Genomic profiling of zebrafish cardiac pacemaker cells

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).