Molecular cloning of pepsinogens A and C from adult newt (Cynops pyrrhogaster) stomach.

The full-length cDNAs of three pepsinogens (Pgs) were cloned from the stomach of newt, Cynops pyrrhogaster, and nucleotide sequences of the full-length cDNAs were determined. Molecular phylogenetic analysis showed that two Pgs, named PgC1 and PgC2, belong to the pepsinogen C group, and one Pg, named PgA, belongs to the pepsinogen A group. The sequences contain an open reading frame (ORF) encoding 385 amino acid residues for PgC1, 383 amino acid residues for PgC2 and 377 amino acid residues for PgA. In addition, all of the three amino acid sequences conserve some unique characteristics such as six cysteine residues and putative active site two aspartic acid residues. All of the pepsinogen mRNAs were detected in the stomach by RT-PCR but not in other organs. Although a slight difference at the time of the start of expression was seen among the three pepsinogen genes, all of them were expressed in the larval stage after hatching. This is the first report on cloning of pepsinogens from urodele stomach.

Retinoic acid metabolizing factor xCyp26c is specifically expressed in neuroectoderm and regulates anterior neural patterning in Xenopus laevis.

Anterior-posterior neural patterning is determined during gastrulation when head structure is induced. Induction of anterior neural structures requires inhibition of Wnt signaling by several Wnt antagonists. We performed microarray analysis to isolate genes regulated by canonical Wnt signaling and abundantly expressed in the anterior neuroectoderm at the early neurula stage. We identified xCyp26c, a Cyp26 (RA-metabolizing protein)-family gene. In situ hybridization showed xCyp26c expression restricted to the anterior region of neurula, while xCyp26a was expressed in both anterior and posterior regions. At the tadpole stage, xCyp26c was also expressed in restricted sets of cranial nerves. Microarray, RT-PCR and in situ hybridization analyses revealed decreased xCyp26c expression with overexpression of beta-catenin, suggesting regulation by Wnt/beta-catenin signaling. We also assessed the effects of retinoic acid (RA) on xCyp26c expression. Embryos treated with 10(-7) M RA showed an anterior shift in the spatial expression of xCyp26c, reflecting a posteriorization effect. Conversely, expression patterns in embryos treated with more than 10(-6) M RA were less affected and remained restricted to the most anterior region. Moreover, injection of xCyp26c mRNA into animal poles caused head defects, and exogenous expression of xCyp26c rescued the posteriorizing effect of RA treatment. Taken together, these results implicated a role for xCyp26c in anterior patterning via RA signaling.

Global expression of simulated microgravity-responsive genes in Xenopus liver cells.

The random positioning machine (RPM) is a method used to generate a simulated-microgravity environment at approximately 0 g. Using an RPM, we analyzed the global gene expression of A8 cells derived from the liver of adult Xenopus laevis. A range of genes on a Xenopus 44K-scale microarray were up- or downregulated two-fold or more: 43 genes (up, 36 genes; down, 7 genes) on culture day 5 in RPM, 74 genes (up, 48 genes; down, 26 genes) on day 8, 105 genes (up, 71 genes; down, 34 genes) on day 10, and 132 genes (up, 98 genes; down, 34 genes) on day 15. Five genes were upregulated two-fold or more throughout culturing in RPM, while only one gene was downregulated over the entire time. We then compared the expression patterns of the RPM-dependent genes in the A8 cells with those in A6 cells established from the kidney of adult Xenopus laevis. Six upregulated genes and three downregulated genes showed the same expression patterns throughout the culturing of A6 and A8 cells in RPM. Such globally responsive genes may play a common role in the cell response to simulated microgravity. We were particularly interested in the downregulation of SPARC in both cell types in RPM, which supported previous observations from simulated-microgravity experiments on earth or microgravity in space. We conclude that SPARC is plays a key role in the response of a cell to microgravity.

Cloning and expression of xP1-L, a new marker gene for larval surface mucous cells of tadpole stomach in Xenopus laevis.

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H(+)/K(+)-ATPase beta subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.

Xenopus galectin-VIa shows highly specific expression in cement glands and is regulated by canonical Wnt signaling.

Anterior-posterior neural patterning of Xenopus embryo is determined during gastrulation and then followed by differentiation of neural structures including brain and eye. The cement gland is a mucus-secreting neural organ located in the anterior end of the neural plate. This study analyzed expression patterns of Xenopus galectin-VIa (Xgalectin-VIa) by whole-mount in situ hybridization, and found highly restricted expression of this gene in the cement gland region. These patterns were similar to those of XAG-1 and XCG, known cement gland-specific genes. In addition, Xgalectin-VIa was expressed in the dorsal edge of eye vesicles, the otic vesicle, and in part of the hatching gland at the tadpole stage. Although the spatial expression pattern was similar, the temporal expression of Xgalectin-VIa differed from that of XAG-1 and XCG. RT-PCR analysis showed only weak Xgalectin-VIa expression in early neurula embryos, whereas both XAG-1 and CGS were strongly expressed at that stage. We also showed that Xgalectin-VIa expression is repressed by enhancement of Wnt signaling and increased by its inhibition. Furthermore, Xgalectin-VIa expression was activated by neural-gene inducer Xotx2, as is the case for XAG-1 and CGS. Together, these results indicated that Xgalectin-VIa possesses different features from other cement gland genes and is a novel and useful marker of the cement gland in developing embryos.

Thyroid hormone-induced expression of a bZip-containing transcription factor activates epithelial cell proliferation during Xenopus larval-to-adult intestinal remodeling.

In the intestine during amphibian metamorphosis, stem cells appear, actively proliferate, and differentiate into an adult epithelium analogous to the mammalian counterpart. To clarify the molecular mechanisms regulating this process, we focused on a bZip-containing transcription factor (TH/bZip). We previously isolated TH/bZip from the Xenopus intestine as one of the candidate genes involved in adult epithelial development. Northern blot and in situ hybridization analyses showed that the transient and region-dependent expression of TH/bZip mRNA correlates well with the growth of adult epithelial primordia originating from the stem cells throughout the Xenopus intestine. To investigate its role in the adult epithelial development, we established an in vitro gene transfer system by using electroporation and organ culture techniques, and we overexpressed TH/bZip in the epithelium of Xenopus tadpole intestines. In the presence of thyroid hormone (TH) where the adult epithelial primordia appeared after 3 days of cultivation, overexpression of TH/bZip significantly increased their proliferating activity. On the other hand, in the absence of TH where the epithelium remained as larval-type without any metamorphic changes, ectopic expression of TH/bZip significantly increased the proliferating activity of the larval epithelium but had no effects on its differentiated state. These results indicate that TH/bZip functions as a growth activator during amphibian intestinal remodeling, although TH/bZip expression in the epithelium alone is not sufficient for inducing the stem cells.

Expression of CCAAT/enhancer binding protein delta is closely associated with degeneration of surface mucous cells of larval stomach during the metamorphosis of Xenopus laevis.

CCAAT/enhancer binding protein delta (C/EBP delta) is one of the transcription factors that have a basic-leucine zipper domain. In mammals, it has been suggested that this transcription factor plays a role in differentiation of adipocytes or in apoptosis of mammary gland epithelial cells. The factor also plays a role in acute-phase response in injury, infection and inflammation. We cloned Xenopus homologues of the C/EBP delta gene from metamorphosing stomach by subtractive hybridization and analyzed spatio-temporal expression pattern of the homologues. Two isoforms of C/EBP delta were isolated and named C/EBP delta-1 and -2. Their deduced amino acid sequences were highly similar to each other (identity, 91.2%). Expression of the C/EBP delta mRNAs in the stomach transiently increased during its metamorphosis-associated remodeling, and the transient up-regulation was also found in thyroid hormone-induced metamorphosis. The C/EBP delta mRNAs were exclusively localized in degenerating larval surface mucous cells, not in newly proliferating and differentiating adult-type epithelial cells. The result suggests a possibility that Xenopus C/EBP delta plays a role in apoptotic cell death of larval-type epithelium during the stomach remodeling.

Stomach remodeling-associated changes of H+/K+-ATPase beta subunit expression in Xenopus laevis and H+/K+-ATPase-dependent acid secretion in tadpole stomach.

Through subtractive hybridization, H+/K+-ATPase beta subunit mRNA, highly expressed in the larval stomach of Xenopus laevis, was isolated. In situ hybridization demonstrated that the H+/K+-ATPase beta subunit mRNA was exclusively expressed in manicotto gland cells of the larval stomach, not in any other cell. Northern blot analysis showed that metamorphosis-associated changes of the H+/K+-ATPase beta subunit mRNA expression in the stomach were characterized by high expression in tadpoles, a considerably lower expression in metamorphosing tadpoles, and a re-increase of expression in froglets. Further in situ hybridization showed that the decrease of expression correlated with the degeneration of larval type epithelium in the manicotto gland, while the re-increase correlated with the differentiation of oxynticopeptic cells of the adult type stomach. Moreover, the H+/K+-ATPase beta subunit mRNA was expressed in adult epithelial primordia. Such changes were found in thyroid hormone-induced precocious metamorphosis. Based on studies using this ATPase as well as xP1 and PgC (pepsinogen C) as molecular markers, this study discusses a probable cell lineage involved in metamorphosis-associated stomach remodeling. The pH of luminal contents of the larval stomach was found to be lower than 2. In addition, the pH of an isolated stomach changed from 7.2 to lower than 4 after incubation in Ringer's solution, suggesting acid production from the larval stomach. This is the first demonstration of the H+/K+-ATPase-mediated acid production and secretion in the larval stomach of Xenopus laevis.

Differential expression of two cathepsin Es during metamorphosis-associated remodeling of the larval to adult type epithelium in Xenopus stomach.

Cathepsin E (CE) was purified from the foregut of Xenopus laevis tadpoles as a mature dimeric form. The purified enzyme was a typical CE among aspartic proteinases with respect to pH dependence of proteolytic activity, susceptibility to pepstatin, and having N-linked high-mannose type oligosaccharide chains. We isolated two cDNAs for the CE (CE1 and CE2) from adult stomach. The amino acid sequence of the N-terminal region of the purified CE coincided with the corresponding sequence predicted from CE1. Northern blot analysis and in situ hybridization were performed. The CE1 mRNA was highly expressed in surface mucous cells and gland cells constituting the larval epithelium of the foregut of pro-metamorphic tadpoles. As metamorphosis began and progressed, CE1 mRNA drastically decreased in amount, and subsequently both CE1 and CE2 mRNAs gradually increased. The increase in CE2 mRNA was detected shortly after the increase in CE1 mRNA. The decrease in CE1 expression correlated with degeneration of the larval type epithelium, while the increases in both CE1 and CE2 expression correlated with formation of the adult type epithelium. Thus, cathepsin E gene expression was differentially regulated during metamorphosis-associated remodeling of the larval to adult type epithelium in stomach.

Molecular cloning of preprocathepsin E cDNA from the stomach of bullfrog Rana catesbeiana.

A cDNA library was constructed from a poly(A)(+) RNA fraction of the gastric mucosa of bullfrog Rana catesbeiana. We cloned a cDNA encoding preprocathepsin E (Pre-Pro-CE) from the library. The present study is the first demonstration of the Pre-Pro-CE cDNA of lower vertebrate such as amphibian. Amino acid sequence deduced from the cDNA was compared with partial amino acid sequence determined by Edman degradation, suggesting that the cDNA comprises an open reading frame encoding a signal peptide (16 amino acids), a pro-sequence (33 amino acids) and a mature protein region (348 amino acids). Two consensus tri-peptide sequences (FDT and VDT) as active site and positions of seven cysteine residues were conserved in this amphibian CE. Although the bullfrog CE was deduced to contain one potential N-linked glycosylation site, its position (Asn(139)-Leu(140)-Thr(141)) was different from that of mammalian CEs. Molecular phylogenetic analysis showed that the bullfrog Pro-CE belongs to the typical Pro-CE group among various aspartic proteinases.