A critical mechanism for generating new branches in the human vascular system is sprouting. In sprouting angiogenesis, endothelial cells from a pre-existing tube are led by actively migrating "tip cells" to form new vessels. Similarly, in the Drosophila respiratory organ (tracheal system), primary branching occurs through a sprouting mechanism in which tip cells play the same essential role. In both models of branch sprouting, the tip cells are morphologically distinct, extending filopodia to sense the local environment and to lead migration up a concentration gradient of a branch-inducing signal (Vascular endothelial growth factor/VEGF and fibroblast growth factor/FGF, respectively). We have shown that tracheal cells compete with each other for tip cell positions in Drosophila, where competition is based on relative levels of FGF Receptor (FGFR) activity and is mediated in part by the lateral inhibitory signal Notch. If Notch signaling is abrogated, a vast excess of tip cells are selected and migrate to the leading end of the sprout while only one or two follower cells comprise the branch stalk. Competition for tip cell positions also occurs during sprouting angiogenesis, with Receptor Tyrosine Kinase (RTK) and Notch signaling playing the same key roles. Despite substantial progress in understanding tip cell selection, the direct targets of Notch signaling that antagonize tip cell behavior in stalk cells remain unknown. Likewise, while RTK signaling promotes tip cell migration, the direct targets of the RTKs in tracheal and endothelial tip cells are not known. We have identified a novel mutation that we have named too many leaders, that confer a tip cell bias to mutant cells, and mutations in the septate/tight junction gene polychaetoid/ZO-1, that confer a Notch-like extra tip cell defect. We propose that tracheal tip cells and endothelial tip cells are selected by a conserved competition-based mechanism, and will systematically dissect the signaling pathways and their downstream targets, with the goal of determining how signaling information is translated into changes in cell shape, cell position within an epithelium, and cell migration, such that a pre- existing tube reorganizes to sprout a side branch.
The specific aims of this project are: 1. To define the RTK core components that mediate tip cell selection, and to determine whether a transcriptional response to RTK signaling is required. 2. To determine how Notch signaling is regulated and transduced during branch sprouting, and to examine how selective loss of Notch ligand expression affects tip cell competition. 3. To test the hypothesis that Polychaetoid and Too Many Leaders are downstream effectors of Notch signaling during tip cell competition.

Public Health Relevance

Angiogenesis-dependent diseases - including age-related macular degeneration, coronary artery disease, stroke, and cancer - result when new blood vessels either grow excessively or insufficiently. New branches in the human vascular system are generated by a process called sprouting angiogenesis, in which endothelial cells from a pre-existing tube are led by actively migrating tip cells to form new vessels. This proposal aims to use Drosophila tracheal branching as a model to uncover the genetic and molecular mechanisms by which tip cells are selected and by which they lead outgrowth of new tubes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM089782-04
Application #
8496076
Study Section
Cardiovascular Differentiation and Development Study Section (CDD)
Program Officer
Hoodbhoy, Tanya
Project Start
2010-07-01
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$285,181
Indirect Cost
$103,664
Name
University of Pennsylvania
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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