PgmNr Z6218: Screening for genetic interactions in the blood-brain barrier in vivo in the zebrafish, Danio rerio.

Authors:
Thomas Clements 1 ; Shinya Hirota 2 ; Sam Kwiatkowski 2 ; Cecilia Guerra 1 ; Aryeh Warmflash 1 ; Joseph McCarty 2 ; Daniel Wagner 1


Institutes
1) Rice University, Houston, TX; 2) University of Texas M. D. Anderson Cancer Center, Houston, TX.


Abstract:

In the blood-brain barrier (BBB), glial cells interact with vascular endothelial cells (ECs) to control blood vessel development and formation. However, the molecular mechanisms of these interactions remain mostly uncharacterized. In order to screen for genetic interactions amongst genes of interest, we used MOs to repress the translation of the zebrafish homologs of these genes in vivo. Morpholinos (MOs) are an established reverse genetic technique that prevents splicing of pre-RNA or translation of mRNA by binding to complementary regions of RNA in vivo. Using MOs allows us to titrate the single doses down to the lowest effective levels and then screen combinations for more drastic effects, which cannot be done using traditional gene knockout methods. The zebrafish is ideal for studying genetic interactions in the blood-brain barrier because fertilization is external and the embryo is transparent, thus allowing for easy visualization of neural development.

It is hypothesized that an uncharacterized signaling network controls gene expression to regulate cerebral blood vessel development mediated by αVβ8 integrin-activated (Igtβ8, IgtαV) TGF-βs. Thus, it is our goal is to confirm crosstalk and coordinated regulation of specific genes in this pathway using conditional systems in mice (done by our collaborator), MO as well as CRISPR-Cas knockouts in zebrafish, and the use of endothelial specific Smad-4 GFP transgenic line. Here we report a synergistic relationship between Igtβ8 and Neuropilin1a (Nrp1a) in vivo as ascertained through the use of MOs, which was done by quantifying loss of transcript through observable brain hemorrhages and RT PCR.



ZFIN Genetics Index
1. nrp1a
2. itgb8