TRANSCRIPTIONAL AND FUNCTIONAL HETEROGENEITY DURING CARDIAC CELLULAR DIFFERENTIATION
Atmanli, Ayhan.
2018
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Abstract: A hallmark
of development and disease is the cellular phenotypic diversification required for
three-dimensional tissue structures. Cellular heterogeneity contributes to the
developmental dynamics of various cell types, such as stem cells, neurons and cancer. In
the heart, the coordinated differentiation, lineage diversification, and functional
maturation of heterogeneous populations ... read moreof cells is a prerequisite for the proper
development of coordinated electrical and contractile function. Although a number of
single-cell transcriptional profiling studies have provided insight into how
intracellular signaling is regulated at the single-cell transcriptional level during
cardiac development, the functional significance of transcriptomic diversity remains
challenging to assess. Collectively, a fundamental goal in the biological sciences is to
determine how individual cells with varied gene expression profiles and diverse
functional characteristics contribute to development, physiology, and disease. Here, we
report a novel strategy to assess gene expression and cell physiology in a multiplexed
fashion in single living cells. Our approach utilizes fluorescently-labeled
mRNA-specific anti-sense RNA probes and dsRNA-binding protein to identify the expression
of specific genes in real-time at single-cell resolution via FRET. In proof-of-principle
experiments, we visualize the β-actin mRNA in single living cells and demonstrate
a heterogeneous mRNA subcellular localization pattern in cardiac myocytes. To employ our
technology for the multiplex analysis of gene expression in individual living cells, we
optimize spectral imaging to faithfully resolve multiple FRET pairs. We then use this
technology to identify multiple distinct myocardial subpopulations expressing the
structural proteins myosin heavy chain α and myosin light chain 2a in real-time
during early differentiation of pluripotent stem cells. We combine this live-cell gene
expression analysis with detailed physiologic phenotyping to capture the functional
evolution of these early myocardial subpopulations during lineage specification and
diversification. We further exploit the multiplex potential of our technology to
identify multiple myocardial subpopulations expressing MHCα, MLC2a and the cardiac
transcription factor NKX2-5 at single-cell resolution. Our results show that the
expression of multiple key players of a cellular differentiation program is directly
linked with the functional outcome at single-cell resolution and further that
progression in cellular differentiation is tightly associated with functional evolution.
This live-cell mRNA imaging approach has the broader potential to study how the
expression of specific genes regulates single-cell physiology and will have wide ranging
application wherever cellular heterogeneity plays an important biological
role.
Thesis (Ph.D.)--Tufts University, 2018.
Submitted to the Dept. of Biomedical Engineering.
Advisors: Ibrahim Domian, and Lauren Black III.
Committee: David Kaplan, and Navin Kapur.
Keywords: Biomedical engineering, and Biology.read less - ID:
- js956s732
- Component ID:
- tufts:24314
- To Cite:
- TARC Citation Guide EndNote
