Abstract 1919: The Identification of the Nkx2–5-regulated Genes Using Purified ES Cell-derived Cardiomyocytes
Introduction: Cardiac transcription factors play crucial roles in cardiac development and differentiation, and to address the target genes they regulate is important for understanding the molecular mechanisms under these conditions.
Objective: The aim of the present study is to identify the Nkx2–5-regulated genes in cardiomyocyte differentiation.
Methods: Nkx2–5 deficient ES cell lines (Nkx2–5 +/− and −/−) were generated by replacing the Nkx2–5 gene with a neomycin resistance gene, and Nkx2–5 overexpressing ES cell line was generated using the knock-in method by inserting the Nkx2–5 cDNA into the Mlc2v locus of Nkx2–5 +/− ES cell line. Purified ES cell-derived cardiomyocytes (ESCM) were obtained by in vitro differentiation and successive neomycin selection of these ES cell lines. The expression profiles were compared among the three lines of purified ESCM for the identification of the Nkx2–5-regulated genes.
Results: After neomycin selection for 7 days from day 10, ESCM survived selectively and showed vigorously beating colonies in all three lines. More than 97% of non-cardiomyocytes were excluded by the neomycin selection. Cardiac gene expression was assessed by realtime PCR and DNA microarray. Nkx2–5 expression was not detected in Nkx2–5 −/− ESCM and was increased to about 125% of Nkx2–5 +/− ESCM in Nkx2–5 overexpressing ESCM. While MHC expression showed no significant difference, most known Nkx2–5 downstream genes, such as ANF, HOP, BMP10 and Cx43 were expressed consistently with the Nkx2–5 expression level in each purified ESCM line, implicating that the present method was appropriate for the identification of unknown downstream genes. The comparison of the gene expression profiles revealed several novel candidates for the Nkx2–5 downstream gene. One of the candidates Cend1, which was reported to be involved with neuronal differentiation, was confirmed to be significantly upregulated in an Nkx2–5 dependent manner among three lines of purified ESCM.
Conclusions: A new system for the identification of downstream target genes of cardiac transcription factors was established. Cend1 was identified as one of the novel Nkx2–5-regulated genes in the ESCM context.