Figure 5.

Dosage effects of Tbx1-GFP activation on heart development. (A) Immunofluorescence with a GFP antibody shows activation of Tbx1-GFP by Tbx1^Cre/+ at E9.5. GFP is detected in the posterior otic vesicle (OV), 1^st and 2^nd branchial arches (BA1, BA2), and mesoderm including the secondary heart field proximal to the outflow tract (OFT). (B) Histological transverse sections at E14.5 shows that Tbx1^Cre/+;Tbx1-GFP^flox/+ mutants do not have outflow tract or ventricular defects. They survive in normal mendelian ratios, unlike Tbx1^Cre/+;Tbx1-GFP^flox/flox mutants which are not viable. (C) Activation of both alleles of Tbx1-GFP by Tbx1^Cre/+ causes outflow tract septation (persistant truncus arteriosis, PTA) and alignment (double outlet right ventricle , DORV) defects with ventricular septal defect (VSD). (D) Western blot of proteins isolated from E9.5 pharyngeal regions to compare protein expression levels of Tbx1-GFP and endogenous Tbx1. For each genotype, we pooled 4 embryos (23–24 somite stage) and loaded equal amounts of protein per lane. The same membrane was probed with antibodies to Tbx1 and β-actin. The expected size of endogenous Tbx1 is ≈50 kD while the Tbx1-GFP fusion protein is ≈75kD. β-actin is detected at ≈40 kD.