A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers

Development ◽  
1996 ◽  
Vol 122 (3) ◽  
pp. 869-880 ◽  
Author(s):  
C. Launay ◽  
V. Fromentoux ◽  
D.L. Shi ◽  
J.C. Boucaut
Keyword(s):  
Neural Induction ◽  
Cement Gland ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Fgf Receptor ◽  
Dorsal Mesoderm ◽  
Poorly Differentiated ◽  
Neural Marker ◽  
Fgf Signalling

We have examined the role of fibroblast growth factor (FGF) signalling in neural induction. The approach takes advantage of the fact that both noggin and the dominant negative mutant activin receptor (delta1XAR1) directly induce neural tissues in the absence of dorsal mesoderm. A truncated FGF receptor (XFD) is co-expressed with noggin or delta1XAR1 in both whole embryos and isolated animal caps. We demonstrate that inhibition of FGF signalling prevents neural induction by both factors. Furthermore, neural induction by organizers (the dorsal lip of blastopore and Hensen's node) is also blocked by inhibiting FGF signalling in ectoderm. It has been proposed that the specification of anterior neuroectoderm, including the cement gland, occurs in a sequential manner as gastrulation proceeds. We show that the specification of the most anterior neuroectoderm by noggin may occur before gastrulation and does not require FGF signalling, since both the cement gland marker XCG-1 and the anterior neural marker Otx-2 are normally expressed in ectodermal explants co-injected with noggin and XFD RNAs, but the cement gland cells are poorly differentiated. In contrast, the expression of both genes induced by CSKA.noggin, which is expressed after the mid-blastula transition, is strongly inhibited by the presence of XFD. Therefore the noggin-mediated neural induction that takes place at gastrula stages is abolished in the absence of FGF signalling. Since inhibition of FGF signalling blocks the neuralizing effect of different neural inducers that function through independent mechanisms, we propose that FGF receptor-related-signalling is required for the response to inducing signals of ectodermal cells from gastrula.

FGF signalling in the early specification of mesoderm in Xenopus

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 477-487 ◽  
Author(s):  
E. Amaya ◽  
P.A. Stein ◽  
T.J. Musci ◽  
M.W. Kirschner
Keyword(s):  
Dorsal Side ◽  
Dominant Negative ◽  
Early Gene ◽  
Immediate Early ◽  
Mesoderm Induction ◽  
Fgf Receptor ◽  
Muscle Formation ◽  
Fgf Signalling ◽  
Lateral Mesoderm

We have examined the role of FGF signalling in the development of muscle and notochord and in the expression of early mesodermal markers in Xenopus embryos. Disruption of the FGF signalling pathway by expression of a dominant negative construct of the FGF receptor (XFD) generally results in gastrulation defects that are later evident in the formation of the trunk and tail, though head structures are formed nearly normally. These defects are reflected in the loss of notochord and muscle. Even in embryos that show mild defects and gastrulate properly, muscle formation is impaired, suggesting that morphogenesis and tissue differentiation each depend on FGF. The XFD protein inhibits the expression of the immediate early gene brachyury throughout the marginal zone, including the dorsal side; it does not, however, inhibit the dorsal lip marker goosecoid, which is expressed in the first involuting mesoderm at the dorsal side that will underlie the head. The XFD protein also inhibits Xpo expression, an immediate early marker of ventral and lateral mesoderm. These results suggest that FGF is involved in the earliest events of most mesoderm induction that occur before gastrulation and that the early dorsal mesoderm is already composed of two cell populations that differ in their requirements for FGF.

Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation

Development ◽  
1996 ◽  
Vol 122 (10) ◽  
pp. 3173-3183 ◽  
Author(s):  
K.L. Kroll ◽  
E. Amaya
Keyword(s):  
Large Scale ◽  
Neural Induction ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mesoderm Induction ◽  
Fgf Signaling ◽  
Fgf Receptor ◽  
Transgenic Expression

We have developed a simple approach for large-scale transgenesis in Xenopus laevis embryos and have used this method to identify in vivo requirements for FGF signaling during gastrulation. Plasmids are introduced into decondensed sperm nuclei in vitro using restriction enzyme-mediated integration (REMI). Transplantation of these nuclei into unfertilized eggs yields hundreds of normal, diploid embryos per day which develop to advanced stages and express integrated plasmids nonmosaically. Transgenic expression of a dominant negative mutant of the FGF receptor (XFD) after the mid-blastula stage uncouples mesoderm induction, which is normal, from maintenance of mesodermal markers, which is lost during gastrulation. By contrast, embryos expressing XFD contain well-patterned nervous systems despite a putative role for FGF in neural induction.

scholarly journals Role of FGF signalling in neural crest cell migration during early chick embryo development

Zygote ◽  
2018 ◽  
Vol 26 (6) ◽  
pp. 457-464 ◽  
Author(s):  
Xiao-tan Zhang ◽  
Guang Wang ◽  
Yan Li ◽  
Manli Chuai ◽  
Kenneth Ka Ho Lee ◽  
...  
Keyword(s):  
Neural Crest ◽  
Neural Tube ◽  
Neural Crest Cell ◽  
Dominant Negative ◽  
Neural Tubes ◽  
Dorsal Neural Tube ◽  
Neural Crest Cell Migration ◽  
Fgf Signalling ◽  
Crest Cell

SummaryFibroblast growth factor (FGF) signalling acts as one of modulators that control neural crest cell (NCC) migration, but how this is achieved is still unclear. In this study, we investigated the effects of FGF signalling on NCC migration by blocking this process. Constructs that were capable of inducing Sprouty2 (Spry2) or dominant-negative FGFR1 (Dn-FGFR1) expression were transfected into the cells making up the neural tubes. Our results revealed that blocking FGF signalling at stage HH10 (neurulation stage) could enhance NCC migration at both the cranial and trunk levels in the developing embryos. It was established that FGF-mediated NCC migration was not due to altering the expression of N-cadherin in the neural tube. Instead, we determined that cyclin D1 was overexpressed in the cranial and trunk levels when Sprouty2 was upregulated in the dorsal neural tube. These results imply that the cell cycle was a target of FGF signalling through which it regulates NCC migration at the neurulation stage.

Induction of the mesendoderm in the zebrafish germ ring by yolk cell-derived TGF-beta family signals and discrimination of mesoderm and endoderm by FGF

Development ◽  
1999 ◽  
Vol 126 (14) ◽  
pp. 3067-3078 ◽  
Author(s):  
A. Rodaway ◽  
H. Takeda ◽  
S. Koshida ◽  
J. Broadbent ◽  
B. Price ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Dominant Negative ◽  
Fibroblast Growth ◽  
Tgf Beta ◽  
Fgf Receptor ◽  
Activin Receptor ◽  
Yolk Cell ◽  
Endogenous Expression ◽  
Vertebrate Embryo ◽  
Fgf Signalling

The endoderm forms the gut and associated organs, and develops from a layer of cells which emerges during gastrula stages in the vertebrate embryo. In comparison to mesoderm and ectoderm, little is known about the signals which induce the endoderm. The origin of the endoderm is intimately linked with that of mesoderm, both by their position in the embryo, and by the molecules that can induce them. We characterised a gene, zebrafish gata5, which is expressed in the endoderm from blastula stages and show that its transcription is induced by signals originating from the yolk cell. These signals also induce the mesoderm-expressed transcription factor no tail (ntl), whose initial expression coincides with gata5 in the cells closest to the blastoderm margin, then spreads to encompass the germ ring. We have characterised the induction of these genes and show that ectopic expression of activin induces gata5 and ntl in a pattern which mimics the endogenous expression, while expression of a dominant negative activin receptor abolishes ntl and gata5 expression. Injection of RNA encoding a constitutively active activin receptor leads to ectopic expression of gata5 and ntl. gata5 is activated cell-autonomously, whereas ntl is induced in cells distant from those which have received the RNA, showing that although expression of both genes is induced by a TGF-beta signal, expression of ntl then spreads by a relay mechanism. Expression of a fibroblast growth factor (eFGF) or a dominant negatively acting FGF receptor shows that ntl but not gata5 is regulated by FGF signalling, implying that this may be the relay signal leading to the spread of ntl expression. In embryos lacking both squint and cyclops, members of the nodal group of TGF-beta related molecules, gata5 expression in the blastoderm is abolished, making these factors primary candidates for the endogenous TGF-beta signal inducing gata5.

Centrosome cohesion is regulated by a balance of kinase and phosphatase activities

2001 ◽  
Vol 114 (20) ◽  
pp. 3749-3757 ◽  
Author(s):  
Patrick Meraldi ◽  
Erich A. Nigg
Keyword(s):  
Protein Phosphatase ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Microtubule Depolymerization ◽  
Drug Induced ◽  
Microtubule Network ◽  
Similar Phenotype ◽  
Underlying Mechanisms

Centrosome cohesion and separation are regulated throughout the cell cycle, but the underlying mechanisms are not well understood. Since overexpression of a protein kinase, Nek2, is able to trigger centrosome splitting (the separation of parental centrioles), we have surveyed a panel of centrosome-associated kinases for their ability to induce a similar phenotype. Cdk2, in association with either cyclin A or E, was as effective as Nek2, but several other kinases tested did not significantly interfere with centrosome cohesion. Centrosome splitting could also be triggered by inhibition of phosphatases, and protein phosphatase 1α (PP1α) was identified as a likely physiological antagonist of Nek2. Furthermore, we have revisited the role of the microtubule network in the control of centrosome cohesion. We could confirm that microtubule depolymerization by nocodazole causes centrosome splitting. Surprisingly, however, this drug-induced splitting also required kinase activity and could specifically be suppressed by a dominant-negative mutant of Nek2. These studies highlight the importance of protein phosphorylation in the control of centrosome cohesion, and they point to Nek2 and PP1α as critical regulators of centrosome structure.

scholarly journals MITOCHONDRIA AS REVERSIBLE REGULATORS OF AGING ASSOCIATED SKIN WRINKLES AND HAIR LOSS IN MICE

2019 ◽  
Vol 3 (Supplement_1) ◽  
pp. S395-S395
Author(s):  
Keshav K Singh
Keyword(s):  
Mouse Model ◽  
Hair Loss ◽  
Transgene Expression ◽  
Skin Aging ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dominant Negative Mutation ◽  
Polymerase Domain ◽  
Skin Wrinkles

Abstract To evaluate the consequences of the decline in mtDNA content associated with aging we have created an inducible mouse model expressing, in the polymerase domain of POLG1, a dominant-negative mutation that induces depletion of mtDNA. We utilized this inducible mouse model to modulate mitochondrial function by depleting and repleting the mtDNA content. We demonstrate that, in mice, ubiquitous expression of dominant-negative mutant POLG1 leads to 1) reduction of mtDNA content in skin, 2) skin wrinkles, and 3) hair loss. By turning off the mutant POLG1 transgene expression in the whole animal, the skin and hair phenotypes revert to normal after repletion of mtDNA. Thus, we have developed whole-animal mtDNA depleter-repleter mice. These mice present evidence that mtDNA homeostasis is involved in skin aging phenotype and loss of hair and provide an unprecedented opportunity to create tissue-specific mitochondrial modulation to determine the role of the mitochondria in a particular tissue.

scholarly journals Foxl2 Up-Regulates Aromatase Gene Transcription in a Female-Specific Manner by Binding to the Promoter as Well as Interacting with Ad4 Binding Protein/Steroidogenic Factor 1

2007 ◽  
Vol 21 (3) ◽  
pp. 712-725 ◽  
Author(s):  
De-Shou Wang ◽  
Tohru Kobayashi ◽  
Lin-Yan Zhou ◽  
Bindhu Paul-Prasanth ◽  
Shigeho Ijiri ◽  
...  
Keyword(s):  
Serum Levels ◽  
Sex Reversal ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Testicular Development ◽  
Aromatase Gene ◽  
Ovarian Differentiation ◽  
Forkhead Domain ◽  
17Β Estradiol

Abstract Increasing evidence suggests the crucial role of estrogen in ovarian differentiation of nonmammalian vertebrates including fish. The present study has investigated the plausible role of Foxl2 in ovarian differentiation through transcriptional regulation of aromatase gene, using monosex fry of tilapia. Foxl2 expression is sexually dimorphic, like Cyp19a1, colocalizing with Cyp19a1 and Ad4BP/SF-1 in the stromal cells and interstitial cells in gonads of normal XX and sex-reversed XY fish, before the occurrence of morphological sex differentiation. Under in vitro conditions, Foxl2 binds to the sequence ACAAATA in the promoter region of the Cyp19a1 gene directly through its forkhead domain and activates the transcription of Cyp19a1 with its C terminus. Foxl2 can also interact through the forkhead domain with the ligand-binding domain of Ad4BP/SF-1 to form a heterodimer and enhance the Ad4BP/SF-1 mediated Cyp19a1 transcription. Disruption of endogenous Foxl2 in XX tilapia by overexpression of its dominant negative mutant (M3) induces varying degrees of testicular development with occasional sex reversal from ovary to testis. Such fish display reduced expression of Cyp19a1 as well as a drop in the serum levels of 17β-estradiol and 11-ketotestosterone. Although the XY fish with wild-type tilapia Foxl2 (tFoxl2) overexpression never exhibited a complete sex reversal, there were significant structural changes, such as tissue degeneration, somatic cell proliferation, and induction of aromatase, with increased serum levels of 17β-estradiol and 11-ketotestosterone. Altogether, these results suggest that Foxl2 plays a decisive role in the ovarian differentiation of the Nile tilapia by regulating aromatase expression and possibly the entire steroidogenic pathway.

scholarly journals Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

2001 ◽  
Vol 12 (7) ◽  
pp. 2171-2183 ◽  
Author(s):  
Juan Ángel Fresno Vara ◽  
Ma Aurora Domı́nguez Cáceres ◽  
Augusto Silva ◽  
Jorge Martı́n-Pérez
Keyword(s):  
Cell Proliferation ◽  
Tyrosine Kinases ◽  
Cellular Proliferation ◽  
Tyrosine Phosphatase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Herbimycin A ◽  
Serine Kinases ◽  
Dependent Cell

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [3H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, andodc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL.

Essential roles of IκB kinases α and β in serum- and IL-1-induced human VSMC proliferation

2000 ◽  
Vol 278 (6) ◽  
pp. H1823-H1831 ◽  
Author(s):  
Sebastian Sasu ◽  
Debbie Beasley
Keyword(s):  
Smooth Muscle ◽  
Fluorescent Protein ◽  
Interleukin 1 ◽  
Fold Increase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mutant Form ◽  
Mobility Shift ◽  
Vsmc Proliferation

Interleukin-1 (IL-1) is a potent vascular smooth muscle cell (VSMC) mitogen, which can stimulate cells via activation of nuclear factor-κB (NF-κB) following phosphorylation of its inhibitory subunit (IκB). Because the proliferative effect of IL-1 is additive with that of serum, the present studies assessed the role of IκB kinases (IKKs) and NF-κB in both IL-1- and serum-induced VSMC proliferation. IL-1β (1 ng/ml) induced marked and persistent NF-κB activation in VSMC that was maximal at 1 h and persisted for 3 days. There was a 3-fold increase in DNA synthesis after acute IL-1 exposure (24–96 h) and a 12-fold increase after chronic IL-1 exposure (>7 days). Electrophoretic mobility shift assay and supershift analysis indicated that IL-1-induced NF-κB complexes consisted of p65/p50 heterodimers and p50 homodimers. Human saphenous vein smooth muscle cells (HSVSMC) were transiently cotransfected with expression plasmids encoding a dominant negative mutant form of either IKKα or IKKβ, in which K44 was mutated to A (K44A), and a green fluorescent protein expression plasmid that allows identification of transfected cells. IL-1 induced nuclear localization of p65 in 95% of cells transfected with vector alone but in only 69% and 26% of cells expressing IKKα (K44A) or IKKβ (K44A), respectively. Likewise, proliferation increased 3.2-fold in IL-1-treated HSVSMC which had been transfected with vector alone, but only 2.2- and 1.5-fold proliferation in HSVSMC expressing IKKα (K44A) or IKKβ (K44A), respectively. Although serum activated NF-κB transiently, serum-induced proliferation was markedly attenuated in HSVSMC expressing IKKα (K44A) and IKKβ (K44A) compared with HSVSMC transfected with vector alone. The results support an essential role of IKKs in the proliferative response of HSVSMC to IL-1 and to serum.

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