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.     

Developmental changes in the pattern of larval beta-globin gene expression in Xenopus laevis. Identification of two early larval beta-globin mRNA sequences

We have analysed beta-globin mRNA sequences in total RNA extracted from embryos and tadpoles of Xenopus laevis at different stages of development and we have identified the most abundantly transcribed beta-globin mRNA (beta T1). The entire nucleotide sequence of a cDNA clone corresponding to this mRNA is known. We have now identified the gene corresponding to this mRNA and we have determined the nucleotide sequences of its immediate 5'-flanking region. Using a DNA fragment from within the coding region of the cloned beta T1 cDNA we show, by primer extension analysis, that beta T1 mRNA is first detectable at stage 28-32 of development. This is the time at which the first presumptive erythropoietic tissue, the ventral blood island, becomes observable histologically. We show that two minor beta-globin genes, distinct from beta T1, are expressed during early stages of development, and that their expression ceases shortly after the beginning of the feeding stage. We term these two early larval genes beta E1 and beta E2. A third minor beta-globin gene is expressed during early development but, unlike beta E1 and beta E2, it is also expressed throughout subsequent larval development. We term this gene beta T2 and show that it corresponds to a gene previously termed beta LII. Finally, using a primer derived from the major adult beta-globin gene (beta 1), we have analysed the accumulation of the major adult beta-globin mRNA during larval development, and we show that this sequence does not accumulate to any significant level before metamorphosis.     

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.     

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 β 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.     

鳜胰岛素样生长因子-ⅠcDNA全长克隆及组织表达分析

采用RT-PCR、cDNA末端快速扩增法(RACE)等技术克隆了鳜(Siniperca chuatsi)肝组织胰岛素样生长因子-I(IGF-I)cDNA全长序列.结果表明,鳜IGF-I cDNA全长1 784 bp,包括5'端非翻译区233bp,3'端非翻译区990 bp和开放阅读框561 bp,共编码186个氨基酸;...     

Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles

The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.     

How does a tadpole know when to metamorphose? A theory linking environmental and hormonal cues

Tadpoles are unusual among free-living amphibians in having an atonic, non-acid secreting, underdeveloped stomach. Morphologically the typical tadpole foregut is most similar to the flaccid, non-acid secreting stomach of adult female of the gastric-brooding frog, Rheobatrachus, during brooding. In Rheobatrachus the brooding condition is induced by prostaglandin E2 secreted from the mouths of brooded larvae. I propose that typical, free-living tadpoles also excrete prostaglandins of the E family in their oral mucus and that these compounds are naturally swallowed with food particles by the tadpoles. According to this hypothesis, when food is abundant larvae swallow a large amount of mucus and, consequently, a lot of hormone, which retards differentiation of the adult, acid secreting, peristaltic stomach. However, when food is less abundant less food and mucus is swallowed. In this situation less prostaglandin passes down the alimentary tract and the gut proceeds to differentiate. If this theory is correct it provides a direct link between an environmental factor--the availability of food--and an endocrinological factor affecting metamorphosis. The theory is consistent with our current understanding of the endocrinology of metamorphosis, as well as the evolution of direct-development in anurans.     

Expression of intermediate filament proteins during development of Xenopus laevis. III. Identification of mRNAs encoding cytokeratins typical of complex epithelia

A Xenopus laevis mRNA encoding a cytokeratin of the basic (type II) subfamily that is expressed in postgastrulation embryos was cDNA-cloned and sequenced. Comparison of the deduced amino acid sequence of this polypeptide (513 residues, calculated mol. wt 55,454; Mr approximately 58,000 on SDS-PAGE) with those of other cytokeratins revealed its relationship to certain type II cytokeratins of the same and other species, but also remarkable differences. Using a subclone representing the 3'-untranslated portion of the 2.4 kb mRNA encoding this cytokeratin, designated XenCK55(5/6), in Northern blot experiments, we found that it differs from the only other Xenopus type II cytokeratin known, i.e. the simple epithelium-type component XenCK1(8), in that it is absent in unfertilized eggs and pregastrulation embryos. XenCK55(5/6) mRNA was first detected at gastrulation (stage 11) and found to rapidly increase during neurulation and further development. It was also identified in Xenopus laevis cultured kidney epithelial cells of the line A6 and in the adult animal where it is a major polypeptide in the oesophageal mucosa but absent in most other tissues examined. The pattern of XenCK55(5/6) expression during embryonic development was similar to that reported for the type I polypeptides of the 'XK81 subfamily' previously reported to be embryo-specific and absent in adult tissues. Therefore, we used a XK81 mRNA probe representing the 3'-untranslated region in Northern blots, S1 nuclease and hybrid-selection-translation assays and found the approximately 1.6 kb XK81 mRNA and the resulting protein of Mr approximately 48,000 not only in postgastrula embryos and tadpoles but also in the oesophagus of adult animals. Our results show that both these type II and type I cytokeratins are synthesized only on gastrulation and are very actively produced in early developmental stages but is continued in at least one epithelium of the adult organism. These observations raise doubts on the occurrence of Xenopus cytokeratins that are strictly specific for certain embryonic or larval stages and absent in the adult. They rather suggest that embryonically expressed cytokeratins are also produced in some adult tissues, although in a restricted pattern of tissue and cell type distribution.     

Cloning,partial purification and in vivo developmental profile of expression of the juvenile hormone epoxide hydrolase of Ctenocephalides felis

cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.     

Xenopus laevis serum albumin: sequence of the complementary deoxyribonucleic acids encoding the 68- and 74-kilodalton peptides and the regulation of albumin gene expression by thyroid hormone during development

In adult Xenopus serum, albumin gene expression is regulated by estrogen through the selective destabilization of its mRNA during the vitellogenic response. The present study reports the cDNA sequence of both the 68K and 74K Xenopus albumin mRNAs, their derived amino acid sequence, and the regulation of albumin gene expression during embryogenesis. Albumin mRNA has a 39 nucleotide 5' untranslated region terminating in a consensus translation initiation site. The derived amino acid sequence yields a 24-amino acid hydrophobic leader sequence (terminating in Lys-Arg) that shares significant homology with the leader peptide of rat albumin. Overall there is 37% sequence identity between rat and frog albumin, with exact conservation of all but one Cys residue and the Pro residues responsible for the three domain structure of the mature protein. The 74K albumin (unlike the 68K albumin) is glycosylated; a point mutation converting Lys256 to Asn introduces an N-linked glycosylation site that is similar to one found in the sequence of mammalian alpha-fetoproteins. A larval albumin-like protein was not detectable by silver staining in serum of tadpoles before the beginning of metamorphosis at stage 48. Albumin mRNA is absent from early tadpoles (stages 22-47); however, it is rapidly induced at stage 48 as one of the earliest manifestations of metamorphosis. Exposure of embryos to 10(-8) M T3, which regulates amphibian metamorphosis, resulted in the premature induction of albumin mRNA, such that it is evident by stage 43.     

Thyroid hormone-dependent repression of α1-microglobulin/bikunin precursor (AMBP) gene expression during amphibian metamorphosis

 A Xenopus AMBP (xAMBP) cDNA clone was isolated from a subtracted liver cDNA library by differential hybridization screening. The deduced amino acid sequence shared 50–60% identity with its mammalian counterparts, which are the precursors of the plasma glycoproteins, α1-microglobulin and bikunin. Both peptide structures were well conserved in xAMBP. Northern and in situ hybridization revealed that the xAMBP gene was specifically expressed in liver parenchymal cells. The gene was activated around embryo hatching and repressed at the metamorphic climax stage. During adult life the mRNA level remained low. Treating the tadpoles with thyroid homone prematurely reduced the mRNA level. Furthermore, thyroid hormone acted on larval hepatocytes in primary culture and reduced the mRNA level. Thus, xAMBP gene expression appears to be repressed through the direct action of thyroid hormone on the hepatocytes at the metamorphic climax stage. On the other hand, adult hepatocytes in thyroid hormone-free culture medium expressed mRNA at a low level, which was not reduced in response to thyroid hormone, suggesting that the repressed xAMBP gene expression in adult hepatocytes was maintained in a thyroid hormone-independent manner. The unique expression profile suggested that the xAMBP gene plays a biological role in the progression of amphibian metamorphosis. Received: 12 April 1996 / Accepted: 19 September 1996     

Thyroid-hormone-dependent and fibroblast-specific expression of BMP-4 correlates with adult epithelial development during amphibian intestinal remodeling

We have identified one of the genes that are up-regulated by thyroid hormone (TH) in Xenopus laevis small intestine as the Xenopus homolog of bone morphogenetic protein-4 (BMP-4). To clarify possible roles of BMP-4 in intestinal remodeling during metamorphosis, we have examined its expression in X. laevis intestine by using in situ hybridization and organ culture techniques. At the beginning of metamorphic climax, BMP-4 mRNA first becomes detectable in the connective tissue, concurrently with the appearance of adult epithelial primordia. Subsequently, when the adult epithelial primordia are actively proliferating, BMP-4 mRNA becomes more abundant only in the connective tissue with a gradient toward the epithelium. Thereafter, as the adult primordia differentiate, the level of BMP-4 mRNA gradually decreases. Thus, BMP-4 expression correlates well with cell proliferation and/or initial differentiation of the adult epithelium, but not with apoptosis of the larval epithelium. Furthermore, the present culture study indicates that (1) TH-induced expression of BMP-4 mRNA is higher in the anterior part of the intestine than in the posterior part, which agrees with the better development of the adult epithelium in the more anterior part, and that (2) the expression of BMP-4 mRNA is up-regulated by TH in the presence of epithelium, but not in its absence. Therefore, BMP-4, which is indirectly induced by TH through some epithelial factor(s), probably plays important roles in adult epithelial development during amphibian intestinal remodeling.     

Temporal and spatial expression of an intestinal Na+/PO43− cotransporter correlates with epithelial transformation during thyroid hormone-dependent frog metamorphosis

The amphibian intestine has two morphologically distinct structures during development. Early embryogenesis generates a simple tube-like intestine in the tadpole whereas after thyroid hormone (T₃)-dependent metamorphosis a newly remodeled adult intestine is formed similar to that of higher vertebrates. This change requires a drastic transformation of the epithelial layer. We have isolated a Na⁺/PO₄³⁻ cotransporter gene that may contribute to this transformation. The deduced amino acid sequence of this gene shows a high degree of homology to the mammalian renal NA⁺/PO₄³⁻ cotransporters, which have little or no expression in organs other than the kidney. The frog gene is highly expressed and regulated by T₃ in the intestine with little expression and/or regulation by T₃ in most other organs. Its mRNA is restricted to the differentiated epithelial cells both in tadpoles and postmetamorphic frogs. Interestingly, its expression is low in premetamorphic tadpoles, but up-regulated when metamorphosis is initiated by endogenous T₃. As the larval epithelium undergoes programmed cell death (apoptosis), the mRNA level drops to a minimum. Subsequently, the gene is reactivated at the tip region of the newly formed adult intestinal folds and a crest-trough polarity of expression is established by the end of metamorphosis. This temporal regulation profile is also reproduced when premetamorphic tadpoles are treated with T₃ to induce precocious intestinal remodeling. These results suggest a possible role of the Na⁺/PO₄³⁻ cotransporter during metamorphosis and demonstrate that the adult epithelial cell differentiation pattern is established in the direction of crest-to-trough of the intestinal fold, concurrent with the epithelial morphogenic process. Dev Genet 20:53–66, 1997. © 1997 Wiley-Liss, Inc.     

Isolation and developmental expression of two nuclear receptors, MHR4 and betaFTZ-F1, in the tobacco hornworm, Manduca sexta

The cDNAs for two members of the nuclear receptor superfamily were isolated from the tobacco hornworm, Manduca sexta. The deduced amino acid sequence of MHR4 shows 93-95% identity in the DNA-binding domain and the first portion of the hinge (D) region with the germ cell nuclear factor (GCNF)-related factors (GRFs) of the silkworm, Bombyx mori, and the mealworm, Tenebrio molitor, and with a genomic sequence from the fruit fly, Drosophila melanogaster. Northern blot hybridization showed that a 7.5 kb MHR4 mRNA appeared in Manduca abdominal epidermis just as the ecdysteroid titer began to decline during the larval molt, disappeared about 12 h later, then transiently reappeared shortly before larval ecdysis. During the pupal and adult molts, a similar pattern of expression was seen (the very end of the adult molt was not studied). At peak times of expression in the epidermis, MHR4 mRNA was also present in fat body and the central nervous system (CNS). The deduced amino acid sequence of Manduca FTZ-F1 is 100% and 96% identical to that of B. mori and Drosophila betaFTZ-F1, respectively, in the DNA-binding domain and the adjacent hinge region including the FTZ-F1 box. Northern blot analysis showed that the >9.5 kb betaFTZ-F1 mRNA appeared in Manduca epidermis during the decline of the ecdysteroid titer in the larval, pupal and adult molts as the first peak of MHR4 mRNA declined, then it disappeared in the larval and pupal molts before the second peak of MHR4 appeared. betaFTZ-F1 mRNA was also found in fat body and the CNS at the time of peak expression in the epidermis during the larval and pupal molts. Both MHR4 and betaFTZ-F1 mRNAs were found in the testis during the onset of spermatogenesis in the prepupal period.