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Disrupted Slit-Robo signalling results in membranous ventricular septum defects and bicuspid aortic valves.

Mommersteeg MT, Yeh ML, Parnavelas JG, Andrews WD - Cardiovasc. Res. (2015)

Bottom Line: Loss of Robo1 or both Robo1 and Robo2 resulted in membranous ventricular septum defects at birth, a defect also found in Slit3, but not in Slit2 mutants.Expression of Notch- and downstream Hey and Hes genes was down-regulated in Robo1 mutants, suggesting that reduced Notch signalling in mice lacking Robo might underlie the defects.Luciferase assays confirmed regulation of Notch signalling by Robo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University College London, 21 University Street, London WC1E 6DE, UK m.mommersteeg@ucl.ac.uk.

No MeSH data available.


Related in: MedlinePlus

Robo activates CBFRE-luciferase activity. (A) Robo and Notch are both membrane-bound receptors with intracellular signalling domains. Cleavage and binding of the intracellular domain of Notch to Cbf1 activates the CBFRE-luciferase reporter construct. (B and C) CBFRE-luciferase activation in the presence of the indicated transfected genes in COS7 cells. (B) P = 0.65, P = 0.045, P = 0.01 compared with NICD1 alone, respectively. Robo1 and 2 with NICD1 compared with Robo1 with NICD1 P = 0.007, to Robo2 with NICD1 P = 0.041, n = 5. (C) P = 0.004, P = 0.004, P = 0.14 compared with NICD1 alone, respectively, n = 5; one-way ANOVA with Bonferroni's multiple comparison test. (D) Scheme showing the similarity of the expression patterns between the Slit-Robo and Notch-Hey genes, with an overview of the observed defects. Luc, luciferase. *P < 0.05, **P < 0.01. For other abbreviations, see the legend of Figure 1.
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CVV040F7: Robo activates CBFRE-luciferase activity. (A) Robo and Notch are both membrane-bound receptors with intracellular signalling domains. Cleavage and binding of the intracellular domain of Notch to Cbf1 activates the CBFRE-luciferase reporter construct. (B and C) CBFRE-luciferase activation in the presence of the indicated transfected genes in COS7 cells. (B) P = 0.65, P = 0.045, P = 0.01 compared with NICD1 alone, respectively. Robo1 and 2 with NICD1 compared with Robo1 with NICD1 P = 0.007, to Robo2 with NICD1 P = 0.041, n = 5. (C) P = 0.004, P = 0.004, P = 0.14 compared with NICD1 alone, respectively, n = 5; one-way ANOVA with Bonferroni's multiple comparison test. (D) Scheme showing the similarity of the expression patterns between the Slit-Robo and Notch-Hey genes, with an overview of the observed defects. Luc, luciferase. *P < 0.05, **P < 0.01. For other abbreviations, see the legend of Figure 1.

Mentions: Next, we tested whether Robo receptors might directly enhance Notch signalling in vitro. Thus, we performed luciferase assays on COS7 cells, which were co-transfected with a plasmid containing four Notch-responsive Cbf1-binding elements and the basal simian virus 40 (SV40) promoter upstream of a luciferase cassette (CBFRE-luc), a plasmid with the Notch1 intracellular domain (NICD1) and/or Robo receptor expression plasmids (Figure 7A–C). In the control experiments, the presence of NICD1 expression strongly stimulated Notch-responsive CBFRE-luciferase activity (Figure 7B). This confirmed that the Notch-responsive element is activated by NICD1, with a 14-fold increase over basal expression levels found with an identical plasmid lacking NICD1 (Figure 7B). Co-transfection of Robo1 or Robo2-expressing plasmid and NICD1 further increased luciferase activation (respectively 18- and 20-fold), albeit Robo1 never reached significant levels. These results indicate that Robo2 is more important for regulating Notch signalling than Robo1. However, co-transfection of both Robo1 and Robo2 with NICD1 resulted in further increase in luciferase activity by 23-fold. This suggests that Notch signalling in the heart is spatially regulated by Robo expression, with maximum regulation when both Robo1 and Robo2 are present. Both Robo1 and Robo2 also minimally activated CBFRE-luciferase activity in the absence of NICD1 (1.6-, 1.5-, or 1.4-fold, respectively, Figure 7C). This might be caused by low levels of endogenous NICD1 present in COS7 cells or by direct regulation of Cbf1-responsive elements by Robo receptors. However, our data indicate that Robo receptors mainly function synergistically with NICD1, possibly by stabilizing N1ICD in the nucleus, although the nature of interaction, be it direct or indirect, will need to be further examined. Combined with the qPCR data, these results show that Robo1 and Robo2 receptors are capable of activating Notch signalling, suggesting that reduced activation of the Notch signalling pathway might underlie the defects observed in the mutants for Slit or Robo.Figure 7


Disrupted Slit-Robo signalling results in membranous ventricular septum defects and bicuspid aortic valves.

Mommersteeg MT, Yeh ML, Parnavelas JG, Andrews WD - Cardiovasc. Res. (2015)

Robo activates CBFRE-luciferase activity. (A) Robo and Notch are both membrane-bound receptors with intracellular signalling domains. Cleavage and binding of the intracellular domain of Notch to Cbf1 activates the CBFRE-luciferase reporter construct. (B and C) CBFRE-luciferase activation in the presence of the indicated transfected genes in COS7 cells. (B) P = 0.65, P = 0.045, P = 0.01 compared with NICD1 alone, respectively. Robo1 and 2 with NICD1 compared with Robo1 with NICD1 P = 0.007, to Robo2 with NICD1 P = 0.041, n = 5. (C) P = 0.004, P = 0.004, P = 0.14 compared with NICD1 alone, respectively, n = 5; one-way ANOVA with Bonferroni's multiple comparison test. (D) Scheme showing the similarity of the expression patterns between the Slit-Robo and Notch-Hey genes, with an overview of the observed defects. Luc, luciferase. *P < 0.05, **P < 0.01. For other abbreviations, see the legend of Figure 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4362403&req=5

CVV040F7: Robo activates CBFRE-luciferase activity. (A) Robo and Notch are both membrane-bound receptors with intracellular signalling domains. Cleavage and binding of the intracellular domain of Notch to Cbf1 activates the CBFRE-luciferase reporter construct. (B and C) CBFRE-luciferase activation in the presence of the indicated transfected genes in COS7 cells. (B) P = 0.65, P = 0.045, P = 0.01 compared with NICD1 alone, respectively. Robo1 and 2 with NICD1 compared with Robo1 with NICD1 P = 0.007, to Robo2 with NICD1 P = 0.041, n = 5. (C) P = 0.004, P = 0.004, P = 0.14 compared with NICD1 alone, respectively, n = 5; one-way ANOVA with Bonferroni's multiple comparison test. (D) Scheme showing the similarity of the expression patterns between the Slit-Robo and Notch-Hey genes, with an overview of the observed defects. Luc, luciferase. *P < 0.05, **P < 0.01. For other abbreviations, see the legend of Figure 1.
Mentions: Next, we tested whether Robo receptors might directly enhance Notch signalling in vitro. Thus, we performed luciferase assays on COS7 cells, which were co-transfected with a plasmid containing four Notch-responsive Cbf1-binding elements and the basal simian virus 40 (SV40) promoter upstream of a luciferase cassette (CBFRE-luc), a plasmid with the Notch1 intracellular domain (NICD1) and/or Robo receptor expression plasmids (Figure 7A–C). In the control experiments, the presence of NICD1 expression strongly stimulated Notch-responsive CBFRE-luciferase activity (Figure 7B). This confirmed that the Notch-responsive element is activated by NICD1, with a 14-fold increase over basal expression levels found with an identical plasmid lacking NICD1 (Figure 7B). Co-transfection of Robo1 or Robo2-expressing plasmid and NICD1 further increased luciferase activation (respectively 18- and 20-fold), albeit Robo1 never reached significant levels. These results indicate that Robo2 is more important for regulating Notch signalling than Robo1. However, co-transfection of both Robo1 and Robo2 with NICD1 resulted in further increase in luciferase activity by 23-fold. This suggests that Notch signalling in the heart is spatially regulated by Robo expression, with maximum regulation when both Robo1 and Robo2 are present. Both Robo1 and Robo2 also minimally activated CBFRE-luciferase activity in the absence of NICD1 (1.6-, 1.5-, or 1.4-fold, respectively, Figure 7C). This might be caused by low levels of endogenous NICD1 present in COS7 cells or by direct regulation of Cbf1-responsive elements by Robo receptors. However, our data indicate that Robo receptors mainly function synergistically with NICD1, possibly by stabilizing N1ICD in the nucleus, although the nature of interaction, be it direct or indirect, will need to be further examined. Combined with the qPCR data, these results show that Robo1 and Robo2 receptors are capable of activating Notch signalling, suggesting that reduced activation of the Notch signalling pathway might underlie the defects observed in the mutants for Slit or Robo.Figure 7

Bottom Line: Loss of Robo1 or both Robo1 and Robo2 resulted in membranous ventricular septum defects at birth, a defect also found in Slit3, but not in Slit2 mutants.Expression of Notch- and downstream Hey and Hes genes was down-regulated in Robo1 mutants, suggesting that reduced Notch signalling in mice lacking Robo might underlie the defects.Luciferase assays confirmed regulation of Notch signalling by Robo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University College London, 21 University Street, London WC1E 6DE, UK m.mommersteeg@ucl.ac.uk.

No MeSH data available.


Related in: MedlinePlus