens were actively expressed throughout the epithelia. These results indicate that the proventricular mesenchyme can regulate c...

The embryonic chicken digestive tract consists of endodermal epithelium and mesenchyme derived from splanchnic mesoderm. Interactions between these two tissues are important for the establishment of regionality and the subsequent differentiation of digestive organs. In the present study we obtained a monoclonal antibody that reacted with mucus-associated antigen and named it the MA antibody. From 6 days of incubation, this antibody reacted with the esophageal, proventricular and gizzard epithelia. In the proventriculus, the MA antigen was expressed in luminal epithelial cells, while pepsinogen-producing gland cells became MA antigen-negative. The intestinal goblet cells, which secrete mucus, became positive to the antibody from day 13 of incubation. When the esophageal, proventricular or gizzard epithelium of a 6 day embryo was associated and cultivated with the proventricular mesenchyme, the luminal epithelial cells remained reactive to the MA antibody while gland cells were negative or only weakly positive. If the small-intestinal epithelium was cultivated with the proventricular or gizzard mesenchyme, the antigen was detected on the apical surface of the epithelium, suggesting that the expression of the MA antigen was induced by mesenchymal influences in the small-intestinal epithelium. These results suggest that spatio-temporally regulated expression of the MA antigen is controlled by the epithelial-mesenchymal interactions.

Localization of DNA-synthesizing cells in the developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens was investigated. DNA-synthesizing cells were scattered throughout the proventricular epithelium during all developmental stages studied. The results indicate that there is no clear proliferative zone in the proventricular epithelium of the chicken. The labeling indices (LI) of proventricular epithelial cells were measured. On the 6.5th day of incubation, the LI of glandular epithelium reached 29.5 ± 1.5%. the highest value of all the stages studied. This extremely rapid cell proliferation can be considered to be a driving force for the elongation of the proventricular glands during the following stages. Just after hatching, the LI of both the glandular and luminal (non-glandular) epithelia significantly increased from those on the 18th day of incubation. It is suggested that the rise in LI possibly reflected proventricular growth to fit in the change in the method of nourishment after hatching. In 2 week old chickens, the LI of both the glandular and luminal epithelia were reduced to approximately 1%. The active production of embryonic chicken pepsinogen in all glandular epithelial cells of the embryonic chicken revealed that proliferation and differentiation are not necessarily exclusive during the embryonic stages of proventricular development.

Ascidian embryogenesis shares several developmental features with vertebrates. Thus, it is presumed that some molecular mechanisms that are critical for vertebrate development may also act in the early development of ascidians. Here, we investigated expression of the ascidian labial group Hox gene HrHox-1 in the development of Halo-cynthia roretzi. HrHox-1 showed a spatially restricted expression pattern along the anterior-posterior axis, which is remarkably similar to that of the vertebrate gene, Hoxb-1. The expression of HrHox-1, however, was exclusively in tissues of ectoderm origin unlike its vertebrate counterpart. Exposure of the embryos to 10−6 M all-trans retinoic acid induced a larval phenotype with elimination of the anteriormost structures, the papillae. In this phenotype, the level of HrHox-1 expression was enhanced and ectopic expression was observed at the anterior terminal epidermis where the papillae are otherwise formed. These observations suggest that there are some conserved mechanisms in the spatial regulation of expression of labial group genes in embryogenesis of ascidians and vertebrates.

Hrlim is a LIM class homeobox gene that was first isolated from the ascidian Halocynthia roretzi. To assess its roles in early development of the ascidian, spatial and temporal expression of Hrlim was examined by whole mount in situ hybridization. This revealed that transcription of Hrlim is activated at the 32-cell stage specifically in the endoderm lineage. Hrlim is also transiently expressed in all notochord precursor cells. Expression in the endoderm lineage continues through to the middle of gastrulation. After gastrulation, Hrlim is expressed in certain lineages that give rise to subsets of cells in the brain and spinal cord. Based on these observations, it is suggested that Hrlim plays multiple distinct roles in ascidian embryogenesis.

Embryonic chick pepsinogen (ECPg) is one of the pepsinogen isozymogens and its expression is restricted to epithelial cells of the embryonic chick proventriculus (glandular stomach). To examine whether DNA methylation is involved in the regulation of organ-specific and developmental stage-specific expression of ECPg gene, we analyzed the extent of methylation of ECPg gene in normal embryonic and hatched chick organs using methylation-sensitive restriction enzymes. In the proventriculus some CCGG sites underwent demethylation in the gene region after the onset of transcription of the ECPg gene. By contrast, these sites were kept methylated throughout the development in the other organs which do not express ECPg gene. GCGC sites in the gene region became methylated in organs which do not express the ECPg gene, after the initiation of transcription of the ECPg gene in the proventriculus. In the proventriculus, GCGC sites, which were methylated in other organs, were kept unmethylated throughout the development. The methylation state of CpG sites showed no change in the proventriculus of a chick 2 weeks after hatching when the expression of the ECPg gene had completely ceased. The data presented here demonstrate that the DNA methylation is involved in the regulation of organ-specific expression, but stage-specific expression might be brought about by some other mechanisms.

It is well established that the differentiation of glandular stomach epithelial cells is affected by many factors including epithelial-mesenchymal interactions. To clarify the control mechanism of their differentiation, we developed a primary culture system for fetal rat glandular stomach epithelial cells, and examined their differentiation in the absence of mesenchyme. Pure glandular stomach epithelial tissues obtained from 16.5-day fetal rats proliferated rapidly, increasing their number about 20 times in the first 7 days. The epithelial nature of the cells was confirmed by the presence of cytokeratin in the cells. Glandular stomach epithelial cells formed simple cuboidal/squamous epithelia with many mucous granules in their cytoplasm, and exhibited epithelial polarity with microvilli on the luminal surface, basal lamina-like material on the basal surface, and junctional complexes in the apical region. Biochemical analysis showed that the cells expressed acid protease activity in culture. Previous studies showed that glandular stomach epithelial cells specifically expressed two types of acid proteases: pepsinogens in chief and mucous neck cells, and cathepsin E in surface mucous cells. Immunohistochemical studies using specific antibodies showed that the cultured cells expressed cathepsin E but not pepsinogens, and the result was confirmed by zymogram and Western blotting analysis. We thus concluded that fetal rat glandular stomach epithelial cells differentiated into surface mucous cells that expressed cathepsin E in primary culture in the absence of mesenchyme.

Epithelial-mesenchymal interaction plays an important role in the differentiation of digestive tract. However, the factors of these mesenchymes involved in induction of the epithelial differentiation of each organs are still unknown. In the present study, we made reconstituted mesenchymal cell aggregates by mixing proventricular mesenchymal cells with other mesenchymal cells, recombined the reconstituted mesenchyme with gizzard epithelium, and observed the differentiation of the gizzard epithelium in the explants with special attention to the appearance of embryonic chicken pepsinogen, one of the molecular marker of the proventricular epithelial cells, in the gizzard epithelium. The results showed that the proventricular mesenchymal cells induce gland formation and pepsinogen in the gizzard epithelium and that the esophageal and gizzard mesenchymal cells have the inhibitory influence on the differentiation of epithelia toward proventricular epithelium. The cells from small-intestinal, lung and dorsal dermal mesenchyme have no such effect. Based on the results obtained so far, a hypothesis was presented to explain the mechanism regulating the differentiation of the epithelium in the digestive tract in the chicken embryo.

In vertebrates, the endoderm gives rise to the epithelial lining of the digestive tract, respiratory system and endocrine organs. After gastrulation, the newly formed endoderm gradually becomes regionalized and differentiates into specific organs. To understand the molecular basis of early endoderm regionalization, which is largely unknown, it is necessary to identify novel region-specific genes as candidates potentially involved in this process. Applying an Affymetrix Array based approach we aimed for the identification of genes specifically upregulated in the foregut or mid-/hindgut endoderm at the onset of regionalization. Several genes exhibiting spatial and temporal restricted expression patterns in the developing early endoderm were identified and their expression was validated via RT-PCR and whole mount in situ hybridization. We report here the detailed gene expression patterns of two novel genes specifically associated with foregut endoderm and of eight novel genes specifically expressed in the mid-/hindgut endoderm at HH stages 10-11. Future functional analysis of these genes may help to elucidate the mechanisms involved in endoderm development and regionalization.

Chicken embryos have been used as a model animal in developmental biology since the time of comparative and experimental embryology. Recent application of gene transfer techniques to the chicken embryo increases their value as an experimental animal. Today, gene transfer into chicken cells is performed by three major systems, lipofection, electroporation and the virus-mediated method. Each system has its own features and applicability. In this overview and the associated four minireviews, the methods and application of each system will be presented.

The endodermal epithelia of esophagus, proventriculus and gizzard of 6-day chicken embryos can form glands and express embryonic chicken pepsinogen (ECPg), when they are subjected to the influence of proventricular mesenchyme, while intestinal epithelium of the same age cannot respond to the inductive influence of proventricular mesenchyme. We attempted in this paper to know whether this regional difference of epithelia to respond to mesenchymal influence originates very early in development or it is established gradually in the course of development of digestive tract. The young presumptive intestinal endoderm taken from embryos having 15–20 somites was associated and cultivated with 6-day proventricular mesenchyme. The presumptive intestinal endoderm never expressed ECPg although it formed gland-like structures. In the control explants composed of presumptive stomach endoderm and proventricular mesenchyme, glands were formed and gland cells expressed ECPg detected by immunocytochemistry and in situ hybridization. These results indicate that the developmental fate of presumptive intestinal endoderm is determined rather strictly at very early developmental stage, and suggest that the segregation of at least two cell lineages occurs early in the development; one which can express ECPg under the influence of proventricular mesenchyme, and another one which cannot express ECPg and differentiates mainly into intestinal epithelium.

Nude mice exhibit athymia and hairlessness by a loss-of-function mutation in the transcription factor Foxn1 gene. Although the immunological functions of Foxn1 have been studied intensively, there have been relatively few studies of its functions in skin. Foxn1 regulates expression of hair keratins, which is essential for normal hair structure; however, how Foxn1 regulates hair keratin expression and hair formation is largely unknown. In the present study, we found that mice lacking phospholipase C (PLC)-delta1, a key molecule in the phosphoinositide signaling pathway, and nude mice show similar hair abnormalities, such as lack of cuticle and bending. We also found that expression of hair keratins was remarkably decreased in skin of PLC-delta1 knockout mice. Furthermore, expression of PLC-delta1 was induced in Foxn1-transfected U2OS cells. In addition, we showed that PLC-delta1 expression was remarkably decreased in skin of nude mice. In skin and keratinocytes of nude mice as well as PLC-delta1 KO mice, activation of PLC downstream effectors, such as PKC and nuclear factor of activated T cells, was impaired. These results indicate that PLC-delta1 is an essential molecule downstream of Foxn1 in normal hair formation, and strongly suggest that hairlessness in nude mice is caused by insufficient expression of PLC-delta1.

In the avian embryo, the endoderm, which forms a simple flat-sheet structure after gastrulation, is regionally specified in a gradual manner along the antero-posterior and dorso-ventral axes, and eventually differentiates into specific organs with defined morphologies and gene expression profiles. In our study, we carried out transplantation experiments using early chick embryos to elucidate the timing of fate establishment in the endoderm. We showed that at stage 5, posteriorly grafted presumptive foregut endoderm expressed CdxA, a posterior endoderm marker, but not cSox2, an anterior endoderm marker. Conversely, anteriorly grafted presumptive mid-hindgut endoderm expressed cSox2 but not CdxA. At stage 8, posteriorly grafted presumptive foregut endoderm also expressed CdxA and not cSox2, but anteriorly grafted presumptive mid-hindgut endoderm showed no changes in its posterior-specific gene expression pattern. At stage 10, both posteriorly grafted foregut endoderm and anteriorly grafted mid-hindgut endoderm maintain their original gene expression patterns. These results suggest that the regional specification of the endoderm occurs between stages 8 and 10 in the foregut, and between stages 5 and 8 in the mid-hindgut.

The tissue interactions between endodermal epithelium and mesenchyme originated from splanchnic mesoderm are essential during the formation of digestive tract. In this review, we introduce a series of works to elucidate the molecular mechanisms of the epithelial-mesenchymal interaction of stomach development in mainly the chicken embryo. We also describe some molecular studies in mouse stomach development.

During development of the chicken proventriculus (glandular stomach), gut endoderm differentiates into glandular and luminal epithelium. We found that Delta1-expressing cells, undifferentiated cells and Notch-activated cells colocalize within the endodermal epithelium during early gland formation. Inhibition of Notch signaling using Numb or dominant-negative form of Su(H) resulted in a luminal differentiation, while forced activation of Notch signaling promoted the specification of immature glandular cells, but prevented the subsequent differentiation and the invagination of the glands. These results suggest that Delta1-mediated Notch signaling among endodermal cells functions as a binary switch for determination of glandular and luminal fates, and regulates patterned differentiation of glands in the chicken proventriculus.

A genetic switch determines whether the indifferent gonad develops into an ovary or a testis. In adult females of many avian species, the left ovary is functional while the right one regresses. In the embryo, bone morphogenetic proteins (BMP) mediate biological effects in many organ developments but their roles in avian sex determination and gonadal differentiation remains largely unknown. Here, we report the sex-specific and left-right (L-R) asymmetric expression pattern of Bmp7 in the chicken gonadogenesis. Bmp7 was L-R asymmetrically expressed at the beginning of genital ridge formation. After sexual differentiation occurred, sex-specific expression pattern of Bmp7 was observed in the ovary mesenchyme. In addition, ovary-specific Bmp7 expression was reduced in experimentally induced female-to-male reversal using the aromatase inhibitor (AI). These dynamic changes of expression pattern of Bmp7 in the gonad with or without AI treatment suggest that BMP may play roles in determination of L-R asymmetric development and sex-dependent differentiation in the avian gonadogenesis.

In vertebrates, the endoderm is established during gastrulation and gradually becomes regionalized into domains destined for different organs. Here, we present precise fate maps of the gastrulation stage chick endoderm, using a method designed to label cells specifically in the lower layer. We show that the first population of endodermal cells to enter the lower layer contributes only to the midgut and hindgut; the next cells to ingress contribute to the dorsal foregut and followed finally by the presumptive ventral foregut endoderm. Grafting experiments show that some migrating endodermal cells, including the presumptive ventral foregut, ingress from Hensen's node, not directly into the lower layer but rather after migrating some distance within the middle layer. Cell transplantation reveals that cells in the middle layer are already committed to mesoderm or endoderm, whereas cells in the primitive streak are plastic. Based on these results, we present a revised fate map of the locations and movements of prospective definitive endoderm cells during gastrulation.

Determination of the developmental fate in the small intestinal epithelium of the chicken embryo has not been fully analyzed up to the present. This study was carried out to analyze the determination time of the developmental fate of the small intestinal epithelium under the influence of other mesenchymes. The small intestinal epithelium reassociated and cultivated with the proventricular or gizzard mesenchyme or the dermis expressed chicken intestinal fatty acid binding protein, sucrase and CdxA as occurs during the normal development of the small intestinal epithelium. The presumptive intestinal endoderm taken from an earlier stage embryo and associated and cultivated with the proventricular or gizzard mesenchyme, showed gene expression patterns which were the same as those found in normal development. However, when the dermis was associated, the epithelium expressed sonic hedgehog, but never expressed intestinal epithelial- or stomach epithelial-markers. These results indicate that the determination of the developmental fate in the small intestinal epithelium and acquisition of autodifferentiation potency occur at the early stage of the gut development. Moreover the presumptive intestinal endoderm needs the supportive influence of the gut mesenchyme in order to differentiate fully into the intestinal epithelium.

Genes encoding pepsinogens, zymogens of digestive enzyme pepsins, are expressed specifically in the gland epithelial cells of the vertebrate stomach, and their expression is also developmentally regulated, therefore providing a good model for the analysis of transcriptional regulation of genes. In the development of chicken embryonic stomach, the epithelium invaginates into the mesenchyme and forms glands and gland epithelial cells then begin to express embryonic chicken pepsinogen (ECPg) gene. It has been shown that cGATA5 binds directly GATA binding sites located within 1.1-kbp upstream of ECPg gene and activates its transcription. To find more precisely the sequences necessary for ECPg gene transcription, we carried out deletion and mutation analysis with 1.1-kbp upstream region. The results suggest that binding of GATA factor to three GATA binding sites within the upstream region -656 to -419 synergistically regulates ECPg expression in the gland epithelial cells.

The oncogene function in primary epithelial cells is largely unclear. Recombination organ cultures in combination with the stable and transient gene transfer techniques by retrovirus and electroporation, respectively, enable us to transfer oncogenes specifically into primary epithelial cells of the developing avian glandular stomach (proventriculus). In this system, the epithelium and mesenchyme are mutually dependent on each other for their growth and differentiation. We report here that either stable or transient expression of v-src in the epithelium causes budding and migration of epithelial cells into mesenchyme. In response to the transient expression of v-Src or a constitutive active mutant of MEK, we observed immediate downregulation of the Sonic hedgehog gene and subsequent elimination of E-cadherine expression in migrating cells, suggesting the involvement of MAP kinase signaling pathway in these processes. v-src-expressing cells that were retained in the epithelium underwent apoptosis (anoikis) and detached from the culture. Continuous expression of v-src by, for example, Rous sarcoma virus (RSV) was required for the epithelial cells to acquire the ability to express type I collagen and fibronectin genes (mesenchymal markers), and finally to establish the epithelial-mesenchymal transition. These observations would partly explain why RSV does not apparently cause carcinoma formation, but induces sarcomas exclusively.