Differential expression of nucleoside diphosphate kinases (NDPK/NM23) during Xenopus early development.

In Xenopus laevis, three nucleoside diphosphate kinase (NDPK) monomers have been described (NDPK X1, X2 and X3) (Ouatas et al., 1997). In eucaryotes, this kinase is known as a hetero- or homohexamer. Here, we examine the distribution of the enzyme and its different subunit mRNAs during oogenesis and early embryogenesis of Xenopus laevis, respectively by immunohistofluorescence and whole-mount in situ hybridization. These analyses show that NDPKs and their mRNAs are differentially distributed throughout the oocyte and early embryos with a high level of transcription in somites and brain. We emphasize two points. First, each mRNA displays a distinct subcellular localization in somites, suggesting a complex regulation of NDPK genes both at the transcriptional and translational level and a possible involvement of NDPK X2 homohexamers in the dorsal muscle differentiation. Second, in oocytes and early embryos, the proteins are mainly localized in the nucleus, suggesting a new mechanism for their nuclear import, since they do not possess any known nuclear import sequences.

Ets-1 and Ets-2 proto-oncogenes exhibit differential and restricted expression patterns during Xenopus laevis oogenesis and embryogenesis.

Xenopus XI-ets-1 and XI-ets-2 are maternally expressed. From late oogenesis to early embryogenesis their transcripts are localized to the animal pole and the intermediate zone, suggesting a function in the differentiation of animal blastomeres and future mesoderm. Their presence at the level of germ plasm suggests also a role in the differentiation of the germinal lineage. Both zygotic genes are expressed ubiquitously beginning at MBT, and then restricted to a circumblastoporal collar. In neurula and tailbud stages, ets-1 and ets-2 transcripts are detected in neural crest cells and their derivatives. Specific transcription can also be observed for ets-1 in the hemangioblastic precursors, in endothelial cells of the forming heart and blood vessels. Ets-2 is itself specifically expressed in the putative pronephros and in the forming pronephric tubules and extending pronephric duct. Like another member of the ets-gene family (XI-fli), both genes are transcribed in regions of the embryo undergoing important morphogenetic modifications, especially in migrating cells and/or along their migration pathways. We postulate that these genes orchestrate modifications of cellular adhesion. Changes in the expression of cadherins and integrins repertories would be consistent with such a role and could account for the phenotypes we reported earlier for XI-fli overexpression. Such a role would be critical for tumor cell dissemination, in addition to the one already ascribed to ets-1 in the expression of proteases specific for the extracellular matrix.

Thyroid hormone regulation of germ cell-specific EF-1 alpha expression during metamorphosis of Xenopus laevis.

In situ hybridization was used to follow the distribution of the mRNAs encoding the somatic form of elongation factor 1 alpha (EF-1 alpha S) and the germinal counterparts of this factor, thesaurin a and EF-1 alpha O, throughout metamorphosis in the gonads of Xenopus laevis tadpoles. EF-1 alpha S mRNA is detected before metamorphosis in both the somatic and germ cells of the gonads. In contrast, thesaurin a and EF-1 alpha O mRNAs are first detected in spermatogonia and oogonia at stages 60-62, corresponding to the climax of metamorphosis and to the peak of circulating thyroid hormone. To determine whether thyroid hormone, the instigator of metamorphosis, is involved in regulating the expression of the germinal gene EF-1 alpha O, Xenopus XTC cells were transfected with an EF-1 alpha O promoter sequence inserted in front of the luciferase reporter gene. Addition of T3 to the cell culture medium induced a dose-dependent increase in transcription from the EF-1 alpha O promoter. This effect was enhanced when the construct was cotransfected with an expression vector for a Xenopus thyroid hormone receptor. Our data show that germ cells switch from a somatic to a germ-cell specific mode of expression during metamorphosis. Furthermore, this switch appears to be induced by thyroid hormone.

The c-ets-1 proto-oncogenes in Xenopus laevis: expression during oogenesis and embryogenesis.

We previously reported the cloning and sequencing of two cDNAs derived from the Xenopus laevis ets-1 gene (Stiegler et al., 1990). The Xl-ets-1a cDNA encodes a polypeptide highly homologous to known ets-1 proteins. The 3'-UTR contains two AATAAA polyadenylation signals together with three copies of the TTTTTAT sequence thought to confer a maturation-specific polyadenylation and implicated in the deadenylation of dormant mRNAs. Several transcripts with maternal characteristics were detected in oogenesis and early embryogenesis. A marked augmentation of the major transcript in the poly(A)+ fraction was detected at fertilization. Ets-1 transcripts were observed at constant levels during the cleavage stages but decreased abruptly at gastrulation, to reappear from neurulation to late embryogenesis. The possible contribution of 3'-UTR sequence elements to this behavior is discussed.

Localization of ras proto-oncogene expression during development in Xenopus laevis.

The expression of the ras protooncogene was investigated in Xenopus laevis, throughout development, by in situ hybridization using a 35S-labelled antisense RNA probe. During oogenesis, the ras RNA was strongly expressed in the cytoplasm of previtellogenic oocytes and further diluted between yolk platelets; no specific localization of transcripts was observed. The signal density was particularly weak over embryo sections until the tailbud stage. On the other hand, a high level of ras RNA expression was detected on sections through the young tadpoles. An intense labelling was observed in several areas, including the branchial apparatus, gut, somites, nervous system, and lens. It is noteworthy that the heterogeneity of labelling increases as tadpoles grow older. Together, these results are discussed in relation to cellular events appearing throughout the early development.

The genes encoding the major 42S storage particle proteins are expressed in male and female germ cells of Xenopus laevis.

As components of the 42S storage particles (thesaurisomes), thesaurin a and thesaurin b are involved in the long-term storage of tRNA and 5S RNA in previtellogenic oocytes of Xenopus laevis. Thesaurin a and thesaurin b are among the most abundant proteins in previtellogenic oocytes. We show here that the mRNAs encoding thesaurin a and thesaurin b are present not only in previtellogenic oocytes but also in pre-meiotic germ cells (oogonia). These mRNAs can also be detected in spermatogonia and early spermatocytes, and are translated into protein in testis, as they are in ovary. We conclude that male germ cells mimic female germ cells in several aspects of gene activity related to RNA accumulation and metabolism.

Germ cell-specific expression of a gene encoding eukaryotic translation elongation factor 1 alpha (eEF-1 alpha) and generation of eEF-1 alpha retropseudogenes in Xenopus laevis.

We have studied by in situ hybridization the expression of the genes encoding the somatic form and the oocyte form of Xenopus laevis eEF-1 alpha. The somatic form of eEF-1 alpha (eEF-1 alpha S) mRNA is virtually undetectable in male and female germ cells of the adult gonad but is very abundant in embryonic cells after the neurula stage. In contrast, another form of eEF-1 alpha (eEF-1 alpha O) mRNA is highly concentrated in oogonia and in previtellogenic oocytes but is undetectable in eggs and embryos. eEF-1 alpha O mRNA is also present in spermatogonia and spermatocytes of adult testis. The latter finding identifies eEF-1 alpha O mRNA as a germ cell-specific gene product. Although germ cells contain very little eEF-1 alpha S mRNA, several eEF-1 alpha S retropseudogenes exist in X. laevis chromosomes. These genes are thought to arise in germ cells from reverse transcription of mRNA and subsequent integration of the cDNA copies into chromosomal DNA. We suggest that eEF-1 alpha S pseudogenes are generated in primordial germ cells of the embryo before they differentiate into oogonia or spermatogonia.

Enhanced c-myc gene expression during forelimb regenerative outgrowth in the young Xenopus laevis.

Analysis of the expression of the c-myc protooncogene has been carried out in the forelimb regenerate of the Xenopus laevis froglet. Northern blot hybridization analysis revealed the presence of a 2.5-kilobase c-myc transcript in the regenerate forelimb at a level at least 7-fold more than the one found in nonregenerating forelimbs or stumps of regenerating forelimbs. In situ hybridization analyses confirmed the relative abundance of c-myc RNA in the regenerate forelimb and provided evidence of spatial localization of high levels of c-myc RNA in specific cell layers. The deepest layers of the wound epithelium of epidermal origin showed a strong signal, whereas virtually no c-myc RNA was detected in the outermost layers. Labeling was also observed in mesenchymal cells of the blastema where it was relatively evenly distributed. This pattern of c-myc RNA in the regenerate might indicate that the expression of c-myc plays a role in the regulation of the continued proliferation of specific cells of the regenerate, whereas repression of this gene in the epidermis correlates with terminal differentiation of keratinocytes.

Genes and mechanisms involved in early embryonic development in Xenopus laevis.

Our laboratory is studying genes involved in the regulation of the balance between cell growth and differentiation during embryonic development in Xenopus. We have analyzed the developmental expression of the proto-oncogenes c-myc, and KiRas 2B, the proliferating cell nuclear antigen (PCNA), and the tumor suppressor gene p53. These genes, usually expressed during cell proliferation, are expressed in the oocyte in large quantities, but the majority of their maternal RNAs are degraded by the gastrula stage. The expression of c-myc and the localization of the protein indicate that c-myc has the characteristics expected for a gene involved in the regulation of the mid-blastula transition, when zygotic expression is turned on in the embryo. Its expression during late development or during regeneration indicates that it enables the cells to remain competent for cycling during organogenesis. In vitro systems that reproduce the principal cellular functions during early development are used as model systems to understand the mechanisms involved in early embryogenesis.

Localization of c-myc expression during oogenesis and embryonic development in Xenopus laevis.

The expression of the proto-oncogene c-myc during oogenesis and embryonic development was followed by in situ hybridization using a cytological protocol adapted to amphibian embryos. The c-myc RNA was highly expressed in the cytoplasm of young oocytes and was further diluted during oocyte growth without specific localization. From the neurula stage on, new myc transcripts were detected and the whole embryo appeared positive with antisense myc RNA probes relative to control sense RNA probes. In addition, a spatial localization of high levels of the transcript was also observed in specific areas of the developing embryo, including the epidermis, gill buds, optic vesicles and lens placodes. These observations might indicate a specific role of the c-myc gene during the differentiation of these tissues. Alternatively, this high level of myc expression might prevent such tissues from entering into terminal differentiation during the growth of the embryo.

Proto-oncogenes and embryonic development.

The role of proto-oncogenes in embryonic development was investigated using one of the most characterized vertebrates, the amphibian Xenopus laevis. Genes which belong to the major proto-oncogene families have been detected in Xenopus genome. The developmental control of the myc gene was assayed using a characterized Xenopus myc probe and specific antibodies. The myc gene is highly expressed as a stable maternal mRNA in oocyte, and an unfertilized egg contains 5 X 10(5)-fold the myc RNA content of a proliferative somatic cell. The myc RNA store is evenly distributed in the oocyte and the egg. Fertilization triggers a post-transcriptional control of the gene and the RNA store is progressively degraded to a constitutive value of 10 to 30 myc RNA copies registered per gastrula embryonic cell. The 62K myc protein is accumulated late in oogenesis. This uncoupling of myc expression and cell proliferation appears as a specific developmental regulation of the myc gene, adapted to the series of rapid cell cleavages occurring after fertilization.

Investigations of endocrine cells in the gastrointestinal tract and pancreas during the metamorphosis of an anuran (Alytes obstetricans L.): histochemical detection of APUD cells.

Endocrine cells were detected at premetamorphosis, prometamorphosis, climax, and juvenile stages using an amine-inducing fluorescence technique with or without previous L-3,4-dihydroxyphenylalanine (L-DOPA) treatment. At premetamorphosis, serotonin cells exhibited yellow fluorescence in the gut primary epithelium of the L-DOPA untreated animals. In the treated animals, green fluorescent APUD cells could be seen in addition to the serotonin cells. In the pancreas, numerous clusters of fluorescent APUD cells were observed. At prometamorphosis the number of fluorescent cells increased in the intestinal primary epithelium and, close to the basal membrane, numerous small regenerative buds devoid of fluorescent cells appeared. In the pancreas of L-DOPA-treated animals, two types of APUD cells could be distinguished by their different fluorescence intensities. At the climax stage, the stomach developed and APUD cells were detectable in the gastric glandular buds. The degenerated primary intestinal epithelium was progressively removed in the intestinal lumen. At this stage, the regenerative buds of the secondary epithelium exhibited APUD cells. In the disorganized pancreas, the induced fluorescence decreased strongly. At the juvenile stage, cords of APUD cells displayed a cytoplasmic green fluorescence in the pancreas. In the stomach and intestine, serotonin and APUD cells were numerous.

In vitro study of the intestinal brush border enzyme activities in developing anuran amphibian: effects of thyroxine, cortisol, and insulin.

The effects of several hormones on intestinal brush border membrane enzymatic activities have been investigated in intestinal explants taken from the amphibian midwife toad at different developmental stages. Explants were treated for at least 2 days with thyroxine (0.1 microgram/ml of culture medium) or for 2 days with cortisol (25 micrograms/ml) or insulin (6 mU/ml). The hydrolases examined were maltase, trehalase, glucoamylase, and alkaline phosphatase. In the explants from tadpoles in prometamorphosis, thyroxine had no effect on hydrolase activities; cortisol increased the activity of only glucoamylase, and insulin increased activity of maltase, glucoamylase, and alkaline phosphatase. When the explants were taken from tadpoles at the beginning of climax, cortisol and insulin generally stimulated the enzyme activities studied. When taken from tadpoles at the end of climax, at the moment when the embryonic cells under the degenerating epithelium divide, cortisol and insulin had little effect on these activities. When the animals terminate their metamorphosis, the intestinal epithelium of the explants is totally newly formed (secondary epithelium). At this time, cortisol stimulated the activities of maltase, glucoamylase, and alkaline phosphatase, while insulin decreased the activities of maltase and glucoamylase.

Hormonal control of the intestinal brush border enzyme activities in developing anuran amphibians. II. Effects of glucocorticoids and insulin during experimental metamorphosis.

The effects of glucocorticoids (hydrocortisone, dexamethasone) and insulin on enzymatic activities of the intestinal brush border membrane were investigated in an anuran amphibian, Alytes obstetricians, before and during experimental metamorphosis produced by immersion into a thyroxine solution. During experimental metamorphosis, a new epithelium (secondary epithelium) replaces the degenerating primary epithelium. The enzymes studied were three glucidases (maltase, glucoamylase, trehalase) and alkaline phosphatase. In tadpoles reaching the end of premetamorphosis, hormones were injected every day (hydrocortisone, dexamethasone: 25 micrograms/g body wt/day; insulin: 5 mU/g body wt/day, for 3 and occasionally 6 consecutive days. Under such conditions, most of the activities in the primary epithelium increased or remained stable. In animals which completed experimental metamorphosis, the secondary epithelium formed. Hydrocortisone (25 micrograms/g body wt/day) and insulin (5 mU/g body wt/day) treatments significantly decreased the enzymatic activities of the new brush border membrane in animals which received one hydrocortisone and/or insulin injection per day, during 3 consecutive days. Such results, which previously had not been obtained systematically in spontaneously metamorphosing tadpoles (El Maraghi-Ater, Mesnard, and Hourdry (1986). Gen. Comp. Endocrinol. 61, 53-63), emphasize the relative independence of the intestinal metabolism during experimental and spontaneous metamorphosis.

Variations of intestinal calcium absorption in adult frogs (Rana esculenta). Effect of lysine.

Intestinal calcium absorption was investigated in an adult frog (Rana esculenta) by injecting a CaCl2 solution containing 45Ca into the lumen. The 45Ca absorption coefficient in the proximal loop was higher than in the distal loop, only when the CaCl2 solution was left for 4 h. This coefficient increased both in the proximal and distal loops when a 4-h treatment was substituted for a 1-h treatment. The coefficient increased in the whole intestine during the first 2 h of treatment (1 h: 21%; 2 h: 55%) and remained stable afterwards in our experimental conditions. The intestinal calcium absorption increase occurred early in the presence of L-lysine (100 mM), since the coefficient already reached its maximum value (52%) after a 1-h treatment.

Phosphorylated proteins from anuran intestinal microvilli membranes--I. Relations with alkaline phosphatase.

The degree of phosphorylation of intestinal microvilli membrane proteins in an adult amphibian, Rana esculenta, was investigated under various experimental conditions. The microvilli protein phosphorylation rate rapidly increases during the first 4 min of incubation in a medium containing [gamma-32P]ATP. This increase is slower afterwards. Cyclic nucleotides (cyclic AMP, cyclic GMP) and sorbitol do not modify the microvilli protein phosphorylation rate. On the contrary, this phosphorylation rate significantly decreases in the presence of L-lysine, when its concentration in the incubation medium is greater than 25 mM. The time course of phosphorylation confirms the inhibitory effects of L-lysine (100 mM). The microvilli membrane proteins were distinguished by polyacrylamide gel electrophoresis. In heated samples, electrophoresis followed by an radioautograph systematically reveals the existence of a very phosphorylated protein with a mol. wt of 86 kDa. The phosphorylation of this protein is partially inhibited by L-lysine (100 mM). The very phosphorylated protein could be the monomer of alkaline phosphatase. The dimer (170 kDa) is visualized on electrophoretograms by its catalytic activity. In mammals, several authors have established a correlation between phosphorylation of the microvilli membrane proteins and the intensity of intestinal calcium absorption. Such a control is presently being investigated in adult Rana esculenta.

Hormonal control of the intestinal brush border enzyme activities in developing anuran amphibians. I. Effects of hydrocortisone and insulin during and after spontaneous metamorphosis.

The effects of hydrocortisone and insulin on the intestinal brush border membrane enzymatic activities in an anuran amphibian, Alytes obstetricans, were investigated at the end of spontaneous metamorphosis and 2 weeks after its completion. At the end of metamorphosis, the brush border is differentiating in the apical region of a developing neoformed epithelium. Two weeks after the completion of metamorphosis, this epithelium is entirely formed. The animals received one hormone injection per day for 2 or 3 days running (hydrocortisone: 1, 5, or 25 micrograms/g body wt/day; insulin: 0.5, 1, or 5 mU/g body wt/day). The hydrolases studied were three glucosidases (maltase, glucoamylase, trehalase), gamma-glutamyl-transferase and alkaline phosphatase. In animals reaching the end of metamorphosis, hormonal treatments rarely modify the three glucosidase activities. Two weeks after metamorphosis, a 5 microgram/g body wt/day hydrocortisone injection usually results in a significant increase of the three glucosidase activities. Conversely, a 0.5 mU/g body wt/day insulin injection induced a marked decrease in these activities. At the end of metamorphosis, hydrocortisone has variable effects on gamma-glutamyl-transferase activity; insulin, however, does not significantly modify this activity. Two weeks later, insulin and sometimes hydrocortisone inhibit gamma-glutamyl-transferase activity. Whatever the developmental stage is, hydrocortisone is able to stimulate alkaline phosphatase activity. At the end of metamorphosis, insulin has no influence on this activity, but 2 weeks after metamorphosis, low doses of the hormone (0.5 mU/g body wt/day) significantly reduce it. These results emphasize the possibility that after spontaneous metamorphosis the enzymatic activities of the new intestinal brush border are hormone controlled. This control could be related to the development of the interrenal and pancreatic islet functions.

Development of peroxisomes in amphibians. III. Study on liver, kidney, and intestine during thyroxine-induced metamorphosis.

This investigation was undertaken to study the ontogeny of hepatic, renal, and intestinal peroxisomes and/or microperoxisomes during thyroxine-induced anuran metamorphosis. Catalase activity was localized cytochemically after incubation in DAB medium, and studied biochemically by a spectrophotometric method. Our morphological and biochemical investigations suggest the formation of a new population of peroxisomes during the hormonal treatment. This is obvious especially for microperoxisomes of the intestinal epithelium since the larval tissue is completely replaced by a new layer during thyroxine-induced metamorphosis. For the peroxisomes of hepatocytes and kidney proximal tubule cells, our assumption is based on the following observations: 1) The number of peroxisomes increases in liver and kidney during thyroxine treatment; 2) this proliferation is accompanied by an enlargement of renal peroxisomes; and 3) 16 days after the beginning of the hormonal treatment, 5.4- and 2.4-fold increases are found for the specific activities of hepatic and renal catalase, respectively. A temporal coordination exists between the structure and the metabolism of peroxisomes and mitochondria during thyroxine-induced metamorphosis.

A histological and dynamic study of the gastric region of Discoglossus pictus larvae, cultured with or without thyroxine.

The organotypic culture of the gastric region is carried out on premetamorphic Discoglossus pictus larvae. Adding thyroxine to the culture medium provokes various transformations. On the cytological level, the reactions observed, which are variable depending on the cell category concerned, can be divided into two types of phenomena: histolytic and histogenetic. Autophagia linked to lysosome intervention is frequently found among the histolytic processes. Autophagic vacuoles and residual bodies are observed. The gastric lumen is filled with deteriorated cells that probably come from the degeneration of the tadpole epithelium (primary epithelium). The incorporation of tritiated thymidine makes it possible to study the evolution of cell proliferation in the control and in the thyroxinated cultures. After a 1-2 day latency period, possibly due to the adjustment of the tissue to the culture environment, the incorporation of the radioprecursor H3-thymidine into the epithelium and the tunica muscularis of thyroxine-treated gut tissue increased on day 3, reached a maximum on day 5, and then dropped slightly on day 7. In the control cultures H3-thymidine incorporation showed the same pattern but lower levels on the same days. The histolytic phenomena induced by thyroxine in vitro are comparable to those of natural metamorphosis. On the other hand, the histogenetic phenomena are incomplete. Proliferating and transitional phases occur but neoformated (or secondary) epithelium does not replace the degenerated primary epithelium, whatever the culture time.