scholarly journals Functional Verification of Novel ELMO1 Variants By Live Imaging in Zebrafish

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3121-3121
Author(s):  
Rongtao Xue ◽  
Ying Wang ◽  
Tienan Wang ◽  
Mei Lyu ◽  
Guiling Mo ◽  
...  
Keyword(s):  
Cell Motility ◽  
Conflicts Of Interest ◽  
Live Imaging ◽  
Dominant Negative ◽  
Functional Verification ◽  
Transient Expression Assay ◽  
Dominant Negative Form ◽  
Reduced Mobility ◽  
Negative Form

Abstract ELMO1 (Engulfment and Cell Motility1) is a gene involved in regulating cell motility through the ELMO1-DOCK2-RAC complex. Contrary to DOCK2 (Dedicator of Cytokinesis 2) deficiency, which has been reported to be associated with immunodeficiency diseases, variants of ELMO1 have been associated with autoimmune diseases, such as diabetes and rheumatoid arthritis (RA). To explore the function of ELMO1 in immune cells and to verify the functions of novel ELMO1 variants in vivo, we established a zebrafish elmo1 mutant model. Live imaging revealed that similar to mammals, the motility of neutrophils and T-cells was largely attenuated in zebrafish mutants. Consequently, the response of neutrophils to injury or bacterial infection was significantly reduced in the mutants. Furthermore, the reduced mobility of neutrophils could be rescued by the expression of constitutively activated Rac proteins, suggesting that zebrafish elmo1 mutant functions via a conserved mechanism. With this mutant, three novel human ELMO1 variants were transiently and specifically expressed in zebrafish neutrophils. Two variants, p.E90K (c.268G>A) and p.D194G (c.581A>G) could efficiently recover the motility defect of neutrophils in the elmo1 mutant; however, the p.R354X (c.1060C>T) variant failed to rescue the mutant. Acts as a dominant-negative form, p.R354X (c.1060C>T) which failed to rescue the elmo1 mutant and inhibited neutrophil movement in siblings. Based on those results, we identified that zebrafish elmo1 played conserved roles in cell motility, similar to higher vertebrates. Using the transient-expression assay, zebrafish elmo1 mutants could serve as an effective model for human variant verification in vivo. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional Verification of Novel ELMO1 Variants by Live Imaging in Zebrafish

2021 ◽  
Vol 9 ◽  
Author(s):  
Rongtao Xue ◽  
Ying Wang ◽  
Tienan Wang ◽  
Mei Lyu ◽  
Guiling Mo ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Bacterial Infection ◽  
Cell Motility ◽  
Immune Cells ◽  
Live Imaging ◽  
Functional Verification ◽  
Transient Expression Assay ◽  
Reduced Mobility

ELMO1 (Engulfment and Cell Motility1) is a gene involved in regulating cell motility through the ELMO1-DOCK2-RAC complex. Contrary to DOCK2 (Dedicator of Cytokinesis 2) deficiency, which has been reported to be associated with immunodeficiency diseases, variants of ELMO1 have been associated with autoimmune diseases, such as diabetes and rheumatoid arthritis (RA). To explore the function of ELMO1 in immune cells and to verify the functions of novel ELMO1 variants in vivo, we established a zebrafish elmo1 mutant model. Live imaging revealed that, similar to mammals, the motility of neutrophils and T-cells was largely attenuated in zebrafish mutants. Consequently, the response of neutrophils to injury or bacterial infection was significantly reduced in the mutants. Furthermore, the reduced mobility of neutrophils could be rescued by the expression of constitutively activated Rac proteins, suggesting that zebrafish elmo1 mutant functions via a conserved mechanism. With this mutant, three novel human ELMO1 variants were transiently and specifically expressed in zebrafish neutrophils. Two variants, p.E90K (c.268G>A) and p.D194G (c.581A>G), could efficiently recover the motility defect of neutrophils in the elmo1 mutant; however, the p.R354X (c.1060C>T) variant failed to rescue the mutant. Based on those results, we identified that zebrafish elmo1 plays conserved roles in cell motility, similar to higher vertebrates. Using the transient-expression assay, zebrafish elmo1 mutants could serve as an effective model for human variant verification in vivo.

scholarly journals Functional Myc-Max heterodimer is required for activation-induced apoptosis in T cell hybridomas.

1994 ◽  
Vol 180 (6) ◽  
pp. 2413-2418 ◽  
Author(s):  
R P Bissonnette ◽  
A McGahon ◽  
A Mahboubi ◽  
D R Green
Keyword(s):  
T Cell ◽  
Apoptotic Cell ◽  
Tolerance Induction ◽  
Dominant Negative ◽  
Binding Partner ◽  
Dominant Negative Form ◽  
Dominant Negative Activity ◽  
Induced Apoptosis ◽  
Negative Form

T cell hybridomas respond to activation signals by undergoing apoptotic cell death, and this is likely to represent comparable events related to tolerance induction in immature and mature T cells in vivo. Previous studies using antisense oligonucleotides implicated the c-Myc protein in the phenomenon of activation-induced apoptosis. This role for c-Myc in apoptosis is now confirmed in studies using a dominant negative form of its heterodimeric binding partner, Max, which we show here inhibits activation-induced apoptosis. Further, coexpression of a reciprocally mutant Myc protein capable of forming functional heterodimers with the mutant Max can compensate for the dominant negative activity and restore activation-induced apoptosis. These results imply that Myc promotes activation-induced apoptosis by obligatory heterodimerization with Max, and therefore, by regulating gene transcription.

FGF suppresses apoptosis and induces differentiation of fibre cells in the mouse lens

Development ◽  
1995 ◽  
Vol 121 (12) ◽  
pp. 4383-4393 ◽  
Author(s):  
R.L. Chow ◽  
G.D. Roux ◽  
M. Roghani ◽  
M.A. Palmer ◽  
D.B. Rifkin ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transgenic Mice ◽  
Dominant Negative ◽  
Fgf Receptor ◽  
Fibre Cell ◽  
Dominant Negative Form ◽  
Lens Fibre ◽  
Mouse Lens ◽  
Negative Form

To determine whether fibroblast growth factor (FGF) has a role in lens development, we have generated transgenic mice expressing a dominant-negative form of the murine FGF receptor-1 (FGFRDN) in the lens. Using the fibre cell-specific alpha A-crystallin promoter to express the FGFRDN, we have asked whether FGF is required for fibre cell differentiation. The transgenic mice display diminished differentiation of fibre cells as indicated by their reduced elongation. In addition, transgenic lenses have an unusual refractile anomaly that morphological and biochemical data show results from the apoptosis of fibre cells in the central region of the lens. These results show that lens fibre cells are dependent on FGF for their survival and differentiation, and demonstrate that growth factor deprivation in vivo can lead to apoptosis.

scholarly journals Myocardial Expression of a Dominant-Negative Form of Daxx Decreases Infarct Size and Attenuates Apoptosis in an In Vivo Mouse Model of Ischemia/Reperfusion Injury

Circulation ◽  
2007 ◽  
Vol 116 (23) ◽  
pp. 2709-2717 ◽  
Author(s):  
François Roubille ◽  
Stéphane Combes ◽  
Juani Leal-Sanchez ◽  
Christian Barrère; ◽  
Frédéric Cransac ◽  
...  
Keyword(s):  
Mouse Model ◽  
Infarct Size ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Dominant Negative ◽  
Dominant Negative Form ◽  
Negative Form

Requirement ofFoxD3-class signaling for neural crest determination inXenopus

Development ◽  
2001 ◽  
Vol 128 (13) ◽  
pp. 2525-2536 ◽  
Author(s):  
Noriaki Sasai ◽  
Kenji Mizuseki ◽  
Yoshiki Sasai
Keyword(s):  
Neural Crest ◽  
Dominant Negative ◽  
Related Factor ◽  
Embryonic Patterning ◽  
Loss Of Function ◽  
Upstream Regulator ◽  
Dominant Negative Form ◽  
Neural Crest Differentiation ◽  
Negative Form

Fox factors (winged-helix transcription factors) play important roles in early embryonic patterning. We show here that FoxD3 (Forkhead 6) regulates neural crest determination in Xenopus embryos. Expression of FoxD3 in the presumptive neural crest region starts at the late gastrula stage in a manner similar to that of Slug, and overlaps with that of Zic-r1. When overexpressed in the embryo and in ectodermal explants, FoxD3 induces expression of neural crest markers. Attenuation of FoxD3-related signaling by a dominant-negative FoxD3 construct (FoxD3delN) inhibits neural crest differentiation in vivo without suppressing the CNS marker Sox2. Interestingly, these loss-of-function phenotypes are reversed by coinjecting Slug. In animal cap explants, neural crest differentiation induced by Slug and Wnt3a is also inhibited by FoxD3delN but not by a dominant-negative form of XBF2. Loss-of-function studies using dominant-negative forms of FoxD3 and Slug indicate that Slug induction by Zic factors requires FoxD3-related signaling, and that FoxD3 and Slug have different requirements in inducing downstream neural crest markers. These data demonstrate that FoxD3 (or its closely related factor) is an essential upstream regulator of neural crest determination.

scholarly journals The Hippo effector TAZ cooperates with oncogenic β-catenin in experimental and human hepatoblastoma development

2019 ◽  
Author(s):  
Junyan Tao ◽  
Shu Zhang ◽  
Jie Zhang ◽  
Katja Evert ◽  
Xiaolei Li ◽  
...  
Keyword(s):  
Sleeping Beauty ◽  
Dominant Negative ◽  
Downstream Effector ◽  
Tail Vein Injection ◽  
Mesenchymal Differentiation ◽  
Dominant Negative Form ◽  
Transcription Factor 1 ◽  
Negative Form

Abstract Backgrounds: Hepatoblastoma (HB) is the most common pediatric liver tumor. Though Wnt/β-catenin and Hippo cascades are implicated in HB development, there is no study on the crosstalk of β-catenin and Hippo downstream effector TAZ in HB. Methods: The expression of TAZ and of β-catenin in human HB specimens was assessed by immunohistochemistry (IHC). The functional interplay between TAZ and β-catenin was tested through delivering either an activated form of TAZ (TAZS89A) alone or co-delivering TAZS89A and an activated form of β-catenin (∆N90-β-catenin) to mouse liver using sleeping beauty transposase via hydrodynamic tail vein injection (SBT-HTVI). In addition, the role of transcriptional enhanced associate domain (TEAD) factors, canonical Notch cascade, Yap, and the tumor modifier heat shock transcription factor 1 (HSF1) along TAZ/β-catenin-driven HB development was studied in vivo and vitro. Results: Activation of TAZ often co-occurred with that of β-catenin in clinical specimens. While overexpression of TAZS89A alone was unable to promote liver tumorigenesis, the concomitant overexpression of TAZ and ∆N90-β-catenin induced the development of HB lesions exhibiting both epithelial and mesenchymal features. Mechanistically, HB development driven by TAZ/β-catenin required TAZ interaction with TEAD factors. Furthermore, TAZ/β-catenin overexpression induced HB development in conditional Yes-associated protein knockout (Yap KO) mice, indicating that Yap activation is dispensable in this model. Activation of the Notch signaling was observed in TAZ/β-catenin mouse lesions, consistent with that reported in human HBs. Blocking of the canonical Notch cascade using the dominant negative form of RBP-J (dnRBP-J) did not inhibit TAZ/β-catenin dependent HB formation in mice, although suppressed the mesenchymal differentiation. Similarly, upregulation of HSF1 was detected in TAZ/β-catenin lesions, but its inactivation did not affect HB development. In human HB cell lines, silencing of TAZ resulted in decreased cell growth, which was reduced more substantially when TAZ knockdown was associated with suppression of either β-catenin or YAP gene. Conclusions: Overall, our study identifies TAZ as a critical oncogene in HB development and progression. Yap, Notch, and HSF1 are dispensable for TAZ/β-catenin induced HB development in mice.

Sign in / Sign up

Export Citation Format

Share Document