Predoctoral-Erin Lambers: The Role of Foxc1 During the Differentiation of Embryonic Stem Cells into Functional Cardiomyocytes

Project: Research project

Description

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.
StatusFinished
Effective start/end date7/1/156/30/17

Funding

  • American Heart Association Midwest Affiliate (15PRE25080006)

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Cardiac Myocytes
Cell Differentiation
Stem Cells
Small Interfering RNA
Transcription Factors
Congenital Heart Defects
Gene Expression Regulation
Luciferases
Transgenes
Genes
Regeneration
Myocardium
Cardiovascular Diseases
Parturition
Calcium
Morbidity
Polymerase Chain Reaction
Messenger RNA
Mutation
Mortality