The mammalian vascular system, lung, and kidney are branched tubular epithelial organs that transport gases or fluids. Although genes required for assembly of these organs have been identified, the developmental mechanisms that determine the shapes and sizes of their tubes are not well understood. The respiratory (tracheal) system of the Drosophila larva has provided a useful genetic model for the study of the development of complex branched tubular networks. Branching morphogenesis in the embryonic tracheal system is controlled by patterned interactions between a fibroblast growth factor (FGF) receptor tyrosine kinase (TK) ortholog, Breathless (Btl), and its FGF ligand, Branchless (Bnl). The developmental logic of the tracheal system is similar to that of the mammalian vascular system, where vascular sprouts expressing the vascular-endothelial growth factor (VEGF) receptor TK grow toward sources of VEGF. How are the airways in tracheal branches sculpted into the appropriate tubular shapes? We obtained an entry point into this problem when we discovered a unique tracheal phenotype caused by a double mutation eliminating both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. The Ptp4E Ptp10D double mutation converts linear unicellular tubes into spherical cysts. Type III RPTPs are highly conserved regulators of receptor TK signaling, and we found that the phenotype involves the loss of negative regulation by the RPTPs of three growth factor receptor TK orthologs: epidermal growth factor receptor (Egfr), Btl, and Pvr (VEGFR ortholog). This phenotype may have never been found in earlier genetic screens because it is only observed when both Ptp4E and Ptp10D are mutated. There may also be no single component downstream of the RPTPs that could be mutated to generate such phenotypes, since the RTKs signal through many pathways. Thus, the identification of genes that regulate tube geometry may require a sensitized genetic screen based on the Ptp4E Ptp10D phenotype. This is the basis of the first specific aim, which describes a systematic screen for recessive mutations that confer enhancement or suppression of the phenotype. Because this is a time-consuming screen, requiring quantitative analysis of individual embryos using confocal microscopy, we will reduce the numbers of lines that need to be screened by using a 'phenotypic screening kit'of deletion (Df) mutations that we have defined. For each deletion that enhances or suppresses the phenotype, we will identify the responsible gene using insertion mutations and RNAi lines, which exist for most Drosophila genes. When we have mutations in individual genes in hand, we will examine their phenotypes in detail and analyze their epistatic relationships with each other, as well as with the RTKs and RPTPs, in order to define genetic pathways. The second specific aim describes a systematic approach by which we can localize and tag the protein products of genes identified in the screen. We can attach the proteins to fluorescent markers of various colors (for localization in live and antibody-stained preparations) and to epitope tags or enzymes (for biochemical characterization). This system will allow us to find proteins that are localized t the regions of cells where tube shape is controlled. We can also analyze tyrosine phosphorylation of the proteins and determine if they physically interact with each other in the embryo.

Public Health Relevance

The developmental logic of the human vascular system is similar to that of the Drosophila respiratory (tracheal system). In both systems, tube growth is driven by interactions between receptor tyrosine kinases (RTKs) and their ligands. In tumor angiogenesis, hypoxic tumor cells recruit new blood vessels through secretion of RTK ligands that are also used during development. An understanding of the RTK signaling pathways that control tracheal tube morphogenesis may help in identification of new targets for research into tumor angiogenesis mechanisms and therapies.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21HD073367-02
Application #
8501610
Study Section
Development - 1 Study Section (DEV1)
Program Officer
Javois, Lorette Claire
Project Start
2012-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
2
Fiscal Year
2013
Total Cost
$156,585
Indirect Cost
$61,685
Name
California Institute of Technology
Department
None
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125