The University of Chicago Research Funding

• Principal Investigator: Marcelo Nobrega, MD, PhD, Assistant Professor, Department of Human Genetics, Committee on Genetics, Genomics & Systems Biology
• Start Date: July 1, 2009
• Total Award Amount: $406,929 (first year) $436,833 (second year)

Public Health Relevance

Congenital heart diseases (CHD) result from the malfunction of protein(s) that build the individual structures of the developing heart or from the presence of these protein(s) in the wrong amounts, at the wrong place and/or at the wrong time. We propose a system to dissect the molecular bases that control the coordinated expression of genes in the developing heart. Our results will further the understanding of heart development and allow us to investigate the roles of mutations in gene regulatory sequences in CHD.

Project Description

The recognition of the hierarchies of genetic pathways coordinating heart development remains incomplete, in part because of the difficulties to identify the critical cis-acting sequences that are required for the regionalized expression of genes during cardiogenesis. Here we propose an integrative system to identify the critical components of the regulatory network of Tbx20, a gene that plays important roles in heart development.

To achieve this, we propose to utilize a combination of a new and validated zebrafish assay to identify heart enhancers in the Tbx20 locus with mouse genetic engineering to characterize the regulatory architecture of Tbx20 and its role in epicardial development. We have recently validated a modified version of traditional zebrafish transgenics to identify mammalian heart enhancers, making this a rapid, efficient and low cost alternative to mouse transgenics in identifying enhancers. We will systematically test sequences in the Tbx20 locus, regardless of their pattern of evolutionary conservation, identifying the critical enhancers that coordinate Tbx20 expression during cardiogenesis.

In a pilot study, we already identified six Tbx20 heart enhancers with modular, and often overlapping territories of expression. We will carry out a detailed characterization of the spatial and temporal domains of these heart enhancers during embryogenesis, leading to a broad understanding of the components of the Tbx20 regulatory network. We will also use an engineered Bacterial Artificial Chromosome (BAC) to delete an enhancer that drives expression in multiple domains of the heart, both unique and redundant with other enhancers. This experimental design will allow us to infer the necessity of individual components of the regulatory network for its proper temporal, spatial and quantitative functional integrity. We will also capitalize on a mutagenized Tbx20 heart enhancer that drives expression exclusively in the epicardium and generate epicardial-specific Cre mouse lines that will be used to map the cellular derivatives of this enhancer and to assess the roles of Tbx20 in this cardiac domain. In summary, we will use a novel zebrafish in vivo assay to characterize the cis-regulatory network that coordinates Tbx20 expression during cardiogenesis, and gain a better understanding of how the components of the network orchestrate the dynamic and complex pattern of Tbx20 expression.

Novel in vivo reagents will be generated and utilized to evaluate the biology of Tbx20 in the developing heart.

This award is funded under the American Recovery and Reinvestment Act of 2009, NIH Award number: 1R01HL088393-01A1