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The ciliary protein Ftm is required for ventricular wall and septal development.

Gerhardt C, Lier JM, Kuschel S, Rüther U - PLoS ONE (2013)

Bottom Line: Despite several studies of the molecular mechanisms involved in ventricular septum (VS) development, very little is known about VS-forming signaling.Since Ftm is a ciliary protein, we investigated presence and function of cilia in murine hearts.Primary cilia could be detected at distinct positions in atria and ventricles at embryonic days (E) 10.5-12.5.

View Article: PubMed Central - PubMed

Affiliation: Institute for Animal Developmental and Molecular Biology, Heinrich Heine University, Düsseldorf, Germany.

ABSTRACT
Ventricular septal defects (VSDs) are the most common congenital heart defects in humans. Despite several studies of the molecular mechanisms involved in ventricular septum (VS) development, very little is known about VS-forming signaling. We observed perimembranous and muscular VSDs in Fantom (Ftm)-negative mice. Since Ftm is a ciliary protein, we investigated presence and function of cilia in murine hearts. Primary cilia could be detected at distinct positions in atria and ventricles at embryonic days (E) 10.5-12.5. The loss of Ftm leads to shortened cilia and a reduced proliferation in distinct atrial and ventricular ciliary regions at E11.5. Consequently, wall thickness is diminished in these areas. We suggest that ventricular proliferation is regulated by cilia-mediated Sonic hedgehog (Shh) and platelet-derived growth factor receptor α (Pdgfrα) signaling. Accordingly, we propose that primary cilia govern the cardiac proliferation which is essential for proper atrial and ventricular wall development and hence for the fully outgrowth of the VS. Thus, our study suggests ciliopathy as a cause of VSDs.

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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.
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pone-0057545-g004: 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.

Mentions: The VS does not fully grow out in Ftm-negative mice (Figure 1B). From other tissue and cell culture experiments, it is known that monocilia mediate proliferative and apoptotic signals [23]. Since ventricular cilia appear at the time, when the muscular VS is growing out [58], and at those regions, where the proliferation of cells effects the outgrowth of the VS [11], we investigated proliferation and apoptosis via bromodeoxyuridine (BrdU) staining to determine the rate of proliferation and by means of TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining to look for cell death at E11.5. Whereas the apoptosis study was inconspicuous (Figure S5A), there were differences in the proliferation rate between wild-type, Ftm-heterozygous and Ftm-homozygous mutant hearts. The proliferation in all Ftm-negative, ciliary areas in ventricles and atria was significantly diminished, while no proliferation differences could be observed in non-ciliary regions (Figure 4B1–B4, C). So in embryonic hearts, cilia seem to be necessary to mediate proliferative signals which in turn are responsible for a part of cardiac cell proliferation. Consequently, when cilia are absent at a later point of time, the rate of proliferation of wild-type, Ftm-heterozygous and Ftm-homozygous mutant embryos should not differ significantly. Performing the same proliferation assays in E14.5 hearts, we found that, indeed, the proliferation in all areas, which were investigated, was similar in wild-type and Ftm-deficient hearts (Figure S5B). The diminished proliferation in Ftm-homozygous mutant hearts is in agreement with the results of semiquantitative Reverse transcription-PCRs. These experiments uncovered a change of expression levels of cyclin E and p27 that are involved in cell cycle regulation and proliferation (Figure S6) substantiating suspicion of a proliferation defect in Ftm-deficient hearts.


The ciliary protein Ftm is required for ventricular wall and septal development.

Gerhardt C, Lier JM, Kuschel S, Rüther U - PLoS ONE (2013)

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.
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Related In: Results  -  Collection

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pone-0057545-g004: 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.
Mentions: The VS does not fully grow out in Ftm-negative mice (Figure 1B). From other tissue and cell culture experiments, it is known that monocilia mediate proliferative and apoptotic signals [23]. Since ventricular cilia appear at the time, when the muscular VS is growing out [58], and at those regions, where the proliferation of cells effects the outgrowth of the VS [11], we investigated proliferation and apoptosis via bromodeoxyuridine (BrdU) staining to determine the rate of proliferation and by means of TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining to look for cell death at E11.5. Whereas the apoptosis study was inconspicuous (Figure S5A), there were differences in the proliferation rate between wild-type, Ftm-heterozygous and Ftm-homozygous mutant hearts. The proliferation in all Ftm-negative, ciliary areas in ventricles and atria was significantly diminished, while no proliferation differences could be observed in non-ciliary regions (Figure 4B1–B4, C). So in embryonic hearts, cilia seem to be necessary to mediate proliferative signals which in turn are responsible for a part of cardiac cell proliferation. Consequently, when cilia are absent at a later point of time, the rate of proliferation of wild-type, Ftm-heterozygous and Ftm-homozygous mutant embryos should not differ significantly. Performing the same proliferation assays in E14.5 hearts, we found that, indeed, the proliferation in all areas, which were investigated, was similar in wild-type and Ftm-deficient hearts (Figure S5B). The diminished proliferation in Ftm-homozygous mutant hearts is in agreement with the results of semiquantitative Reverse transcription-PCRs. These experiments uncovered a change of expression levels of cyclin E and p27 that are involved in cell cycle regulation and proliferation (Figure S6) substantiating suspicion of a proliferation defect in Ftm-deficient hearts.

Bottom Line: Despite several studies of the molecular mechanisms involved in ventricular septum (VS) development, very little is known about VS-forming signaling.Since Ftm is a ciliary protein, we investigated presence and function of cilia in murine hearts.Primary cilia could be detected at distinct positions in atria and ventricles at embryonic days (E) 10.5-12.5.

View Article: PubMed Central - PubMed

Affiliation: Institute for Animal Developmental and Molecular Biology, Heinrich Heine University, Düsseldorf, Germany.

ABSTRACT
Ventricular septal defects (VSDs) are the most common congenital heart defects in humans. Despite several studies of the molecular mechanisms involved in ventricular septum (VS) development, very little is known about VS-forming signaling. We observed perimembranous and muscular VSDs in Fantom (Ftm)-negative mice. Since Ftm is a ciliary protein, we investigated presence and function of cilia in murine hearts. Primary cilia could be detected at distinct positions in atria and ventricles at embryonic days (E) 10.5-12.5. The loss of Ftm leads to shortened cilia and a reduced proliferation in distinct atrial and ventricular ciliary regions at E11.5. Consequently, wall thickness is diminished in these areas. We suggest that ventricular proliferation is regulated by cilia-mediated Sonic hedgehog (Shh) and platelet-derived growth factor receptor α (Pdgfrα) signaling. Accordingly, we propose that primary cilia govern the cardiac proliferation which is essential for proper atrial and ventricular wall development and hence for the fully outgrowth of the VS. Thus, our study suggests ciliopathy as a cause of VSDs.

Show MeSH
Related in: MedlinePlus