Building the cardiovascular system in early embryos

MUNSTERBERG_U21DTP

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.

We previously identified cis-regulatory elements (CRE) associated with Nkx2.5 and Cer1, two genes that are essential for normal heart formation. 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 student will characterize 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.

The student will be fully integrated with wider efforts in the lab to identify cis-regulatory elements in different mesoderm lineages by characterizing chromatin accessibility genome-wide. The student will focus on testing the hypothesis that Nkx2.5 and Cer1 – two key factors for cardiogenesis, are co-regulated by signalling networks and their downstream TFs. This ultimately leads to specification of mesoderm progenitors towards the cardiac fate and the activation of cell differentiation programmes.

References:

Characterising open chromatin identifies novel cis-regulatory elements important for paraxial mesoderm formation and axis extension. Mok GF, Folkes L, Weldon S, Maniou E, Martinez-Heredia V, Williams R, Godden A, Wheeler GN, Moxon S and Münsterberg AE, bioRxiv 2020 DOI: 10.1101/2020.01.20.912337

miR-133-mediated regulation of the Hedgehog pathway orchestrates embryo myogenesis. Mok GF, Lozano-Velasco E, Maniou E, Viaut C, Moxon S, Wheeler G, Münsterberg A. Development 145 (2018), doi: 10.1242/dev.159657

Smad1 transcription factor integrates BMP2 and Wnt3a signals in migrating cardiac progenitor cells. Song J, McColl J, Camp E, Kennerley N, Mok GF, McCormick D, Grocott T, Wheeler GN and Münsterberg A. PNAS 111(20): 7337-42 (2014)