Analysis of competence and of Brachyury autoinduction by use of hormone-inducible Xbra

Development ◽  
1997 ◽  
Vol 124 (11) ◽  
pp. 2225-2234 ◽  
Author(s):  
M. Tada ◽  
M.A. O'Reilly ◽  
J.C. Smith
Keyword(s):  
Glucocorticoid Receptor ◽  
Target Genes ◽  
Early Embryo ◽  
Fate Map ◽  
Animal Pole ◽  
Reading Frame ◽  
Xenopus Development ◽  
Series Of Experiments ◽  
Inducing Factors ◽  
Mesoderm Inducing Factors

Analysis of gene function in Xenopus development frequently involves over-expression experiments, in which RNA encoding the protein of interest is microinjected into the early embryo. By taking advantage of the fate map of Xenopus, it is possible to direct expression of the protein to particular regions of the embryo, but it has not been possible to exert control over the timing of expression; the protein is translated immediately after injection. To overcome this problem in our analysis of the role of Brachyury in Xenopus development, we have, like Kolm and Sive (1995; Dev. Biol. 171, 267–272), explored the use of hormone-inducible constructs. Animal pole regions derived from embryos expressing a fusion protein (Xbra-GR) in which the Xbra open reading frame is fused to the ligand-binding domain of the human glucocorticoid receptor develop as atypical epidermis, presumably because Xbra is sequestered by the heat-shock apparatus of the cell. Addition of dexamethasone, which binds to the glucocorticoid receptor and releases Xbra, causes formation of mesoderm. We have used this approach to investigate the competence of animal pole explants to respond to Xbra-GR, and have found that competence persists until late gastrula stages, even though by this time animal caps have lost the ability to respond to mesoderm-inducing factors such as activin and FGF. In a second series of experiments, we demonstrate that Xbra is capable of inducing its own expression, but that this auto-induction requires intercellular signals and FGF signalling. Finally, we suggest that the use of inducible constructs may assist in the search for target genes of Brachyury.

scholarly journals The Xenopus MyoD gene: an unlocalised maternal mRNA predates lineage-restricted expression in the early embryo

Development ◽  
1990 ◽  
Vol 108 (4) ◽  
pp. 669-680 ◽  
Author(s):  
R.P. Harvey
Keyword(s):  
Gene Expression ◽  
Cultured Cells ◽  
Early Response ◽  
Early Embryo ◽  
Mesoderm Induction ◽  
Animal Pole ◽  
Early Expression ◽  
Inducing Factors ◽  
Mesoderm Inducing Factors ◽  
Myod Gene

Expression of the mouse MyoD gene appears to represent a critical point in the commitment of cultured cells to muscle. In Xenopus, myogenic commitment begins during mesoderm induction which is initiated early in development by endogenous growth factors. To study MyoD gene expression during induction, a Xenopus MyoD gene and homologous cDNAs were selected from Xenopus libraries and analysed. Two different cDNAs have been sequenced. They code for proteins closely related to each other and to mouse MyoD and are likely to be expressed from duplicated Xenopus MyoD genes. Surprisingly, MyoD mRNA is first detected during oogenesis and the maternal species is not localized exclusively to the region of the blastula fated to muscle. Zygotic MyoD mRNA accumulates slowly above maternal levels beginning at the MBT and new transcripts are localized to the somitic mesoderm. Expression outside of somites has been detected in developing heads of tailbud embryos and can be induced in blastula animal pole explants treated with mesoderm-inducing factors. The early expression of MyoD in Xenopus development suggests that it may play a part in the induction of muscle mesoderm and generally strengthens the evidence that MyoD is determinative in muscle commitment. In addition, the initiation of MyoD transcription at the MBT and its stimulation by mesoderm-inducing factors implies that MyoD gene expression is an immediate early response to mesoderm induction.

Bix1, a direct target of Xenopus T-box genes, causes formation of ventral mesoderm and endoderm

Development ◽  
1998 ◽  
Vol 125 (20) ◽  
pp. 3997-4006 ◽  
Author(s):  
M. Tada ◽  
E.S. Casey ◽  
L. Fairclough ◽  
J.C. Smith
Keyword(s):  
Target Genes ◽  
Regulatory Region ◽  
Homeobox Gene ◽  
Subtractive Hybridization ◽  
Vertebrate Development ◽  
Transcription Activator ◽  
T Box ◽  
Endodermal Differentiation ◽  
Inducing Factors ◽  
Mesoderm Inducing Factors

Brachyury, a member of the T-box gene family, is required for posterior mesoderm and notochord differentiation in vertebrate development, and mis-expression of Xenopus Brachyury causes ectopic mesoderm formation. Brachyury is a transcription activator, and its ability to activate transcription is essential for its biological function, but Brachyury target genes have proved difficult to identify. Here we employ a hormone-inducible Brachyury construct and subtractive hybridization to search for such targets. Using this approach we have isolated Bix1, a homeobox gene expressed both in the marginal zone of Xenopus and in the vegetal hemisphere. Expression of Bix1 is induced in an immediate-early fashion by mesoderm-inducing factors such as activin as well as by the products of the T-box genes Xbra and VegT (also known as Antipodean, Brat and Xombi). Activation of Bix1 in response to Xbra is direct in the sense that it does not require protein synthesis, and both Xbra and VegT activate expression of a reporter gene driven by the Bix 5′ regulatory region, which contains an Xbra/VegT binding site. Mis-expression of low levels of Bix1 causes formation of ventral mesoderm, while high levels induce endodermal differentiation. These results suggest that Bix1 acts downstream of both VegT and Xbra to induce formation of mesoderm and endoderm.

Normal fates and states of specification of different regions in the axolotl gastrula

Development ◽  
1985 ◽  
Vol 86 (1) ◽  
pp. 247-269
Author(s):  
J. Herman Cleine ◽  
Jonathan M. W. Slack
Keyword(s):  
Marginal Zone ◽  
Culture Conditions ◽  
Early Embryo ◽  
Ambystoma Mexicanum ◽  
Specific Marker ◽  
Fate Map ◽  
Animal Pole ◽  
Glycoprotein Synthesis ◽  
Early Gastrula

A fate map was constructed for four regions of the early gastrula of Ambystoma mexicanum using orthotopic grafts from donors labelled with FLDx (fluoresceinated-lysinated-dextran). The region around the animal pole gave rise to epidermis only and did not include prospective neural plate. The dorsal marginal zone contributed to cephalic endoderm and to the whole length of the axial mesoderm (notochord and somites), the lateral marginal zone to lateroventral and somitic mesoderm, and the ventral marginal zone to lateroventral mesoderm. It was found that the dorsal marginal zone contributed relatively more to the anterior regions of the mesodermal mantle and the ventral marginal zone more to its posterior parts. The same regions of the gastrula and also vegetal yolky tissue were cultured as explants and labelled with tritiated mannose. Their glycoprotein synthesis pattern was compared to those of the neurula tissues to which they contribute in vivo. Animal pole explants synthesized large amounts of the epidermis-specific marker epimucin. Dorsal marginal zone explants did not synthesize epimucin but did make amounts of S2 and S6 indicative of mesoderm, as well as the notochord-specific markers S2·2 and S3·2. Lateral marginal zone explants showed the same pattern as the dorsal marginal zone including the two notochord-specific markers, although they do not contribute to notochord in vivo. Ventral marginal zone explants were more variable in their behaviour. Yolky tissue from the vegetal hemisphere of the gastrula or the archenteron floor of the neurula synthesized mainly polydisperse material of high molecular weight rather than discrete glycoproteins. The results indicate that at the early gastrula stage states of specification exist which correspond to the three germ layers, ecto-, meso- and endoderm. The ectodermal specification of animal pole explants is quite robust and cannot easily be changed by variation of the culture conditions. However treatment with a concentrated pellet of vegetalizing factor does induce a change to mesodermal specification, which is clearly detectable in the pattern of glycoprotein synthesis. Similar inductive interactions between different regions of the early embryo are thought to occur during normal development.

scholarly journals Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

2008 ◽  
Vol 22 (8) ◽  
pp. 1754-1766 ◽  
Author(s):  
Weiwei Chen ◽  
Thoa Dang ◽  
Raymond D. Blind ◽  
Zhen Wang ◽  
Claudio N. Cavasotto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Divalent Cations ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

scholarly journals Quantitative comparison of mesoderm inducing factors in xenopus

1989 ◽  
Vol 27 ◽  
pp. 53
Author(s):  
J.B.A. Green ◽  
G. Howes ◽  
M. Yaqoob ◽  
J. Cooke ◽  
J.C. Smith
Keyword(s):  
Quantitative Comparison ◽  
Inducing Factors ◽  
Mesoderm Inducing Factors

Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis

1992 ◽  
Vol 12 (2) ◽  
pp. 589-597
Author(s):  
E S Dieken ◽  
R L Miesfeld
Keyword(s):  
Transcriptional Regulation ◽  
Glucocorticoid Receptor ◽  
Target Genes ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Transactivation Domain ◽  
Lymphocyte Apoptosis ◽  
Murine Cell Line ◽  
N Terminus ◽  
Simplex Virus

Genetic studies have suggested that transcriptional regulation of specific target genes (by either induction or repression) is the molecular basis of glucocorticoid-mediated lymphocyte apoptosis. To examine the role of transcriptional regulation more directly, we developed a complementation assay utilizing stable transfection of wild-type (wt) and mutant (nti) glucocorticoid receptor (GR) cDNA constructs into a GR-deficient S49 murine cell line (7r). Our data confirm that the level of functional GR is rate limiting for S49 apoptosis and moreover that the GR amino terminus (N terminus), which as been deleted from the nti GR, is absolutely required for complementation in this system. Surprisingly, we found that at physiological levels of receptor, expression of the nti GR in cells containing wt GR results in enhanced dexamethasone sensitivity rather than a dominant negative phenotype. One interpretation of these data is that DNA binding by wt-nti heterodimers may be functionally similar to that of wt-wt homodimers, indicating that GRE occupancy by at least one transactivation domain may be sufficient to induce the hormonal response. To determine whether acidic activating sequences such as those localized to the GR N terminus are important in the induction of lymphocyte apoptosis, we tested the activity of a chimeric receptor in which we replaced the entire GR N terminus with sequences from the herpes simplex virus VP16 protein. Our results demonstrate that 7r cells expressing VP-GR fusions are indeed steroid sensitive, strongly supporting the idea that S49 apoptosis is dependent on transcriptional regulation of specific genes which respond to acidic activating domains, implying that induction, rather than repression, may be the critical initiating event.

scholarly journals From The Cover: Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes

2004 ◽  
Vol 101 (44) ◽  
pp. 15603-15608 ◽  
Author(s):  
J.-C. Wang ◽  
M. K. Derynck ◽  
D. F. Nonaka ◽  
D. B. Khodabakhsh ◽  
C. Haqq ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Chromatin Immunoprecipitation ◽  
Target Genes

scholarly journals Aberrant corticosteroid metabolism in tumor cells enables GR takeover in enzalutamide resistant prostate cancer

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jianneng Li ◽  
Mohammad Alyamani ◽  
Ao Zhang ◽  
Kai-Hsiung Chang ◽  
Michael Berk ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Glucocorticoid Receptor ◽  
Tumor Cells ◽  
Target Genes ◽  
Hydroxysteroid Dehydrogenase ◽  
Mouse Xenograft ◽  
Ubiquitin E3 Ligase ◽  
Metabolic Mechanism ◽  
Stimulation Of

Prostate cancer is driven by androgen stimulation of the androgen receptor (AR). The next-generation AR antagonist, enzalutamide, prolongs survival, but resistance and lethal disease eventually prevail. Emerging data suggest that the glucocorticoid receptor (GR) is upregulated in this context, stimulating expression of AR-target genes that permit continued growth despite AR blockade. However, countering this mechanism by administration of GR antagonists is problematic because GR is essential for life. We show that enzalutamide treatment in human models of prostate cancer and patient tissues is accompanied by a ubiquitin E3-ligase, AMFR, mediating loss of 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2), which otherwise inactivates cortisol, sustaining tumor cortisol concentrations to stimulate GR and enzalutamide resistance. Remarkably, reinstatement of 11β-HSD2 expression, or AMFR loss, reverses enzalutamide resistance in mouse xenograft tumors. Together, these findings reveal a surprising metabolic mechanism of enzalutamide resistance that may be targeted with a strategy that circumvents a requirement for systemic GR ablation.

Intercellular communication in the early embryo of the ascidian Ciona intestinalis

Development ◽  
1988 ◽  
Vol 102 (1) ◽  
pp. 55-63 ◽  
Author(s):  
F. Serras ◽  
C. Baud ◽  
M. Moreau ◽  
P. Guerrier ◽  
J.A.M. Van den Biggelaar
Keyword(s):  
Intercellular Communication ◽  
Lucifer Yellow ◽  
Ciona Intestinalis ◽  
Early Embryo ◽  
Cell Stage ◽  
Early Embryos ◽  
Junctional Conductance ◽  
Voltage Dependent ◽  
Cytoplasmic Bridges ◽  
Series Of Experiments

We have studied the intercellular communication pathways in early embryos of the ascidian Ciona intestinalis. In two different series of experiments, we injected iontophoretically the dyes Lucifer Yellow and Fluorescein Complexon, and we analysed the spread of fluorescence to the neighbouring cells. We found that before the 32-cell stage no dye spread occurs between nonsister cells, whereas sister cells are dye-coupled, possibly via cytoplasmic bridges. After the 32-cell stage, dye spread occurs throughout the embryo. However, electrophysiological experiments showed that nonsister cells are ionically coupled before the 32-cell stage. We also found that at the 4-cell stage junctional conductance between nonsister cells is voltage dependent, which suggests that conductance is mediated by gap junctions in a way similar to that observed in other embryos.

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