Robert W. Old

Analysis of Fcγ receptor haplotypes in rheumatoid arthritis: FCGR3A remains a major susceptibility gene at this locus, with an additional contribution from FCGR3B

The Fcγ receptors play important roles in the initiation and regulation of many immunological and inflammatory processes, and genetic variants (FCGR) have been associated with numerous autoimmune and infectious diseases. The data in rheumatoid arthritis (RA) are conflicting and we previously demonstrated an association between FCGR3A and RA. In view of the close molecular proximity with FCGR2A, FCGR2B and FCGR3B, additional polymorphisms within these genes and FCGR haplotypes were examined to refine the extent of association with RA. Biallelic polymorphisms in FCGR2A, FCGR2B and FCGR3B were examined for association with RA in two well characterized UK Caucasian and North Indian/Pakistani cohorts, in which FCGR3A genotyping had previously been undertaken. Haplotype frequencies and linkage disequilibrium were estimated across the FCGR locus and a model-free analysis was performed to determine association with RA. This was followed by regression analysis, allowing for phase uncertainty, to identify the particular haplotype(s) that influences disease risk. Our results reveal that FCGR2A, FCGR2B and FCGR3B were not associated with RA. The haplotype with the strongest association with RA susceptibility was the FCGR3A–FCGR3B 158V-NA2 haplotype (odds ratio 3.18, 95% confidence interval 1.13–8.92 [P = 0.03] for homozygotes compared with all genotypes). The association was stronger in the presence of nodules (odds ratio 5.03, 95% confidence interval 1.44–17.56; P = 0.01). This haplotype was also more common in North Indian/Pakistani RA patients than in control individuals, but not significantly so. Logistic regression analyses suggested that FCGR3A remained the most significant gene at this locus. The increased association with an FCGR3A–FCGR3B haplotype suggests that other polymorphic variants within FCGR3A or FCGR3B, or in linkage disequilibrium with this haplotype, may additionally contribute to disease pathogenesis.

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 differnetiation, 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.

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).

VegT activation of the early zygotic gene Xnr5 requires lifting of Tcf-mediated repression in the Xenopus blastula

Xenopus Nodal-related (Xnr) 5 is one of the earliest expressed components of a network of TGF-beta factors participating in endoderm and mesoderm formation. Zygotic gene expression is not required for induction of Xnr5; rather, expression is dependent on the maternal factors VegT, localised throughout the vegetal pole, and beta-catenin, functional in the future dorsal region of the embryo. Using transient assays with a luciferase reporter in Xenopus embryos, we have defined a minimal promoter, which mimics the response of the endogenous gene to applied factors. Expression of luciferase from the minimal promoter is dorsal-specific and requires two T-box half sites and a functional beta-catenin/XTcf-3 pathway. Mutation of two Tcf/Lef sites in the minimal promoter permits induction by VegT to wild-type promoter levels in the presence of a dominant-negative XTcf-3, indicating that beta-catenin/XTcf-3 are repressive and are not required as transactivators of Xnr5 transcription. The activity of the Tcf/Lef mutant promoter is similar in both ventral and dorsal sides of the embryo. In transgenic experiments, the dorsal specificity of expression of a beta-gal reporter driven by the wild-type minimal promoter is abolished upon mutation of these Tcf/Lef sites. We propose a model in which XTcf-3 functions as a repressor of Xnr5 throughout the blastula embryo, except where repression is lifted by the binding of beta-catenin in the dorsal region. This removal of repression allows activation of the promoter by VegT in the dorsal vegetal region. Subsequently, zygotically expressed LEF1 supersedes the role of beta-catenin/XTcf-3.

Isolation of a novel RXR from Xenopus that most closely resembles mammalian RXRβ 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.

Non-β-cell progenitors of β-cells in pregnant mice

Pregnancy is a normal physiological condition in which the maternal β-cell mass increases rapidly about two-fold to adapt to new metabolic challenges. We have used a lineage tracing of β-cells to analyse the origin of new β-cells during this rapid expansion in pregnancy. Double transgenic mice bearing a tamoxifen-dependent Cre-recombinase construct under the control of a rat insulin promoter, together with a reporter Z/AP gene, were generated. Then, in response to a pulse of tamoxifen before pregnancy, β-cells in these animals were marked irreversibly and heritably with the human placental alkaline phosphatase (HPAP). First, we conclude that the lineage tracing system was highly specific for β-cells. Secondly, we scored the proportion of the β-cells marked with HPAP during a subsequent chase period in pregnant and non-pregnant females. We observed a dilution in this labelling index in pregnant animal pancreata, compared to non-pregnant controls, during a single pregnancy in the chase period. To extend these observations we also analysed the labelling index in pancreata of animals during the second of two pregnancies in the chase period. The combined data revealed statistically-significant dilution during pregnancy, indicating a contribution to new beta cells from a non-β-cell source. Thus for the first time in a normal physiological condition, we have demonstrated not only β-cell duplication, but also the activation of a non-β-cell progenitor population. Further, there was no transdifferentiation of β-cells to other cell types in a two and half month period following labelling, including the period of pregnancy.

Unequal representation of different unique genomic DNA sequences in the cell-free plasma DNA of individual donors

OBJECTIVES To assess whether different genomic cell-free DNAs are equally abundant in the plasma of individual donors, and any relationship between DNA methylation and representation in plasma. DESIGN AND METHODS The concentrations of DNA in plasma were determined by real-time PCR. RESULTS Different DNA sequences were not equally represented. The relative abundances were similar in different donors. CONCLUSIONS Different DNA sequences are not equally abundant in plasma, with no relationship between DNA methylation and abundance.

A novel MAP kinase phosphatase is localised in the branchial arch region and tail tip of Xenopus embryos and is inducible by retinoic acid

Using a differential display strategy, we have isolated a cDNA corresponding to a mRNA which is induced by retinoic acid treatment of late gastrula Xenopus embryos, and much more strongly induced by retinoic acid and cycloheximide. The cDNA, designated X17C, encodes a novel mitogen-activated protein (MAP) kinase phosphatase of 378 amino acid residues which is only distantly related to other known MAP kinase phosphatases. In normal embryogenesis, the X17C mRNA is expressed after the midblastula transition and accumulates during gastrulation. In neurula and tailbud stage embryos the mRNA is localised in two domains, one in the anterior region of the embryo, and one at the tail tip. When expressed from synthetic mRNA injected into oocytes, the X17C protein is found within the cytosolic fraction and not in the nucleus. The X17C protein dephosphorylates and inactivates Xenopus MAP kinase in oocytes stimulated to undergo maturation by progesterone. We indicate the application of X17C as a tool for interfering with MAP kinase signaling in somatic cells of embryos, using FGF receptor-mediated MAP kinase activation in animal cap explants.

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 of a dominant negative retinoic acid receptor γ in Xenopus embryos leads to partial resistance to retinoic acid

The administration of the teratogen retinoic acid (and other retinoids) to vertebrate embryos causes a range of developmental abnormalities. It remains to be shown how these teratogenic effects are mediated, and whether or not they reflect any morphogenetic roles retinoids may have in normal development. The most intensively studied cellular action of retinoids has been the activation of retinoid receptors (RARs and RXRs), which are members of the steroid/thyroid family of ligand-modulated transcription factors. Here we report experiments designed to investigate whether the teratogenic effects of retinoic acid on early Xenopus embryos are mediated transcriptionally by receptors, and if these receptors are necessary for normal early Xenopus development. We have demonstrated transcriptional activation of injected reporter genes by exogeneously supplied retinoic acid in Xenopus embryos, presumably as a consequence of the activation of endogenous retinoid receptors. This assay system has been used to demonstrate functional expression, from injected mRNA of (1) a wild-type RARγ (2) a domain-swapped receptor in which the retinoic acid binding domain has been replaced by a thyroid hormone domain to create a thyroid hormone responsive receptor, and (3) a dominant negative from of the RARγ. The wild-type RARγ increases the severity of retinoic acid-mediated defects. In the presence of thyroid hormone the domain-swapped receptor causes abnormalities of gastrulation. The dominant negative decreases the severity of retinoic acid-mediated defects. We conclude that the teratogenic effects of exogenous retinoic acid on Xenopus embryos are mediated, at least in part, transcriptionally via retinoid receptors. It is notable that the dominant negative has no effect on normal development in the absence of exogenous retinoic acid. This is despite observations that this receptor completely blocks transcriptional activation of reporter genes by exogenous retinoic acid up to the beginning of gastrulation, and substantially relieves the teratogenic effects of retinoic acid.