Building the cardiovascular system in early embryos

The cardiovascular system forms early in development to support growth and survival of the embryo. This project aims to identify transcriptional mechanisms governing early steps of progenitor cell specification using an avian model of heart development. Evolutionary conservation of these mechanisms will be assessed in the amphibian, Xenopus laevis.
Cis-regulatory elements (CRE) associated with Nkx2.5 and Cer1, two genes that are essential for normal heart formation, have previously been identified. Pilot experiments, using Citrine-fluorescent reporters, show that the CREs become active early - in prospective cardiovascular cell lineages that will contribute to the heart and vessels.
To identify upstream regulators of early fate decisions, the project will involve characterizing these CREs in vivo. Electroporation of Citrine-reporters followed by time-lapse imaging will determine the timing of activation and cell types will be identified using co-staining with known markers. The Wnt and BMP signaling pathways are important for cardiac development and their effect on CRE activity will be examined. Motif search and footprint analysis will identify candidate transcription factor (TF) binding sites and mutagenesis will determine their functional importance. Furthermore, important TF sites will be edited by using CRISPR approaches. Finally, whether CREs are functionally conserved across species will be tested using microinjections in Xenopus as well as transgenic embryos and tadpoles.