Abstract 938: Cardiac-Specific Deletion of Cited2 Reveals a Cell Autonomous Role in Positively Regulating Vegfa Expression in the Developing Heart
The transcriptional co-activator Cited2 plays a key role in cardiac development and left-right patterning, regulating the expression of diverse targets of Nodal-signaling in the lateral plate mesoderm such as Pitx2c. Cited2 recruits co-activator CREBBP/EP300 to chromatin via DNA-bound transcription factors such as TFAP2 and SMADs. Cited2 also inhibits hypoxia-activated transcription by blocking CREBBP/EP300 recruitment to HIF1A. Concomitantly, global loss of Cited2 is associated with increased cardiac expression of the HIF1A target Vegfa indicating that it negatively regulates Vegfa expression. To dissect the cell-autonomous role of Cited2 in cardiac development we examined the effects of a cardiogenic mesoderm specific knockout of Cited2. Methods: We compared embryos with Cited2flox/-;Nkx2–5Cre (cardiogenic-mesoderm specific deletion) and Cited2flox/+;Nkx2–5Cre (littermate control) genotype. We examined cardiac morphology using magnetic resonance imaging and histology at 15.5 days post-coitum (dpc) and cardiac transcript expression using quantitative RT-PCR at 13.5 dpc. The recombined Cited2 allele activated lacZ expression, permitting assessment of recombination efficiency using X-gal cleavage between 8.5 - 9.5 dpc. Results: Cardiac-specific conditional Cited2 deletion resulted in 6/11 (55%) embryos showing ventricular septal defect. LacZ staining revealed efficient recombination in cardiogenic mesoderm and in cardiac myocytes. Cited2 mRNA expression was reduced 4.7 fold (p<0.001) in conditionally deleted hearts. Surprisingly, there was a 1.54 fold reduction in Vegfa level (p<0.018) in hearts lacking Cited2. No difference in Hif1a or Pitx2c level was seen. Conclusion: Cardiac-specific conditional deletion of Cited2 revealed it has a hitherto unsuspected cell-autonomous role in positively regulating Vegfa expression in heart development. As Vegfa loss is associated with cardiac malformation including VSD, the observed reduction in Vegfa may explain, at least in part, heart defects observed with cardiac-specific Cited2 deletion. Our results suggest the increased cardiac expression of Vegfa in the global knockout likely results from a non cell-autonomous mechanism such as embryonic hypoxia.