Figure 1.
Ftm-deficient murine embryos show perimembranous and muscular ventricular septal defects.
(A, B) Hematoxylin and Eosin stainings at E14.5 on transverse heart sections. (A) In wild-type mouse embryos, the ventricular septum consists of a muscular part (muVS) and a membranous part (meVS). (B) In Ftm−/− embryos, the muscular VS displays a shorter and thinner shape and the membranous VS is missing (indicated by the asterisk) representing a perimembranous ventricular septal defect. (C) While in Ftm+/+ (n = 23) and Ftm+/− mice (n = 21) the heart develops normally, 33% of Ftm−/− mice (n = 27) show perimembranous ventricular septal defects. This statistics is based on investigations of mice at E13.5, E14.5, E15.5, E16.5 and E17.5. (D) Ftm+/+ mouse embryos (n = 23) do not suffer from muscular ventricular septal defects. 81.5% of all analyzed Ftm−/− embryos (n = 27) display muscular ventricular septal defects. Embryos at E13.5, E14.5, E15.5, E16.5 and E17.5 were examined in this context. LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle; meVS, membranous ventricular septum; muVS, muscular ventricular septum; AS, atrial septum.
Figure 2.
Distribution of primary cilia in murine embryonic hearts.
(A, B, C, D, E, A1−5, B1, C1+2, D1, E1, A1i−A5i, B1i, C1i, C2i, D1a, E1i) Immunofluorescence on transverse heart sections at E12.5. (A, B, C) Tubulin cytoskeleton of cardiac cells is stained in green by acetylated α-tubulin resulting in a total view of ventricles (A) and atria (B, C). Cilia are stained in green by acetylated α-tubulin, basal bodies in red by pericentrin and cell nuclei in blue by DAPI (A1−5, B1, C1+2, A1i−A5i, B1i, C1i, C2i). Scale bars (in white) represent a length of 0.5 mm (A, B, C, D, E), 10 µm (A1−5, B1, C1+2, D1, D1a, E1) or 2 µm (A1i−A5i, B1i, C1i, C2i, E1i). (A, B, C, D, E) Coloured squares mark cardiac regions which are presented magnified in A1−5, B1, C1+2, D1+1a and E1. (A1−A5, B1, C1+2, E1) Coloured squares mark cardiac regions which are shown magnified in A1i-A5i, B1i, C1i, C2i and E1i. (A, B, C, D, E, A1−A5, B1, C1, C2, D1, D1a, E1, A1i−A5i, B1i, C1i+2i, E1i) The colour of the square correlates with the colour of the number of the magnified figures. (D) Arl13b is stained in red resulting in a total view of ECCs. (D1) Arl13b staining reveals ciliary presence on ECCs, while these cilia cannot be detected by staining acetylated α-tubulin in green (D1a). (E) Tubulin cytoskeleton of cardiac cells is stained in green by detyrosinated tubulin resulting in a total view of the ECCs. (E1, E1i) Cilia are stained in green by detyrosinated tubulin, basal bodies in red by γ-tubulin and cell nuclei in blue by DAPI. (F) Schematic illustration of ciliary distribution in embryonic mouse hearts. We found cilia exclusively at E10.5–12.5 and solely in distinct ventricular and atrial regions (blue lines) and on ECCs (turquoise staining). ECC, endocardial cushion cells; LA, left atrium; RA, right atrium; LV, left ventricle; LVa, left ventricle apical; LVm/b left ventricle medial/basal; RV, right ventricle; TB, trabecular formations; VS, ventricular septum.
Figure 3.
Loss of Ftm leads to shorter cardiac cilia.
(A, B) Immunofluorescence on transverse heart sections at E11.5. Cilia are stained in green by acetylated α-tubulin and cell nuclei in blue by DAPI. Scale bars (in white) represent a length of 2 µm. Ftm is localised at cardiac cilia (A), but is absent from Ftm-negative cilia (B). (C) Comparison of wild-type and Ftm-deficient ciliary length in ventricles and atria (n = 50 cilia, respectively). Ftm-negative cilia are significantly shorter in both ventricles (p = 3.41E−12) and atria (p = 2.79E−06).
Figure 4.
Reduced proliferation in ciliary regions of Ftm-deficient murine hearts and thickness decrease of Ftm-negative walls.
(A, B, A1–4, B1–4) Immunofluorescence on transverse ventricular sections at E11.5. Dividing cells (red staining) are marked by BrdU and cell nuclei (blue staining) by DAPI. Scale bars (in white) represent a length of 0.5 mm (A, B) or 20 µm (A1–4, B1–4). (A, B) Coloured squares mark cardiac regions which are presented magnified in A1–4 and B1–4, respectively. The colour of the square correlates with the colour of the number of the magnified figures. (C) Proliferation rate is determined by the relation of dividing (BrdU-marked) cells to the number of all cells in this heart region at E11.5 (Ftm+/+: n = 6; Ftm+/−: n = 11; Ftm−/−: n = 5). There is significantly less proliferation in the ciliary regions of ventricles and atria compared to non-ciliary regions. (D) Cardiac wall thickness measurements of wild-type (n = 6) and Ftm-deficient (n = 6) atria and ventricles in former ciliary and non-ciliary regions at E14.5. Walls are significantly thinner in all former ciliary regions. Additionally, ventricular, non-ciliary regions show a reduction in wall thickness, while atrial, non-ciliary regions do not differ significantly. LA, left atrium; RA, right atrium; LV, left ventricle; cLV, ciliary region of the left ventricle; ncLV, non-ciliary region of the left ventricle; RV, right ventricle; ciliary region of the right ventricle; VS, ventricular septum.
Figure 5.
Shh and Pdgfrα signals are downregulated in Ftm-negative murine hearts.
(A–D) Real-time PCR analysis of wild-type and Ftm-deficient ventricular (A, B) and atrial tissue (C, D) at E11.5 (A, C) and E14.5 (B, D). (A) Shh target gene expression of Ptc1 and Pdgfrα target gene expression of Hif1α are significantly downregulated in E11.5 Ftm−/− ventricles (n = 3, respectively; Ptc1: p = 0.013; Hif1α: p = 0.005). (B) At E14.5, both signaling pathways are unaffected in Ftm-negative ventricles (n = 6, respectively). (C, D) In Ftm−/− atria, Shh and Pdgfrα signaling are not significantly altered at E11.5 (n = 6 atria, respectively; C) and at E14.5 (n = 6 atria, respectively; D).
Figure 6.
Shh and Pdgfrα signaling components localize at cardiac cilia.
(A+B) Immunofluorescence on transverse heart sections at E11.5. Cilia are stained in green by acetylated α-tubulin and cell nuclei in blue by DAPI. Scale bars (in white) represent a length of 2 µm. (A) Ciliary Gli3-190 localisation (red staining) in wild-type ventricles demonstrates that Shh signaling is transduced by ventricular cilia. (B) Pdgfrα (red staining) is distributed along cardiac cilia in wild-type ventricles.
Figure 7.
Shh signaling acts upstream of Pdgfrα signals in cardiac cilia.
(A–D) Immunofluorescence on transverse heart sections at E11.5. Cilia are stained in green by acetylated α-tubulin and cell nuclei in blue by DAPI. Scale bars (in white) represent a length of 2 µm. (A) Gli3-190 (red staining) still shows a ciliary localisation in ventricular Ftm−/− cilia. (B) Pdgfrα (red staining) is absent in cilia of Ftm-deficient ventricles. (C) Gli3-190 protein is not observed at cilia of Shh−/− ventricles. (D) Pdgfrα cannot be detected in cilia of Shh-negative ventricles.
Figure 8.
Ftm-negative hearts display a disturbance in Gli3 processing.
(A) Western blot analysis of E11.5 embryo and heart protein lysates. Actin serves as loading control. In Ftm-negative embryos (n = 3), there is more Gli3-190 protein than in wild-type littermates, but an equal amount of Gli3-83. The amount of Gli3-190 protein is higher in Ftm-deficient than in wild-type hearts (n = 12, respectively), while conversely, there is less Gli3-83 in Ftm−/− hearts indicating a processing defect in Ftm-negative hearts. (B, C) Graphical evaluation of the Gli3-190/Gli3-83 ratio in wild-type and Ftm-deficient embryos and hearts, respectively. (B) The ratio of Gli3-190/Gli3-83 is 2.64 fold elevated in Ftm-homozygous mutant embryos. (C) Gli3-190/Gli3-83 ratio is 10.94 fold increased in Ftm-negative hearts.
Figure 9.
Myocardial (violett), endocardial (orange) and most likely trabecular cilia (turquoise) regulate proliferation at distinct cardiac regions. In the ventricles, the cell proliferation in these regions results in wall thickness control, trabecular formation and a push of cells toward the base of the muscular ventricular septum (indicated by pink arrows). ECC cilia (yellow) seem to be different from the other cardiac cilia, since they do not regulate ECC proliferation. LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle; muVS, muscular ventricular septum.