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. Author manuscript; available in PMC: 2015 Jun 16.

Published in final edited form as: J Cardiovasc Dev Dis. 2014 Nov 20;1(3):214–236. doi: 10.3390/jcdd1030214

Ectopic Noggin in the Nfatc1 endocardial lineage is embryonic lethal. (A–F): Resultant phenotype of Nog^End mutant embryos (B,D,F) compared to wildtype littermate controls (A,C,E) at E9.5, 10.5 and 12. Wholemount right sided views show that E9.5 and 10.5 Nog^End embryos are grossly unaffected but that E12 Nog^End mutants are runted and exhibit pooling of blood in both blood vessels (indicated via arrow in F) and heart itself when compared to wildtype control littermates; (G–I): Non-radioactive in situ hybridization analysis using an anti-Noggin DIG-labelled probe confirms absent endogenous mRNA expression in control E10 heart (G) but robust endocardially-restricted transgenic Noggin (blue signal) in Nog^End mutant heart within cells lining the atria, atrioventricular cushions and ventricle. G’ and H’ inserts represent low power images of whole heart sections; (J): Semi-quantitative RT-PCR measurement of Noggin levels in control and mutant E9.5 pooled hearts (n = 4 of each genotype) confirmed elevated Noggin expression levels (×6.4) in the mutant hearts (data shown from 34 PCR cycles). Note, GAPDH loading control (upper panel J) is similar in both samples (data shown from 20 PCR cycles); (K): Western analysis of TGFβ superfamily downstream effector Smad signaling in pooled E10.5 hearts (n = 4 of each genotype) revealed Nog^End mutant isolated hearts exhibit reduced pSmad1/5/8 levels (only ~35% of wildtype levels) when normalized to Tubulin levels, indicative of suppressed downstream BMP ligand signaling. Additionally, pSmad3 is similarly reduced in mutant hearts (only ~40% of wildtype levels), but pSmad2 levels remain unchanged. This suggests that downstream TGFβ ligand signaling is also partially suppressed; (L–O): Immunohistochemical analysis of pSmad1/5/8 in E10.5 control (L,N) and mutant (M,O) hearts demonstrates that expression is unaffected in cardiomyocytes but that pSmad1/5/8 expression (brown nuclear staining) is reduced in Nog^End mutant cushions compared to controls (* in (L)) and that pSmad1/5/8 expression is diminished within mutant endocardial cells (arrow in (O)) compared to wildtype controls (arrows in (N)). Scale bars: (G,H) = 20 μm; I = 50 μm. Abbreviations: lv, left ventricle; rv, right ventricle.

Ectopic Noggin in the Nfatc1 endocardial lineage is embryonic lethal. (A–F): Resultant phenotype of Nog^End mutant embryos (B,D,F) compared to wildtype littermate controls (A,C,E) at E9.5, 10.5 and 12. Wholemount right sided views show that E9.5 and 10.5 Nog^End embryos are grossly unaffected but that E12 Nog^End mutants are runted and exhibit pooling of blood in both blood vessels (indicated via arrow in F) and heart itself when compared to wildtype control littermates; (G–I): Non-radioactive in situ hybridization analysis using an anti-Noggin DIG-labelled probe confirms absent endogenous mRNA expression in control E10 heart (G) but robust endocardially-restricted transgenic Noggin (blue signal) in Nog^End mutant heart within cells lining the atria, atrioventricular cushions and ventricle. G’ and H’ inserts represent low power images of whole heart sections; (J): Semi-quantitative RT-PCR measurement of Noggin levels in control and mutant E9.5 pooled hearts (n = 4 of each genotype) confirmed elevated Noggin expression levels (×6.4) in the mutant hearts (data shown from 34 PCR cycles). Note, GAPDH loading control (upper panel J) is similar in both samples (data shown from 20 PCR cycles); (K): Western analysis of TGFβ superfamily downstream effector Smad signaling in pooled E10.5 hearts (n = 4 of each genotype) revealed Nog^End mutant isolated hearts exhibit reduced pSmad1/5/8 levels (only ~35% of wildtype levels) when normalized to Tubulin levels, indicative of suppressed downstream BMP ligand signaling. Additionally, pSmad3 is similarly reduced in mutant hearts (only ~40% of wildtype levels), but pSmad2 levels remain unchanged. This suggests that downstream TGFβ ligand signaling is also partially suppressed; (L–O): Immunohistochemical analysis of pSmad1/5/8 in E10.5 control (L,N) and mutant (M,O) hearts demonstrates that expression is unaffected in cardiomyocytes but that pSmad1/5/8 expression (brown nuclear staining) is reduced in Nog^End mutant cushions compared to controls (* in (L)) and that pSmad1/5/8 expression is diminished within mutant endocardial cells (arrow in (O)) compared to wildtype controls (arrows in (N)). Scale bars: (G,H) = 20 μm; I = 50 μm. Abbreviations: lv, left ventricle; rv, right ventricle.