Figure 1. Loss of centrosome integrity during heart development.

(A) Analysis of centriole (γ-tubulin) configuration in E15- or P5-isolated ventricular rat cardiomyocytes (Troponin I). Nuclei: DAPI. PC: paired-centrioles. SC: split-centrioles. Scale bar: 5 µm. (B) Frequency of cells with paired-centrioles during development. Bi: Binucleated. (C) Analysis of the localization of the centrosome proteins PCM1, PCNT (Pericentrin), and CEP135 in isolated cardiomyocytes. (D) PCNT localization frequency in cardiomyocytes isolated from different developmental stages. (E) Centrosomal PCNT signal intensity in P3-isolated cardiomyocytes with paired- and split-centrioles relative to E15-isolated cardiomyocytes. (F) Frequency of paired-centrioles in P0-isolated cardiomyocytes after siRNA-mediated Pcnt knockdown. scr: scrambled. (G) Representative images of the analysis in (F). (H) PCM1 localization frequency in cardiomyocytes isolated from different developmental stages. (I) Analysis of PCM1 and PCNT localization in E15-isolated cardiomyocytes cultured for either 1 or 8 days. (J) Frequency of P0-isolated cardiomyocytes with paired-centrioles after 1 day, 3 days, or 6 days in culture. (K) RT-PCR analysis of Pcnt B and S isoform expression during rat heart development in vivo. (L) Localization of PCNT isoforms. P3-isolated cardiomyocytes immunostained with antibodies against either both PCNT B and S isoforms or only the PCNT B isoform. Yellow arrows: cardiomyocyte nuclei. Red arrows: non-myocyte nuclei. Unless otherwise noted, scale bars: 10 µm; red arrowheads: centrioles; data are mean ± SD, n = 3, *: p < 0.05. For the experiments ≥ 10 cells (E), ≥ 50 cells (B, F, J), ≥ 100 (D, H) cells were analyzed per experimental condition.

DOI: http://dx.doi.org/10.7554/eLife.05563.003

Figure 1—figure supplement 1. Loss of centrosome integrity during heart development.

(A) Representative images of centrosomes (γ-tubulin) in heart cryosections of P0 rat heart ventricles. Nuclei: DAPI. Cardiac nuclei: Nkx2.5. Arrowheads indicate centrioles. γ-tubulin signals separated by a distance greater than 2 µm were considered as singlets. Scale bars: 10 µm. (B) Quantitative analysis of centriole signals and configurations in cardiomyocytes and non-myocytes from cyrosections of E15-, P0-, P3-, P5, or adult (2 months) rat heart ventricles. CM: cardiomyocyte. NCM: non-myocyte. Results are from three independent animals, data are mean ± SD, ≥ 100 cells were analyzed per experimental condition, *: p < 0.05 (refers to doublets). (C) Representative images of P3-isolated cardiomyocytes immunostained for mother centriole (Odf2) and daughter centrioles (Centrobin) (green arrowheads). SC: split-centrioles. Colored ratios equal daughter or mother centriole: all centrioles. (D) Analysis of the localization of the centrosome proteins PCM1, PCNT (Pericentrin), and CEP135 in isolated cardiomyocytes. Red arrowheads indicate centrioles. (E) Representative images of Cdk5Rap2 localization in E15- and P3-isolated rat cardiomyocytes. Yellow arrowhead: Cdk5Rap2 at the nuclear envelope. (F) Quantitative analysis of centriolar Cdk5Rap2 signal intensity in E15- and P3-isolated cardiomyocytes. Data are mean ± SD, n = 3, ≥ 10 cells were analyzed per experimental condition, *: p < 0.05. (G) Representative images of PCM1 localization in E15-isolated cardiomyocytes (Troponin I). Centrioles: γ-tubulin.

DOI: http://dx.doi.org/10.7554/eLife.05563.004

Figure 1—figure supplement 2. Loss of centrosome integrity during heart development.

(A) Localization of Pericentrin (PCNT) B-GFP in non-myocytes and cardiomyocytes from different developmental stages. Cardiomyocyte (Troponin I), centriole (γ-tubulin). Arrowheads indicate centrioles. Yellow asterisk: cardiomyocytes. Blue asterisk: non-myocyte. (B) Frequency of PCNT B-GFP-positive centrioles in non-myocytes (NCM) and cardiomyocytes (CM) from different developmental stages. ≥ 50 cells pooled from several experiments were analyzed per time point. (C) Representative images of centriole configuration and PCNT and PCM1 localization in mouse iPSC-derived cardiomyocytes. Yellow scale bars: 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.05563.005