Abstract 1246: The Cardiac Crescent Exhibits Nkx2–5 Dependent Left-Right Asymmetry
Previous studies have defined a role for Nodal, Cripto and Pitx2c in left-right (LR) asymmetry. The transcriptional patterning of the cardiac crescent that is populated by cardiac precursor cells has not been previously described. We have engineered and utilized a 6 kb Nkx2–5-EYFP transgenic mouse that directs reporter expression in cardiac progenitors and combinatorially mated them into the wildtype (WT) or Nkx2–5 mutant backgrounds. Staged embryos were bisected, cardiac progenitors were collected using FACS and the signature of gene expression of cardiac progenitors that populate the left vs. the right regions of the cardiac crescent were examined. We observed that the left side of the WT cardiac crescent had increased expression of Nodal, Cripto and Pitx2, transcripts that are known to have functions critical to future chamber myocardium as well as Nkx2–5. The right side of the crescent was not enriched for a specific gene program. We analyzed the same LR transcriptional program in the Nkx2–5 mutant cardiac crescent. Nkx2–5 nulls exhibited a loss of the left-sided enrichment for Nodal, Cripto and Pitx2c and the cardiac transcripts that were enriched on the left side of the WT crescent revealed equal expression in the Nkx2–5 null crescents. In contrast, many right-side enriched transcripts retained the patterns they exhibited in the WT. Since the left-sided program was enriched in genes predicted to function in ventricular chamber myocardium, which appears to be defective in the Nkx2.5 nulls, the disruption of this program in the nulls supports a functional role for early LR asymmetry in patterning future myocardial lineages. Analysis of Nodal and Cripto promoters revealed the presence of evolutionary conserved Nkx2–5 binding sites (NKE). We utilized EMSA, mutagenesis, supershift, ChIP assays and a Nkx2–5 inducible ES/EB system to verify that Nodal and Cripto are direct Nkx2–5 downstream targets. Our data support the hypothesis that Nkx2–5 functions in the cardiac progenitor cell population to maintain the left-right patterning of the cardiac crescent.