The research in our lab focus on two levels of gene regulation: transcriptional and translational. At the level of transcriptional regulation, we seek to understand the mechanism by which transcription factors (TFs) and epigenetic landscape interact to regulate heart development. At the level of translation, we investigate the mechanism of translational control of maternal mRNAs though cytoplasmic polyadenylation during early embryonic development.
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 have been shown to interact with chromatin modifying factors, and loss of function of several histone modifying enzymes have been found to affect various aspects of cardiac development. Such high degree of complexity in developmental regulation in vivo necessitates an approach which takes into account both genetic and epigenetic factors. Using a genomics approach and capitalizing on the advantages of zebrafish, we want to uncover genetic and epigenetic factors contributing to the process of heart development and elucidate their regulatory mechanism.
Transcriptional regulatory network of heart development
The vertebrate heart undergoes three key stages of morphogenesis: specification and migration of cardiac progenitors, formation of the beating linear heart tube, and looping to form a multi-chambered organ. In each of these stages, TFs play a crucial role in initiating transcription of cardiac genes, leading to a cascade of genetic regulation. At the core of this regulation machinery is the interaction between cardiac TFs Nkx2.5, Gata5, Tbx5, and Hand2 which is necessary for the establishment of cardiac identity in cells of the embryonic mesoderm, their subsequent diversification into atrial and ventricular progenitors, and their migration to the midline to form the linear heart tube.
Building upon our experience in using ChIP-seq on zebrafish whole embryos and FACS-sorted cells to study transcriptional regulation during zebrafish development , we are focusing our current effort to characterize the downstream regulatory network of cardiac TFs during key phases of heart development. In parallel to this, we are developing tools for tissue-specific analysis of transcriptional regulation in the form of transgenic lines expressing fusion tagged TFs.
Epigenome profile of heart development
Epigenetic marks in the form of modified histones have been commonly used to identify chromatin states, indicating the transcriptional status or activity of particular genetic elements, such as enhancers and promoter. A systematic catalogue of these marks, combined with the information on TF binding sites in the genome, would provide a comprehensive and unbiased view of transcriptional regulatory landscape during heart development in vivo. Together with functional analysis in zebrafish mutants, we aim to identify genome-wide elements associated with heart defects, and to characterize epigenetic contributions to heart development.
During embryogenesis, a silent transcriptional period exists from the moment of fertilization up to the time of zygotic genome activation known as the maternal to zygotic transition (MZT). During this period of transcriptional silence, development is regulated by maternally deposited mRNAs which consisted of two different subpopulations: those which exist in a polyadenylated form and those with very short or no poly(A) tail at fertilization and are gradually polyadenylated with developmental progression . The latter cohort of maternal mRNAs is thought to undergo a form of translational control known as cytoplasmic polyadenylation, which involves its initial de-adenylation at the point of fertilization and subsequent re-adenylation to activate its translation. In support of this, the 3’UTR of this cohort of maternal mRNAs contain signals known to indicate delayed cytoplasmic polyadenylation. We observed that pan-embryonic inhibition of cytoplasmic polyadenylation resulted in the inability of the embryo to undergo MZT, suggesting that this process is a crucial mechanism underlying the maternal control of pre-MZT development.
Our research in this topic focuses on two lines of investigation:
- to characterize the molecular mechanism of cytoplasmic polyadenylation during pre-MZT development, and
- to understand how cytoplasmic polyadenylation contributes to the regulation of MZT.
In our previous RNA-seq data , the transcripts of at least three different cytoplasmic element binding proteins (CPEBs) were present during pre-MZT period. These factors are known to regulate both cytoplasmic polyadenylation and translation initiation. Currently, functional study of the CPEBs in zebrafish embryogenesis is in progress, as well as the development of methods and tools for the analysis of RNA binding by these factors.
- . Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish. PLoS Genet. 2013 ;9(10):e1003852.
- Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition. Genome Res. 2011 ;21(8):1328-38.