Detailed information about the project:
Project Duration: 01.09.2016-31.08.2018
Project number: 2015/19/P/NZ3/03613
Project title: “Genomic profiling of zebrafish cardiac pacemaker cells”
Project leader: Dr. Rashid Minhas
Source of funding: National Science Centre, Poland, POLONEZ
Budget: 921 064 PLN

Objective:

The cardiac conduction system (CCS) is an essential component of the heart. It is responsible for initiating and coordinating the electrical signals that cause rhythmic and synchronized contractions of the atria and ventricles. The CCS is evolutionarily conserved in the building plan of the heart, and it indicates that the cellular and molecular mechanisms that drive the formation of pacemaker tissues are almost similar among vertebrates. Components of the mammalian conduction system are morphologically well defined in mouse and human. However, the molecular mechanisms by which the CCS cells are set apart and specified from a common cardiomyocyte cell are not thoroughly understood to date.

The study of heart development is often hindered by the fact that the organ is absolutely required for survival in most organisms. In zebrafish (Danio rerio), a functioning cardiac system develops at 24 hpf, but is not essential for the survival of early embryos and thus zebrafish poses a unique advantage in this respect. Furthermore, zebrafish is highly amenable to genetic modifications, and has a short generation time, which allows convenient and rapid analysis of gene function and modelling of human genetic defects. Using zebrafish as an in vivo system, we would like to carry out transcriptomic profiling of these highly specialized cells, coupled with profiling of chromatin state, to indicate important regulatory regions implicated in CCS development. Expression profiling of these cells would help us identify key genes expressed specifically in pacemaker cells. While profiling of chromatin state will allow us to identify regulatory regions active specifically in the CCS. A combination of these profiles will thus become the basis for the assembly of a gene regulatory network underlying the development of the CCS.

Abstract
Congenital heart disease (CHD) is one of the most common type of birth defect, accounting for one-third of all major congenital anomalies. Among CHDs, one of the major group of cardiac patients have disorders of the cardiac conduction system (CCS) and its associated tissues, causing life threatening severe arrhythmias. CCS cells are present in two different locations in the heart: the sinoatrial node (SAN) and the atrioventricular node (AVN). The zebrafish is a well-established vertebrate model for cardiovascular studies. To understand the molecular mechanism underlying the development of the pacemakers cells, we use a zebrafish transgenic line with GFP expression in CCS cells. Zebrafish hearts were isolated at 72 hpf and GFP positive cells were sorted using FACS. High quality RNA was extracted and subjected to RNA-Seq to profile their transcriptome.  This study will shed light on novel CCS-specific molecular markers and highlight the active regulatory regions responsible for the specialized function of these cells. This knowledge will further improve the understanding of sinus node dysfunction and facilitate the development of novel therapies.

Posters at the international conferences:

(1) Genomic interaction responsible for pacemaker-specific GFP expression in zebrafish heart

Gene-regulatory systems in development Parador de Carmona, Spain 

(2) Genomic profiling of zebrafish cardiac pacemaker cells
Fishmed 2018 (International Institute of Molecular and Cell Biology (IIMCB), Warsaw, Poland

(3) Deciphering the zebrafish cardiac pacemaker development with RNA-Seq
18th International Zebrafish Conference University of Medicine Wisconsin, Madison, USA

Activities towards the general public:
(1) Be Heathy as a Fish workshop, 10-14 July 2018
Be Heathy as a Fish workshops as a part of the International Biology School for young, talented students from Ukraine. The objective of the educational program is to teach children basic knowledge about the life of fish and about possibilities of their use in studies on certain human diseases. During the workshops children watch the Healthy as a Fish movie, perform simple biological experiments, take part in a discussion about the genetic similarities of humans and fish, and receive a copy of the Be Healthy as a Fish book.
Since December 2012, our Institute has been implementing the FishMed project, supported by the European Union. Within this project, IIMCB has opened one of the largest facilities to grow and breed lines of zebrafish as research models in this part of Europe. We are pioneering research, in which we use zebrafish as an attractive alternative to studies in which higher vertebrates are used as the model organisms. Using this species as an example, children have the opportunity to learn how important fish are for us, as well as discover what we as a society can gain through the work of biologists in the future.
(2) The Biologists Night, 12 January 2018
The event was held on January 12, 2018 at the Faculty of Biology University of Warsaw, and at the Center of Biological-Chemical Sciences University of Warsaw. The program included lectures, laboratories, workshops and exhibitions. About 600 participants attended the event. It was a great opportunity to present zebrafish to broader audience.

Publications:

Minhas, R., Paterek, A., Łapiński, M., Bazała, M., Korzh, V., Winata, C.L. 2018. A novel conserved enhancer at zebrafish zic3 and zic6 loci drives neural expression. Developmental Dynamics. Manuscript Submitted.

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 665778 (National Science Centre (NCN), Poland).

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.

Publications:

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.

Heart arrhythmia is a condition where the rhythm of heart contraction becomes irregular. This can lead to the formation of blood clots with devastating consequences such as heart attack and stroke. However, despite its seriousness, heart arrhythmia is very little understood compared to other types of heart diseases. The genetic factors which cause the condition is not well known and there is very little medical treatment available to treat such conditions, the most common being surgery and artificial pacemakers. Therefore, heart arrhythmia patients often have to live with the condition and experience a decrease in quality of life and often need to undergo continuous and costly medical procedures due to common recurrence of the condition. Our research aims to improve the understanding of the genetic mechanism causing heart arrhythmia, and with this knowledge, we hope to contribute to the medical field in improving the diagnosis as well as treatment methods of heart arrhythmia.

The pacemaker is a group of cells in the heart which spontaneously generate small electrical current at a regular rhythm and propagate this current throughout the heart, causing its rhythmic contraction. If the pacemakers develops abnormally, the rhythm of heart contraction is affected, leading to different types of arrhythmia depending on which pacemaker is affected and the nature of the defect. To study how the pacemaker develops and functions, we use the zebrafish as a model organism due to its similarity in heart physiology and genetics to that of humans. Using a genomics approach, we will perform a study to elucidate the molecular mechanism underlying pacemaker development in the zebrafish, followed by initiating the groundworks for establishing the zebrafish as a model organism to study pacemakers function and diseases associated with their dysfunction. Besides identifying factors in common between human and zebrafish, our results will also suggest novel genetic factors with potential implications in heart arrhythmia. This knowledge is envisaged to contribute to future development of molecular diagnosis as well as inform the design of future clinical therapies for heart arrhythmia.

The project is carried-out within the First-TEAM programme of the Foundation for Polish Science and is co-financed from the European Union under the European Regional Development Fund.

Cardiovascular progenitors originate in the lateral plate mesoderm (LPM) and express overlapping sets of genes, among which is nkx2.5 which encodes a transcription factor responsible for driving cardiac fate from mesodermal progenitor. Interestingly, Nkx2.5 have also been shown to play a role in specifying the blood and vascular lineage and a subpopulation of nkx2.5-expressing cells in the LPM were found to give rise to pharyngeal arch endothelial cells. Our previous study (Pawlak et al., Genome Res. 2019) also suggests the same, in which we observed that nkx2.5-expressing cells express genes implicated in the development of, not only heart, but also haematopoiesis and vasculogenesis. This is also accompanied by the enrichment of motifs for GATA, Fli, ETS, ERG, and ETV family of TFs in open chromatin regions associated with these genes, suggesting that they are regulated at both genetic and epigenetic levels. An attractive hypothesis that arises is that a group of cells exist within the pool of nkx2.5-expressing progenitors which possess alternative potential to become cardiac or blood/vascular lineage. In order to dissect into the molecular mechanism behind this duality of function, we aim to pinpoint at which stage the segregation between cardiac and hemoangiogenic fates start to become segregated, the pathway and intermediate states these progenitors go through during lineage specification, and finally to determine the epigenetic contribution to this process. The project employs single cell transcriptomics and epigenomics analysis coupled with computational modelling of cell lineage trajectory to elucidate the mechanism of Nkx2.5-driven lineage specification of cardiac and hemoangiogenic fates. 

This project is supported by the OPUS grant from the National Science Center 2019/35/B/NZ2/02548.