Stefania Nicoli Ph.D.
Assistant Professor of Medicine (Cardiology)
Zebrafish; Post-transcriptional regulation; Small RNAs; Angiogenesis; Artery-vein differentiation; Vessels stability; Hematopoiesis; Lymphatic development
Identification and regulation of miRNAs-mRNA during the development of zebrafish embryonic and adult vascular system. We are extending the repertoire of small RNAs and mRNAs expressed in Zebrafish endothelial cells. We will illuminate ultrahigh throughput sequencing approach in order to profile the transcriptome and miRNAome of endothelial cells isolated from early to later stage of development. The goal of this project will be to identify different temporal and spatial requirements of specific miRNAs-RNA interactions throughout the formation of the cardiovascular system.
Generation of endothelial miRNAs mutant. To dissect the function of a specific miRNA during vascular development we are generating Zebrafish embryos carrying mutations within the pre-miRNA genome sequence in order to alter their biogenesis and induce miRNA loss of function. In order to do so we are utilizing both Zinc Finger Nucleases and TALEN. Both strategies are based on engineering specific chimeric DNA binding proteins,fused to the nuclease domain of the restriction enzyme Fok1 . Upon binding and dimerization they cleave DNA creating a double strand break repaired by non-homologous end joining, often resulting in a micro-deletion or insertion in the genome sequence. The goal of this project is to assess the function of a specific miRNA with endothelial expression in order to discover and dissect the defect associated with the miRNA loss of function.
Role of miRNAs-mRNAs during artery–vein differentiation and function. Starting from human arterial and venous primary cells we will capture miRNA-mRNAs by immunoprecipitation of the RISC complex using the HITS-CLIP strategy. This approach will reveal specific RNAs target by miRNAs differentially required between arteries and vein. We will use the zebrafish system in order to validate in vivo conserved miRNA-RNA interactions required for proper differentiation and maintenance of arterial and vein fate.
Extensive Research Description
The vascular system is fundamental for embryonic development and adult life, and aberrant vascularization is associated with numerous diseases, including cancer,atherosclerosis and stroke. Since the processes that govern blood vessel formation are conserved, it is possible to use model systems to gain novel insights on vascular development and function. The Zebrafish (Danio rerio) is an ideal model to study blood vessel formation during embryonic development. The transparency and external development of the zebrafish embryo allow an unprecedented level of observation and experimental manipulation. In parallel, numerous techniques allow forward and reverse genetic analysis of signaling pathways in the zebrafish.These genetic approaches coupled with the ability to easily visualize circulatory patterns and blood vessel morphology, make the zebrafish an ideal in vivo platform to assay gene function during vascular development.
microRNAs (miRNAs) are highly conserved non-coding small RNAs that post-transcriptionally regulate gene expression by binding to the 3’UTR of target mRNAs and inhibit their translation, or promote their degradation. miRNAs are autonomously transcribed in a large mRNA transcript (pri-mRNA), or are found in introns of coding genes. In both cases,mature miRNAs are formed by sequential processing into a primary stem loop precursor (pre-miRNAs) by the endonucleases Drosha and Dicer. In vertebrates,the 22 base pair duplex miRNAs are unwound and a single mature strand is loaded onto Argonaute 2 (Ago2). The Ago2/miRNA complex (the RNA-induced silencing complex, or RISC), leads to translational repression and decreased transcript stability, through deadenylation. miRNAs function in a number of different biological processes, including cardiogenesis, muscle development, oncogenesis, brain morphogenesis, and hematopoiesis.
Despite recent findings, several critical barriers remain that hamper the study of miRNAs. First, identification of relevant miRNA targets, especially cell-specific target transcripts in vivo,can be difficult. Second, genetic manipulation (i.e. targeted knockout) of miRNA sequences in the vertebrate genome can be challenging and, until recently, had been limited to mice. Third, in many cases loss of miRNA function leads to subtle phenotypic changes, which can be difficult to observe and characterize during embryonic stages in mouse. Finally, the genetic interaction of miRNAs and their targets can be difficult to dissect in vivo in the mouse system.
The lab takes advantage of the zebrafish as a model system to overcome these barriers. Our goal is to elucidate how miRNAs participated in the genetic network driving arteries-veins differentiation, angiogenesis, neuro-vascular and lymphatic development.