Blood vessels are required for organ formation and homeostasis during embryonic development and throughout adult life. Since the signaling mechanisms that govern blood vessel formation are largely conserved and are similar during embryonic and adult stages, it is possible to utilize model organisms to gain insight into this process. Blood vessel formation occurs through the coordination of a complex array of cellular behaviors. Recent evidence indicates that the Notch signaling pathway plays an essential role during this process to determine cell fates and modulate signaling output during vascular development. Notably, Notch is essential for promoting arterial endothelial cell fate while during angiogenesis it helps to limit an endothelial cell's response to pro-angiogenic cues. However, little is known about when and where Notch activation occurs during the early stages of vascular development or how Notch activation affects endothelial cell fates at different steps of blood vessel formation. Furthermore, few Notch target genes have been identified that may mediate the effects of Notch activation in endothelial cells. In the studies proposed here, we will take advantage of the zebrafish as a model system to address the dynamic role of Notch signaling during blood vessel development. Using a transgenic indicator line that is transcriptionally responsive to Notch signaling, we will visualize the temporal and spatial dynamics of Notch activation in developing blood vessels through time-lapse analysis in live zebrafish embryos. In addition, we will establish transgenic lines expressing a photoconvertible fluorescent protein in all endothelial cells or in Notch-positive endothelial cells. These lines will allow detailed fate mapping of Notch-positive and Notch-negative endothelial cells during multiple stages of vascular development. We will also characterize the function of putative direct targets of Notch in endothelial cells by taking advantage of our ability to generate zebrafish knockout lines using zinc finger nucleases. This will enable the rapid generation of null alleles in target genes of interest to determine their role downstream of Notch. Importantly, this technique will also allow us to delete endogenous Notch responsive cis elements in genes of interest to definitively characterize the role of Notch in regulating downstream targets. Finally, we will combine global expression profiling and genome-wide assay of occupancy at Notch binding sites to characterize the Notch-responsive transcriptional network in endothelial cells.

Public Health Relevance

A crucial step in the formation of new blood vessels is the establishment of proper blood vessel identity (e.g. artery versus vein). Recent evidence suggests that loss of blood vessel identity in a number of congenital human diseases can lead to vascular malformations. The studies in this proposal will address how blood vessel identities are determined during development.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL093467-05
Application #
8453420
Study Section
Cardiovascular Differentiation and Development Study Section (CDD)
Program Officer
Schramm, Charlene A
Project Start
2009-04-06
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
5
Fiscal Year
2013
Total Cost
$387,595
Indirect Cost
$151,975
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
Quillien, Aurelie; Moore, John C; Shin, Masahiro et al. (2014) Distinct Notch signaling outputs pattern the developing arterial system. Development 141:1544-52
Villefranc, Jacques A; Nicoli, Stefania; Bentley, Katie et al. (2013) A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development. Development 140:1497-506
Dang, Lan T H; Lawson, Nathan D; Fish, Jason E (2013) MicroRNA control of vascular endothelial growth factor signaling output during vascular development. Arterioscler Thromb Vasc Biol 33:193-200
Cheung, Isla D; Bagnat, Michel; Ma, Taylur P et al. (2012) Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b. Dev Biol 361:68-78
Nicoli, Stefania; Knyphausen, Carl-Philipp; Zhu, Lihua J et al. (2012) miR-221 is required for endothelial tip cell behaviors during vascular development. Dev Cell 22:418-29
Aday, Aaron W; Zhu, Lihua Julie; Lakshmanan, Abirami et al. (2011) Identification of cis regulatory features in the embryonic zebrafish genome through large-scale profiling of H3K4me1 and H3K4me3 binding sites. Dev Biol 357:450-62
Clements, Wilson K; Kim, Albert D; Ong, Karen G et al. (2011) A somitic Wnt16/Notch pathway specifies haematopoietic stem cells. Nature 474:220-4