Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos

Development ◽  
1998 ◽  
Vol 125 (14) ◽  
pp. 2677-2685 ◽  
Author(s):  
E.M. Joseph ◽  
D.A. Melton
Keyword(s):  
Gene Function ◽  
Ectopic Expression ◽  
Embryonic Cells ◽  
Xenopus Embryos ◽  
Maternal Mrna ◽  
Dorsal Mesoderm ◽  
Mesoderm Formation

The Xenopus Vg1 gene, a TGFbeta superfamily member, is expressed as a maternal mRNA localized to prospective endoderm, and mature Vg1 protein can induce both endodermal and mesodermal markers in embryonic cells. Most previous work on embryonic inducers, including activin, BMPs and Vg1, has relied on ectopic expression to assay for gene function. Here we employ a mutant ligand approach to block Vg1 signaling in developing embryos. The results indicate that Vg1 expression is essential for normal endodermal development and the induction of dorsal mesoderm in vivo.

Quantitative Analysis of Gene Function in the Drosophila Embryo

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 273-284
Author(s):  
William D Tracey ◽  
Xiangqun Ning ◽  
Martin Klingler ◽  
Sunita G Kramer ◽  
J Peter Gergen
Keyword(s):  
Gene Function ◽  
Model Systems ◽  
Drosophila Embryo ◽  
Direct Role ◽  
Developmental Context ◽  
Maternal Mrna ◽  
Early Drosophila Embryo ◽  
Segment Polarity ◽  
Pair Rule

Abstract The specific functions of gene products frequently depend on the developmental context in which they are expressed. Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined. Here we describe a system that allows for genetically controlled overexpression of any gene of interest under normal physiological conditions in the early Drosophila embryo. This regulated expression is achieved through the use of Drosophila lines that express a maternal mRNA for the yeast transcription factor GAL4. Embryos derived from females that express GAL4 maternally activate GAL4-dependent UAS transgenes at uniform levels throughout the embryo during the blastoderm stage of embryogenesis. The expression levels can be quantitatively manipulated through the use of lines that have different levels of maternal GAL4 activity. Specific phenotypes are produced by expression of a number of different developmental regulators with this system, including genes that normally do not function during Drosophila embryogenesis. Analysis of the response to overexpression of runt provides evidence that this pair-rule segmentation gene has a direct role in repressing transcription of the segment-polarity gene engrailed. The maternal GAL4 system will have applications both for the measurement of gene activity in reverse genetic experiments as well as for the identification of genetic factors that have quantitative effects on gene function in vivo.

scholarly journals Ectopic Expression of SPARC in Xenopus Embryos Interferes with Tissue Morphogenesis: Identification of a Bioactive Sequence in the C-terminal EF Hand

1997 ◽  
Vol 45 (5) ◽  
pp. 643-655 ◽  
Author(s):  
Sashko Damjanovski ◽  
Xantha Karp ◽  
Sarah Funk ◽  
E. Helene Sage ◽  
Maurice J. Ringuette
Keyword(s):  
Cultured Cells ◽  
Ectopic Expression ◽  
Copper Binding ◽  
Tissue Morphogenesis ◽  
Obvious Effect ◽  
Developmental Defects ◽  
Xenopus Embryos ◽  
Axial Defects

SPARC is a matricellular Ca2+-binding glycoprotein that exhibits both counteradhesive and antiproliferative effects on cultured cells. It is secreted by cells of various tissues as a consequence of morphogenesis, response to injury, and cyclic renewal and/or repair. In an earlier study with Xenopus embryos we had shown a highly specific and regulated pattern of SPARC expression. We now show that ectopic expression of SPARC before its normal embryonic activation produces severe anomalies, some of which are consistent with the functions of SPARC proposed from studies in vitro. Microinjection of SPARC RNA, protein, and peptides into Xenopus embryos before endogenous embryonic expression generated different but overlapping phenotypes. (a) Injection of SPARC RNA into one cell of a two-cell embryo resulted in a range of unilateral defects. (b) Precocious exposure of embryos to SPARC by microinjection of protein into the blastocoel cavity was associated with certain axial defects comparable to those obtained with SPARC RNA. (c) SPARC peptides containing follistatin-like and copper-binding sequences were without obvious effect, whereas SPARC peptide 4.2, corresponding to a disulfide-bonded, Ca2+-binding domain, was associated with a reduction in axial structures that led eventually to complete ventralization of the embryos. Histological analysis of ventralized embryos indicated that the morphogenetic events associated with gastrulation might have been inhibited. Microinjection of other Ca2+-binding glycoproteins, such as osteopontin and bone sialoprotein, resulted in phenotypes that were unique. We probed further the structural correlates of this region of SPARC in the context of tissue development. Co-injection of peptide 4.2 with Ca2+ or EGTA, and injection of peptide 4.2K (containing a mutated consensus Ca2+-binding sequence), demonstrated that the developmental defects associated with peptide 4.2 were independent of Ca2+. However, the disulfide bridge in this region of SPARC was found to be critical, as injection of peptide 4.2AA, a mutant lacking the cystine, generated no axial defects. We have therefore shown for the first time in vivo that the temporally inappropriate presence of SPARC is associated with perturbations in tissue morphogenesis. Moreover, we have identified at least one bioactive region of SPARC as the C-terminal disulfide-bonded, Ca2+-binding loop that was previously shown to be both counteradhesive and growth-inhibitory.

Xrel3/XrelA attenuates β-catenin-mediated transcription during mesoderm formation in Xenopus embryos

2011 ◽  
Vol 435 (1) ◽  
pp. 247-257 ◽  
Author(s):  
Mark W. L. Kennedy ◽  
Kenneth R. Kao
Keyword(s):  
Cell Fate ◽  
Target Genes ◽  
Marginal Zone ◽  
Ectopic Expression ◽  
Nuclear Factor Κb ◽  
Mesoderm Induction ◽  
Mesoderm Cell ◽  
Mesoderm Formation

In Xenopus laevis embryonic development, activation of the Wnt/β-catenin pathway promotes mesoderm cell fate determination via Xnr (Xenopus nodal-related) expression. We have demonstrated previously that Rel/NF-κB (nuclear factor κB) proteins expressed in presumptive ectoderm limit the activity of Xnrs to the marginal zone of embryos during mesoderm induction, which assists to distinguish mesoderm from ectoderm. The mechanism of this regulation, however, is unknown. In the present study, we investigated whether Rel/NF-κB proteins are able to modulate mesoderm formation by mediating Wnt/β-catenin signalling. We determined that ectopic expression of XrelA or Xrel3 in the dorsal marginal zone perturbed dorsal mesoderm formation by down-regulating multiple Wnt/β-catenin target genes including Xnr3, Xnr5 and Xnr6. Ventral co-expression of XrelA or Xrel3 with either wild-type β-catenin or constitutively active β-cateninS37A abrogated β-catenin-induced axis duplication and attenuated β-catenin-stimulated reporter transcription. Lastly, we provide evidence that Xrel3, but not XrelA, can interact with β-catenin without affecting the association of β-catenin with other transcriptional co-activators in vitro. Both Xrel3 and XrelA, however, prevented the accumulation, in nuclei, of exogenously expressed and endogenous β-catenin in vivo. These results suggest that Rel proteins are able to bind β-catenin and attenuate β-catenin-mediated transcription by nuclear exclusion.

scholarly journals Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development.

1996 ◽  
Vol 133 (5) ◽  
pp. 1123-1137 ◽  
Author(s):  
M A Torres ◽  
J A Yang-Snyder ◽  
S M Purcell ◽  
A A DeMarais ◽  
L L McGrew ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Signaling Pathway ◽  
Ectopic Expression ◽  
Functional Class ◽  
Dominant Negative ◽  
Beta Catenin ◽  
Embryonic Cells ◽  
Xenopus Embryos ◽  
Secondary Axis ◽  
Functional Classes

When overexpressed in Xenopus embryos, Xwnt-1, -3A, -8 and -8b define a functional class of Wnts (the Wnt-1 class) that promotes duplication of the embryonic axis, whereas Xwnt-5A, -4, and -11 define a distinct class (the Wnt-5A class) that alters morphogenetic movements (Du, S., S. Purcell, J. Christian, L. McGrew, and R. Moon. 1995. Mol. Cell. Biol. 15:2625-2634). Since come embryonic cells may be exposed to signals from both functional classes of Wnt during vertebrate development, this raises the question of how the signaling pathways of these classes of Wnts might interact. To address this issue, we coexpressed various Xwnts and components of the Wnt-1 class signaling pathway in developing Xenopus embryos. Members of the Xwnt-5A class antagonized the ability of ectopic Wnt-1 class to induce goosecoid expression and a secondary axis. Interestingly, the Wnt-5A class did not block goosecoid expression or axis induction in response to overexpression of cytoplasmic components of the Wnt-1 signaling pathway, beta-catenin or a kinase-dead gsk-3, or to the unrelated secreted factor, BVg1. The ability of the Wnt-5A class to block responses to the Wnt-1 class may involve decreases in cell adhesion, since ectopic expression of Xwnt-5A leads to decreased Ca2+-dependent cell adhesion and the activity of Xwnt-5A to block Wnt-1 class signals is mimicked by a dominant negative N-cadherin. These data underscore the importance of cell adhesion in modulating the responses of embryonic cells to signaling molecules and suggest that the Wnt-5A functional class of signaling factors can interact with the Wnt-1 class in an antagonistic manner.

scholarly journals TEAD4 modulated LncRNA MNX1-AS1 contributes to gastric cancer progression partly through suppressing BTG2 and activating BCL2

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
You Shuai ◽  
Zhonghua Ma ◽  
Weitao Liu ◽  
Tao Yu ◽  
Changsheng Yan ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Mechanistic Model ◽  
Ectopic Expression ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Tissue Samples ◽  
Reporter Assays

Abstract Background Gastric cancer (GC) is the third leading cause of cancer-related mortality globally. Long noncoding RNAs (lncRNAs) are dysregulated in obvious malignancies including GC and exploring the regulatory mechanisms underlying their expression is an attractive research area. However, these molecular mechanisms require further clarification, especially upstream mechanisms. Methods LncRNA MNX1-AS1 expression in GC tissue samples was investigated via microarray analysis and further determined in a cohort of GC tissues via quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. Cell proliferation and flow cytometry assays were performed to confirm the roles of MNX1-AS1 in GC proliferation, cell cycle regulation, and apoptosis. The influence of MNX1-AS1 on GC cell migration and invasion was explored with Transwell assays. A xenograft tumour model was established to verify the effects of MNX1-AS1 on in vivo tumourigenesis. The TEAD4-involved upstream regulatory mechanism of MNX1-AS1 was explored through ChIP and luciferase reporter assays. The mechanistic model of MNX1-AS1 in regulating gene expression was further detected by subcellular fractionation, FISH, RIP, ChIP and luciferase reporter assays. Results It was found that MNX1-AS1 displayed obvious upregulation in GC tissue samples and cell lines, and ectopic expression of MNX1-AS1 predicted poor clinical outcomes for patients with GC. Overexpressed MNX1-AS1 expression promoted proliferation, migration and invasion of GC cells markedly, whereas decreased MNX1-AS1 expression elicited the opposite effects. Consistent with the in vitro results, MNX1-AS1 depletion effectively inhibited the growth of xenograft tumour in vivo. Mechanistically, TEAD4 directly bound the promoter region of MNX1-AS1 and stimulated the transcription of MNX1-AS1. Furthermore, MNX1-AS1 can sponge miR-6785-5p to upregulate the expression of BCL2 in GC cells. Meanwhile, MNX1-AS1 suppressed the transcription of BTG2 by recruiting polycomb repressive complex 2 to BTG2 promoter regions. Conclusions Our findings demonstrate that MNX1-AS1 may be able to serve as a prognostic indicator in GC patients and that TEAD4-activatd MNX1-AS1 can promote GC progression through EZH2/BTG2 and miR-6785-5p/BCL2 axes, implicating it as a novel and potent target for the treatment of GC.

scholarly journals Ectopic Expression of Inhibitors of Protein Phosphatase Type 1 (PP1) Can Be Used to Analyze Roles of PP1 in Drosophila Development

Genetics ◽  
2003 ◽  
Vol 164 (1) ◽  
pp. 235-245
Author(s):  
Daimark Bennett ◽  
Balázs Szöőr ◽  
Sascha Gross ◽  
Natalia Vereshchagina ◽  
Luke Alphey
Keyword(s):  
Protein Phosphatase ◽  
Muscle Development ◽  
Ectopic Expression ◽  
High Specificity ◽  
Type I ◽  
Protein Phosphatase Type 1 ◽  
Mitotic Figures ◽  
Protein Phosphatase Type

Abstract We have identified two proteins that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. We show that modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants. These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGFβ superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, we show that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

scholarly journals Two Drosophila Innexins Are Expressed in Overlapping Domains and Cooperate to Form Gap-Junction Channels

2000 ◽  
Vol 11 (7) ◽  
pp. 2459-2470 ◽  
Author(s):  
Lucy A. Stebbings ◽  
Martin G. Todman ◽  
Pauline Phelan ◽  
Jonathan P. Bacon ◽  
Jane A. Davies
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ectopic Expression ◽  
In Vivo Studies ◽  
Electrophysiological Properties ◽  
Gap Junction Channels ◽  
Overlapping Domains ◽  
In Vitro Data

Members of the innexin protein family are structural components of invertebrate gap junctions and are analogous to vertebrate connexins. Here we investigate two Drosophila innexin genes,Dm-inx2 and Dm-inx3 and show that they are expressed in overlapping domains throughout embryogenesis, most notably in epidermal cells bordering each segment. We also explore the gap-junction–forming capabilities of the encoded proteins. In pairedXenopus oocytes, the injection of Dm-inx2mRNA results in the formation of voltage-sensitive channels in only ∼ 40% of cell pairs. In contrast, Dm-Inx3 never forms channels. Crucially, when both mRNAs are coexpressed, functional channels are formed reliably, and the electrophysiological properties of these channels distinguish them from those formed by Dm-Inx2 alone. We relate these in vitro data to in vivo studies. Ectopic expression ofDm-inx2 in vivo has limited effects on the viability ofDrosophila, and animals ectopically expressingDm-inx3 are unaffected. However, ectopic expression of both transcripts together severely reduces viability, presumably because of the formation of inappropriate gap junctions. We conclude that Dm-Inx2 and Dm-Inx3, which are expressed in overlapping domains during embryogenesis, can form oligomeric gap-junction channels.

scholarly journals A MYBL2 complex for RRM2 transactivation and the synthetic effect of MYBL2 knockdown with WEE1 inhibition against colorectal cancer

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Qian Liu ◽  
Lijuan Guo ◽  
Hongyan Qi ◽  
Meng Lou ◽  
Rui Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Dna Synthesis ◽  
Ectopic Expression ◽  
Building Blocks ◽  
Small Subunit ◽  
S Phase ◽  
Clinical Samples ◽  
Dependent Manner

AbstractRibonucleotide reductase (RR) is a unique enzyme for the reduction of NDPs to dNDPs, the building blocks for DNA synthesis and thus essential for cell proliferation. Pan-cancer profiling studies showed that RRM2, the small subunit M2 of RR, is abnormally overexpressed in multiple types of cancers; however, the underlying regulatory mechanisms in cancers are still unclear. In this study, through searching in cancer-omics databases and immunohistochemistry validation with clinical samples, we showed that the expression of MYBL2, a key oncogenic transcriptional factor, was significantly upregulated correlatively with RRM2 in colorectal cancer (CRC). Ectopic expression and knockdown experiments indicated that MYBL2 was essential for CRC cell proliferation, DNA synthesis, and cell cycle progression in an RRM2-dependent manner. Mechanistically, MYBL2 directly bound to the promoter of RRM2 gene and promoted its transcription during S-phase together with TAF15 and MuvB components. Notably, knockdown of MYBL2 sensitized CRC cells to treatment with MK-1775, a clinical trial drug for inhibition of WEE1, which is involved in a degradation pathway of RRM2. Finally, mouse xenograft experiments showed that the combined suppression of MYBL2 and WEE1 synergistically inhibited CRC growth with a low systemic toxicity in vivo. Therefore, we propose a new regulatory mechanism for RRM2 transcription for CRC proliferation, in which MYBL2 functions by constituting a dynamic S-phase transcription complex following the G1/early S-phase E2Fs complex. Doubly targeting the transcription and degradation machines of RRM2 could produce a synthetic inhibitory effect on RRM2 level with a novel potential for CRC treatment.

scholarly journals The ectopic expression of Arabidopsis glucosyltransferase UGT74D1 affects leaf positioning through modulating indole-3-acetic acid homeostasis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shanghui Jin ◽  
Bingkai Hou ◽  
Guizhi Zhang
Keyword(s):  
Acetic Acid ◽  
Ectopic Expression ◽  
Leaf Angle ◽  
Kinase Substrate ◽  
Auxin Distribution ◽  
Leaf Tissues ◽  
Photosynthesis Efficiency ◽  
Indole 3 Acetic Acid

AbstractLeaf angle is an important agronomic trait affecting photosynthesis efficiency and crop yield. Although the mechanisms involved in the leaf angle control are intensively studied in monocots, factors contribute to the leaf angle in dicots are largely unknown. In this article, we explored the physiological roles of an Arabidopsis glucosyltransferase, UGT74D1, which have been proved to be indole-3-acetic acid (IAA) glucosyltransferase in vitro. We found that UGT74D1 possessed the enzymatic activity toward IAA glucosylation in vivo and its expression was induced by auxins. The ectopically expressed UGT74D1 obviously reduced the leaf angle with an altered IAA level, auxin distribution and cell size in leaf tissues. The expression of several key genes involved in the leaf shaping and leaf positioning, including PHYTOCHROME KINASE SUBSTRATE (PKS) genes and TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) genes, were dramatically changed by ectopic expression of UGT74D1. In addition, clear transcription changes of YUCCA genes and other auxin related genes can be observed in overexpression lines. Taken together, our data indicate that glucosyltransferase UGT74D1 could affect leaf positioning through modulating auxin homeostasis and regulating transcription of PKS and TCP genes, suggesting a potential new role of UGT74D1 in regulation of leaf angle in dicot Arabidopsis.

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