Cardiovascular disease is the number one cause of morbidity and mortality in the developing world. Embryonic Stem Cells (ESCs) hold great potential for regeneration of damaged myocardium, however the molecular circuitry that guides ESC differentiation into cardiac lineages remains poorly understood. This is exemplified by the elusive role of the developmental transcription factor, Foxc1 during cardiac development. When one FoxC1 allele is mutated in humans it causes Axenfeld-Rieger syndrome characterized by ocular defects and often heart defects. Foxc1 homozygous null mice are either embryonic lethal or die soon after birth with the majority of these mice containing severe heart malformations. The only known foxc1 target during heart development is tbx1, a transcription factor necessary for the proper alignment of the outflow tract. However, because foxc1 homozygous null mice contain heart mutations that are far more severe than tbx1 homozygous null mice it is probable that foxc1 has additional regulatory roles in heart development. It is also unknown in which specific cell types of the heart foxc1 functions. I have recently generated preliminary data, using ESCs, showing that knockdown of Foxc1 reduces early cardiomyocyte differentiation and that overexpression of foxc1 increases expression of early cardiomyocyte transcripts and enhances beating. These data taken together, has lead to the central hypothesis that Foxc1 is critical for ESC differentiation into cardiomyocytes and their function through the proper regulation of direct downstream target genes. This hypothesis will be tested with 3 Specific Aims. Aim 1 is to test the hypothesis that Foxc1 is necessary for the early differentiation of ESCs into functional cardiomyocytes. I will differentiate Nkx2.5-GFP reporter ESCs in which foxc1 has been stably knocked down with lentiviral shRNA constructs. Compared to a scramble shRNA control, cardiac differentiation will be assessed with cardiomyocyte mRNA and protein markers, in addition to quantification of Nkx2.5GFP+ cells. Function of cardiomyocytes will be assessed by quantification of beating and analysis of calcium handling. Aim2 is to test the hypothesis that overexpression of Foxc1 is sufficient to increase ESC differentiation into functional cardiomyocyte lineages. Foxc1 overexpression will be induced in ESCs containing a foxc1 doxycycline-controllable transgene. After ESC differentiation is induced in endogenous vs overexpressing foxc1 cells, I will assess cardiomyocyte differentiation and function as described above in aim 1. Aim 3 is to test the hypothesis that Foxc1 directly regulates downstream targets during ESC differentiation into cardiomyocytes. ChIP sequencing analysis will be performed after differentiation of ESCs into cardiomyocytes to test for the direct gene targets bound by Foxc1 during early cardiac differentiation. Putative targets will be validated using ChIP-PCR. Using luciferase assays we will confirm Foxc1 regulation of target gene expression.
|Effective start/end date||7/1/15 → 6/30/17|
- American Heart Association Midwest Affiliate (15PRE25080006)
Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.