Cardia bifida is known to occur in animal models lacking the cardiogenic transcriptional factor GATA-4. The downstream target genes responsible for this cardiac deformity remain unknown, however. Treatment with small interfering RNAs (siRNA) specifically targeting GATA-4 into cardiac mesodermal cells led to the development of cardia bifida in chick embryos. RT-PCR using mRNAs extracted from cardiac tubes revealed that the GATA-4-specific siRNA selectively suppresses expression of N-cadherin mRNA, one of the genes essential for the single heart formation, without affecting other cardiac marker mRNAs. Analysis of the N-cadherin gene promoter activity using a luciferase reporter gene system and electrophoretic mobility shift assays revealed that GATA-4 binds directly to the N-cadherin gene promoter region, thereby transactivating its expression. We therefore concluded that the cardia bifida observed in the GATA-4-deleted model is caused by the transcriptional down-regulation of N-cadherin expression. (C) 2003 Elsevier Inc. All rights reserved.