Transcription of embryonic chick pepsinogen gene is affected by mesenchymal signals through its 5'-flanking region.
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Biomedical subjects
S Yasugi
Publications and source records attributed to S Yasugi.
Spatially and temporally regulated expression of the LIM class homeobox gene Hrlim suggests multiple distinct functions in development of the ascidian, Halocynthia roretzi.
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.
Amino Acid Sequence↗
Expression of the labial group Hox gene HrHox-1 and its alteration induced by retinoic acid in development of the ascidian Halocynthia roretzi.
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 Halocynthia 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.
Amino Acid Sequence↗
The role of gravity in chick embryogenesis.
Thirty fertilized chick eggs preincubated for 0, 7 and 10 days on earth (10 eggs each) were flown in the space shuttle 'Endeavour' and further incubated for 7 days under microgravity. Twenty out of thirty eggs (9/10 ten-day-old; 10/10 seven-day-old; 1/10 zero-day-old) were recovered alive after landing. The only living embryo of the zero-day-old group died 24 days after launch, and was comparable to a 16-day-old embryo. The high mortality of the 0-day-old eggs appeared to be related to the specific inner structure of the egg. Simulation experiments performed on earth indicated that when yolk stayed in the albumen for more than 2 days, most of the embryos died. The subtle difference in specific gravity between the yolk (1.029) and albumen (1.040) plays a critical role in early chick embryogenesis.
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Fetal rat glandular stomach epithelial cells differentiate into surface mucous cells which express cathepsin E in the absence of mesenchymal cells in primary culture.
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.
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Developmental changes of DNA methylation pattern of embryonic chick pepsinogen gene.
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.
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Mesenchymal regulation of epithelial gene expression in developing avian stomach: 5'-flanking region of pepsinogen gene can mediate mesenchymal influence on its expression.
The expression of a gene encoding an embryonic chick pepsinogen was investigated in developing avian gut. Expression is restricted to the epithelial layer of the embryonic proventriculus (glandular stomach). We can therefore regard this gene as a marker gene for proventricular epithelial differentiation. There is some considerable evidence in favour of epithelial-mesenchymal interactions being important during the development of the gastrointestinal system; for example, pepsinogen expression is induced in proventricular and gizzard (muscular stomach) epithelial by the proventricular mesenchyme but is suppressed by the gizzard mesenchyme. In the present paper, we studied how the mesenchymes influence this gene expression pattern. For this we produced constructs containing various portions of the 5'-flanking region of the embryonic chick pepsinogen gene, driving reporter sequences (beta-galactocidase or luciferase), and these constructs were transfected into dissociated epithelial cells either from the proventriculus or gizzard. We then recombined these cells with mesenchymal cells and cultured them as cell aggregates. In this way, we were able to dissect the timing and other requirements of the epithelial-mesenchymal interactions for expression of embryonic chick pepsinogen gene. We also report that 1.1 kb of 5'-flanking sequence is sufficient to drive correct expression of embryonic chick pepsinogen gene, although further enhancement was seen if the constructs contained 3.2 kb of upstream sequence.
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Regulation of pepsinogen gene expression in epithelial cells of vertebrate stomach during development.
Pepsinogens are zymogens of pepsins, aspartic proteases working as digestive enzymes in the vertebrate stomach, of which biological and molecular properties have been extensively studied. In developmental biology, pepsinogens offer excellent molecular markers of differentiation of stomach epithelial cells, since their expression is strictly limited to those cells and there are some isozymes that are expressed in developmental stage-specific manner. It is now well established that the expression of embryonic chicken pepsinogen (ECPg) gene is regulated by epithelial-mesenchymal interactions: it is mesenchyme that determines the expression pattern of ECPg along the digestive tract, by supporting or inhibiting the intrinsically endowed ability of epithelial cells to express it. In the present review article, I will describe recent molecular biological and experimental embryological consequences of our studies on the regulation of ECPg expression by mesenchymal cells, with special attention to the nature of mesenchymal factors and the molecular mechanisms of reactivity of epithelial cells to the mesenchymal influences.
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Tissue- and cell-specific control of guinea pig cathepsin E gene expression.
Northern blotting of RNAs from normal guinea pig tissues revealed that the tissue distribution of cathepsin E mRNA was relatively limited and the highest level of the mRNA was observed in the stomach mucosa. Expression of the mRNA was also observed in the spleen, although the level was very low. These results were in good agreement with the distribution of the cathepsin E-producing cells as revealed by immunohistochemistry. In the separated fractions of dispersed mucosal cells prepared from the stomach by centrifugal elutriation, the extent of cathepsin E mRNA expression was closely correlated with the enrichment of the producing cells. In addition, both CCGG and GCGC sites within the gene region were hypomethylated to a greater extent in the producing tissues than elsewhere, reflecting specific hypomethylation in the producing cells. The observed tissue- and cell-specific transcriptional control of cathepsin E gene, which is correlated with a decreased level of methylation in the gene region, suggests that the enzyme is probably involved in specific functions of particular differentiated cells, especially those of the stomach mucosa.
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Susceptibility of epithelia to directive influences of mesenchymes during organogenesis: uncoupling of morphogenesis and cytodifferentiation.
Morphogenesis and functional cytodifferentiation are two major events in organogenesis, and normally they take place inseparably either in vivo or in vitro conditions. In this article, we reviewed a series of our recent results on mesenchymal-epithelial interactions in organogenesis of digestive organs, urogenital organs and the skin of avian and mammalian embryos, giving special attention to the importance of the responses of epithelia to the directive influences of mesoderms and also to the uncoupling of morphogenesis and cytodifferentiation, which has often been observed during the course of these studies.
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Isolation and structural analysis of embryonic chicken pepsinogen gene: avian homologue of prochymosin gene.
Embryonic chicken pepsinogen gene was isolated from a chicken genomic library. This gene occupied approximately 3.5 kb of the genomic DNA and was separated into nine exons by eight introns. The positions of exon-intron junctions coincided with those in the human pepsinogen A gene and the bovine prochymosin gene. Southern blot analysis of chicken genomic DNA revealed that the structure of the isolated gene reflects the original structure in the chicken chromosome. At the same time, the presence of another copy of embryonic pepsinogen gene was suggested. 5'-flanking region of the isolated ECPg gene was analyzed.
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Presence of pepsinogens immunoreactive to anti-embryonic chicken pepsinogen antiserum in fish stomachs: possible ancestor molecules of chymosin of higher vertebrates.
1. Stomachs of adult teleosts and elasmobranchs reacted to an anti-embryonic chicken pepsinogen antiserum (anti-ECPg) as well as to an anti-adult chicken pepsinogen antiserum (anti-ACPg). 2. Zymograms and immunoblots of stomach extracts revealed that anti-ECPg- and anti-ACPg-reactive substances possess peptic activity. 3. The possible relationship between anti-ECPg-reactive pepsinogens in fish and prochymosins in higher vertebrates is discussed.
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Molecular cloning and the nucleotide sequence of cDNA for embryonic chicken pepsinogen: phylogenetic relationship with prochymosin.
Embryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a cDNA clone by screening a lambda gt11 library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this cDNA clone hybridized to a mRNA of 1,600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequencing longer cDNAs obtained by screening the same library with the 200-base pair cDNA and primer extension with a synthetic primer. The cDNA consisted of 1,281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other.
Amino Acid Sequence↗
Pepsinogen gene transcription induced in heterologous epithelial-mesenchymal recombinations of chicken endoderms and glandular stomach mesenchyme.
Proventricular (glandular stomach) mesenchyme of chicken embryos can induce endoderms of some parts of embryonic digestive tract to produce embryonic chicken pepsinogen (ECPg), a marker protein for the differentiation of embryonic proventricular epithelium. In the present study, we investigated the production of ECPg mRNA in the course of epithelial-mesenchymal interactions between endoderms of digestive tract and proventricular mesenchyme. ECPg mRNA was detected by Northern hybridization with ECPg cDNA as a probe. In normal development of the proventriculus, ECPg mRNA was first detected at day 7 of incubation, and it ceased to be produced by day 21. Embryonic esophagus, gizzard and small intestine did not contain ECPg mRNA. When 6-day esophagus, gizzard or proventricular endoderm was associated and cultured with 6-day proventricular mesenchyme, the recombinates formed proventricular-gland-like complex glands and produced ECPg mRNA in almost equal quantity. However, 6-day small intestinal or 3.5-day allantoic endoderm did not produce pepsinogen mRNA under the same conditions, though the recombinates formed complex glands. These results indicate that the proventricular mesenchyme can induce de novo transcription of ECPg gene in esophagus, proventricular and gizzard endoderms, and that ECPg gene in small intestinal and allantoic endoderms fails to react to the inducing signal.
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Pepsinogen-like immunoreactivity among vertebrates: occurrence of common antigenicity to an anti-chicken pepsinogen antiserum in stomach gland cells of vertebrates.
Stomachs of 14 species selected from five classes of vertebrate were surveyed concerning the reactivity to an anti-adult chicken pepsinogen antiserum (anti-ACPg) with indirect immunofluorescence method. Gland cells of all these stomachs showed reactivity to the antiserum. Crude extract of stomachs from five representatives of mammals, birds, amphibians and fish showed peptic activity (at pH 2.2) of which 70-90% were pepstatin-sensitive. Zymogram and immunoblotting of crude extract revealed that the anti-ACPg-reactive proteins have peptic activity. Molecular weights of anti-ACPg-reactive proteins determined by immunoblotting coincided with the values of purified pepsinogens previously reported for these animals. These results indicate that pepsinogens have been conserved well during vertebrate evolution.
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[The course of pepsinogens during growth in mice].
In the mouse stomach exist two acid proteases separable on the polyacrylamide gel electrophoresis. One of them increases in activity after weaning and is a pepsinogen immunologically related to the pepsinogens of other vertebrates. The other is predominant before weaning and is presumed to be a chymosin-like acid protease.
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[Gland formation and pepsinogen expression in the gizzard endoderm as affected by the proventricular mesenchyme in the chick embryo].
Studies of epithelio-mesenchymal recombination between the proventriculus and gizzard of 6-day chick embryos revealed that the proventricular mesenchyme can induce the morphogenesis of complex glands and cause the expression of embryonic pepsinogen in the gizzard endoderm, while the gizzard mesenchyme inhibits both of them.
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Differentiation of allantoic endoderm implanted into the presumptive digestive area in avian embryos. A study with organ-specific antigens.
Quail allantoic endoderm was implanted into the presumptive digestive-tract area of chick embryos, and the differentiation of the endoderm was examined morphologically and immunocytochemically with antisera against pepsinogens and sucrase. The allantoic endoderm was incorporated into the host digestive organs. It often became continuous with the host endoderm and formed a chimaeric digestive-tract epithelium. It differentiated morphologically into the epithelium of the digestive organ into which it was incorporated, showing the morphological inductive ability in situ of the digestive-tract mesenchyme against the allantoic endoderm. However, the allantoic endoderm did not produce pepsinogens even when it was incorporated into the host proventricular mesenchyme and formed well-developed proventricular glands. This result indicates that the heterotypic morphogenesis of the allantoic endoderm is not necessarily accompanied by the heterotypic cytodifferentiation. In contrast, the anti-sucrase antiserum-reactive cells often differentiated in the allantoic endoderm incorporated into not only the intestine but also other organs. This confirmed our previous observation that the allantoic endoderm has a tendency to differentiate into the intestinal epithelium in the heterologous environment.
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