PubMed Health⌕ Search

Biomedical subjects

H R Woodland

Publications and source records attributed to H R Woodland.

At least 19 recordsLinked to original sources
PubMed2001

Initiation and early patterning of the endoderm.

We review the early stages of endoderm formation in the major animal models. In Amphibia maternal molecules are important in initiating endoderm formation. This is followed by successive signaling events that establish and then pattern the endoderm. In other organisms there are differences in endodermal development, particularly in the initial, prephylotypic stages. Later many of the same key families of transcription factors and signaling cassettes are used in all animals, but more work will be needed to establish exact evolutionary homologies.

Amphibians↗
PubMed2000

Changes in embryonic cell fate produced by expression of an endodermal transcription factor, Xsox17.

Many molecules induce the ectopic expression of tissue-specific genes in Xenopus embryos. Conversely, interfering with their activity disrupts patterns of gene expression, implicating them in normal development. Does this mean that they control cell fate (i.e. position, as well as differentiation)? Xsox17alpha and beta can induce ectopic expression of endodermal markers; inhibiting their function suppresses expression of endodermal marker genes in the developing gut (Cell 91 (1997) 397). Here we show the effect of these manipulations on cell lineage. Expressing Xsox17 in a cells normally fated to become ectoderm causes their descendants either to relocate into the embryonic gut or to die at a late developmental stage. Conversely, disrupting Xsox17 activity in cells normally fated to be endodermal causes them to enter mesodermal and ectodermal lineages.

Animals↗
PubMed1999

Mode of action of VegT in mesoderm and endoderm formation.

mRNA encoding the T-box transcription factor VegT is located throughout the vegetal pole of the Xenopus egg and is believed to play an important part in endoderm and mesoderm formation. We find that VegT generates endoderm both by cell-autonomous action and by generating TGF-beta family signals, the latter being entirely responsible for its mesoderm-inducing activity. Signalling molecules induced cell-autonomously by VegT include derrière, Xnr4 and activin B. Xnr1 and Xnr2 are also induced, but primarily in a non-autonomous manner. All of these signalling molecules are found in the blastula and gastrula vegetal pole and induce both endoderm and mesoderm in the animal cap assay, and hence are good candidates both for the endogenous zygotic mesoderm-inducing signal and for reinforcing the vegetal expression of endoderm markers.

Activins↗
PubMed1998

Xpat, a gene expressed specifically in germ plasm and primordial germ cells of Xenopus laevis.

cDNAs specific to vegetal poles of Xenopus gastrula embryos were used as a probe to screen a gastrula vegetal pole cDNA library. One of the novel clones isolated had an RNA expression pattern consistent with it being a component of germ plasm and it was thus named Xpat (Xenopus primordial germ cell associated transcript). The open reading frame encodes a 35 kDa protein with no clear homologies. The RNA is localised to the vegetal pole throughout oogenesis and early cleavage. During gastrulation cells containing this message move internally and at tailbud stages they migrate in an antero-dorsal direction. Xpat mRNA is not detectable once the dorsal mesentery forms. We show that the 3'-UTR is required and is sufficient for localisation of exogenous RNA to the vegetal pole of oocytes. We propose that Xpat UTR-containing transcripts can be localised by the Vg1 or late pathway of mRNA localisation during stage III of oogenesis, but endogenous Xpat appears to be localised earlier by a mitochondrial cloud mechanism similar to that proposed for Xcat-2.

Amino Acid Sequence↗
PubMed1998

Involvement of NF-kappaB associated proteins in FGF-mediated mesoderm induction.

In this report, we have used mRNA injection to study the action of mutants of XrelA, a Xenopus homolog of the RelA (p65) component of NF-kappaB, on the induction of mesoderm in Xenopus embryos. A region of the rel homology domain of XrelA was deleted to create XrelA deltaSP, which retains the dimerization and activation domains, but no longer binds to DNA. We also made an analogous derivative of mammalian NF-kappaB1 (p50). We show that both constructs have dominant inhibitory activity. When message encoding either is injected into eggs or oocytes, DNA binding of rel family members is suppressed, as is transactivation of a kappaB-dependent promoter in embryos. Expression of XrelA deltaSP in animal caps blocks the induction of mesoderm by bFGF. In addition, this mutant prevents elongation movements generated by activin, but has little effect on posterior dorsal cytodifferentiation, which in marked contrast is blocked by inhibition of the FGF signal transduction pathway between the receptor and MAP kinase. The specificity of the XrelA deltaSP effect on FGF signaling is shown by rescue of mesodermal marker expression when XrelA deltaSP is co-expressed with a specific rel inhibitor. The target of these dominant negative constructs seems to be neither XrelA itself, nor p50, but rather some other molecule with which XrelA, rather than NF-kappaB1, heterodimerizes. We show that XrelA deltaSP blocks FGF induction of mesoderm downstream of MAP kinase and Xbra expression. Thus it prevents the maintenance of Xbra expression by inhibiting its autoregulation by embryonic FGF (eFGF). We suggest that XrelA deltaSP differs from other reported inhibitors of FGF signaling because it inhibits only gastrula stage FGF signaling and not the maternally programmed signaling at the blastula stage. Our results therefore suggest that zygotic FGF action is required for cell movements rather than dorsal differentiation.

Activins↗
PubMed1997

Xsox17alpha and -beta mediate endoderm formation in Xenopus.

We have isolated two Xenopus relatives of murine Sox17 expressed in gastrula presumptive endoderm. Xsox17alpha and -beta expression can be induced in animal caps by activin, but not by FGF. Ectopic expression of these genes in animal caps induces the expression of endoderm markers; this induction is blocked by overexpression of a fusion of the Xsox17beta HMG domain to the Drosophila Engrailed repressor domain, as is induction of endoderm markers by activin and the expression of endodermal markers in whole embryos and isolated vegetal poles. These experiments, as well as the effects of the mRNAs on embryo phenotypes, suggest that the Xsox17 genes mediate an activin-induced endoderm differentiation pathway in animal caps and are involved in normal endoderm differentiation in embryos.

Activins↗
PubMed1996

N-acetyl-cysteine causes a late re-specification of the anteroposterior axis in the Xenopus embryo.

N-acetyl cysteine is an agent which has been shown to interrupt signal transduction processes linking a wide range of stimuli to the activation of NF-kappa B in mammalian cells. We have investigated its effect on the early development of Xenopus embryos by injecting it into blastulae, using concentrations comparable to those effective on cultured cells. High concentrations at the late blastula or early gastrula stage suppress posterior and enhance anterior development, yielding embryos with enlarged cement glands and otherwise consisting of little except head in extreme cases. Reducing the amount of N-acetyl cysteine injected leads to progressively more posterior structures developing. Injection into one- or two-cell embryos gives similar phenotypes, but of reduced severity and the cement gland is not so enlarged. Explants of animal cap cells taken several hours after injection develop to give large amounts of cement gland material. We have examined the expression of a number of genes in the anteriorised embryos. Posterior markers and Xsna are reduced. Noggin and Goosecoid mRNA are up-regulated through the gastrula and persist at these levels until at least the late neurula stage, whereas in controls Noggin is much lower and Goosecoid is absent at these stages. The most anteriorised phenotype may be a consequence of this changed expression.

Acetylcysteine↗
PubMed1995

Developmental effects of over-expression of normal and mutated forms of a Xenopus NF-kappa B homologue.

High level over-expression of XrelA1, a homologue of the p65 sub-unit of NF-kappa B and of Drosophila dorsal, arrests Xenopus development at the gastrula stage, producing a reduction in the levels of expression of various genes of developmental interest without general reduction in transcription or cessation of cell division. There is little Goosecoid expression, even though a dorsal lip forms. At lower levels XrelA1 mRNA primarily produces disruption of the mid-dorsal axis. A dominant interference gene product, delta 222, produces mainly posterior, but also anterior abnormalities. On the basis of these results we postulate that the role of XrelA1 in the vertebrate embryo is unlikely to be in dorsoventral development, but more likely in the formation of the termini.

Animals↗
PubMed1994

XrelA, a Xenopus maternal and zygotic homologue of the p65 subunit of NF-kappa B. Characterisation of transcriptional properties in the developing embryo and identification of a negative interference mutant.

We have isolated two clones (XrelA.1 and XrelA.2) from Xenopus ovary representing differentially processed mRNAs homologous throughout their translated regions to the mammalian p65 subunit of NF-kappa B. The transcripts are ubiquitously present throughout development, but are most abundant in late blastulae and gastrulae. Overproduced protein shows nuclear localisation in both oocytes and early embryos. The XrelA.2 product bound to DNA as an oligomer which was not detected in the normal embryo. Two endogenous kappa B-binding complexes were present, showing no stage-specific variation, although one was relatively deficient in posterior regions of the early neurula. They were not disrupted by dimerization with over-expressed XrelA, suggesting that they were not produced by NF-kappa B/Rel/dorsal family members. The transcriptional properties of the cloned XrelA were assayed in intact embryos by co-injecting XrelA mRNA and a linear HIV LTR-driven CAT reporter gene. CAT levels were stimulated 20-30-fold by XrelA mRNA levels in the 100 pg range, and this was wholly dependent on NF-kappa B binding sites, and largely dependent on those for SP-1. These results were remarkably reproducible and show that quantitative analysis of transcription factor function is possible in intact developing Xenopus embryos A mutant lacking the transcriptional activation domain antagonised co-injected wild-type XrelA, providing a potential dominant negative p65 mutant for interfering with NF-kappa B function in analysing NF-kappa B function in normal development.

Amino Acid Sequence↗
PubMed1993

The MyoD binding site is dispensable for cardiac actin gene expression in the somites of later stage Xenopus embryos.

We have studied the transcription directed by the promoter of a Xenopus borealis cardiac actin gene fused to a globin reporter sequence after injection into X. laevis embryos. This promoter is known to be effective specifically in somites of neurula stage embryos, and work on a very similar X. laevis gene has indicated the importance of an upstream MyoD binding site (E box) in this process. We show that, although in the absence of the MyoD site there is a small amount of tissue-specific expression in neurulae, transcription is abundant in the tailbud embryo a few hours later. Thus, although the E box is initially essential, other sequences must adopt the same role soon afterwards.

Actins↗
PubMed1993

Determination of the sequence requirements for the expression of a Xenopus borealis embryonic/larval skeletal actin gene.

In this study, we demonstrate that all sequences necessary and sufficient for the expression of a Xenopus borealis alpha 3B embryonic/larval skeletal actin gene, reside in a 156-nucleotide fragment of the promoter that spans nucleotides -197 to -42. This region of the promoter contains three imperfect repeats of the CC(A/T)6GG (CArG) box motif that have been demonstrated to be important in the expression of other sarcomeric actin genes. Deletion of the actin promoter, using Xenopus microinjection techniques as a transient assay system for promoter activity, shows that the most distal CArG box (CArG box 3) is essential for the full expression of the gene. Under our assay conditions, the most proximal CArG box (CArG box 1) exhibits two binding activities using bandshift analysis. One of these binding activities contains components antigenically related to a serum-response factor (transcription factor), whilst the second does not. In contrast, CArG box3 produces only a single retarded band using electrophoretic mobility-shift analysis. Although the shifted complex coelectrophoreses with the CArG box 1/serum-response factor complex, the band produced by CArG box3 appears to be distinct from SRF. In addition to the CArG motifs, a further upstream regulatory element has been identified in the actin promoter between nucleotides -197 and -167. In the actin promoter, a downstream region can apparently fulfil this function.

Actins↗
PubMed1992

Regulation of expression of a Xenopus borealis embryonic/larval alpha 3 skeletal-actin gene.

We have isolated a genomic clone, related in sequence to the skeletal-actin gene sub-family. It is expressed in the skeletal muscle of embryos from the neurula stage onwards and in tadpoles, but not in adults. The equivalent Xenopus laevis gene is expressed as a major transcript in adult muscle, as well as at earlier stages. The intron/exon structure is typical of vertebrate skeletal-actin genes, as is the possession of multiple copies of three serum-response elements in the promoter of this gene. The Xenopus actin and beta-globin genes were fused in their second introns. This construct, which contained 2.4 kb of upstream sequence, was injected into fertilized eggs at the two-cell stage. It showed the normal pattern of tissue-specific transcription. Thus all of the information necessary for appropriate expression of this actin gene in the embryo is contained in the region that extends from a point 2.4 kb upstream of transcription initiation to the centre of the second exon. A series of enhancer constructs were made in which upstream regions of the actin gene were placed upstream of a X. laevis beta-globin gene. The region immediately adjacent to the promoter, containing the three serum-response elements, was able to drive muscle-specific expression, and there was also a general enhancement of transcription by regions further upstream.

Actins↗
PubMed1991

Mutational analysis of the galactose binding ability of recombinant ricin B chain.

Ricin B chain (RTB) is an N-glycosylated galactose-specific lectin which folds into two globular domains. Each domain binds one galactoside. The x-ray crystallographic structure has shown that the two binding sites are structurally similar and contain key binding residues which hydrogen bond to the sugar, and a conserved tripeptide, Asp-Val-Arg. We have used oligonucleotide site-directed mutagenesis to change either the binding residues or the homologous tripeptide in one or other or in both of the sites. The 5' signal sequence and RTB coding region were excised from preproricin cDNA and fused in frame to generate preRTB cDNA. Transcripts synthesized in vitro from wild-type or mutant preRTB cloned into the Xenopus transcription vector pSP64T using SP6 RNA polymerase, were microinjected into Xenopus oocytes. The recombinant products were segregated into the oocyte rough endoplasmic reticulum and core-glycosylated, and the N-terminal signal peptide was removed. Mutating sugar binding sites individually did not abrogate the lectin activity of RTB. When both sites were changed simultaneously, RTB was produced which was soluble and stable but no longer able to bind galactose. Changing the Asn residues of the two RTB N-glycosylation sites to Gln showed that oligosaccharide side chains were essential for both the stability and biological activity of recombinant RTB.

Animals↗
PubMed1991

The effects of N-glycosylation on the lectin activity of recombinant ricin B chain.

Soluble, biologically-active recombinant ricin B chain has been produced by expressing B chain-encoding DNA in heterologous eukaryotic or prokaryotic hosts. N-Glycosylated recombinant ricin B chain expressed in Xenopus oocytes bound to both immobilized asialofetuin and immobilized lactose. Non-glycosylated ricin B chain expressed in either E. coli or in tunicamycin-treated oocytes did not bind to immobilized lactose. However, it did bind to asialofetuin, and increasing concentrations of free lactose did not reduce this asialofetuin binding dramatically, in contrast to the effect of free lactose on the binding of either glycosylated recombinant B chain or native ricin B chain.

Amino Acid Sequence↗
PubMed1989

Spatial aspects of neural induction in Xenopus laevis.

A monoclonal antibody, 2G9, has been identified and characterised as a marker of neural differentiation in Xenopus. The epitope is present throughout the adult central nervous system and in peripheral nerves. Staining is first detected in embryos at stage 21 in the thoracic region. By stage 29 it stains the whole central nervous system, except the tail tip. The epitope is present in a 65K Mr protein, and includes sialic acid. The antibody also reacts with neural tissue in mice and axolotls and newts. 2G9 was used to show that both notochord and somites are capable of neural induction, and the stimulus is present as late as stage 22. Attempts to demonstrate the induction of nervous system by developing nervous system (homoiogenetic induction) were unsuccessful. The view that the lateral extent of the nervous system might be determined by that of the inductive stimulus is discussed. Neural induction was detected as early as stage 10 and occurs in embryos without gastrulation and without cell division from stage 7 1/2.

Animals↗
PubMed1988

The organisation and expression of histone genes from Xenopus borealis.

We have isolated genomic clones from Xenopus borealis representing 3 different types of histone gene cluster. We show that the major type (H1, H2B, H2A, H4, H3), present at about 60-70 copies per haploid genome (1), is tandemly reiterated with a repeat length of 15 kb. In situ hybridization to mitotic chromosomes shows that the majority of histone genes in Xenopus borealis are at one locus. This locus is on the long arm of one of the small sub-metacentric chromosomes. A minor cluster type with the gene order H1, H3, H4, H2A is present at about 10-15 copies. The genome also contains rare or unique cluster types present at less than 5 copies having other types of organisation. An isolate of this type had the gene order H1, H4, H2B, H2A, H1 (no H3 cloned). Microinjection of all of the clones into Xenopus laevis oocyte nuclei shows that most of the genes present are functional or potentially functional and a number of variant histone proteins have been observed. S1 mapping experiments confirm that the genes of the major cluster are expressed in all tissues and at all developmental stages examined.

Animals↗