Fig. 3. (A) To obtain restricted expression of the transgene, two electrodes were placed in a cross configuration. With this arrangement, electric pulses hit only a limited region of the precardiac mesoderm. (B) Morphology of a heart, in which the Tbx5-EGFP expression construct was electroporated at stage 5. Abnormal indentation is shown by a red arrowhead. GFP fluorescence signals (C) are observed predominantly in the restricted part of the right ventricle (red arrowhead). (D) Morphology of the electroporated heart at E7 showing an abnormal broad indentation (red arrowhead) that shows GFP fluorescence (E, arrowhead). (F) Schematic representation of the experimental design. At stage 5, Tbx5-EGFP gene was electroporated at a restricted region of the rostral precardiac field. At stage 10, ectopic misexpression was evident in a rostral part of the prospective right ventricle, making an ectopic boundary of Tbx 5 expression. (G) At E7, when in situ hybridization using cANF probe was performed in electroporated heart, ectopic induction of this gene was evident in a restricted of the prospective right ventricle between two red arrowheads. Normal boundary of cANF expression is indicated by a yellow arrowhead. (H,I) At E5, serial sections showed that misexpression of Tbx5 resulted in (H) abnormal growth of trabeculae (red arrowheads). At the tip of each trabecula, ectopic BMP2 expression was induced, albeit weakly (red arrowheads). (I) cANF gene was ectopically induced in the right side of the abnormal trabecula growth (red arrowheads). Normal cANF expression is indicated by black arrowheads. Note that abnormal trabecula growth is evident between the ectopic cANF expression and the normal cANF expression. (J-N) At E6, serial sections were made and examined both histologically (Hematoxylin and Eosin staining) and by marker expression. In the Tbx5-misexpressed heart, an ectopic septum-like structure (IVS*) was observed (J) in addition to the normal septum (IVS). Higher magnification of this section revealed that trabeculae amalgamated to form both the ectopic and the normal septums (K). When in situ hybridization was carried out, BMP2 (L), Tll1 (M) and VEGF (N) genes were found to be expressed in the ectopic septum (red arrowheads) as in the normal septum (black arrowheads).