Cytochemical characterization of mucosubstances in the chick glandular stomach during embryonary and postnatal development

The chick stomach is composed of two parts: glandular or proventriculus and muscular or gizzard which are morphological and functionally different. Since characterization of the mucosubstances in the adult stomach and its comparison with the embryonary stomach have not been made, we performed the study of the cytochemical characteristics of mucosubstances in the chick glandular stomach during the embryonic and post-natal periods, to obtain information on changes produced in these components during functional differentiation. In this work we established that during development, the epithelial cells of the superficial layer content predominantly glycoproteins and the glycosaminoglycans of the glands decrease when they begin to secrete other compounds, such as proteolytic enzymes necessary for digestion. This sequence of mucosubstances appearance is concordant with the increase of carbonic anhydrase, which reaches its highest specific activity from 15 to 20 days of incubation. In this period hydrochloric acid secretion increases and therefore, glycoprotein secretion becomes necessary to protect the mucous membranes.     

Gizzard epithelium of chick embryos can express embryonic pepsinogen antigen,a marker protein of proventriculus

Summary The avian stomach is composed of two distinct organs, the proventriculus and the gizzard. Pepsinogen expression in the proventricular and gizzard epithelia of chick embryos was investigated immunohistochemically with anti-embryonic chick pepsinogen (anti-ECPg) antiserum. In normal development, the ECPg antigen was expressed only in the glandular epithelial cells of the embryonic proventriculus from the 8th day of incubation onwards. However, both proventricular and gizzard epithelia of 6-day embryos expressed the ECPg antigen when recombined and cultured with the proventricular mesenchyme. Chronological studies revealed that the ECPg antigen was first detected in a few epithelial cells at 3 days of cultivation. The percentage of ECPg-positive cells among the total epithelial cells in each recombinant increased with the length of the culture period and all the glandular epithelial cells were positive at 9 days. During this process, the percentage of ECPg-positive cells in each cultured recombinant was similar in proventricular and gizzard epithelia. Moreover, both epithelia could express the ECPg antigen when recombined and cultured with the oesophageal or small-intestine mesenchyme for 9 days, though the percentage of ECPg-positive cells in each cultured recombinant was much lower than that in the cultured recombinant with the proventricular mesenchyme. These results indicate that the gizzard epithelium of 6-day chick embryos possesses a similar potential for pepsinogen expression as the proventricular epithelium of the same age.     

Involvement of the signal transduction pathway mediated by epidermal growth factor receptor in the differentiation of chicken glandular stomach

During the development of the chicken proventriculus (glandular stomach), the initially undifferentiated epithelium differentiates into two distinct cell populations: the glandular epithelium, cells of which secrete embryonic chicken pepsinogen (ECPg), and luminal epithelial cells, which express the chicken spasmolytic polypeptide gene (cSP). Based on knowledge of the adult mouse stomach, the ligands of epidermal growth factor (EGF) receptor (EGFR) were expected to affect differentiation of the proventricular epithelium. When EGF was added to the medium in which proventriculi were cultured in vitro, gland formation was suppressed in a dose-dependent manner and the amount of ECPg mRNA decreased, whereas morphological differentiation of luminal epithelium was stimulated. Simultaneous treatment of the proventriculus with EGF and tyrphostin 47 resulted in the attenuation of the effect of EGF, suggesting that EGF, or other ligands of EGFR, may actually be involved in the normal course of development of the proventricular epithelium.     

Pepsinogen Induction in Chick Stomach Epithelia by Reaggregated Proventricular Mesenchymal Cells In Vitro

In vitro organ culture system which permits embryonic chick proventriculus (glandular stomach) to synthesize pepsinogen de novo was developed. Explants of the proventricular rudiment were cultured on Millipore filters in Medium 199 with Earle's salts supplemented with 50% 12-day embryo extract at 38°C in 95% air and 5% CO₂.
In these culture conditions, pepsinogen, a functional marker protein of proventriculus, was first detected after 3 days of cultivation of 6-day chick proventricular rudiment. When recombined and cultured with 6-day proventricular mesenchyme, 6-day oesophageal, proventricular or gizzard (muscular stomach) epithelium expressed pepsinogen while small intestinal epithelium did not. These results were consistent with the previous results obtained by chorioallantoic membrane (CAM) grafting, and showed that the culture conditions are permissive for pepsinogen expression.
When recombined and cultured with reaggregated mesenchymal cells isolated from 6-day proventricular mesenchymal fragments, both 6-day proventricular and gizzard epithelia formed glandular structure and expressed pepsinogen. This indicates that the proventricular mesenchymal cells retain the ability to induce morphogenesis and cytodifferentiation of the proventricular epithelium even if the normal organization of proventricular mesenchyme is once destroyed.     

Tissue interaction regulates expression of a spasmolytic polypeptide gene in chicken stomach epithelium

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 luminal 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.     

Notch signaling functions as a binary switch for the determination of glandular and luminal fates of endodermal epithelium during chicken 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.     

Down-regulation of endodermal Shh is required for gland formation in chicken stomach

During the development of the proventriculus (glandular stomach) of the chicken embryo, the endodermal epithelium invades into the surrounding mesenchyme and forms glands. The glandular epithelial cells produce pepsinogen, while the non-glandular (luminal) epithelial cells secrete mucus. Sonic hedgehog is expressed uniformly in the proventricular epithelium before gland formation, but its expression ceases in gland cells. Here we present evidence that down-regulation of Sonic hedgehog is necessary for gland formation in the epithelium using a specific inhibitor of Sonic hedgehog signaling and virus mediated overexpression of Sonic hedgehog. We also show that gland formation is not induced by down-regulation of Sonic hedgehog alone; a mesenchymal influence is also required.     

In vitro analysis of mesenchymal influences on the differentiation of stomach epithelial cells of the chicken embryo

It is well established that epithelial-mesenchymal interactions play important roles in the differentiation of stomach epithelial cells in the chicken embryo. To analyze mesenchymal influences on the differentiation of the epithelial cells, we developed a tissue culture system for stomach (proventriculus and gizzard) epithelia of chicken embryo, and examined their differentiation in the presence or absence of mesenchyme. Stomach epithelium from 6-day chicken embryo did not express embryonic chicken pepsinogen (ECPg), a marker molecule of glandular epithelial cells of proventriculus, while it expressed marker molecules of epithelial cells of the luminal surface of stomach, when cultured alone on the Millipore filter, covered with the gel consisting of extracellular matrix components. When the epithelium was recombined with mesenchyme separated by the filter, differentiation of the epithelium was affected by the recombined mesenchyme. Proventricular and lung mesenchymes induced the expression of ECPg in epithelial cells, and the expression was extensive when the gel contained basement membrane components. Proventricular and gizzard epithelia showed different responses to the mesenchymal action. We tested the effects of some growth factors on the differentiation of epithelial cells using this culture system. Furthermore we devised a "conditioned semi-solid medium experiment" for analysis of the inductive properties of proventricular and lung mesenchymes. The results of this experiment clearly demonstrated for the first time that diffusible factors from mesenchyme induce the differentiation of glandular epithelial cells in the absence of mesenchymal cells.     

Chronological analysis of the intestinalization of chick stomach endoderm induced in vitro by duodenal mesenchyme

Summary Inductive action of duodenal mesenchyme on stomach endoderm in the chick embryo was chronologically analysed in vitro by the use of electron microscopy and immunofluorescence techniques. The behaviour of the endoderm-mesenchyme interfaces was particularly studied during the induction. In recombinates of 4-day stomach endoderm and 6-day duodenal mesenchyme, all the endodermal cells were undifferentiated at the start of cultivation. Small-intestinal sucrase antigen could first be detected on the 5th day of cultivation in one-third of the stomach endoderm, and a striated border on the 7th day. With a longer cultivation period, intestine-type cells increased in number in the stomach endoderm and the density of microvilli on the apical surface became higher. At the endoderm-mesenchyme interfaces a number of direct contacts between endodermal and mesenchymal cells were observed from the beginning to the end of cultivation. These were especially abundant in the early period before the appearance of signs of intestinal cytodifferentiation. These results suggest that the mesenchymal cells adjacent to the endodermal tissue play an important role in the intestinal induction which occurs during the early period of cultivation, probably via direct cell-to-cell contracts.     

The development of the guinea pig gallbladder epithelial cells. I. Light microscopical and carbohydrate-histochemical investigations (author's transl)]

The development of the guinea pig gallbladder epithelium was studied from the 19th day of intrauterine life to the 31st postnatal day by means of histological and histochemical staining reactions. At first, the epithelium is a columnar pseudostratified one. Its transformation into a simple columnar eptihelium is terminated by the 31st day of the intrauterine life. Then the epithelial cells become more columnar and their nuclei acquire a basal position. Somewhat later the epithelium invaginates the underlying mesenchyme. Up to the 57th day the epithelium contains much glycogen. Neutral and carboxylated mucosubstances are demonstrable after the 30th day. From the 48th day onwards sulphated mucosubstances can be visualized in some cells in the depth of the invaginations and from the 51st day in the epithelial cells of the gallbladder. "Light" mucoid cells appear first in the epithelium of day 58. After the 6th postnatal day the "light" cells are rarely seen in the invaginations. The development of the gallbladder epithelium is completed about the 10th postnatal day. The epithelial mucosubstances of the gallbladder of the adult animal could be classified as GC- mucins and S-mucinsA, 1.0 MgCl2.     

BMPs are necessary for stomach gland formation in the chicken embryo: a study using virally induced BMP-2 and Noggin expression

Epithelial-mesenchymal interactions are necessary for the normal development of various digestive organs. In chicken proventriculus (glandular stomach), morphogenesis and differentiation of the epithelium depend upon the inductive signals coming from underlying mesenchyme. However, the nature of such signals is still unclear despite extensive analyses carried out using experimental tissue recombinations. In this study we have examined the possible involvement of bone morphogenetic proteins (BMPs) in the formation of stomach glands in the chicken embryo. Analysis of the expression patterns of BMP-2, -4 and -7 showed that these BMPs were present in the proventricular mesenchyme prior to the initiation of the proventricular gland formation. BMP-2 expression, in particular, was restricted to the proventriculus among anterior digestive organs. Virus-mediated BMP-2 overexpression resulted in an increase in the number of glands formed. Moreover, ectopic expression of Noggin, which antagonizes the effect of BMPs, in the proventricular mesenchyme or epithelium, led to the complete inhibition of gland formation, indicating that BMP signals are necessary for the proventricular gland formation. These findings suggest that BMPs are of prime importance as mesenchymal signals for inducing proventricular glands.     

Apoptosis during chick inner ear development: some observations by TEM and TUNEL techniques

In order to clarify the occurrence, distribution and possible role of apoptosis during inner ear development, the ultrastructural aspects (by TEM) (at 9-19 incubation day and 1 day after hatching) and the distribution of the apoptotic phenomenon (by the TdT-mediated dUTP nick end-labeling technique), were studied in the crista ampullaris of chick embryo at 5-19 days of incubation to hatching and of postnatal 1-day old chick. We found, in the sensorial epithelium, dark supporting cells in chick embryos and mainly dark hair cells in postnatal chicks, both with ultrastructural features consistent with those of apoptosis. The presence of apoptotic phenomena was confirmed by the TUNEL technique. According to our findings, it is hypothesized that apoptosis in the inner ear may be involved: 1) at first, in macroscopic remodelling of the membranous labyrinth in early developmental stages, 2) later, in the correct differentiation of the hair and of the supporting cells, leading to characteristic cellular pattern formation and 3) finally, in physiological cell turnover of the postnatal chicken sensorial epithelium of the crista.     

扬子鳄胚胎背腺的发生及退化

本文在１４例扬子鳄Ａｌｌｉｇａｔｏｒｓｉｎｅｎｓｉｓ胚胎中观察了背腺的发生及退化过程。孵化第２８天,背中线左右两侧第二行鳞片处的表皮内陷形成实心的背腺腺芽;第３８天,背腺腺泡明显,腺上皮为复层上皮;从第４６天开始,腺上皮出现明显的退化,大量增殖的腺管上皮细胞逐渐堵塞腺管及腺孔。扫描电镜观察表明,孵化第３２—３６天的胚胎背部第二行鳞的各列鳞片表面均有背腺腺孔,以后逐渐出现少数不规则的退化,孵化第５８天以后,绝大多数背腺腺孔消失。对扬子鳄背腺的发生及退化现象作了讨论。     

Cell Proliferation during Bud Formation of the Quail Uropygial Gland

A localized increase of labeling index (L.I.) was observed in bud-forming epithelium of the uropygial gland of the Japanese quail embryo through computer-aided analysis. At day 10 glandular buds are not yet formed anywhere in the epithelium and the distribution of proliferative activity in the epithelium is not differential. On the following day the peripheral buds are being formed at the externo-lateral end of the basal luminal epithelium, L.I. increases locally in these buds. At day 12 a localized increase of L.I. takes place also at the median end; extremely high labeling indices are seen at the tip of the peripheral buds and developing central buds.     

FGF10 is required for cell proliferation and gland formation in the stomach epithelium of the chicken embryo

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.     

Expression and function of Wnt5a in the development of the glandular stomach in the chicken 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.     

Cytodifferentiation of the adenohypophysis of the domestic fowl

Summary In the chick embryo the first membrane-bound secretory granules occur in the cytoplasm of occasional cells in the cephalic lobe of pars distalis at the 7th day of incubation. On the 8th day most of the cells in both the cephalic and caudal lobes contain secretory granules that are variable in size, form and density.On the 9th day at least two types of glandular cells are distinguishable in the cephalic and in the caudal lobes; however, these cells are not comparable with those of the adult gland. Differentiation of acidophils and basophils occurs, apparently simultaneously, in 11-day embryos.The cells of the cephalic and caudal lobes are morphologically distinct from their first appearance. Thus it is concluded that these two lobes develop independently and differently from an early stage of ontogenesis.The secretory granules are formed in the Golgi area of the hypophysial cells after the 8th day of incubation. However, secretory material may be synthesized also by a process not involving the Golgi apparatus.Nerve fibers containing granules first appear in the superficial layer of the median eminence on the 8th embryonic day and by the 12th day three types of granules and two types of clear vesicles are identifiable.The investigation reported herein was supported by grant from Japan-U.S. Cooperative Science Program of Japan Association for Science Promotion to Professor Mikami and by U.S.-Japan Cooperative Science Program Grant No. GF-33334 to Professor Farner.     

Localization of DNA-synthesizing cells and cell proliferation pattern in developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens

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.     

Chicken keratin-19: Cloning of cDNA and analysis of expression in the chicken embryonic gut

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.