Figure 4

Overexpression of Wnt signaling rescues Cdh2 mutant phenotype. (A) Representative traverse section showing Wnt signaling activity in AHF progenitors as revealed by TCL/LEF-lacZ mice. Black arrows denote cells that have active Wnt signaling within pharyngeal mesoderm, whereas red arrows denote cells with inactive Wnt signaling within heart tube. Scale bar, 500 μm. (B) Representative images of AHF-CPCs isolated from wild type (AHF-Cre; AHF-GFP; Cdh2^fl/+) and mutant (AHF-Cre; AHF-GFP; Cdh2^fl/fl) stained red for either nuclear β-catenin or membrane-bound β-catenin. The average mean intensities were tabulated from 60 fields. Nuclei are marked by DAPI. A cut-off value >10 was imposed to remove background. Scale bar, 50 μm. Error bars indicate SD, n = 3 replicates, ^**P < 0.001, evaluated by Student's t-test. (C) Quantitative PCR analysis of mRNA isolated from sorted progenitor cells (AHF-GFP+ CPCs) carrying AHF-GFP reporter transgene. The expression levels show that Wnt signaling ligands, receptors and effector genes (Axin2 and Lef1) were downregulated in Cdh2-deficient progenitor cells, while upregulation of differentiation genes such as Mlc2a and Tnnt2 were observed. Samples were normalized against the housekeeping gene, β-actin. Error bars indicate SD, n = 3 replicates. (D) Representative sagittal sections of single (AHF-Cre; Cdh2^fl/+; bCat(e3)^fl/+) and double (AHF-Cre; Cdh2^fl/fl; bCat(e3)^fl/+) mutants at E9.5. Sections stained for cTnT (red) and Isl1 (green) revealed similar expression patterns. Premature differentiation of Isl1+ CPCs into cTnT+ cells at the pharyngeal mesoderm was not observed in the double mutant as compared to single N-cadherin mutant (AHF-Cre; Cdh2^fl/fl) (Figure 3D). Nuclei are marked by DAPI. Scale bar, 100 μm. (E) Graphical representation of the number of cardiomyocytes (cTnT+ cells) in the AHF in various mutants (AHF-Cre; Cdh2^fl/fl, AHF-Cre; Cdh2^fl/+; bCat(e3)^fl/+ and AHF-Cre; Cdh2^fl/fl; bCat(e3)^fl/+) compared to control at E9.5 (somite number = 23-29). GOF, gain of function; LOF, loss of function. cTnT+ cells within the AHF, the region between OFT and the anterior portion of pharyngeal mesoderm, were counted. A total of 7-9 serial sections for each embryo were used. Error bars indicate SD, n = 6 embryos. ^***P = 0.001, evaluated by Student's t-test.