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Rho-kinase-dependent actin turnover and actomyosin disassembly are necessary for mouse spinal neural tube closure.

Escuin S, Vernay B, Savery D, Gurniak CB, Witke W, Greene ND, Copp AJ - J. Cell. Sci. (2015)

Bottom Line: Preventing actomyosin cross-linking, F-actin assembly or myosin II contractile activity did not disrupt spinal closure.Co-exposure to Blebbistatin rescued the neurulation defects caused by RhoA inhibition, whereas an inhibitor of myosin light chain kinase, ML-7, had no such effect.We conclude that regulation of RhoA, Rho kinase, LIM kinase and cofilin signalling is necessary for spinal neural tube closure through precise control of neuroepithelial actin turnover and actomyosin disassembly.

View Article: PubMed Central - PubMed

Affiliation: Newlife Birth Defects Research Centre, Institute of Child Health, University College London, WC1N 1EH, UK.

No MeSH data available.


Related in: MedlinePlus

Stabilization of F-actin delays spinal neural tube closure. (A) PNP length is significantly increased at the 15–19 and 20–24 somite stage after 18–20 h culture in 10 nM jasplakinolide (Jasp), compared with DMSO controls (*P<0.05). (B) Biochemical fractionation shows proportionately increased F-actin and reduced G-actin in Jasp-treated embryos relative to DMSO controls (**P<0.001). (C) Immunohistochemistry (phalloidin, red; anti-MHCB, green) reveals actomyosin accumulation at the apical neuroepithelial surface (asterisks) and on some lateral cell surfaces (arrowheads) after Jasp treatment (embryos have 20–21 somites). Right, enlargement of the boxed areas. Scale bars: 30 µm. (D–F) Intensity profile scans along the neuroepithelial basal-to-apical axis. Jasp-treated embryos show an extension of phalloidin staining (intensity normalized to 100%) towards the basal surface (arrows in D), which is confirmed by quantification in the most apical 30% of the neuroepithelium (E; **P<0.001). MHCB staining intensity (non-normalized) is greater apically in Jasp-treated embryos than in DMSO controls (F). Significance values were calculated with a Student's t-test, compared with DMSO control.
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JCS164574F3: Stabilization of F-actin delays spinal neural tube closure. (A) PNP length is significantly increased at the 15–19 and 20–24 somite stage after 18–20 h culture in 10 nM jasplakinolide (Jasp), compared with DMSO controls (*P<0.05). (B) Biochemical fractionation shows proportionately increased F-actin and reduced G-actin in Jasp-treated embryos relative to DMSO controls (**P<0.001). (C) Immunohistochemistry (phalloidin, red; anti-MHCB, green) reveals actomyosin accumulation at the apical neuroepithelial surface (asterisks) and on some lateral cell surfaces (arrowheads) after Jasp treatment (embryos have 20–21 somites). Right, enlargement of the boxed areas. Scale bars: 30 µm. (D–F) Intensity profile scans along the neuroepithelial basal-to-apical axis. Jasp-treated embryos show an extension of phalloidin staining (intensity normalized to 100%) towards the basal surface (arrows in D), which is confirmed by quantification in the most apical 30% of the neuroepithelium (E; **P<0.001). MHCB staining intensity (non-normalized) is greater apically in Jasp-treated embryos than in DMSO controls (F). Significance values were calculated with a Student's t-test, compared with DMSO control.

Mentions: To test whether apical actomyosin accumulation, as seen after Y27632 treatment, might be responsible for defective spinal neurulation we used Jasplakinolide (Jasp), a drug that blocks actin depolymerization and increases the F-actin pool (Cramer, 1999). Culture in Jasp for 18–21 h to the 15–19 and 20–24 somite stages caused significant closure delay compared with DMSO controls (Fig. 3A; supplementary material Fig. S2E). More than 70% of actin was filamentous in these embryos (Fig. 3B) with a threefold reduction in monomeric G-actin (supplementary material Fig. S2F). Sections showed a massive accumulation of colocalized F-actin and MHCB at the apical surface as well as patches more basally within the neuroepithelium (Fig. 3C). Quantification confirmed the mainly apical accumulation of actomyosin (Fig. 3D–F), which was reproducible between embryos (supplementary material Fig. S2G). We considered the possibility that inhibition of ROCK might lead to a RhoA-dependent inhibition of embryonic cell proliferation or increase in cell death. However, measurement of the percentage of neuroepithelial cells positive for phosphorylated histone H3 staining and activated caspase 3 showed that neither cell proliferation nor programmed cell death was altered in Y27632- or Jasp-treated embryos (supplementary material Fig. S2H,I). We conclude, therefore, that F-actin stabilization by Jasp is sufficient to cause apical actomyosin accumulation and to inhibit spinal neural tube closure.Fig. 3.


Rho-kinase-dependent actin turnover and actomyosin disassembly are necessary for mouse spinal neural tube closure.

Escuin S, Vernay B, Savery D, Gurniak CB, Witke W, Greene ND, Copp AJ - J. Cell. Sci. (2015)

Stabilization of F-actin delays spinal neural tube closure. (A) PNP length is significantly increased at the 15–19 and 20–24 somite stage after 18–20 h culture in 10 nM jasplakinolide (Jasp), compared with DMSO controls (*P<0.05). (B) Biochemical fractionation shows proportionately increased F-actin and reduced G-actin in Jasp-treated embryos relative to DMSO controls (**P<0.001). (C) Immunohistochemistry (phalloidin, red; anti-MHCB, green) reveals actomyosin accumulation at the apical neuroepithelial surface (asterisks) and on some lateral cell surfaces (arrowheads) after Jasp treatment (embryos have 20–21 somites). Right, enlargement of the boxed areas. Scale bars: 30 µm. (D–F) Intensity profile scans along the neuroepithelial basal-to-apical axis. Jasp-treated embryos show an extension of phalloidin staining (intensity normalized to 100%) towards the basal surface (arrows in D), which is confirmed by quantification in the most apical 30% of the neuroepithelium (E; **P<0.001). MHCB staining intensity (non-normalized) is greater apically in Jasp-treated embryos than in DMSO controls (F). Significance values were calculated with a Student's t-test, compared with DMSO control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4510849&req=5

JCS164574F3: Stabilization of F-actin delays spinal neural tube closure. (A) PNP length is significantly increased at the 15–19 and 20–24 somite stage after 18–20 h culture in 10 nM jasplakinolide (Jasp), compared with DMSO controls (*P<0.05). (B) Biochemical fractionation shows proportionately increased F-actin and reduced G-actin in Jasp-treated embryos relative to DMSO controls (**P<0.001). (C) Immunohistochemistry (phalloidin, red; anti-MHCB, green) reveals actomyosin accumulation at the apical neuroepithelial surface (asterisks) and on some lateral cell surfaces (arrowheads) after Jasp treatment (embryos have 20–21 somites). Right, enlargement of the boxed areas. Scale bars: 30 µm. (D–F) Intensity profile scans along the neuroepithelial basal-to-apical axis. Jasp-treated embryos show an extension of phalloidin staining (intensity normalized to 100%) towards the basal surface (arrows in D), which is confirmed by quantification in the most apical 30% of the neuroepithelium (E; **P<0.001). MHCB staining intensity (non-normalized) is greater apically in Jasp-treated embryos than in DMSO controls (F). Significance values were calculated with a Student's t-test, compared with DMSO control.
Mentions: To test whether apical actomyosin accumulation, as seen after Y27632 treatment, might be responsible for defective spinal neurulation we used Jasplakinolide (Jasp), a drug that blocks actin depolymerization and increases the F-actin pool (Cramer, 1999). Culture in Jasp for 18–21 h to the 15–19 and 20–24 somite stages caused significant closure delay compared with DMSO controls (Fig. 3A; supplementary material Fig. S2E). More than 70% of actin was filamentous in these embryos (Fig. 3B) with a threefold reduction in monomeric G-actin (supplementary material Fig. S2F). Sections showed a massive accumulation of colocalized F-actin and MHCB at the apical surface as well as patches more basally within the neuroepithelium (Fig. 3C). Quantification confirmed the mainly apical accumulation of actomyosin (Fig. 3D–F), which was reproducible between embryos (supplementary material Fig. S2G). We considered the possibility that inhibition of ROCK might lead to a RhoA-dependent inhibition of embryonic cell proliferation or increase in cell death. However, measurement of the percentage of neuroepithelial cells positive for phosphorylated histone H3 staining and activated caspase 3 showed that neither cell proliferation nor programmed cell death was altered in Y27632- or Jasp-treated embryos (supplementary material Fig. S2H,I). We conclude, therefore, that F-actin stabilization by Jasp is sufficient to cause apical actomyosin accumulation and to inhibit spinal neural tube closure.Fig. 3.

Bottom Line: Preventing actomyosin cross-linking, F-actin assembly or myosin II contractile activity did not disrupt spinal closure.Co-exposure to Blebbistatin rescued the neurulation defects caused by RhoA inhibition, whereas an inhibitor of myosin light chain kinase, ML-7, had no such effect.We conclude that regulation of RhoA, Rho kinase, LIM kinase and cofilin signalling is necessary for spinal neural tube closure through precise control of neuroepithelial actin turnover and actomyosin disassembly.

View Article: PubMed Central - PubMed

Affiliation: Newlife Birth Defects Research Centre, Institute of Child Health, University College London, WC1N 1EH, UK.

No MeSH data available.


Related in: MedlinePlus