Arterial and venous (A-V) patterning is critical for the establishment of functional embryonic and adult vasculature. Proper A-V differentiation is critical for the formation of functional vessels during tissue repair processes such as wound healing and is associated with a number of pathological conditions including A-V malformations and tumor induced angiogenesis. Therefore understanding the mechanisms of A-V differentiation will lead to new treatments in tumorigenesis, wound repair and multiple vascular disorders. While it is difficult to study A-V patterning in mammalian systems, transparent zebrafish embryos are easily accessible for observation and experimental manipulations. During formation of the major vessels, vascular endothelial progenitor cells (EPCs) assume arterial or venous identity prior to the initiation of circulation. However, it is not understood how EPCs choose among the arterial or venous fates if they are all exposed to the same signaling molecules such as Vegf and Hh. We have found that the expression timing of an evolutionarily conserved master regulator of vasculogenesis Etsrp / Etv2 is one of the critical factors in A-V differentiation. Furthermore, arterial and venous progenitors may originate at different spatial locations, the inner and the outer lines and thus experience different Vegf concentrations. We hypothesize that the arterial- venous fate of EPCs is determined by the combination of etsrp-dependent timing of Vegf receptor flk1 and hyaluronan (HA) receptor stabilin 2 (stab2) expresion and their spatial location within Vegf gradient. To demonstrate that the timing of etsrp expression affects A-V differentiation, photoactivatable morpholinos will be used to inhibit etsrp function at different developmental stages in zebrafish embryos. Fate mapping and time- lapse imaging will be performed to characterize the cell movements and to determine the arterial-venous fates of the inner and outer lines of EPCs. To determine if Vegf and Hh gradients play a role in the activation of Notch signaling and arterial differentiation within the inner line angioblasts, overexpression and loss of function approaches will be used to inhibit Vegf, Hh and Notch signaling combined with lineage tracing to determine the A-V cell fates. To determine if Etsrp downstream target Stab2 functions as a receptor for HA and induces activation of Notch signaling during A-V differentiation, A-V defects in Stab2 and HA synthase Has2 knockdown embryos will be investigated. It will be analyzed if HA-Stab2 signaling leads to Tyr-phosphorylation of Stab2 and ERK phosphorylation, resulting in Notch pathway activation and arterial marker expression. Upon completion of this study, we will have identified the molecular mechanism how the timing of etsrp expression and Vegf gradient lead to the differential expression of arterial and venous genes. The acquired knowledge will be critical in our understanding of molecular mechanisms of A-V differentiation and will have an impact on developing treatments for multiple diseases and pathological conditions related to vasculature formation which include A-V malformations, diabetic retinopathy, wound healing and tumorigenesis.!

Public Health Relevance

The proposed project will utilize a zebrafish model system to investigate molecular mechanisms of arterial-venous specification. It will determine how the timing and location of endothelial cell specification translate into differential arterial and venous cell fates. It will investigate how different inputs from transcription factors such as Etsrp / Etv2 and morphogens such as Vegf are integrated to induce or inhibit expression of arterial or venous specific genes. The acquired knowledge will be critical in our understanding of fundamental mechanisms of vascular differentiation, which will lead to new treatments in tumorigenesis, wound repair and multiple vascular disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL107369-04
Application #
8645705
Study Section
Cardiovascular Differentiation and Development Study Section (CDD)
Program Officer
Reid, Diane M
Project Start
2011-04-01
Project End
2016-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
4
Fiscal Year
2014
Total Cost
$374,850
Indirect Cost
$129,850
Name
Cincinnati Children's Hospital Medical Center
Department
Type
DUNS #
071284913
City
Cincinnati
State
OH
Country
United States
Zip Code
45229
Rost, Megan S; Sumanas, Saulius (2014) Hyaluronic acid receptor Stabilin-2 regulates Erk phosphorylation and arterial--venous differentiation in zebrafish. PLoS One 9:e88614
Glenn, Nicole O; Schumacher, Jennifer A; Kim, Hyon J et al. (2014) Distinct regulation of the anterior and posterior myeloperoxidase expression by Etv2 and Gata1 during primitive Granulopoiesis in zebrafish. Dev Biol 393:149-59
Schumacher, Jennifer A; Zhao, Emma J; Kofron, Matthew J et al. (2014) Two-color fluorescent in situ hybridization using chromogenic substrates in zebrafish. Biotechniques 57:254-6
Kohli, Vikram; Schumacher, Jennifer A; Desai, Sharina Palencia et al. (2013) Arterial and venous progenitors of the major axial vessels originate at distinct locations. Dev Cell 25:196-206
Kohli, Vikram; Rehn, Kira; Sumanas, Saulius (2011) Single cell fate mapping in zebrafish. J Vis Exp :