Identification of putative targets of Nkx2-5 in Xenopus laevis using gene expression analysis and cross-species annotation

While the heart is the first organ to form during development, the earliest molecular mechanisms surrounding specification and differentiation are not clear. In vertebrates, the first marker of cardiac specification is the expression of the homeobox transcription factor Nkx2-5 and its paralogs. While some downstream targets of Nkx2-5 have been discovered, they alone are not sufficient to completely rescue Nkx2-5 knock-downs, suggesting that other targets of Nkx2-5 regulation remain unknown. In order to identify early targets of Nkx2-5, Xenopus laevis embryos were injected with synthetic Nkx2-5 mRNA and changes in gene expression measured using Affymetrix GeneChips. To interpret the data, X. laevis genomic annotation was augmented using cross-species information from the CrossGene database followed by gene ontology enrichment, network analysis, spatial expression, and Nkx2-5 binding site predictions. This allowed us to compile a list of 99 UniGene clusters representing likely early targets of Nkx2-5. Nkx2-5+GFP or GFP mRNA was injected into 8 cell Xenopus embryos. Total RNA was collected at St. 11.5. comparison of gene expression profiles for treated vs, control