Investigations in Cardiac Development and Cardiac Regeneration

Abstract

Cardiovascular disease and congenital heart disease impose a massive burden on society around the world. From the cost in terms of lost human lives and diminished quality of life, to the financial expense of ongoing medical treatment, the heart’s inability to effectively repair and regenerate itself presents a major challenge for medical research. The research conducted within this thesis hoped to contribute to our knowledge of the molecular pathways of myocardial development, and to explore the potential of olfactory derived stem cells to repopulate insulted myocardium. A combination of molecular biology and classical embryology techniques were first used to characterise two novel cDNAs identified in an earlier study as being upregulated in the regions of cardiac development within the chick embryo. cDNA and genomic library screening along with RACE (rapid amplification of cDNA ends) produced products which were sequenced to identify both the transcript and genomic sequence for both of the genes. Protein expression constructs were then used to identify the localisation of the encoded proteins, and whole mount in situ hybridisation utilised to identify the temporal and spatial expression patterns of the genes. The first cDNA was identified as the vertebrate homologue of the Drosophila e(y)2 gene, and produces a transcript of approximately 600 bp in the chick with a genomic structure consisting of 5 exons covering approximately 6 Kb. The encoded protein localises to the nucleus. Its expression is ubiquitous both temporally and spatially, which is at odds with its initial method of identification. The second cDNA remains novel at the time of submission, and shows no homology to any characterised genes. This cDNA, named C1-3C, produces two alternative transcripts, one of approximately 700 bp, and a second of 9.9 Kb, with a genomic structure showing no introns within the 2 Kb of analysed sequence. The encoded protein again localises to the nucleus. Expression of the C1-3C gene demonstrated a discrete pattern, though this pattern is again contrary to an up-regulation within the cardiogenic regions. Whilst unfortunately neither of the investigated genes appear to play a direct role in cardiac development, the aim of characterisation of these novel cDNAs was achieved.