Gene targeting strategy and validation of conditional deletion of <i>Nkx2-5</i> in ventricular trabeculae.

<p>(A) Schematic illustration of the gene targeting strategy. Conditional <i>Nkx2-5</i> inactivation (<i>Nkx2-5</i>^<i>-/-</i><i>)</i> was obtained by crossing mice containing an <i>Nkx2-5</i> floxed allele with tamoxifen-inducible <i>Cx40-cre-ERT2</i> mice. Time course of tamoxifen injections and cardiac phenotyping analyses are represented on a time scale and the corresponding <i>Nkx2-5</i> mutant groups, <i>i</i>.<i>e</i>. ΔTrbE10 and ΔTrbE14 are indicated below the scale. (B) Nkx2-5 immunofluorescence on transversal sections of adult hearts from Control (Ctrl) and <i>Nkx2-5</i>^{<i>ΔTrbE10</i>} (∆TrbE10) and <i>Nkx2-5</i>^{<i>ΔTrbE14</i>} (∆TrbE14) mutant mice. (C) Genetic tracing of ventricular trabeculae in <i>Cx40-creERT2</i>::<i>R26-YFP</i> mice at P20 showing the distribution of YFP+ in the apex and mid ventricle after Cre induction at E10 or E14. (D) <i>Nkx2-5</i> deletion was quantified by counting the percentage of Nkx2-5-positive cardiomyocytes per frame at the subendocardial surface of transverse sections of adult Control (CTL), <i>Nkx2-5</i>^{<i>ΔTrbE10</i>} (∆TrbE10) and <i>Nkx2-5</i>^{<i>ΔTrbE14</i>} (∆TrbE14) mutant mice. n = 20–30 frames per heart; N = 3 mice per group; Mean±SEM; ***p<0.001 <i>Nkx2-5</i>^{<i>ΔTrbE10</i>} <i>vs</i> control, ***p<0.001 <i>Nkx2-5</i>^{<i>ΔTrbE14</i>} <i>vs</i> control (E) Schematic representation of the <i>Nkx2-5</i>-deleted cardiac regions in the apex or in mid-ventricle after tamoxifen injection at embryonic stages (∆TrbE10) or fetal stages (∆TrbE14). IVS: interventricular septum; pm: papillary muscle.