New epidermal keratin genes from Xenopus laevis: hormonal and regional regulation of their expression during anuran skin metamorphosis

Xenopus larval keratin (XLK) was isolated by gel electrophoresis of proteins of tadpole skin. Screening of an expression cDNA library of tail tissues by specific polyclonal antibodies against XLK produced XLK cDNA (xlk). Its complete nucleotide and predicted amino acid sequences revealed that XLK was a new member of type II keratin. Screening of a cDNA library of adult Xenopus skin using an oligonucleotide probe which had been designed from well-conserved N-terminal amino acid sequences of the rod domain of type I keratin produced two cDNAs, xak-a and xak-b, which were found to be new members of type I keratin gene. Northern blot analysis showed that xlk was expressed exclusively in the larval skin whereas xak-a and xak-b were expressed exclusively in the adult skin. Their expression level was regulated in a region- and metamorphic stage- dependent manner during larval skin development. mRNA in situ hybridization experiments identified the cells that expressed xlk, and xak-a and xak-b as larva- specific epidermal cells (skein cells and basal cells), and adult suprabasal epidermal cells, respectively. These three genes were found to be late responsive to thyroid hormone. Phylogenetic relationships of these keratins with known ones are discussed.     

Hormonal regulation of adult type keratin gene expression in larval epidermal cells of the frog Xenopus laevis

Triiodothyronin (T3) is known to induce amphibian metamorphosis but other hormones such as glucocorticoids accelerate T3 action. The increase in plasma concentration of both T3 and glucocorticoids during metamorphic climax is correlated with the transformation of the epidermis from larval type (uncornified) to adult type (cornified). Previously we have shown that T3 induced adult-type 63 Kd keratin gene expression and cornification of the larval epidermis. In this study, we have examined the effects of T3 and hydrocortisone (HC) on the conversion of larval to adult epidermal cells in vitro. When larval epidermal cells were treated with both T3 and HC, they had a synergistic effect on adult-type keratin synthesis (both 63 Kd and 49 Kd keratins) and epidermal cornification. The synergistic effect between T3 and HC required a pretreatment with T3 for 3 days. During this time, addition of HC to cultures containing T3 did not change the amount of 63 Kd keratin mRNA. Thus, HC did not reduce the lag time for epidermal cells to respond to T3. After 4 days of hormone treatment, T3 increased the amount of 63 Kd keratin mRNA 9-fold while T3 and HC induced it 18-fold. When cultures were pretreated with T3 for 3 days, a 1 day treatment with HC was sufficient to obtain the synergistic effect. Thus the induction of 63 Kd keratin gene expression by T3 required a much longer lag (3 days) than the lag required for the synergistic action of T3 and HC (less than 1 day).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium regulation of epidermal cell differentiation in the frog Xenopus laevis.

Adult frogs have a stratified epidermis with a keratinized stratum corneum. Since the extracellular calcium concentration is known to regulate differentiation of mammalian epidermal cells in vitro, we studied the effects of calcium on the terminal differentiation of frog epidermal cells. Exposure of the epidermal cells to a high concentration of calcium (greater than 0.2 mM) induced cornification and the synthesis of a 51 Kd acidic keratin. These data are very similar to the results from mammalian epidermal cell cultures, suggesting that the mechanism of terminal differentiation is conserved throughout the evolution of terrestrial vertebrates.     

Differential keratin gene expression during the differentiation of the cement gland of Xenopus laevis.

The expression of both epidermal and nonepidermal keratins has been detected in the cement gland of Xenopus laevis by antibody staining. Northern blot and in situ hybridizations with gene-specific probes indicated the expression of the nonepidermal keratin, XK endo B, and the embryonic epidermal keratin, XK70, in the cement gland. Furthermore, since explanted animal pole cells can be induced to differentiate into cement gland cells in vitro by incubation in NH4Cl, we have demonstrated the in vitro induction of XK endo B, maintenance of XK70, and repression of another embryonic epidermal keratin, XK81. This is the first report of keratin gene expression in the cement gland.     

Spatial and temporal expression pattern of a novel gene in the frog Xenopus laevis: correlations with adult intestinal epithelial differentiation during metamorphosis

We report the cloning of a novel gene (ID14) and its expression pattern in tadpoles and adults of Xenopus laevis. ID14 encodes a 315-amino acid protein that has a signal peptide and a nidogen domain. Even though several genes have a nidogen domain, ID14 is not the homolog of any known gene. ID14 is a late thyroid hormone (TH)-regulated gene in the tadpole intestine, and its expression in the intestine does not begin until the climax of metamorphosis, correlating with adult intestinal epithelial differentiation. In contrast, ID14 is expressed in tadpole skin and tail and is not regulated by TH. In situ hybridization revealed that this putative extracellular matrix protein is expressed in the epithelia of the tadpole skin and tail and in the intestinal epithelium after metamorphosis. In the adult, ID14 is found predominantly in the intestine with weak expression in the stomach, lung, and testis. Its exclusive expression in the adult intestinal epithelial cells makes it a useful marker for developmental studies and may give insights into cell/cell interactions in intestinal metamorphosis and adult intestinal stem cell maintenance.     

Modulation of Na,K-ATPase expression during early development of Xenopus laevis.

In amphibian and mammalian systems, regulation of Na+ transport via the Na,K-ATPase plays an important role in distinct developmental processes such as blastocoele formation and neurulation. In this study, we have followed the Na,K-ATPase activity, the biosynthesis, and the cellular accumulation of catalytic alpha-subunits after fertilization of Xenopus laevis eggs up to neurula formation. Our data show that Na,K-ATPase activity increases significantly between stages 4 and 6 and again between stages 13 and 24. The four-fold rise in Na,K-ATPase activity during blastocoele formation is not mediated by an increased cellular pool of alpha-subunits. On the other hand, a five-fold increase of the biosynthesis rate around midblastula precedes a progressive accumulation up to neurula stage mainly of alpha 1-subunits and to a lesser extent of a second alpha-immunoreactive species. In contrast, newly synthesized glycoproteinic beta 1-subunits of Na,K-ATPase cannot be detected up to late neurula. These data indicate that (1) upregulation of Na,K-ATPase activity during blastocoele and neurula formation are mediated by different regulation mechanisms and (2) alpha- and possibly beta-isoforms are expressed in a developmentally regulated fashion during early Xenopus development.     

The expression of epidermal antigens in Xenopus laevis

Five kinds of monoclonal antibodies that are specific for the epidermis of Xenopus embryos were produced. Epidermis-specific antibodies were used to investigate the spatial and temporal expressions of epidermal antigens during embryonic and larval development. The cells that were recognized by the antibodies at the larval stage are as follows: all of the outer epidermal cells and cement gland cells were recognized by the antibody termed XEPI-1, all of the outer and inner epidermal cells, except the cement gland cells, were recognized by XEPI-2 antibody, the large mucus granules and the apical side of the outer epidermal cells, except for the ciliated epidermal cells, were recognized by XEPI-3 antibody, the large mucus granules and basement membrane were recognized by XEPI-4 antibody, and the small mucus granules contained in the outer epidermal cells as well as extracellular matrices were recognized by the antibody termed XEPI-5. All of the epidermal antigens, except XEPI-4, were first detected in the epidermal region of the late gastrula or early neurula. The XEPI-4 antigen was first detected in stage-26 tail-bud embryos. None of these antigens were expressed by the neural tissues at any time during embryonic development. Only the XEPI-2 antigen continued to be expressed after metamorphosis, while the expression of the other antigens disappeared during or before metamorphosis. The specificity of the antibodies allowed us to classify the epidermal cells into four types in early epidermal development. The four types of epidermal cells are (1) the outer epidermal cells that contain small mucus granules, (2) the ciliated epidermal cells, (3) the outer epidermal cells that contain large mucus granules and (4) the inner sensorial cells.     

Sexual differentiation and hormonal regulation of the laryngeal synapse in Xenopus laevis

In Xenopus laevis frogs, sex differences in adult laryngeal synapses contribute to sex differences in vocal behavior. This study explores the development of sex differences in types of neuromuscular synapses and the development and hormone regulation of sex differences in transmitter release. Synapses in the juvenile larynx have characteristics not found in adults: juvenile muscle fibers can produce subthreshold or suprathreshold potentials in response to the same strength of nerve stimulation and can also produce multiple spikes to a single nerve stimulus. Juvenile laryngeal muscle also contains the same synapse types (I, II, and III) as are found in adult laryngeal muscle. The distribution of laryngeal synapse types in juveniles is less sexually dimorphic than the distribution in adults. Analysis of quantal content indicates that laryngeal synapses characteristically release low amounts of transmitter prior to sexual differentiation. Quantal content values from male and female juveniles are similar to values for adult males and are lower than values for adult females. When juveniles are gonadectomized and treated with exogenous estrogen, quantal content values increase significantly, suggesting that this hormone may increase transmitter release at laryngeal synapses during development. Gonadectomy alone does not affect quantal content of laryngeal synapses in either sex. Androgen treatment decreases quantal content in juvenile females but not males; the effect is opposite to and smaller than that of estrogen. Thus, muscle fiber responses to nerve stimulation and transmitter release are not sexually dimorphic in juvenile larynges. Transmitter release is strengthened, or feminized, by the administration of estradiol, an ovarian steroid hormone. © 1995 John Wiley & Sons, Inc.     

Spatial and temporal localization of FGF receptors in Xenopus laevis

Summary Mesoderm formation is a result of cell-cell interactions between the vegetal and animal hemisphere and is thought to be mediated by inducing peptide growth factors including members of the FGF and TGF superfamilies. Our immunochemical study analyses the distribution of FGF receptors coded by the human flg gene during embryogenesis of Xenopus laevis. Immunostaining was detected in the dorsal and ventral ectoderm and also in the marginal zone of early cleavage, blastula and gastrula stages. Signals were very strong in the mid and late blastula (stage 8 and 9) and declined slightly in the early gastrula (stage 10). A dramatic decrease was observed up to the late gastrula (stage 11⁺). In stage 13 embryos, immunostaining was only found in cells around the blastopore. Isolated ectoderm cultured in vitro showed a similar temporal expression and decrease of the signal as the normal embryos. These results indicate that receptor expression is independent of the interaction of the animal cells with the vegetal part of the embryo. Of interest is the fact that the signal cannot only be found at or near the cell surface but also within the cell. This suggests the presence of an intracellular isoform of the receptor resulting from the endogenous expression of splice variants and the internalization of transmembrane receptor. Taken together our results suggest that the loss of competence (for bFGF around stage 10) is not directly correlated with the presence of receptors. The possible roles of heparan sulphate glucosaminoglycans (low affinity receptors) and control mechanisms in the intracellular signalling pathway downstream of the receptor level should be taken into consideration.     

Posttranscriptional regulation of c-myc RNA during early development of Xenopus laevis

The remarkable stability of c-myc during oogenesis contrasts with its degradation during the early developmental period in Xenopus laevis. Three evolutionary conserved motifs found in the 3'-untranslated region of Xenopus c-myc RNAs have been analyzed for a possible role in c-myc RNA degradation. No specific degradation was observed when these sequences were cloned downstream of a reporter gene and the corresponding RNAs were injected into fertilized eggs. The relation between polyadenylation and degradation of c-myc mRNA has been examined during early development. c-myc is adenylated during early oogenesis, and a dramatic de-adenylation occurs in full grown oocytes. Consequently, the de-adenylation of c-myc mRNA that occurs in eggs might be a requirement for its degradation after fertilization, but is not sufficient to trigger its degradation.     

Regulated gene expression of hyaluronan synthases during Xenopus laevis development

Here reported is the developmental gene expression pattern of the three known vertebrate hyaluronan synthases (XHas1, XHas2 and XHas3) and a comparative analysis of their mRNAs spatio-temporal distribution during Xenopus laevis development. We found that while XHas2 shows a steady-state expression from gastrula to late tailbud stage, XHas1 is mainly present in the early phases of development while XHas3 is predominantly transcribed in tailbud embryos. XHas1, XHas2 and XHas3 show distinct tissue expression patterns. In particular, XHas1 is localized in ectodermal derivatives and in cranial neural crest cells, whereas XHas2 is mainly found in mesoderm-derived structures and in trunk neural crest cells. Moreover, the expression pattern of XHas2 overlaps that of MyoD in cells committed to a muscle fate. Unlike the other hyaluronan synthases, XHas3 mRNA distribution is very restricted. In particular, XHas3 is expressed in the otic vesicles and closely follows the inner ear development. In conclusion, XHas1, XHas2 and XHas3 mRNAs have distinct and never overlapping spatial expression domains, which would suggest that these three enzymes may play different roles during embryogenesis.     

Nephroblastoma in the clawed frog Xenopus laevis.

One 2.5-3 year old female clawed frog (Xenopus laevis), out of a consignment of 4,000 frogs, was found to have an abnormal abdominal growth, weighing 7.9 g. The growth was examined histologically and on the basis of the abundant stroma and serially-arranged tubules nephroblastoma was diagnosed. The growth is not considered to be transmissible.     

Spatial and temporal properties of ventral blood island induction in Xenopus laevis.

Questions of dorsoventral axis determination and patterning in Xenopus seek to uncover the mechanisms by which particular mesodermal fates, for example somite, are specified in the dorsal pole of the axis while other mesoderm fates, for example, ventral blood island (VBI), are specified at the ventral pole. We report here that the genes Xvent-1, Xvent-2, and Xwnt-8 do not appear to be in the pathway of VBI induction, contrary to previous reports. Results from the selective inhibition of bone morphogenetic protein (BMP) activity, a key regulator of VBI induction, by ectopic Noggin, Chordin, or dominant negative BMP ligands and receptors suggest an alternative route of VBI induction. Injection of noggin or chordin RNA into animal pole blastomeres effectively inhibited VBI development, while marginal zone injection had no effect. Cell autonomous inhibition of BMP activity in epidermis with dominant negative ligand dramatically reduced the amount of (&agr;)T3 globin expression. These results indicate that signaling activity from the Spemann Organizer alone may not be sufficient for dorsoventral patterning in the marginal zone and that an inductive interaction between presumptive VBIs and ectoderm late in gastrulation may be crucial. In agreement with these observations, other results show that in explanted blastula-stage marginal zones a distinct pattern develops with a restricted VBI-forming region at the vegetal pole that is independent of the patterning activity of the Spemann Organizer.