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.     

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.     

Thyroid hormone directly induces hepatocyte competence for estrogen-dependent vitellogenin synthesis during the metamorphosis of Xenopus laevis

Hepatocytes competent for estrogen-dependent vitellogenin synthesis appeared and increased in number in the liver at the metamorphic climax of Xenopus laevis (A. Kawahara, S. Kohara, Y. Sugimoto, and M. Amano, 1987, Dev. Biol. 122, 139-145). The present study was conducted to determine whether cells competent for vitellogenin synthesis could be induced by thyroid hormone in a primary culture of larval hepatocytes. The thyroid hormone, triiodothyronine (T3), directly induced the competent cells in a primary culture of premetamorphic larval hepatocytes in a dose- and duration-dependent manner. The competency acquired in response to T3 persisted after removal of the hormone. Aphidicholin, an inhibitor of DNA synthesis, failed to block this induction, suggesting the presence of a "precursor cell fraction." This cell fraction in the hepatocyte population increased with the progress of metamorphosis. The thyroid hormone is thus considered the cause of competent cell formation at metamorphic climax.     

Histone gene expression in early development of Xenopus laevis

Abstract. This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis , and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3.
Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.     

Spatial, temporal, and hormonal regulation of epidermal keratin expression during development of the frog, Xenopus laevis.

To study the mechanism of hormone-induced keratin expression in the epidermis during Xenopus metamorphosis, a monospecific antibody was raised against a unique carboxy-terminal peptide of the 63-kDa keratin. Immunohistological analysis demonstrated that the onset of 63-kDa keratin expression showed distinct regional and temporal differences. The expression started at stage 54 in the hindlimb epidermis, at stage 57 in the head, and over 1 month later at stage 63 in the tail. The amount of 63-kDa keratin was further regulated during epidermal stratification and differentiation. The 63-kDa keratin was expressed first in basal epidermal cells before stratification began. The outer layer of the larval epidermis (periderm) did not express the 63-kDa keratin. As the cells moved out of basal layer, they stained more intensely with the anti-keratin antibody indicating that 63-kDa keratin synthesis is up-regulated during differentiation. Similar results were obtained with cultures of purified epidermal cells grown in high calcium conditions. Since we have shown that thyroid hormone (T3) induces 63-kDa keratin gene expression and hydrocortisone (HC) modulates T3 action we examined the effects of T3 and HC at the single cell level with the anti-keratin antibody. Immunostaining demonstrated that T3 alone and T3 plus HC increased the number of 63-kDa keratin-positive cells as well as the amount of 63-kDa keratin per cell. Unexpectedly these hormones had the same effects on head and tail epidermal cells even though the latter cells degenerate during metamorphosis. The major difference between tail and head cells was that the percentage 63-kDa keratin-producing cells was much greater in the head than in the tail.     

Sexually dimorphic expression of a laryngeal-specific, androgen-regulated myosin heavy chain gene during Xenopus laevis development.

Masculinization of the larynx in Xenopus laevis frogs is essential for the performance of male courtship song. During postmetamorphic (PM) development, the initially female-like phenotype of laryngeal muscle (slow and fast twitch fibers) is converted to the masculine form (entirely fast twitch) under the influence of androgenic steroids. To explore the molecular basis of androgen-directed masculinization, we have isolated cDNA clones encoding portions of a new Xenopus myosin heavy chain (MHC) gene. We have detected expression of this gene only in laryngeal muscle and specifically in males. All adult male laryngeal muscle fibers express the laryngeal myosin (LM). Adult female laryngeal muscle expresses LM only in some fibers. Expression of LM during PM development was examined using Northern blots and in situ hybridization. Males express higher levels of LM than females throughout PM development and attain adult levels by PM3. In females, LM expression peaks transiently at PM2. Treatment of juvenile female frogs with the androgen dihydrotestosterone masculinizes LM expression. Thus, LM appears to be a male-specific, testosterone-regulated MHC isoform in Xenopus laevis. The LM gene will permit analysis of androgen-directed sexual differentiation in this highly sexually dimorphic tissue.     

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.     

Transforming growth factor-beta5 expression during early development of Xenopus laevis

Members of the transforming growth factor-beta (TGF-beta) superfamily play various roles during development in both vertebrates and invertebrates. Two isoforms, TGF-beta2 and -beta5, have been isolated from Xenopus laevis. We describe here the localization of TGF-beta5 mRNA in early embryos of X. laevis, assessed by whole-mount in situ hybridization. The first detectable expression of TGF-beta5 was seen in the stage 14 embryo at the posterior tip of notochord, which continued to later stages, accompanied by the expression in bilateral regions of posterior wall in the tail region next to the notochord. At later stages, transient expression was seen in the cement gland (around stage 21) and in the somites (stages 24-27). In addition, expression was present in the branchial arches (stage 29-36) and olfactory placodes (stage 36).     

Tetracycline-regulated gene expression switch in Xenopus laevis

Xenopus is a well-characterized model system for the investigation of biological processes at the molecular, cellular, and developmental level. The successful application of a rapid and reliable method for transgenic approaches in Xenopus has led to renewed interest in this system. We have explored the applicability of tetracycline-regulated gene expression, first described by Gossen and Bujard in 1992, to the Xenopus system. By optimizing conditions, tetracycline repressor induced expression of a luciferase reporter gene was readily and reproducibly achieved in both the Xenopus oocyte and developing embryo. This high level of expression was effectively abrogated by addition of low levels of tetracycline. The significance of this newly defined system for studies of chromatin dynamics and developmental processes is discussed.