Figure_1.tif (4.58 MB)
Download file

A kinome-wide screen for modifiers of EGFR- and PI3K-dependent glial neoplasia.

Download (0 kB)
posted on 14.02.2013, 02:14 authored by Renee D. Read, Tim R. Fenton, German G. Gomez, Jill Wykosky, Scott R. Vandenberg, Ivan Babic, Akio Iwanami, Huijun Yang, Webster K. Cavenee, Paul S. Mischel, Frank B. Furnari, John B. Thomas

(A) Optical projections of whole brain-nerve cord complexes from late 3rd instar larvae approximately 130 hrs old, displayed at the same scale. Dorsal view; anterior up. GFP labels glia (green). Each brain is composed of 2 hemispheres and a nerve cord. Knockdown of strong suppressor loci decreased brain size, even relative to wild-type controls, as in CG11859dsRNA;dEGFRλ;dp110CAAX and CG11660dsRNA;dEGFRλ;dp110CAAX animals. Glial-specific overexpression of ΔEGFR drives increased glial cell numbers, brain enlargement, and lethality, and knockdown of strong suppressor loci grossly decreased brain size relative to controls, as in CG11859dsRNA;ΔEGFR and CG11660dsRNA;ΔEGFR animals. (B–H) 3 µm optical projections of brain hemispheres from late 3rd instar larvae approximately 130 hrs old, displayed at the same scale. Frontal sections, midway through brains. Anterior up; midline to left. Glial cell nuclei labeled with Repo (red); glial cell bodies labeled with GFP (green). Brains counter-stained with anti-HRP (blue), which reveals neuropil at high intensity and neuronal cell bodies at low intensity. Dark areas contain unstained neuronal precursor cells. dEGFRλ;dp110CAAX (C) and ΔEGFR (F) brains showed a dramatic increase in glial cell number (red nuclei, green) relative to wild-type (B). Upon suppression, as in CG11859dsRNA;dEGFRλ;dp110CAAX (D), CG11859dsRNA;ΔEGFR (G), and CG11660dsRNA;ΔEGFR (H), there are few excess glia (red nuclei), and remaining glial cells show abnormal development (green). Reduction in both glial (green) and neuronal cell types (low intensity blue) account for reduced brain size upon CG11660 and CG11859 knockdown in the context of dEGFRλ;dp110CAAX or ΔEGFR, which suggests that remaining abnormal glia do not properly support neuronal cell survival. Modifier constructs were also tested for effects in wild-type glia, as in CG11859dsRNA animals (E). (I) Breakdown of screen results by kinases tested. Unconfirmed modifiers are defined by only one RNAi construct each. (J) Functional classifications of confirmed modifiers. Individual kinases noted in Table S6.