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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

A spectrum of valve malformations in Slit mutants. (A–J) Analysis of the cardiac valves at the indicated developmental stages in Slit2+/+ (A, B, and D), Slit2−/− (A, C, and E), Slit3+/+ (F, G, and I), Slit3−/− (F, H, and J) embryos. (A and F) Measurements of the total valve (Total), atrioventricular valve (AV), aortic valve (Ao), and pulmonary trunk valve (PT) volume corrected for ventricular volume. (A) Total, n = 5, P = 0.08; AV, P = 0.92; Ao, P = 0.016; PT, P = 0.025. (F) Total, WT n = 5, KO n = 3, P = 0.025; AV, P = 0.025; Ao, P = 0.46; PT, P = 0.10; Mann–Whitney U test. Note that while there is overall increased valve volume in Robo1−/−;Robo2−/− mutants, Slit2−/− mutants only show increased semilunar (A) and Slit3−/− only increased total and atrioventricular valve volume (F). (B, C, G, and H) Three-dimensional reconstructions of the semilunar valves, seen from the ventricular side. Black arrowhead, note the absence of the posterior aortic valve in the Slit2−/− (C) while this valve is hypoplastic in the Slit3−/− (H) embryos. (D, E, I, and J) Immunohistochemistry sections (cTnI and DAPI) showing the atrioventricular valves. White arrowheads, Slit3−/− (J) embryos show thickened valves. R/L/PAo, right/left/posterior aortic valves; R/L/APT, right/left/anterior pulmonary trunk valve; AVVs, atrioventricular valves. *P < 0.05. For other abbreviations, see the legend of Figure 1. Scale bars depict 100 µm.
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CVV040F5: A spectrum of valve malformations in Slit mutants. (A–J) Analysis of the cardiac valves at the indicated developmental stages in Slit2+/+ (A, B, and D), Slit2−/− (A, C, and E), Slit3+/+ (F, G, and I), Slit3−/− (F, H, and J) embryos. (A and F) Measurements of the total valve (Total), atrioventricular valve (AV), aortic valve (Ao), and pulmonary trunk valve (PT) volume corrected for ventricular volume. (A) Total, n = 5, P = 0.08; AV, P = 0.92; Ao, P = 0.016; PT, P = 0.025. (F) Total, WT n = 5, KO n = 3, P = 0.025; AV, P = 0.025; Ao, P = 0.46; PT, P = 0.10; Mann–Whitney U test. Note that while there is overall increased valve volume in Robo1−/−;Robo2−/− mutants, Slit2−/− mutants only show increased semilunar (A) and Slit3−/− only increased total and atrioventricular valve volume (F). (B, C, G, and H) Three-dimensional reconstructions of the semilunar valves, seen from the ventricular side. Black arrowhead, note the absence of the posterior aortic valve in the Slit2−/− (C) while this valve is hypoplastic in the Slit3−/− (H) embryos. (D, E, I, and J) Immunohistochemistry sections (cTnI and DAPI) showing the atrioventricular valves. White arrowheads, Slit3−/− (J) embryos show thickened valves. R/L/PAo, right/left/posterior aortic valves; R/L/APT, right/left/anterior pulmonary trunk valve; AVVs, atrioventricular valves. *P < 0.05. For other abbreviations, see the legend of Figure 1. Scale bars depict 100 µm.

Mentions: To investigate a role for defective cushion maturation in the formation of the ventricular septum defects, we next analysed the volume of the different cardiac cushion regions. We did not observe any differences in morphology or volume of the outflow tract or atrioventricular cushions in Robo1, Robo2, Slit2, or Slit3 mutant embryos at E14.5 (data not shown). However, in all E12.5 Robo1;Robo2 mutants, although cushion volume was not changed, the just forming aortic posterior cushion seemed absent (Figure 3H and I). In contrast, the pulmonary anterior cushion was forming normally. At E15.5, all Robo1;Robo2 double mutants showed thickening of both the semilunar and atrioventricular valves, in combination with now clearly visible bicuspid aortic valves (Figure 4K–O; see Supplementary material online, Figure S2E and F). While the overall aortic valve volume was similar between Robo1;Robo2 double mutants and wild-type littermates, this was caused by the absence of the posterior non-coronary aortic valve; the two remaining valves were thicker than their wild-type counterparts (Figure 4K–M). At E18.5, single Robo1 or Robo2 mutant valves remained indistinguishable from their wild-type littermates (Figure 4A–J). In contrast, E18.5 mice mutant for Slit2 showed thickening of both the aortic and pulmonary semilunar valves, similar to the Robo1;Robo2 double mutants; however, the atrioventricular valves retained a normal size (Figure 5A–E). As only one Slit2 mutant recapitulated the bicuspid aortic valves as seen in the absence of both Robo1 and Robo2, total aortic valve volume was significantly increased in mice lacking Slit2 (Figure 5A–C). The thickness of the semilunar valves in Slit3 mutants was relatively normal, but now the atrioventricular valves were strikingly thicker (Figure 5F–J). The posterior aortic semilunar leaflet, missing in the Robo1;Robo2 double mutants, was highly hypoplastic in the absence of Slit3, but never completely absent (Figure 5G and H). These data indicate a role for Slit-Robo signalling during valve maturation and functional redundancy and requirement of Robo1 and Robo2 in both the outflow tract and the atrioventricular valve regions, whereas the Slit ligands show region-specific functions. Furthermore, these results underline the possible multi-causal origin of the ventricular septum defects.Figure 4


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)

A spectrum of valve malformations in Slit mutants. (A–J) Analysis of the cardiac valves at the indicated developmental stages in Slit2+/+ (A, B, and D), Slit2−/− (A, C, and E), Slit3+/+ (F, G, and I), Slit3−/− (F, H, and J) embryos. (A and F) Measurements of the total valve (Total), atrioventricular valve (AV), aortic valve (Ao), and pulmonary trunk valve (PT) volume corrected for ventricular volume. (A) Total, n = 5, P = 0.08; AV, P = 0.92; Ao, P = 0.016; PT, P = 0.025. (F) Total, WT n = 5, KO n = 3, P = 0.025; AV, P = 0.025; Ao, P = 0.46; PT, P = 0.10; Mann–Whitney U test. Note that while there is overall increased valve volume in Robo1−/−;Robo2−/− mutants, Slit2−/− mutants only show increased semilunar (A) and Slit3−/− only increased total and atrioventricular valve volume (F). (B, C, G, and H) Three-dimensional reconstructions of the semilunar valves, seen from the ventricular side. Black arrowhead, note the absence of the posterior aortic valve in the Slit2−/− (C) while this valve is hypoplastic in the Slit3−/− (H) embryos. (D, E, I, and J) Immunohistochemistry sections (cTnI and DAPI) showing the atrioventricular valves. White arrowheads, Slit3−/− (J) embryos show thickened valves. R/L/PAo, right/left/posterior aortic valves; R/L/APT, right/left/anterior pulmonary trunk valve; AVVs, atrioventricular valves. *P < 0.05. For other abbreviations, see the legend of Figure 1. Scale bars depict 100 µm.
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CVV040F5: A spectrum of valve malformations in Slit mutants. (A–J) Analysis of the cardiac valves at the indicated developmental stages in Slit2+/+ (A, B, and D), Slit2−/− (A, C, and E), Slit3+/+ (F, G, and I), Slit3−/− (F, H, and J) embryos. (A and F) Measurements of the total valve (Total), atrioventricular valve (AV), aortic valve (Ao), and pulmonary trunk valve (PT) volume corrected for ventricular volume. (A) Total, n = 5, P = 0.08; AV, P = 0.92; Ao, P = 0.016; PT, P = 0.025. (F) Total, WT n = 5, KO n = 3, P = 0.025; AV, P = 0.025; Ao, P = 0.46; PT, P = 0.10; Mann–Whitney U test. Note that while there is overall increased valve volume in Robo1−/−;Robo2−/− mutants, Slit2−/− mutants only show increased semilunar (A) and Slit3−/− only increased total and atrioventricular valve volume (F). (B, C, G, and H) Three-dimensional reconstructions of the semilunar valves, seen from the ventricular side. Black arrowhead, note the absence of the posterior aortic valve in the Slit2−/− (C) while this valve is hypoplastic in the Slit3−/− (H) embryos. (D, E, I, and J) Immunohistochemistry sections (cTnI and DAPI) showing the atrioventricular valves. White arrowheads, Slit3−/− (J) embryos show thickened valves. R/L/PAo, right/left/posterior aortic valves; R/L/APT, right/left/anterior pulmonary trunk valve; AVVs, atrioventricular valves. *P < 0.05. For other abbreviations, see the legend of Figure 1. Scale bars depict 100 µm.
Mentions: To investigate a role for defective cushion maturation in the formation of the ventricular septum defects, we next analysed the volume of the different cardiac cushion regions. We did not observe any differences in morphology or volume of the outflow tract or atrioventricular cushions in Robo1, Robo2, Slit2, or Slit3 mutant embryos at E14.5 (data not shown). However, in all E12.5 Robo1;Robo2 mutants, although cushion volume was not changed, the just forming aortic posterior cushion seemed absent (Figure 3H and I). In contrast, the pulmonary anterior cushion was forming normally. At E15.5, all Robo1;Robo2 double mutants showed thickening of both the semilunar and atrioventricular valves, in combination with now clearly visible bicuspid aortic valves (Figure 4K–O; see Supplementary material online, Figure S2E and F). While the overall aortic valve volume was similar between Robo1;Robo2 double mutants and wild-type littermates, this was caused by the absence of the posterior non-coronary aortic valve; the two remaining valves were thicker than their wild-type counterparts (Figure 4K–M). At E18.5, single Robo1 or Robo2 mutant valves remained indistinguishable from their wild-type littermates (Figure 4A–J). In contrast, E18.5 mice mutant for Slit2 showed thickening of both the aortic and pulmonary semilunar valves, similar to the Robo1;Robo2 double mutants; however, the atrioventricular valves retained a normal size (Figure 5A–E). As only one Slit2 mutant recapitulated the bicuspid aortic valves as seen in the absence of both Robo1 and Robo2, total aortic valve volume was significantly increased in mice lacking Slit2 (Figure 5A–C). The thickness of the semilunar valves in Slit3 mutants was relatively normal, but now the atrioventricular valves were strikingly thicker (Figure 5F–J). The posterior aortic semilunar leaflet, missing in the Robo1;Robo2 double mutants, was highly hypoplastic in the absence of Slit3, but never completely absent (Figure 5G and H). These data indicate a role for Slit-Robo signalling during valve maturation and functional redundancy and requirement of Robo1 and Robo2 in both the outflow tract and the atrioventricular valve regions, whereas the Slit ligands show region-specific functions. Furthermore, these results underline the possible multi-causal origin of the ventricular septum defects.Figure 4

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