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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

Proliferation is normal in the BMPER-/- cushions.Proliferation was assessed in the atrioventricular cushions (AVCs) and outflow tract (OFT) cushions. Proliferative cells were detected via phosphohistone H3 expression (green), and sagittal sections were colabeled with the myocardial marker MF20 (red) and nuclear marker DAPI (blue). For each sample, all mesenchymal cells in at least 3 sections or a minimum of 100 cells were counted. (A-G) Wild-type (A, C, E) and BMPER-/- (B, D, F) AVCs were evaluated at E9.5 (A, B), E10.5 (C, D), and E11.5 (E, F). (A, B) At E9.5, no significant differences were observed between genotypes. (C, D) By E10.5, the proliferation remained similar in both the BMPER-/- and wild-type AVCs. (E, F) By E11.5, the proliferation rate increased similarly in both genotypes. (G) The proliferation rates for each group were quantified. n = 2, 4, and 3 for wild-type AVCs and 2, 5, and 3 for BMPER-/- AVCs at E9.5, E10.5, and E11.5, respectively. (H-N) Wild-type and BMPER-/- OFT cushions were evaluated in the same manner. (H, I) At E9.5, proliferation was increased, though not significantly, in the OFT cushions of BMPER-/- embryos compared with wild-type embryos. (J, K) By E10.5, the proliferation rate decreased in the BMPER-/- OFT cushions and was comparable to that in the wild-type OFT cushions. (L, M) As EMT ended and the OFT cushions entered the proliferative phase, the proliferation rate increased similarly in both genotypes. (N) The proliferation rates for each group were quantified. n = 2, 4, and 3 for wild-type OFT cushions and 2, 5, and 3 for BMPER-/- OFT cushions at E9.5, E10.5, and E11.5, respectively. Scale bars in A, B, H, and I = 100 μm; scale bars in C, D, J, and K = 110 μm and apply to E, F, L, and M.
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pone.0139209.g003: Proliferation is normal in the BMPER-/- cushions.Proliferation was assessed in the atrioventricular cushions (AVCs) and outflow tract (OFT) cushions. Proliferative cells were detected via phosphohistone H3 expression (green), and sagittal sections were colabeled with the myocardial marker MF20 (red) and nuclear marker DAPI (blue). For each sample, all mesenchymal cells in at least 3 sections or a minimum of 100 cells were counted. (A-G) Wild-type (A, C, E) and BMPER-/- (B, D, F) AVCs were evaluated at E9.5 (A, B), E10.5 (C, D), and E11.5 (E, F). (A, B) At E9.5, no significant differences were observed between genotypes. (C, D) By E10.5, the proliferation remained similar in both the BMPER-/- and wild-type AVCs. (E, F) By E11.5, the proliferation rate increased similarly in both genotypes. (G) The proliferation rates for each group were quantified. n = 2, 4, and 3 for wild-type AVCs and 2, 5, and 3 for BMPER-/- AVCs at E9.5, E10.5, and E11.5, respectively. (H-N) Wild-type and BMPER-/- OFT cushions were evaluated in the same manner. (H, I) At E9.5, proliferation was increased, though not significantly, in the OFT cushions of BMPER-/- embryos compared with wild-type embryos. (J, K) By E10.5, the proliferation rate decreased in the BMPER-/- OFT cushions and was comparable to that in the wild-type OFT cushions. (L, M) As EMT ended and the OFT cushions entered the proliferative phase, the proliferation rate increased similarly in both genotypes. (N) The proliferation rates for each group were quantified. n = 2, 4, and 3 for wild-type OFT cushions and 2, 5, and 3 for BMPER-/- OFT cushions at E9.5, E10.5, and E11.5, respectively. Scale bars in A, B, H, and I = 100 μm; scale bars in C, D, J, and K = 110 μm and apply to E, F, L, and M.

Mentions: As a potential mechanism to explain the sudden increase in Sox9-positive cells in the BMPER-/- atrioventricular cushions at E10.5, we examined proliferation at E9.5-E11.5. Surprisingly, though, no differences were observed in the proliferation rates in the BMPER-/- atrioventricular cushions compared with their wild-type counterparts at any time point examined (Fig 3A–3G). Additionally, no statistically significant differences in the proliferation rate were observed between genotypes in the outflow tract cushions at any time point examined; however, there was a non-significant increase at E9.5 in the BMPER-/- outflow tract cushions (Fig 3H–3N). These results suggest that the increase in Sox9-positive cell number at E10.5 is not due to an increase in cell proliferation. Other unknown mechanisms involved in this process remain to be determined.


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)

Proliferation is normal in the BMPER-/- cushions.Proliferation was assessed in the atrioventricular cushions (AVCs) and outflow tract (OFT) cushions. Proliferative cells were detected via phosphohistone H3 expression (green), and sagittal sections were colabeled with the myocardial marker MF20 (red) and nuclear marker DAPI (blue). For each sample, all mesenchymal cells in at least 3 sections or a minimum of 100 cells were counted. (A-G) Wild-type (A, C, E) and BMPER-/- (B, D, F) AVCs were evaluated at E9.5 (A, B), E10.5 (C, D), and E11.5 (E, F). (A, B) At E9.5, no significant differences were observed between genotypes. (C, D) By E10.5, the proliferation remained similar in both the BMPER-/- and wild-type AVCs. (E, F) By E11.5, the proliferation rate increased similarly in both genotypes. (G) The proliferation rates for each group were quantified. n = 2, 4, and 3 for wild-type AVCs and 2, 5, and 3 for BMPER-/- AVCs at E9.5, E10.5, and E11.5, respectively. (H-N) Wild-type and BMPER-/- OFT cushions were evaluated in the same manner. (H, I) At E9.5, proliferation was increased, though not significantly, in the OFT cushions of BMPER-/- embryos compared with wild-type embryos. (J, K) By E10.5, the proliferation rate decreased in the BMPER-/- OFT cushions and was comparable to that in the wild-type OFT cushions. (L, M) As EMT ended and the OFT cushions entered the proliferative phase, the proliferation rate increased similarly in both genotypes. (N) The proliferation rates for each group were quantified. n = 2, 4, and 3 for wild-type OFT cushions and 2, 5, and 3 for BMPER-/- OFT cushions at E9.5, E10.5, and E11.5, respectively. Scale bars in A, B, H, and I = 100 μm; scale bars in C, D, J, and K = 110 μm and apply to E, F, L, and M.
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pone.0139209.g003: Proliferation is normal in the BMPER-/- cushions.Proliferation was assessed in the atrioventricular cushions (AVCs) and outflow tract (OFT) cushions. Proliferative cells were detected via phosphohistone H3 expression (green), and sagittal sections were colabeled with the myocardial marker MF20 (red) and nuclear marker DAPI (blue). For each sample, all mesenchymal cells in at least 3 sections or a minimum of 100 cells were counted. (A-G) Wild-type (A, C, E) and BMPER-/- (B, D, F) AVCs were evaluated at E9.5 (A, B), E10.5 (C, D), and E11.5 (E, F). (A, B) At E9.5, no significant differences were observed between genotypes. (C, D) By E10.5, the proliferation remained similar in both the BMPER-/- and wild-type AVCs. (E, F) By E11.5, the proliferation rate increased similarly in both genotypes. (G) The proliferation rates for each group were quantified. n = 2, 4, and 3 for wild-type AVCs and 2, 5, and 3 for BMPER-/- AVCs at E9.5, E10.5, and E11.5, respectively. (H-N) Wild-type and BMPER-/- OFT cushions were evaluated in the same manner. (H, I) At E9.5, proliferation was increased, though not significantly, in the OFT cushions of BMPER-/- embryos compared with wild-type embryos. (J, K) By E10.5, the proliferation rate decreased in the BMPER-/- OFT cushions and was comparable to that in the wild-type OFT cushions. (L, M) As EMT ended and the OFT cushions entered the proliferative phase, the proliferation rate increased similarly in both genotypes. (N) The proliferation rates for each group were quantified. n = 2, 4, and 3 for wild-type OFT cushions and 2, 5, and 3 for BMPER-/- OFT cushions at E9.5, E10.5, and E11.5, respectively. Scale bars in A, B, H, and I = 100 μm; scale bars in C, D, J, and K = 110 μm and apply to E, F, L, and M.
Mentions: As a potential mechanism to explain the sudden increase in Sox9-positive cells in the BMPER-/- atrioventricular cushions at E10.5, we examined proliferation at E9.5-E11.5. Surprisingly, though, no differences were observed in the proliferation rates in the BMPER-/- atrioventricular cushions compared with their wild-type counterparts at any time point examined (Fig 3A–3G). Additionally, no statistically significant differences in the proliferation rate were observed between genotypes in the outflow tract cushions at any time point examined; however, there was a non-significant increase at E9.5 in the BMPER-/- outflow tract cushions (Fig 3H–3N). These results suggest that the increase in Sox9-positive cell number at E10.5 is not due to an increase in cell proliferation. Other unknown mechanisms involved in this process remain to be determined.

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