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BMPER Promotes Epithelial-Mesenchymal Transition in the Developing Cardiac Cushions.

Dyer L, Lockyer P, Wu Y, Saha A, Cyr C, Moser M, Pi X, Patterson C - PLoS ONE (2015)

Bottom Line: In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation.These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development.Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.

View Article: PubMed Central - PubMed

Affiliation: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America.

ABSTRACT
Formation of the cardiac valves is an essential component of cardiovascular development. Consistent with the role of the bone morphogenetic protein (BMP) signaling pathway in cardiac valve formation, embryos that are deficient for the BMP regulator BMPER (BMP-binding endothelial regulator) display the cardiac valve anomaly mitral valve prolapse. However, how BMPER deficiency leads to this defect is unknown. Based on its expression pattern in the developing cardiac cushions, we hypothesized that BMPER regulates BMP2-mediated signaling, leading to fine-tuned epithelial-mesenchymal transition (EMT) and extracellular matrix deposition. In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation. However, proliferation is not impaired in the developing BMPER-/- valves. In vitro data show that BMPER directly binds BMP2. In cultured endothelial cells, BMPER blocks BMP2-induced Smad activation in a dose-dependent manner. In addition, BMP2 increases the Sox9 protein level, and this increase is inhibited by co-treatment with BMPER. Consistently, in the BMPER-/- embryos, semi-quantitative analysis of Smad activation shows that the canonical BMP pathway is significantly more active in the atrioventricular cushions during EMT. These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development. Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.

No MeSH data available.


Related in: MedlinePlus

EMT is dysregulated in the BMPER-/- cushions.EMT markers Nfat-c1 (red) and Sox9 (green) were assessed in the atrioventricular cushions (AVCs; A-G) and outflow tract (OFT) cushions (H-N) at E9.5-E11.5. In wild-type (A, C, E) and BMPER-/- (B, D, F) AVCs at E9.5 (A, B), E10.5 (C, D), and E11.5 (E, F), Nfat-c1 expression appears identical between genotypes at each time point. In contrast, BMPER-/- embryos tend to have fewer Sox9-positive cells at E9.5 (B; p<0.1, quantified in G). By E10.5, the number of Sox9-positive cells is significantly higher in the BMPER-/- AVCs (D; *, p<0.01) compared with the wild-type AVCs (C). This pattern is not maintained, with a non-significant reduction in Sox9-positive cells present in the BMPER-/- AVCs by E11.5 (compare F with wild-type in E). (G) The number of Sox9-positive cells was counted in 2–4 sagittal sections, and the two most populated AVCs per embryo were averaged and used to compare how the cushions were populated. n = 6, 5, and 4 WT AVCs (black bars) and 6, 7, and 4 BMPER-/- AVCs (white bars) at E9.5, E10.5, and E11.5, respectively. (H-N) Nfat-c1 expression in the OFT cushions appears identical between genotypes at each time point. Though not statistically significant, BMPER-/- OFT cushions show the same pattern of Sox9-positive cell counts as the AVCs, with a tendency toward fewer Sox9-positive cells at E9.5 (I compared with WT in H, quantified in N), more Sox9-positive cells at E10.5 (K compared with WT in J), and fewer Sox9-positive cells at E11.5 (M compared with WT in L). (N) The number of Sox9-positive cells was counted as describe in (G). n = 6, 7, and 4 WT OFT cushions (black bars) and 6, 8, and 4 BMPER-/- OFT cushions (white bars) at E9.5, E10.5, and E11.5, respectively. Student’s t-test was used to compare means between genotypes. Scale bars in C and D = 120 μm and apply to A-F and H-M.
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pone.0139209.g002: EMT is dysregulated in the BMPER-/- cushions.EMT markers Nfat-c1 (red) and Sox9 (green) were assessed in the atrioventricular cushions (AVCs; A-G) and outflow tract (OFT) cushions (H-N) at E9.5-E11.5. In wild-type (A, C, E) and BMPER-/- (B, D, F) AVCs at E9.5 (A, B), E10.5 (C, D), and E11.5 (E, F), Nfat-c1 expression appears identical between genotypes at each time point. In contrast, BMPER-/- embryos tend to have fewer Sox9-positive cells at E9.5 (B; p<0.1, quantified in G). By E10.5, the number of Sox9-positive cells is significantly higher in the BMPER-/- AVCs (D; *, p<0.01) compared with the wild-type AVCs (C). This pattern is not maintained, with a non-significant reduction in Sox9-positive cells present in the BMPER-/- AVCs by E11.5 (compare F with wild-type in E). (G) The number of Sox9-positive cells was counted in 2–4 sagittal sections, and the two most populated AVCs per embryo were averaged and used to compare how the cushions were populated. n = 6, 5, and 4 WT AVCs (black bars) and 6, 7, and 4 BMPER-/- AVCs (white bars) at E9.5, E10.5, and E11.5, respectively. (H-N) Nfat-c1 expression in the OFT cushions appears identical between genotypes at each time point. Though not statistically significant, BMPER-/- OFT cushions show the same pattern of Sox9-positive cell counts as the AVCs, with a tendency toward fewer Sox9-positive cells at E9.5 (I compared with WT in H, quantified in N), more Sox9-positive cells at E10.5 (K compared with WT in J), and fewer Sox9-positive cells at E11.5 (M compared with WT in L). (N) The number of Sox9-positive cells was counted as describe in (G). n = 6, 7, and 4 WT OFT cushions (black bars) and 6, 8, and 4 BMPER-/- OFT cushions (white bars) at E9.5, E10.5, and E11.5, respectively. Student’s t-test was used to compare means between genotypes. Scale bars in C and D = 120 μm and apply to A-F and H-M.

Mentions: At E18.5, the BMPER-/- embryo displays a mitral valve phenotype that is consistent with mitral valve prolapse [12]. Thus, in combination with the increased expression of BMPER in the developing cushions when EMT takes place and BMP2’s critical role in EMT [4], we hypothesized that BMPER would affect EMT. We evaluated the expression of two EMT markers: Nfat-c1, which is an early marker of EMT [20], and Sox9, which is a late marker of EMT and a known BMP2 target [7]. In the atrioventricular cushions, Nfat-c1 expression was observed in the overlying endocardium at E9.5–11.5 in both the wild-type and BMPER-/- embryos, with no apparent differences (Fig 2A–2F, red signal). In contrast, non-significantly fewer Sox9-positive cells were initially observed in the BMPER-/- atrioventricular cushions at E9.5 (Fig 2B, compared with 2A, green signal, and 2G). However, by E10.5, significantly more Sox9-positive cells were present in the BMPER-/- atrioventricular cushions (Fig 2D, green signal, and 2G). Interestingly, although the total number of Sox9-positive cells increased at E10.5, the percentage of mesenchymal cells that expressed Sox9 non-significantly decreased at E10.5 (82.5% in the BMPER-/- vs. 86.8% in the wild-type atrioventricular cushions), indicating that more Sox9-negative mesenchymal cells were also present in the BMPER-/- atrioventricular cushions. The increase in total Sox9-positive cells was short-lived; between E10.5 and E11.5, the number of wild-type Sox9-positive cells increased greatly, whereas the BMPER-/- Sox9-positive cells showed only a slight increase, leading to fewer overall Sox9-positive mesenchymal cells in the BMPER-/- atrioventricular cushions compared with the wild-type cushions (Fig 2E and 2F, green signal and 2G). Additionally, the percentage of mesenchymal cells that were Sox9-positive was also significantly decreased (72.2% in the BMPER-/- vs. 92.3% in the wild-type cushions, p<0.01), suggesting that a subset of mesenchymal cells have prematurely stopped expressing Sox9. A similar, though non-significant, pattern was observed in the outflow tract cushions (Fig 2H–2N). The disruption of Sox9 expression, but not Nfat-c1 expression, is consistent with BMPER’s expression within the forming mesenchymal cells but exclusion from the overlying endocardium. Together, these data suggest that EMT is dysregulated in the absence of BMPER but that a secondary mechanism quickly compensates for the lack of BMPER.


BMPER Promotes Epithelial-Mesenchymal Transition in the Developing Cardiac Cushions.

Dyer L, Lockyer P, Wu Y, Saha A, Cyr C, Moser M, Pi X, Patterson C - PLoS ONE (2015)

EMT is dysregulated in the BMPER-/- cushions.EMT markers Nfat-c1 (red) and Sox9 (green) were assessed in the atrioventricular cushions (AVCs; A-G) and outflow tract (OFT) cushions (H-N) at E9.5-E11.5. In wild-type (A, C, E) and BMPER-/- (B, D, F) AVCs at E9.5 (A, B), E10.5 (C, D), and E11.5 (E, F), Nfat-c1 expression appears identical between genotypes at each time point. In contrast, BMPER-/- embryos tend to have fewer Sox9-positive cells at E9.5 (B; p<0.1, quantified in G). By E10.5, the number of Sox9-positive cells is significantly higher in the BMPER-/- AVCs (D; *, p<0.01) compared with the wild-type AVCs (C). This pattern is not maintained, with a non-significant reduction in Sox9-positive cells present in the BMPER-/- AVCs by E11.5 (compare F with wild-type in E). (G) The number of Sox9-positive cells was counted in 2–4 sagittal sections, and the two most populated AVCs per embryo were averaged and used to compare how the cushions were populated. n = 6, 5, and 4 WT AVCs (black bars) and 6, 7, and 4 BMPER-/- AVCs (white bars) at E9.5, E10.5, and E11.5, respectively. (H-N) Nfat-c1 expression in the OFT cushions appears identical between genotypes at each time point. Though not statistically significant, BMPER-/- OFT cushions show the same pattern of Sox9-positive cell counts as the AVCs, with a tendency toward fewer Sox9-positive cells at E9.5 (I compared with WT in H, quantified in N), more Sox9-positive cells at E10.5 (K compared with WT in J), and fewer Sox9-positive cells at E11.5 (M compared with WT in L). (N) The number of Sox9-positive cells was counted as describe in (G). n = 6, 7, and 4 WT OFT cushions (black bars) and 6, 8, and 4 BMPER-/- OFT cushions (white bars) at E9.5, E10.5, and E11.5, respectively. Student’s t-test was used to compare means between genotypes. Scale bars in C and D = 120 μm and apply to A-F and H-M.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4587915&req=5

pone.0139209.g002: EMT is dysregulated in the BMPER-/- cushions.EMT markers Nfat-c1 (red) and Sox9 (green) were assessed in the atrioventricular cushions (AVCs; A-G) and outflow tract (OFT) cushions (H-N) at E9.5-E11.5. In wild-type (A, C, E) and BMPER-/- (B, D, F) AVCs at E9.5 (A, B), E10.5 (C, D), and E11.5 (E, F), Nfat-c1 expression appears identical between genotypes at each time point. In contrast, BMPER-/- embryos tend to have fewer Sox9-positive cells at E9.5 (B; p<0.1, quantified in G). By E10.5, the number of Sox9-positive cells is significantly higher in the BMPER-/- AVCs (D; *, p<0.01) compared with the wild-type AVCs (C). This pattern is not maintained, with a non-significant reduction in Sox9-positive cells present in the BMPER-/- AVCs by E11.5 (compare F with wild-type in E). (G) The number of Sox9-positive cells was counted in 2–4 sagittal sections, and the two most populated AVCs per embryo were averaged and used to compare how the cushions were populated. n = 6, 5, and 4 WT AVCs (black bars) and 6, 7, and 4 BMPER-/- AVCs (white bars) at E9.5, E10.5, and E11.5, respectively. (H-N) Nfat-c1 expression in the OFT cushions appears identical between genotypes at each time point. Though not statistically significant, BMPER-/- OFT cushions show the same pattern of Sox9-positive cell counts as the AVCs, with a tendency toward fewer Sox9-positive cells at E9.5 (I compared with WT in H, quantified in N), more Sox9-positive cells at E10.5 (K compared with WT in J), and fewer Sox9-positive cells at E11.5 (M compared with WT in L). (N) The number of Sox9-positive cells was counted as describe in (G). n = 6, 7, and 4 WT OFT cushions (black bars) and 6, 8, and 4 BMPER-/- OFT cushions (white bars) at E9.5, E10.5, and E11.5, respectively. Student’s t-test was used to compare means between genotypes. Scale bars in C and D = 120 μm and apply to A-F and H-M.
Mentions: At E18.5, the BMPER-/- embryo displays a mitral valve phenotype that is consistent with mitral valve prolapse [12]. Thus, in combination with the increased expression of BMPER in the developing cushions when EMT takes place and BMP2’s critical role in EMT [4], we hypothesized that BMPER would affect EMT. We evaluated the expression of two EMT markers: Nfat-c1, which is an early marker of EMT [20], and Sox9, which is a late marker of EMT and a known BMP2 target [7]. In the atrioventricular cushions, Nfat-c1 expression was observed in the overlying endocardium at E9.5–11.5 in both the wild-type and BMPER-/- embryos, with no apparent differences (Fig 2A–2F, red signal). In contrast, non-significantly fewer Sox9-positive cells were initially observed in the BMPER-/- atrioventricular cushions at E9.5 (Fig 2B, compared with 2A, green signal, and 2G). However, by E10.5, significantly more Sox9-positive cells were present in the BMPER-/- atrioventricular cushions (Fig 2D, green signal, and 2G). Interestingly, although the total number of Sox9-positive cells increased at E10.5, the percentage of mesenchymal cells that expressed Sox9 non-significantly decreased at E10.5 (82.5% in the BMPER-/- vs. 86.8% in the wild-type atrioventricular cushions), indicating that more Sox9-negative mesenchymal cells were also present in the BMPER-/- atrioventricular cushions. The increase in total Sox9-positive cells was short-lived; between E10.5 and E11.5, the number of wild-type Sox9-positive cells increased greatly, whereas the BMPER-/- Sox9-positive cells showed only a slight increase, leading to fewer overall Sox9-positive mesenchymal cells in the BMPER-/- atrioventricular cushions compared with the wild-type cushions (Fig 2E and 2F, green signal and 2G). Additionally, the percentage of mesenchymal cells that were Sox9-positive was also significantly decreased (72.2% in the BMPER-/- vs. 92.3% in the wild-type cushions, p<0.01), suggesting that a subset of mesenchymal cells have prematurely stopped expressing Sox9. A similar, though non-significant, pattern was observed in the outflow tract cushions (Fig 2H–2N). The disruption of Sox9 expression, but not Nfat-c1 expression, is consistent with BMPER’s expression within the forming mesenchymal cells but exclusion from the overlying endocardium. Together, these data suggest that EMT is dysregulated in the absence of BMPER but that a secondary mechanism quickly compensates for the lack of BMPER.

Bottom Line: In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation.These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development.Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.

View Article: PubMed Central - PubMed

Affiliation: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America.

ABSTRACT
Formation of the cardiac valves is an essential component of cardiovascular development. Consistent with the role of the bone morphogenetic protein (BMP) signaling pathway in cardiac valve formation, embryos that are deficient for the BMP regulator BMPER (BMP-binding endothelial regulator) display the cardiac valve anomaly mitral valve prolapse. However, how BMPER deficiency leads to this defect is unknown. Based on its expression pattern in the developing cardiac cushions, we hypothesized that BMPER regulates BMP2-mediated signaling, leading to fine-tuned epithelial-mesenchymal transition (EMT) and extracellular matrix deposition. In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation. However, proliferation is not impaired in the developing BMPER-/- valves. In vitro data show that BMPER directly binds BMP2. In cultured endothelial cells, BMPER blocks BMP2-induced Smad activation in a dose-dependent manner. In addition, BMP2 increases the Sox9 protein level, and this increase is inhibited by co-treatment with BMPER. Consistently, in the BMPER-/- embryos, semi-quantitative analysis of Smad activation shows that the canonical BMP pathway is significantly more active in the atrioventricular cushions during EMT. These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development. Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.

No MeSH data available.


Related in: MedlinePlus