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Experiment details for myh6

Hempel A et al. (2017) Assay

The CapZ interacting protein Rcsd1 is required for cardiogenesis downstream of Wnt11a in Xenopus laevis.

Gene Clone Species Stages Anatomy
myh6.L laevis NF stage 28 heart , primary heart field

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  Fig. 4. : Rcsd1 depletion interferes with cardiac differentiation. A. Expression of nkx2-5, isl1 and tbx20 at stage 20 upon Rcsd1 down-regulation. B. Quantitative presentation of data shown in A. C. Unilateral injection Rcsd1 MO leads to reduced cardiac marker gene expression at stage 28 (ventral views, red arrowheads). D. Quantitative presentation of data shown in C. E-F. qPCR approaches with cardiac explants at stage 28 (E) and stage 33/34 (F) confirmed the down-regulation of tnni3 and myh6 upon Rcsd1 depletion. N, number of single explants used for qPCR. G. Unilateral inhibition of cardiac marker gene expression upon Rcsd1 MO injection (ventral views, red arrowheads) is restored by the co-injection of full-length Xenopus rcsd1 RNA (ventral views, black arrowheads). H. Quantitative presentation of data shown in G. n, number of independent experiments; N, number of analysed embryos. Error bars indicate standard error of the means (s.e.m.). *, p≤0.05, **; p≤0.01; ***, p≤0.001; ****, p≤0.0001; calculated by a non-parametric Mann-Whitney rank sum test.

Gene Clone Species Stages Anatomy
myh6.L laevis NF stage 28 heart , primary heart field

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  Fig. 5. : Rcsd1 acts downstream of Wnt11a. A. Unilateral injection of Wnt11a MO leads to inhibition of cardiac marker gene expression (red arrowheads), which is restored by the co-injection of Xenopus full-length rcsd1 RNA (black arrowheads). B. Quantitative presentation of data shown in A. C. Unilateral injection of Wnt11a MO leads to inhibition of rcsd1 expression (red arrowheads) D. Quantitative presentation of data shown in C. n, number of independent experiments; N, number of embryos analysed. Error bars indicate standard error of the means (s.e.m.). *, p≤0.05, **; p≤0.01; ***; p≤0.001; ****, p≤0.0001; calculated by a non-parametric Mann-Whitney rank sum test.

Gene Clone Species Stages Anatomy
myh6.L laevis NF stage 28 heart , primary heart field

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  Fig. 6. : Rcsd1 functionally interacts with CapZ in the cytosol. A. Schematic overview of used murine Rcsd1 and deletion constructs. Rcsd1 contains a CPI (capping protein interaction) and a NLS (nuclear localization signal) motif. Numbers indicate amino acids in the Rcsd1 protein. B. Amino acid sequence of murine CPI and NLS motifs. Numbers indicate amino acids in the Rcsd1 protein. C. Transfection of the full-length EGFP-Rcsd1 fusion construct revealed that Rcsd1 is localized in the nucleus and cytosol (upper row). Deletion of CPI (middle row) or NLS (lower row) in Rcsd1 leads to a cytosolic localization. Scale bar: 100 μm. D-E. Split YFP complementation assays. Murine full-length Rcsd1 and Rcsd1δNLS (labelled as δNLS) interact with CapZa and CapZb whereas Rcsd1δCPI (labelled as δCPI) does not. Scale bars D, E: 10 μm. F. Rcsd1 MO was unilaterally injected along with different murine Rcsd1 constructs and the expression of the cardiac markers tnni3, actc1, myh6 and alcam was monitored at stage 28 (ventral views). Reduced marker gene expression upon loss of Rcsd1 (red arrowheads) was rescued by co-injection with the full-length murine Rcsd1 construct (Rcsd1, dark grey arrowheads) and Rcsd1δNLS (dark blue arrowheads). Rcsd1δCPI however (orange arrowheads) was not able to restore marker gene expression. G. Quantitative presentation of data shown in F. n, number of independent experiments; N, number of analysed embryos. Error bars indicate standard error of the means (s.e.m.). n.s., not significant, *, p≤0.05; **, p≤0.01; ***, p≤0.001; calculated by a non-parametric Mann-Whitney rank sum test.