Elucidating the gene regulatory network of zebrafish heart development using genomics

Detailed information about the project:

Project number: 2014/13/B/NZ2/03863
Project title: “Elucidating the gene regulatory network of zebrafish heart development using genomics”
Project leader: Cecilia Winata, PhD, Investigators: Katarzyna Nieścierowicz PhD, Michal Pawlak PhD
Source of funding: National Science Centre, Poland, OPUS
Budget: 955 500 PLN

Project decription:

The heart muscle or myocardium makes up most of the heart tissues and is mainly responsible for its function. Upon completion of gastrulation, heart muscle cells or cardiomyocytes (CMs) are specified from a pool of mesodermal progenitors at the anterior portion of the embryonic lateral plate mesoderm (Stainier et al. 1993; Stainier and Fishman 1994; Kelly et al. 2014). As development proceeds, heart progenitors migrate to the midline and form a tube structure known as the primitive heart tube (Stainier et al. 1993). This structure subsequently expands through cell division and addition of more cells originating from the progenitor pool (Kelly et al. 2014; Knight and Yelon 2016). Looping of the heart tube then gives rise to distinct chambers of the heart, namely, the atria and ventricles. Although the vertebrate heart can have between two to four chambers, the step-wise morphogenesis of progenitors specification, migration, tube formation, and looping, are highly conserved between species (Jensen et al. 2013).

CMs are specified early during embryogenesis and undergo various cellular processes of proliferation, migration, and differentiation which collectively give rise to a fully formed and functioning heart. Crucial to regulating each step of heart morphogenesis are cardiac transcription factors (TFs) which include Nkx2.5, Gata5, Tbx5, and Hand2 (Clark et al. 2006; Nemer 2008). These TFs are known to play a role in establishing the CM identity of mesodermal progenitor cells, regulating the formation and looping of the heart tube, as well as the specification of atrial and ventricular CMs. Specification and differentiation of the cardiac progenitors are regulated by the interactions between several key TFs - Nkx2.5, Gata5, Tbx5, and Hand2. Members of the GATA family of TFs, Gata4, Gata5, and Gata6, are responsible for the earliest step of cardiac progenitor specification (Jiang and Evans 1996; Jiang et al. 1998; Reiter et al. 1999; Singh et al. 2010; Lou et al. 2011; Turbendian et al. 2013). Gata factors activate the expression of nkx2.5, another early marker of CMs (Chen and Fishman 1996; Lien et al. 1999). Although not essential for specification of CMs, Nkx2.5 plays an important role in initiating the expression of many cardiac genes in mouse and regulating the numbers of atrial and ventricular progenitors (Searcy et al. 1998; Targoff et al. 2008). Similarly, another TF expressed in CM progenitors, Hand2, is responsible for proliferation of ventricular progenitors (Yelon et al. 2000). Hand2 also induces and maintains the expression of Tbx5, which is necessary for atrial specification in the mouse (Liberatore et al. 2000; Bruneau et al. 2001).

Despite the established knowledge of key TFs regulating the various steps of heart morphogenesis, considerable challenges to understand the mechanism of heart development still exist as little is known about their molecular mechanism and downstream targets. Transcription is modulated by cis regulatory elements that are located in non-coding regions of the genome, which serve as binding sites for TFs (Farnham 2009; Shlyueva et al. 2014). Although these regulatory elements equally contribute to the molecular mechanism controlling development, there is still a lack of systematic resources and understanding of their roles in heart development. Moreover, cardiac TFs have been shown to interact with chromatin-modifying factors, and the loss of function of several histone-modifying enzymes has been found to affect various aspects of cardiac development (Miller et al. 2008; Nimura et al. 2009; Lou et al. 2011; Takeuchi et al. 2011). Therefore, the chromatin landscape is another factor which needs to be considered when studying the process of heart development. Importantly, the lack of understanding how heart development proceeds makes it difficult to determine the cause of different forms of congenital heart disease (CHD). Here we seek to understand the nature of interaction between TFs and epigenomic landscape, how this landscape changes throughout development, and how it affects heart development.

The study of heart development poses a unique challenge due to the importance of the organ for survival. The disruption of factors regulating the early steps of heart formation can result in early embryonic lethality. The use of zebrafish as a model organism alleviates this problem by allowing access to developing embryos immediately after fertilization and its ability to survive without a functioning heart up to a comparatively late stage of development (Stainier 2001; Staudt and Stainier 2012). To elucidate the dynamics of the transcriptional regulatory landscape during heart development, we isolated CMs directly from the developing wild-type zebrafish heart at three key stages of morphogenesis: linear heart tube formation (24 hpf), chamber formation and differentiation (48 hpf), and heart maturation (72 hpf). Similarly, we isolated CMs from cardiac TF mutants of gata5, tbx5a and hand2 at 72 hpf. We then combined transcriptome profiling (RNA-seq) with an assay for chromatin accessibility (ATAC-seq) (Buenrostro et al. 2013) to capture the dynamics of regulatory landscape throughout the progression of heart morphogenesis in vivo. Our results unravelled the gene regulatory network driving key processes of heart development.


Dynamics of cardiomyocyte transcriptome and chromatin landscape demarcates key events of heart development. Pawlak M, Kedzierska KZ, Migdal M, Nahia KA, Ramilowski JA, Bugajski L, Hashimoto K, Marconi A, Piwocka K, Carninci P, Winata CL. Genome Res. 2019 Mar;29(3):506-519. doi: 10.1101/gr.244491.118. Epub 2019 Feb 13.

Decoding the Heart through Next Generation Sequencing Approaches. Pawlak M, Niescierowicz K, Winata CL. Genes (Basel). 2018 Jun 7;9(6). pii: E289. doi: 10.3390/genes9060289. Review.