We use a number of genetic approaches in the two most well-suited living organisms for cardiac and vascular biology: the mouse and the zebrafish model systems.
We use loss-of-function strategies (knockout and inducible, conditional knockout models) for a number of genes of interest to understand genetic and cellular events in a mammalian setting. Using these tools permits us to investigate cell-type specification and tissue morphogenesis in vivo. In addition, we have a number of lineage-restricted and inducible Cre strains to perform tissue-specific autonomy studies and fate-mapping experiments. Finally, we have begun to undertake a pilot forward genetic screen in a lymphatic-specific reporter mouse strain to identify and characterise novel mutants that fail to form a functional lymphatic vasculature during embryogenesis.
We use zebrafish to perform large forward genetic screens that will identify mutants and ultimately key genes and pathways involved in vascular development and heart morphogenesis in the embryo. To speed up this gene discovery approach, we have optimized mutant mapping by whole-genome sequencing at the IMB. In addition to unbiased genetic screening, we use TALEN genome editing to generate zebrafish mutants for key genes of interest. This allows us to rapidly delineate genetic pathways using classical genetic interaction and epistasis experiments. Combined with traditional reverse genetics tools , such as morpholino knockdown and gene overexpression, we are now able to use the zebrafish model to rapidly understand gene function in cardiac and vascular biology.
We use a range of technologies to generate novel transgenic lines in the zebrafish. Cloning methodologies include traditional cloning, the Gateway system and recombineering technologies. Using these methods, we have generated reporter lines that are tissue specific as well as lines that label subcellular compartments or tag proteins of interest. We have also generated transgenic lines capable of expressing wild-type and modified proteins under spatial and/or temporal control.
The fields of developmental and cell biology have become more closely entwined in the last decade. We study both cultured cells of the cardiovascular system and cells within the developing embryo (in vivo cell biology).
We utilize cultured endothelial cells routinely to examine signaling pathways, cell behaviour, transcriptional pathways and mechanisms of relevance to development and pathogenesis.
In vivo cell biology
We use the zebrafish as a model for in vivo cell biology. As well as utilizing the fish embryo to image whole tissue-resolution processes, we examine cellular events that shape organs.
A range of microscopy techniques are used within the IMBs ACRF Imaging facility. These include confocal imaging for high-resolution visualization of cellular behavior in live embryos (zebrafish and mouse) and fixed tissues as well as spinning disk confocal microscopy for capturing processes at high-speed and fine detail. We also perform optical projection tomography to model in 3D the vascular network of larger samples, including whole mouse embryos. Finally, we also perform live mouse imaging to assess disease progression (e.g. tumour metastasis).
We have access to a wealth of chemical libraries that are housed within the IMB. Using microfluidics, reporter cell lines, transgenic zebrafish reporter lines and embryonic morphology, we are capable of performing medium- and high-throughput chemical screens to identify drugs that inhibit a morphological process or signaling pathway of interest. We also have collaborative avenues for following up the structure-function of these compounds and related analogues.