Abstract 178 Cardiac Development and Gene Regulation Platform, Sunday, 5/2

The regulated formation of the heart is conserved through expression of similar control molecules in different organisms. One of these important control molecules is the homeodomain containing transcription factor Nkx2.5. Studies by others have identified the Nkx2.5 homologue in fruit flies, fish, frog, chicken, mouse and human. Flies without the gene (called tinman) never develop a heart. Mice, homozygous null for Nkx2.5, die during embryogenesis with small misformed hearts, and the overexpression of Nkx2.5 in tadpoles leads to cardiomegaly. Recently a number of human cardiac defects, including atrial ventricular septal defects, tetralogy of Fallot, and AV block, could be traced to dominant mutations in the Nkx2.5 gene. The frog Xenopus laevis provides a simplified system for the study of early heart development, as the formation of the heart, which is complete within several days, can be observed outside the mother. We have used Xenopus to further investigate the function of Nkx2.5 and Nkx 2.3, a related protein present in the Xenopus heart.

First, the endogenous expression of Nkx2.3 and 2.5 in Xenopus embryos was reduced using antisense oligonucleotides. These studies were carried out using a novel chemical modification of the oligonucleotide. Briefly, phosphodiester internucleoside linkages at several terminal residues were modified using diethylethylenediamine, rendering the oligonucleotide resistant to exonuclease and imparting a net positive charge. Antisense oligonucleotides directed against either Nkx2.3 or 2.5 were injected into fertilized eggs. The hearts of the resulting embryos were visualized using immunohistochemical staining for muscle and vascular tissue, coupled with confocal microscopy to allow optical sectioning of the hearts. The hearts were found to be smaller than control hearts, and to have abnormal left-right axis development. These findings suggest that the level of expression of Nkx2.3 and 2.5 may influence the number of cells that ultimately form the heart, and that Nkx2.3 and 2.5 also impart spatial information.

Second, experiments underway will determine if the expression of Nkx2.5 mutations that are associated with congenital heart defects in humans will lead to similar defects in a frog heart. Plasmids containing the Xenopus Nkx2.5 cDNA have been mutated to encode the human Nkx2.5 mutations in preparation for injection of altered mRNA into Xenopus embryos. These studies may provide further insight into mechanisms of normal and abnormal cardiac development.