The formation of new blood vessels is required for embryonic development as well as the progression of numerous diseases. The processes that govern blood vessel formation and remodeling are conserved allowing the use model systems to investigate this process. The zebrafish has emerged as an ideal genetic system to dissect the molecular mechanisms involved in vascular development. Furthermore, the transparency of the zebrafish embryos facilitates direct visualization of endothelial cell behaviors in vivo during blood vessel development. Recent work has implicated small non- coding RNAs, referred to as microRNAs, in the proper development of the vascular system. However, little is known about the signaling pathways that miRNAs control during this process. Using deep sequencing methods, I have identified a number of endothelial expressed miRNAs. Furthermore, initial functional characterization of these miRNAs suggests that they play distinct and specific roles in vascular development. In this proposal, I will investigate the role of two endothelial miRNAs, miR-221 and miR-222, by generating zebrafish bearing targeted deletions within miRNA seed sequences using zinc finger nucleases. In parallel, I will generate an endothelial cell-specific transgenic line expressing myc tagged Argonaute 2 (Ago2). This line will allow me to purify miR-221 and miR-222 target complexes specifically from endothelial cells through immunoprecipitation of Ago2. The detailed phenotypic analysis of miRNA-knockout zebrafish, along with the ability to identify relevant endothelial-cell specific miRNA targets will allow me to determine what pathways and cell behaviors miR-221 or miR-222 may be controlling. These findings will then enable me to dissect the genetic networks regulated by endothelial miRNAs during vascular development. Together, the ability to combine targeted genetic manipulation of miRNAs along with the detailed imaging approaches available in the zebrafish, will allow me to gain novel insights onto the role of miRNAs in vascular development.

Public Health Relevance

We are developing technology to facilitate the in vivo study of microRNAs in the vascular system. The resulting data will allow us to gain new knowledge concerning microRNA function in controlling endothelial cell function and blood vessel formation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Career Transition Award (K99)
Project #
1K99HL105791-01
Application #
8029302
Study Section
Special Emphasis Panel (ZHL1-CSR-Z (O1))
Program Officer
Roltsch, Mark
Project Start
2011-02-04
Project End
2013-01-31
Budget Start
2011-02-04
Budget End
2012-01-31
Support Year
1
Fiscal Year
2011
Total Cost
$87,172
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Genetics
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Lopez-Ramirez, Miguel A; Calvo, Charles-Félix; Ristori, Emma et al. (2016) Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres. J Vis Exp :53617
Santoro, Massimo M; Nicoli, S (2013) miRNAs in endothelial cell signaling: the endomiRNAs. Exp Cell Res 319:1324-30