| Summary: | <p>The human heart is known to have limited regeneration capability, and myocardial
infarction leads to irreparable cardiac damage and pathological remodelling. The epicardial
cell layer has been identified as a viable resident tractable source of multipotent
progenitors in the heart which may be harnessed for cardiac regeneration. During
development, the epicardium is known to be an important source of cardiomyogenic
paracrine factors as well as supportive cardiac progenitors and thereafter becoming
quiescent in adulthood. Interestingly, embryonic epicardial genes are re-expressed
following cardiac injury and have been shown to be an important part of the zebrafish
cardiac regeneration process. However, little is known about the upstream elements which
underlie both epicardial development and regeneration processes, which may be utilized to
improve current treatment regimes. In this report, my primary focus is centred on
elucidating the cis-regulatory and trans-regulatory elements acting on an epicardial gene
locus of interest during development. Previously generated RNA sequencing results show
that the G-protein coupled receptor, Vaso-intestinal peptide receptor 2 (vipr2) was
expressed in epicardial cells at 5 days post fertilization (5dpf). Vipr2 is also differentially
expressed during cardiac regeneration at 3 days post-injury (dpi) of cryoinjured zebrafish
hearts. Here, I show with in situ techniques that vipr2 is expressed in both a rare
subpopulation of epicardial cells as well as in most cardiomyocytes in the myocardium.
Chromatin accessibility assays and in vivo validation revealed a functional enhancer within
vipr2 intronic regions, enhancer 14, which was found to be active from the 21-somite stage
and exhibiting heart-specific activity from 3dpf to 5dpf during development. Enhancer 14
was further shown to be active in vipr2-expressing epicardial cells, as well as the
cardiomyocytes of the primordial layer of the heart. Through combined transcription factor
3
binding motif identification, site-directed mutagenesis and transient transgenesis, two
transcription factors, myeloid ecotropic viral integration site 1 homolog (MEIS1) and
helicase-like transcription factor (HLTF), were found to be critical for enhancer 14
activity. In the future, vipr2 and enhancer 14 knockout zebrafish lines could be generated
to elucidate gene and enhancer function during epicardial and myocardial development and
regeneration.</p>
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