The aim of our research is to understand the mechanism of gene regulation during embryonic development in vivo using zebrafish (Danio rerio) as a model organism. Our main research interests center around the regulation of gene expression in embryonic development. In particular, we seek to understand the mechanism by which transcription factors (TFs) and the chromatin landscape interact to regulate the development of an organ. In addition, we investigate the biological consequences of post-transcriptional modifications on maternal mRNAs, which include cytoplasmic polyadenylation and RNA editing.
Our main line of research attempts to understand the mechanism of transcriptional regulation through the interaction between TFs and the epigenetic landscape in heart development and disease. Although the heart in different species of vertebrates can have two to four chambers, the step-wise morphogenesis of progenitor specification, migration, tube formation, and looping has been shown to be highly conserved. The study of heart development poses a unique challenge due to the importance of the organ for survival. Disruption to factors regulating the early steps of heart formation cause early embryonic lethality. The zebrafish (Danio rerio) alleviates this problem by allowing access to developing embryos right after fertilization and its ability to survive without a functioning heart up to a comparatively late stage of development. Taking advantage of this model organism, many genes regulating heart development have been identified. However, despite these advances, considerable challenges to understand the mechanism of heart development still exist. Firstly, there is still a lack of knowledge on molecular mechanism and downstream targets of cardiac TFs. Secondly, the transcription of genes are modulated by cis regulatory elements located in non-coding regions of the genome, which also serve as binding sites for TFs. Thus, mutations in these regulatory elements equally affect developmental outcome as mutations in coding regions. However, there is still a lack of systematic resource for these elements and understanding of their roles in heart development. Thirdly, an additional layer of regulation exists in the form of epigenetics. Cardiac TFs interact with chromatin modifying factors, and loss of function of several histone modifying enzymes have been found to affect various aspects of cardiac development. Using a genomics approach and capitalizing on the advantages of zebrafish, we aim to uncover genetic and epigenetic factors contributing to the process of heart development and elucidate their regulatory mechanism.
Transcriptional regulatory landscape in developing cardiomyocytes
Heart muscle cells or CMs are specified early during embryogenesis from a pool of mesodermal progenitors. At the core of the machinery that regulates each step of heart morphogenesis are cardiac TFs, including Nkx2.5, Gata5, Tbx5, and Hand2. 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 and the specification of atrial and ventricular CMs.To elucidate the dynamics of the transcriptional regulatory landscape during heart development, we employed a combination of transcriptome profiling (RNA-seq) and an assay for chromatin accessibility (ATAC-seq) at several key stages of heart development. Our analyses revealed the dynamic regulatory landscape throughout heart development and identified interactive molecular networks driving key events of heart morphogenesis (Pawlak et al., in revision). In addition, we applied in silico TF footprinting methodology on our ATAC-seq data to identify genome-wide binding sites of Nkx2.5, Gata5, Tbx5, and Hand2. Ultimately, we aim to characterize the contribution of the dynamic transcriptional regulatory landscape to heart development and identify novel elements associated with heart defects.
Genomics dissection of pacemaker development
The cardiac conduction system is responsible for generating and propagating the electrical impulses that are required for the contraction of heart muscle tissues. The cardiac conduction system consists of pacemaker cells, specialized heart muscle cells that serve to ensure rhythmic contractions of the heart. Pacemaker cells possess distinctive morphological and electrophysiological properties that are specialized for their function. They are set apart early from CMs in the course of heart development through the induction of expression of TFs which prevents their differentiation into CMs. Once specified, pacemaker progenitor cells further develop low conductance properties through the expression of gap junction proteins that are distinct from CMs. Despite the knowledge of key genetic factors that are required for pacemaker cell specification, the molecular mechanisms that regulate their development are still insufficiently understood. Important questions remain with regard to the ways in which the underlying molecular mechanism translates into the proper functioning of pacemaker cells and the consequences of their dysregulation. Moreover, inherited forms of arrhythmia are often associated with more common forms of congenital heart malformations that affect other tissue types of the heart, including CMs, implying interconnectivity of the gene regulatory networks that govern their development and function.
The zebrafish heart exhibits remarkable similarities to the human heart in terms of basal heart rate, electrophysiological properties, and action potential shape and duration. Thus, it is an ideal model organism to study the heart pacemaker and model human clinical conditions that affect pacemaker function. We utilized transgenic lines which express GFP in subpopulations of pacemaker cells to characterize the morphology of the zebrafish pacemaker and to isolate pacemaker cells. Transcriptome profiling of isolated pacemaker cells revealed distinct molecular profiles defining cardiomyocytes and pacemaker which provide important insights into the mechanism of their diversification. We aim to identify regulatory pathways underlying pacemaker development and function and to establish zebrafish as a model for pacemaker dysfunction.
During embryogenesis, a silent transcriptional period exists from the moment of fertilization to the time of zygotic genome activation, known as the mid-blastula transition (MBT) in zebrafish and frogs. During this period of transcriptional silence, development is regulated by maternally deposited mRNAs that undergo various forms of posttranscriptional modifications to regulate their expression.
Translational control by cytoplasmic polyadenylation
Maternal mRNAs are initially deposited in the immature oocyte in a translationally dormant state, with a very short poly(A) tail. Two major waves of cytoplasmic polyadenylation occur during oocyte maturation and upon fertilization, resulting in the translational activation of distinct subpopulations of maternal mRNAs. Through profiling of polysome-associated transcriptome, we established cytoplasmic polyadenylation as a prominent mode of the temporal activation of maternal mRNAs that is necessary for MBT (Winata et al., Development, 2018). Current work in the laboratory focuses on studying the mechanistic basis of cytoplasmic polyadenylation through functional analyses of cytoplasmic polyadenylation element binding proteins (CPEBs) that are known to regulate cytoplasmic polyadenylation and translation. The transcripts of at least three different CPEBs (cpeb1b, cpeb4a, and cpeb4b) are present as maternal mRNAs and associated with polysomes between fertilization and MBT. Functional studies of these CPEBs are currently underway, and we are developing methods and tools for the analysis of RNA binding by these factors in the form of CRISPR-generated transgenic lines.
RNA editing in early embryogenesis
RNA editing refers to the post-transcriptional modification of RNA sequences, the most common form of which is A-to-I conversion that occurs through the deamination of adenosine (A) at the C6 position, converting it to an inosine (I). In humans, the misregulation of A-to-I RNA editing in somatic tissues may lead to neurological and metabolic disorders, autoimmune diseases, and cancer. A mode of post-transcriptional gene regulation, such as RNA modification, would nicely fit into the scenario that occurs at the earliest stages of embryonic development, during which the embryo contains an abundant supply of maternal mRNAs and zygotic transcription is still absent. Surprisingly, despite this, RNA editing has been seldom considered in the context of embryonic development.
Two paralogs of the A-to-I RNA editing enzyme, adar and adarb1, are present in the form of maternally deposited transcript at the earliest stages of development (Winata et al., Development, 2018). To reliably detect RNA editing events, we developed a new and improved method for RNA editing discovery. Our preliminary analyses suggested the presence of RNA editing in both maternally deposited and zygotic transcripts. Currently, we have generated a zebrafish mutant line for the adar gene using CRISPR/Cas9. This line will be used for more detailed functional studies of RNA editing. Furthermore, we are also attempting to identify a correlation between editing and structure through the prediction of secondary structures in conserved domains in selected mRNA candidates that will be identified through comparative transcriptome analyses with several closely related fish species from the Carp family.