Abstract 3403: Mechanisms of Nkx2–5 Dependent Left-Right Asymmetry of the Cardiac Crescent
Previous studies have defined a role for Nodal, Cripto and Pitx2c in left-right (LR) asymmetry. The transcriptional patterning of the cardiac crescent 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 however enrichment was noted for Hoxa9 and Hoxa10. 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 and revealed equal expression for Nodal, Cripto and Pitx2c and the cardiac transcripts that were enriched on the left side of the WT crescent. In contrast, many right-side enriched transcripts retained the patterns they exhibited in the WT including Hoxa9 and Hoxa10. Since the left-sided program was enriched in genes predicted to function in ventricular chamber myocardium, which is 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 Nkx2–5 binding sites. We utilized EMSA, ChIP assays, luciferase transcriptional assays, mutagenesis and an Nkx2–5 inducible ES/EB system to verify that Nodal and Cripto are direct Nkx2–5 downstream targets. Furthermore, studies utilizing the Nkx2–5 ES/EB system and luciferase transcriptional assays also supported further mechanisms regulating LR asymmetry involving interactions of Hoxa9/a10 and Nkx2–5. 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.
This research has received full or partial funding support from the American Heart Association, AHA National Center.