The homeodomain transcription factor, NKX2-5, is the most commonly mutated gene associated with congenital heart defects (CHDs), accounting for 1-4% of specific malformations with a predilection for abnormalities at the arterial and venous poles of the heart. The underlying molecular mechanisms responsible for cardiac malformations found in patients with NKX2-5 mutations remain poorly understood. To improve diagnostic and therapeutic measures in these cases, a more precise understanding of early cardiac developmental processes at the outflow (OFT) and inflow (IFT) tracts is critical. The embryonic heart begins as a linear tube derived from first heart field (FHF) progenitors, with expansion occurring through accretion of late-differentiating cells of the second heart field (SHF) to the arterial and venous poles. Nkx2-5 is expressed in both the FHF and SHF and, while vital functions of Nkx genes in the FHF have been implicated in cardiac specification and morphogenesis, little is known about the distinct mechanisms regulated by Nkx genes in the anterior (aSHF) and posterior (pSHF) SHFs. By exploiting benefits of the zebrafish model, we recently published evidence demonstrating that nkx2.5 and nkx2.7, two NKX2-5 homologs expressed in the zebrafish heart, play essential roles in maintaining ventricular identity and display similar chamber-specific functions in SHF cardiomyocytes. Furthermore, our preliminary data provide new evidence that nkx genes exhibit previously unappreciated, crucial functions in regulating SHF progenitor populations through discrete mechanisms at OFT and IFT. We find that Nkx genes promote aSHF progenitor augmentation at the arterial pole and restrict isl1, a LIM homeodomain transcription factor, to the sinus venosus. In this proposal, we test the novel hypothesis that nkx genes are required to recruit aSHF progenitors to the OFT and to restrict pSHF progenitors via isl1 to the IFT.
In Aim 1, we will dissect the cellular and temporal roles of Nkx genes in specification, accretion, proliferation, and identity maintenance in the aSHF and pSHF employing heat-shock inducible overexpression of nkx2.5, time series analysis, EdU incorporation studies, developmental timing assays, and state-of-the-art microscopy.
In Aim 2, we will utilize the zebrafish model along with CRISPR and ChIP methodologies to examine previously unrecognized direct and indirect downstream effectors of Nkx genes and also new candidates associated with CHD in humans. Combining the tools available in zebrafish and human genomics data, our research will uncover the developmental mechanisms regulated by Nkx genes that are responsible for SHF-derived CHDs, some of the most severe and lethal malformations associated with NKX2-5 mutations.

Public Health Relevance

Nkx2-5 is a key causative gene associated with congenital heart defects (CHDs) in patients, yet we have limited understanding of its distinct functions in the developing arterial and venous poles of the heart. This project will examine the cellular and molecular mechanisms downstream of Nkx genes in the second heart field of the zebrafish embryo that are responsible for promoting arterial pole formation and limiting venous pole development. These studies will also investigate mutations in novel Nkx targets found in patients with CHDs to enhance our understanding of the molecular etiologies of cardiac outflow and inflow tract anomalies and ultimately to assist in remediation of fetal and neonatal heart pathology.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL131438-03
Application #
9623275
Study Section
Cardiovascular Differentiation and Development Study Section (CDD)
Program Officer
Schramm, Charlene A
Project Start
2017-01-01
Project End
2021-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
3
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Pediatrics
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Hillman, Elizabeth Mc; Voleti, Venkatakaushik; Patel, Kripa et al. (2018) High-speed 3D imaging of cellular activity in the brain using axially-extended beams and light sheets. Curr Opin Neurobiol 50:190-200
Shi, Lingyan; Zheng, Chaogu; Shen, Yihui et al. (2018) Optical imaging of metabolic dynamics in animals. Nat Commun 9:2995
Colombo, Sophie; de Sena-Tomás, Carmen; George, Vanessa et al. (2018) Nkx genes establish second heart field cardiomyocyte progenitors at the arterial pole and pattern the venous pole through Isl1 repression. Development 145:
Pradhan, Arjana; Zeng, Xin-Xin I; Sidhwani, Pragya et al. (2017) FGF signaling enforces cardiac chamber identity in the developing ventricle. Development 144:1328-1338
Iklé, Jennifer M; Tavares, Andre L P; King, Marisol et al. (2017) Nkx2.5 regulates endothelin converting enzyme-1 during pharyngeal arch patterning. Genesis 55:
Harrington, Jamie K; Sorabella, Robert; Tercek, Abigail et al. (2017) Nkx2.5 is essential to establish normal heart rate variability in the zebrafish embryo. Am J Physiol Regul Integr Comp Physiol 313:R265-R271