# Mechanisms of second heart field development regulated by Nkx genes

> **NIH NIH R01** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2021 · $429,539

## Abstract

PROJECT SUMMARY
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.

## Key facts

- **NIH application ID:** 10092207
- **Project number:** 5R01HL131438-05
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** KIMARA L TARGOFF
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $429,539
- **Award type:** 5
- **Project period:** 2017-01-01 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10092207

## Citation

> US National Institutes of Health, RePORTER application 10092207, Mechanisms of second heart field development regulated by Nkx genes (5R01HL131438-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10092207. Licensed CC0.

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