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Ectopic Noggin in a Population of Nfatc1 Lineage Endocardial Progenitors Induces Embryonic Lethality.

Snider P, Simmons O, Wang J, Hoang CQ, Conway SJ - J Cardiovasc Dev Dis (2014)

Bottom Line: This is coupled with hypoplastic endocardial cushions, reduced trabeculation and fewer mature contractile fibrils in mutant hearts.Molecular analysis demonstrated that endocardial Noggin resulted in a specific alteration of TGFβ/BMP-mediated signal transduction, in that, both Endoglin and ALK1 were downregulated in mutant endocardium.Combined, these results demonstrate the cell-autonomous requirement of the endocardial lineage and function of unaltered BMP levels in facilitating endothelium-cardiomyocyte cross-talk and promoting endocardial cushion formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Biology and Neonatal Medicine Program, HB Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

ABSTRACT

The initial heart is composed of a myocardial tube lined by endocardial cells. The TGFβ superfamily is known to play an important role, as BMPs from the myocardium signal to the overlying endocardium to create an environment for EMT. Subsequently, BMP and TGFβ signaling pathways synergize to form primitive valves and regulate myocardial growth. In this study, we investigated the requirement of BMP activity by transgenic over-expression of extracellular BMP antagonist Noggin. Using Nfatc1(Cre) to drive lineage-restricted Noggin within the endocardium, we show that ectopic Noggin arrests cardiac development in E10.5-11 embryos, resulting in small hearts which beat poorly and die by E12.5. This is coupled with hypoplastic endocardial cushions, reduced trabeculation and fewer mature contractile fibrils in mutant hearts. Moreover, Nfatc1(Cre) -mediated diphtheria toxin fragment-A expression in the endocardium resulted in genetic ablation and a more severe phenotype with lethality at E11 and abnormal linear hearts. Molecular analysis demonstrated that endocardial Noggin resulted in a specific alteration of TGFβ/BMP-mediated signal transduction, in that, both Endoglin and ALK1 were downregulated in mutant endocardium. Combined, these results demonstrate the cell-autonomous requirement of the endocardial lineage and function of unaltered BMP levels in facilitating endothelium-cardiomyocyte cross-talk and promoting endocardial cushion formation.

No MeSH data available.


Related in: MedlinePlus

qPCR mRNA profiling and in situ verification of expression alterations. (A): Quantitative PCR using a custom array of 84 genes responsive to TGFß superfamily signal transduction revealed statistically significant changes in transcript levels of the 13 listed genes. Data are presented as a logarithmic plot of relative expression (mutant/wildtype), where a value of 1 indicates no difference between E10.5 NogEnd mutant and wildtype pooled hearts and values <1 indicate reduced expression. The biggest alterations were observed in Noggin levels, which were x4 fold elevated in mutant hearts; and Endoglin levels, which were×3.7 fold reduced in mutant hearts. Error bars represent SEM; (B,C): Reduced Endoglin expression levels were verified by radioactive in situ hybridization, as the E10.5 mutant heart (B) exhibits significantly less Endoglin mRNA throughout the endocardial lineage when compared to wildtype littermate (B); (D–G): As qPCR profiling did not reveal any alterations in either endocardially-restricted Tgfβ1 or myocardial Msx2 expression levels, we used in situ hybridization to examine whether spatial alterations were present in mutant hearts; Radioactive in situ hybridization revealed comparable Tgfβ1 mRNA expression in control (D) and mutants (E), and Msx2 was normally expressed within the controls (F) and NogEnd mutant (F) myocardium adjacent to the AV cushions. Abbreviations: avc, atrioventricular cushion; lv, left ventricle; rv, right ventricle.
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Figure 5: qPCR mRNA profiling and in situ verification of expression alterations. (A): Quantitative PCR using a custom array of 84 genes responsive to TGFß superfamily signal transduction revealed statistically significant changes in transcript levels of the 13 listed genes. Data are presented as a logarithmic plot of relative expression (mutant/wildtype), where a value of 1 indicates no difference between E10.5 NogEnd mutant and wildtype pooled hearts and values <1 indicate reduced expression. The biggest alterations were observed in Noggin levels, which were x4 fold elevated in mutant hearts; and Endoglin levels, which were×3.7 fold reduced in mutant hearts. Error bars represent SEM; (B,C): Reduced Endoglin expression levels were verified by radioactive in situ hybridization, as the E10.5 mutant heart (B) exhibits significantly less Endoglin mRNA throughout the endocardial lineage when compared to wildtype littermate (B); (D–G): As qPCR profiling did not reveal any alterations in either endocardially-restricted Tgfβ1 or myocardial Msx2 expression levels, we used in situ hybridization to examine whether spatial alterations were present in mutant hearts; Radioactive in situ hybridization revealed comparable Tgfβ1 mRNA expression in control (D) and mutants (E), and Msx2 was normally expressed within the controls (F) and NogEnd mutant (F) myocardium adjacent to the AV cushions. Abbreviations: avc, atrioventricular cushion; lv, left ventricle; rv, right ventricle.

Mentions: To identify the endocardial targets that are altered via ectopic Noggin and the factors that may mediate endocardial-cardiomyocyte signaling during cardiac morphogenesis, we examined expression of TGFβ superfamily targets and genes known to be involved in BMP and TGFβ ligand downstream signaling by qPCR in isolated E10.5 hearts (n = 3 pooled isolated NogEnd and wildtype hearts, carried out in duplicate samples). The E10.5 stage was chosen as NogEnd mutants do not exhibit an overt phenotype at this age (Figure 1D), but do show pSMAD deviations (Figure 1K). Quantitative PCR using a custom array of 84 genes related to TGFβ /BMP-mediated signal transduction, including SMAD, SMAD target genes, adhesion and extracellular molecules and transcription factors revealed significant changes in transcript levels of the 13 listed genes (Figure 5A). As proof of principle, it was significant that expression of Noggin was elevated almost×4 fold in NogEnd hearts. Meaningfully, neither Bmp2 nor Bmp4 were affected, and endocardially-restricted Tgfβ1, myocardial Tgfβ2 and Msx2 levels were unaltered. Similarly, Smad1-5 expression levels were normal. However, Tgfβ3 mRNA was downregulated ×1.9 fold in NogEnd hearts (Figure 5A).


Ectopic Noggin in a Population of Nfatc1 Lineage Endocardial Progenitors Induces Embryonic Lethality.

Snider P, Simmons O, Wang J, Hoang CQ, Conway SJ - J Cardiovasc Dev Dis (2014)

qPCR mRNA profiling and in situ verification of expression alterations. (A): Quantitative PCR using a custom array of 84 genes responsive to TGFß superfamily signal transduction revealed statistically significant changes in transcript levels of the 13 listed genes. Data are presented as a logarithmic plot of relative expression (mutant/wildtype), where a value of 1 indicates no difference between E10.5 NogEnd mutant and wildtype pooled hearts and values <1 indicate reduced expression. The biggest alterations were observed in Noggin levels, which were x4 fold elevated in mutant hearts; and Endoglin levels, which were×3.7 fold reduced in mutant hearts. Error bars represent SEM; (B,C): Reduced Endoglin expression levels were verified by radioactive in situ hybridization, as the E10.5 mutant heart (B) exhibits significantly less Endoglin mRNA throughout the endocardial lineage when compared to wildtype littermate (B); (D–G): As qPCR profiling did not reveal any alterations in either endocardially-restricted Tgfβ1 or myocardial Msx2 expression levels, we used in situ hybridization to examine whether spatial alterations were present in mutant hearts; Radioactive in situ hybridization revealed comparable Tgfβ1 mRNA expression in control (D) and mutants (E), and Msx2 was normally expressed within the controls (F) and NogEnd mutant (F) myocardium adjacent to the AV cushions. Abbreviations: avc, atrioventricular cushion; lv, left ventricle; rv, right ventricle.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4469290&req=5

Figure 5: qPCR mRNA profiling and in situ verification of expression alterations. (A): Quantitative PCR using a custom array of 84 genes responsive to TGFß superfamily signal transduction revealed statistically significant changes in transcript levels of the 13 listed genes. Data are presented as a logarithmic plot of relative expression (mutant/wildtype), where a value of 1 indicates no difference between E10.5 NogEnd mutant and wildtype pooled hearts and values <1 indicate reduced expression. The biggest alterations were observed in Noggin levels, which were x4 fold elevated in mutant hearts; and Endoglin levels, which were×3.7 fold reduced in mutant hearts. Error bars represent SEM; (B,C): Reduced Endoglin expression levels were verified by radioactive in situ hybridization, as the E10.5 mutant heart (B) exhibits significantly less Endoglin mRNA throughout the endocardial lineage when compared to wildtype littermate (B); (D–G): As qPCR profiling did not reveal any alterations in either endocardially-restricted Tgfβ1 or myocardial Msx2 expression levels, we used in situ hybridization to examine whether spatial alterations were present in mutant hearts; Radioactive in situ hybridization revealed comparable Tgfβ1 mRNA expression in control (D) and mutants (E), and Msx2 was normally expressed within the controls (F) and NogEnd mutant (F) myocardium adjacent to the AV cushions. Abbreviations: avc, atrioventricular cushion; lv, left ventricle; rv, right ventricle.
Mentions: To identify the endocardial targets that are altered via ectopic Noggin and the factors that may mediate endocardial-cardiomyocyte signaling during cardiac morphogenesis, we examined expression of TGFβ superfamily targets and genes known to be involved in BMP and TGFβ ligand downstream signaling by qPCR in isolated E10.5 hearts (n = 3 pooled isolated NogEnd and wildtype hearts, carried out in duplicate samples). The E10.5 stage was chosen as NogEnd mutants do not exhibit an overt phenotype at this age (Figure 1D), but do show pSMAD deviations (Figure 1K). Quantitative PCR using a custom array of 84 genes related to TGFβ /BMP-mediated signal transduction, including SMAD, SMAD target genes, adhesion and extracellular molecules and transcription factors revealed significant changes in transcript levels of the 13 listed genes (Figure 5A). As proof of principle, it was significant that expression of Noggin was elevated almost×4 fold in NogEnd hearts. Meaningfully, neither Bmp2 nor Bmp4 were affected, and endocardially-restricted Tgfβ1, myocardial Tgfβ2 and Msx2 levels were unaltered. Similarly, Smad1-5 expression levels were normal. However, Tgfβ3 mRNA was downregulated ×1.9 fold in NogEnd hearts (Figure 5A).

Bottom Line: This is coupled with hypoplastic endocardial cushions, reduced trabeculation and fewer mature contractile fibrils in mutant hearts.Molecular analysis demonstrated that endocardial Noggin resulted in a specific alteration of TGFβ/BMP-mediated signal transduction, in that, both Endoglin and ALK1 were downregulated in mutant endocardium.Combined, these results demonstrate the cell-autonomous requirement of the endocardial lineage and function of unaltered BMP levels in facilitating endothelium-cardiomyocyte cross-talk and promoting endocardial cushion formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Developmental Biology and Neonatal Medicine Program, HB Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

ABSTRACT

The initial heart is composed of a myocardial tube lined by endocardial cells. The TGFβ superfamily is known to play an important role, as BMPs from the myocardium signal to the overlying endocardium to create an environment for EMT. Subsequently, BMP and TGFβ signaling pathways synergize to form primitive valves and regulate myocardial growth. In this study, we investigated the requirement of BMP activity by transgenic over-expression of extracellular BMP antagonist Noggin. Using Nfatc1(Cre) to drive lineage-restricted Noggin within the endocardium, we show that ectopic Noggin arrests cardiac development in E10.5-11 embryos, resulting in small hearts which beat poorly and die by E12.5. This is coupled with hypoplastic endocardial cushions, reduced trabeculation and fewer mature contractile fibrils in mutant hearts. Moreover, Nfatc1(Cre) -mediated diphtheria toxin fragment-A expression in the endocardium resulted in genetic ablation and a more severe phenotype with lethality at E11 and abnormal linear hearts. Molecular analysis demonstrated that endocardial Noggin resulted in a specific alteration of TGFβ/BMP-mediated signal transduction, in that, both Endoglin and ALK1 were downregulated in mutant endocardium. Combined, these results demonstrate the cell-autonomous requirement of the endocardial lineage and function of unaltered BMP levels in facilitating endothelium-cardiomyocyte cross-talk and promoting endocardial cushion formation.

No MeSH data available.


Related in: MedlinePlus