Isolation of a novel RXR from Xenopus that most closely resembles mammalian RXR beta and is expressed throughout early development.

In a search for nuclear receptors that may mediate the teratogenic effects of the potential morphogen, retinoic acid, on the early development of Xenopus we have isolated a novel Xenopus RXR that most closely resembles the mammalian beta-type RXR. Xenopus RXR beta mRNA is expressed throughout early embryogenesis, and functions as an accessory protein to enhance the DNA-binding of other members of the nuclear receptor superfamily.

Activity of a cloned Xenopus albumin gene promoter in the homologous frog oocyte system.

The activity of the liver-specific promoter from the Xenopus laevis 68 kDa albumin gene in homologous oocytes has been analysed by microinjection. We find that the albumin promoter functions relatively efficiently in oocytes, directing the synthesis of correctly initiated transcripts, and deletion analysis reveals that only a small amount of upstream sequence is required for full activity.

Expression pattern of a novel hyaluronidase during Xenopus embryogenesis.

We have isolated a cDNA encoding a novel hyaluronidase, which is expressed during embryogenesis. The encoded protein was expressed as a fusion polypeptide with glutathione S-transferase, and the affinity-purified fusion protein was shown to possess hyaluronidase activity with a pH optimum about pH 4.0. The expression of the XEH1 gene was analysed by in situ hybridization, and was first apparent in scattered cells in a broad ventral region of late gastrula embryos. As development proceeded through to tailbud stages, the domain of expression became progressively more restricted, eventually being located in the developing liver rudiment near the primary hepatic cavity. The results reveal the dynamic regulation of the contrasting activities of hyaluronan synthesis and degradation during early morphogenetic movements.

Xenopus Enhancer of Zeste (XEZ); an anteriorly restricted polycomb gene with a role in neural patterning.

We have identified the Xenopus homologue of Drosophila Enhancer of Zeste using a differential display strategy designed to identify genes involved in early anterior neural differentiation. XEZ codes for a protein of 748 amino acids that is very highly conserved in evolution and is 96% identical to both human and mouse EZ(H)2. In common with most other Xenopus Pc-G genes and unlike mammalian Pc-G genes, XEZ is anteriorly restricted. Zygotic expression of XEZ commences during gastrulation, much earlier than other anteriorly localized Pc-G genes; expression is restricted to the anterior neural plate and is confined later to the forebrain, eyes and branchial arches. XEZ is induced in animal caps overexpressing noggin; up-regulation of XEZ therefore represents a response to inhibition of BMP signalling in ectodermal cells. We show that the midbrain/hindbrain junction marker En-2,and hindbrain marker Krox-20, are target genes of XEZ and that XEZ functions to repress these anteroposterior marker genes. Conversely, XEZ does not repress the forebrain marker Otx-2. XEZ overexpression results in a greatly thickened floor of the forebrain. These results implicate an important role for XEZ in the patterning of the nervous system.

Expression pattern of BXR suggests a role for benzoate ligand-mediated signalling in hatching gland function.

The Xenopus laevis nuclear receptor BXR has recently been shown to be activated by a class of endogenous benzoate metabolites, indicating the presence of a novel and unsuspected benzoate ligand-dependent signalling pathway. The receptor is expressed ubiquitously in blastula and gastrula stage embryos, and its expression declines during neurula stages. In order to examine further this novel vertebrate signalling system, we have examined the expression of the BXR gene in tailbud stage embryos and adults. We show here that in Xenopus tailbud stage embryos expression is restricted to the hatching gland, suggesting a role in hatching gland function. Neither BXR nor a BXR-VP16 fusion is sufficient to specify hatching gland in neurally-induced tissue. In adults, BXR expression is abundant in the brain and gonads. This expression pattern in adults is distinct from any of the putative mammalian homologues. A nuclear receptor that mediates benzoate signalling has yet to be found in mammals.

Effects of retinoic acid on Xenopus embryos.

Retinoic acid (RA) has powerful dose-dependent dysmorphogenic effects on Xenopus embryos. The defects produced are dependent upon the stage and duration of treatment. Characteristic dysmorphogenic effects occur in the visceral (branchial) arch region and the tail. These regions correspond to the domains of expression of the retinoid receptors RAR gamma and RXR beta. By expressing domain-swapped and dominant negative derivatives of RAR gamma in embryos, we have shown that the effects of RA are mediated transcriptionally. Furthermore, the expression of dominant negative receptors in the absence of exogenous ligand has no apparent harmful effect upon early development. Finally, we have identified a novel MAP kinase phosphatase whose expression pattern is localized to regions similar to RAR gamma, and which is upregulated by RA.

Xenopus laevis Oct-1 does not bind to certain histone H2B gene promoter octamer motifs for which a novel octamer-binding factor has high affinity.

Oct-1 and a second, previously unidentified octamer-binding protein (Oct-R) have been identified in extracts of Xenopus laevis oocytes and embryos. Oct-1 does not bind to the octamer motif associated with certain Xenopus laevis histone H2B gene promoters, whereas Oct-R binds well to this motif, but only in the sequence context of the H2B gene promoter.

Trans-activation of transcription, from promoters containing immunoglobulin gene octamer sequences, by myeloma cell mRNA in Xenopus oocytes.

To study factors required for immunoglobulin gene transcription hybrid promoters were made by linking octamer elements to a Xenopus albumin gene construct containing only 50bp of the albumin gene promoter. When injected into oocytes these hybrid promoters directed transcription far less efficiently than the unmodified 50bp albumin gene promoter fragment. Activity of the hybrid promoter, but not the unmodified albumin promoter, could be stimulated by preinjection of poly(A)+ RNA from NS1 myeloma cells. This stimulation may be caused by translation of the NS1 poly(A)+ RNA into transcription factors that act on the octamer. Both the reduction in transcription caused by octamer insertion and the extent of the inducibility by NS1 RNA are greater when two, rather than one, octamers are inserted.

Regulation of the early expression of the Xenopus nodal-related 1 gene, Xnr1.

The Xenopus nodal related-1 (Xnr1) gene has a complex expression pattern in embryos, with two temporal phases. In the first phase, transcripts are first detected in perinuclear sites in the vegetal region of the blastula. During gastrulation, this expression disappears and transcripts become localised to the dorsal marginal zone. Expression stops and then restarts in a second phase at neurula and tailbud stages, firstly in two symmetric patches near the posterior end of the notochord, and then asymmetrically in a large domain in the left lateral plate mesoderm. In this study, we have investigated the regulation of the early phase of expression of Xnr1. We show that the T-box transcription factor VegT can induce Xnr1. It had previously been shown that Xnr1 can induce VegT in ectoderm cells and we show that the early expression of Xnr1 is regulated by an autoregulatory loop. By inspection of the Xnr1 promoter sequence, we have identified two non-palindromic T-box-binding sites, which are 10 bp apart. Using mutational analysis, we have shown that these elements are required for the VegT induction of Xnr1. The Xnr1 promoter shows striking homologies with the Xnr3 promoter. In particular, two elements that are required for Wnt signaling are conserved between these two promoters, but the two T-box sites are not conserved, and Xnr3 is not induced by VegT. A region of the promoter containing the T-box sites and the Wnt sites is sufficient to drive expression of a reporter gene in a dorsal domain in transgenic Xenopus at the gastrula stage. We show that this pattern of expression of the transgene in gastrulae is not dependent on the T-box sites.

In situ hybridization to lampbrush chromosomes: a potential source of error exposed.

Denatured 3H-labelled DNAs containing Xenopus or human globin sequences hybridize to RNA transcripts on a single pair of lateral loops on lampbrush chromosome IX of Triturus cristatus carnifex, and to no other loops on this chromosome or the rest of the complement. However they do so, not because of the globin sequences in the probes, but rather because the plasmids from which the probes were prepared were constructed with G.C homopolymer tails. Simple sequence poly d(C/G)n probes also hybridize with RNA transcripts on this same pair of loops, and with no others.

Structure and functional expression of a cloned Xenopus thyroid hormone receptor.

A clone corresponding to a thyroid hormone receptor was isolated from a Xenopus laevis cDNA library prepared from folliculated oocytes. The cDNA encodes a protein of 418 amino acid residues with a domain structure, including a putative DNA binding region with two zinc fingers, similar to other members of the v-erbA-related superfamily of receptors. The encoded protein resembles the TR alpha 1-type receptor of the rat. When expressed in COS cells the protein product binds triiodothyronine with a Kd of 0.12 nM. The receptor mediates thyroid-hormone-inducible expression of a reporter gene which includes a thyroid hormone response element in its upstream region.

Neural induction and patterning by fibroblast growth factor, notochord and somite tissue in Xenopus.

Two natural neural inducing sources have been used, the notochord and the somites together with the growth factor bFGF, to investigate the anterior/posterior patterning of neural tissue in an animal cap explant model in Xenopus laevis. Notochord and somite tissue from stages 12.5/13 and 16, respectively, were manually isolated, and combined heterochronically with responding animal cap ectoderm aged to gastrula stages. Somite recombinants were also constructed with animal caps injected with noggin mRNA. The responses of the ectoderm were analyzed by reverse transcription polymerase chain reaction (RT-PCR) detection of marker gene expression, and in some cases by in situ hybridization. The requirement for FGF receptor function was analyzed using the dominant negative FGF receptor (XFD). The experiments showed that bFGF is capable of direct neural induction in caps aged to stage 10.5. It was also shown that notochords are capable of inducing anterior neural tissue in gastrula stage animal cap ectoderm, and this induction is sensitive to XFD in the responding tissue. Injection of noggin mRNA results in the induction of anterior neural differentiation, and it was demonstrated that this induction was insensitive to the expression of XFD in the responding tissue. It was also shown that somite tissue recombined with gastrula stage animal cap ectoderm, can induce both anterior and posterior nervous tissue and can also posteriorize noggin-induced anterior neural tissue when combined with noggin-injected animal cap ectoderm. This response is partially sensitive to XFD expression. The results shed light on the role of competence of animal cap ectoderm and the signals from postgastrulation axial and paraxial mesoderm in the patterning of the amphibian nervous system.

Nitric oxide synthase expression and steroid regulation in the uterus of women with menorrhagia.

Menorrhagia (excessive menstrual bleeding) is a common clinical problem of unknown aetiology. The free-radical and vasodilator nitric oxide (NO) relaxes the myometrial smooth muscle and is a strong candidate for the cause of excessive blood loss in menorrhagic patients. The aim of this study was to measure NO production in women with and without menorrhagia to detect nitric oxide synthase (NOS) isoforms in uterine cells and to investigate any steroid effects on myometrial NOS expression. We showed for the first time that menorrhagic endometrium produces significantly higher amounts of NOx (the sum of NO(2-) and NO(3-)) than control endometrium (P < 0.01). Inducible NOS (iNOS) protein was detected by immunoblotting in endometrial and myometrial tissue extracts. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) experiments revealed an induction of myometrial smooth muscle endothelial NOS (eNOS) expression by progesterone and 17beta-oestradiol, while myometrial iNOS expression was unaffected by steroid hormones. These results are consistent with the hypothesis that NO plays a role in excessive menstrual bleeding and provide the first evidence on steroid regulation of eNOS in the human non-pregnant uterus.

Localization of histone gene transcripts in newt lampbrush chromosomes by in situ hybridization.

Denatured 3H-labelled DNAs containing histone gene sequences originating from the echinoderms Psammechinus miliaris, Echimus esculentus and Strongylocentrotus purpuratus have been in situ hybridized to RNA transcripts on newt lampbrush chromosomes. Autoradiographs of the hybridized lampbrush preparations show labelling restricted to four or fewer lateral loop pairs all lying within the heteromorphic regions of chromosome I, also one or two loop pairs on chromosome VI, one loop pair on chromosome X and one loop pair on chromosome XI. For oocytes from a single newt, coincident label distribution is found with DNA's of diverse echinoderm origin; however different newts show some specific individual diversity in label distribution, including heterozygosity in the case of loops on bivalents VI and X. The more conspicuously labelled loops, particularly those on chromosome I, show a pattern of labelling which is explicable if the newt histone DNA sequences are confined to short intercepts of lateral loop axis. Transcription is initiated prior to the histone DNA sequences, proceeds through the histone DNA sequences, and beyond, and the histone RNA sequences are cut from the transcripts before the termination of transcription.

A novel nuclear receptor superfamily member in Xenopus that associates with RXR, and shares extensive sequence similarity to the mammalian vitamin D3 receptor.

We report the isolation of xONR1, a novel member of the nuclear receptor superfamily from Xenopus laevis. xONR1 shares a high degree of amino acid sequence identity with the mammalian receptor for 1 alpha, 25-dihydroxy vitamin D3, particularly within the DNA-binding domain, although it does not bind this ligand. xONR1 DNA binding is stimulated by association with retinoid X receptor gamma (RXR gamma).

Mesoderm induction in Xenopus caused by activation of MAP kinase.

Mesoderm induction is a critical early step in vertebrate development, involving changes in gene expression and morphogenesis. In Xenopus, normal mesoderm formation depends on signalling through the fibroblast growth factor (FGF) tyrosine kinase receptor. One important signalling pathway from receptor tyrosine kinases involves p21ras (ref. 5). Ras associates with the serine kinase c-Raf-1 in a GTP-dependent manner, and this complex phosphorylates and activates MAPK/ERK kinase (MEK), a protein kinase with dual specificity. MEK then activates p42mapk and (at least in mammals) p44mapk, members of the mitogen-activated protein (MAP) kinase family. FGF activates MAP kinase during mesoderm induction, and the use of dominant-negative constructs suggests that mesoderm induction by FGF requires both Ras and Raf. However, these experiments do not reveal whether Ras and Raf do act through MAP kinase to induce mesoderm or whether another pathway, such as the phosphatidylinositol 3-kinase cascade, is involved. Here we show that expression of active forms of MEK or of MAP kinase induces ventral mesoderm of the kind elicited by FGF. Overexpression of a Xenopus MAP kinase phosphatase blocks mesoderm induction by FGF, and causes characteristic defects in mesoderm formation in intact embryos, whereas inhibition of the P13 kinase and p70 S6 kinase pathways has no effect on mesoderm induction by FGF. FGF induces different types of mesoderm in a dose-dependent manner; strikingly, this is mimicked by expressing different levels of activated MEK. Together, these experiments demonstrate that activation of MAP kinases is necessary and sufficient for mesoderm formation.

Lipopolysaccharide treatment in vivo induces widespread tissue expression of inducible nitric oxide synthase mRNA.

Nitric oxide (NO) may mediate the hypotension of septic shock, but the effect of endotoxin on inducible NO synthase (iNOS) mRNA expression remains unclear. We studied the effects of lipopolysaccharide (LPS) treatment in vivo on iNOS mRNA expression using reverse transcription and polymerase chain reaction. The iNOS mRNA was absent or negligible in any tissue studied from control rats, but was markedly increased in lung, liver, spleen, skeletal muscle and kidney from LPS-treated rats. The LPS-induced increase in iNOS mRNA was prevented by dexamethasone. Our results indicate that LPS treatment in vivo induces the expression of an iNOS mRNA via a dexamethasone-sensitive mechanism, and thus provide direct molecular evidence for the involvement of NO in septic shock.

Nucleotide sequences of H1 histone genes from Xenopus laevis. A recently diverged pair of H1 genes and an unusual H1 pseudogene.

Four clones containing H1 histone gene sequences were previously isolated from a Xenopus laevis genomic library (1) and we now present the complete nucleotide sequences of these H1 genes and their flanking regions. Two of these genes code for minor H1 proteins, probably H1C, when expressed in the oocyte transcription/translation system and are present on clones with almost identical overall organization. However, at the nucleotide level these genes differ in showing base insertions and deletions, as well as substitutions. A third gene sequence which is more related to the major X. laevis H1A, corresponds to the 3' two thirds of an H1 gene. This gene has in place of a 5' coding region at least 1800 bp of apparently noncoding sequence, some of which is A-T rich. The junction does not correspond to the consensus sequence of an intron/exon boundary and therefore this H1 sequence is more likely to represent a pseudogene. Comparisons of the coding and flanking regions of these X. laevis H1 genes indicate the kind of differences which can occur among H1 subtypes within a species. A region of homology noted in the 3' noncoding portion of vertebrate histone genes is discussed in relation to the mechanism of termination of transcription.